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Biochemical studies on the expression of overdominance at the phosphoglucomutase-2 locus in the Pacific… Pogson, Grant H. 1988

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BIOCHEMICAL STUDIES ON THE EXPRESSION OF AT  THE PHOSPHOGLUCOMUTASE-2  OVERDOMINANCE  LOCUS  IN THE P A C I F I C OYSTER, CRASSOSTREA GIGAS  (THUNBERG)  by GRANT H. POGSON B.Sc.  Carleton  University,  Ottawa  1980  A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY in THE FACULTY OF GRADUATE STUDIES (Department o f Z o o l o g y ) We a c c e p t t h i s t h e s i s required  THE  as conforming standard  UNIVERSITY OF BRITISH December 1988 ©  G r a n t H. Pogson,  to the  COLUMBIA  1988  In  presenting this  degree at the  thesis  in  University of  partial  fulfilment  of  of  department  this thesis for or  by  his  or  requirements  British Columbia, I agree that the  freely available for reference and study. I further copying  the  representatives.  an advanced  Library shall make  it  agree that permission for extensive  scholarly purposes may be her  for  It  is  granted  by the  understood  that  head of copying  my or  publication of this thesis for financial gain shall not be allowed without my written permission.  Department  of  Zoology  The University of British Columbia Vancouver, Canada Date  DE-6 (2/88)  December 20, 1988  ii  ABSTRACT  Numerous  studies  between m u l t i p l e - l o c u s  have documented heterozygosity  in n a t u r a l populations, remain  unknown.  examine  the  mechanism  2 (Pqm-2)  merits  g e n o t y p e s were  of  confined  to  enzymic  because  displayed  of  allozymes,  frequencies  The  were  strict  the  mantle  most  extremely  its  of  ranges  seven of  detected  adult  the  locus the  between Pqm-2 these  Pqm-2-92  were  allele,  for  The  all  expression  was  considered  observed  incompatibility  Pqm-2  temperature  but  examined.  Pqm-2  the  variation  with  allelic  populations.  common  heterozygotes  u n u s u a l p r o p e r t y of  The m a g n i t u d e and  as  to  gigas.  intermediacy  l i m i t e d s c o p e of  and  in natural  three  activities.  the  the  was  phosphoglucomutase-  parameters,  and s t r u c t u r a l p r o p e r t i e s  study  Crassostrea  genotypes p o s s e s s i n g  displayed  patterns  hypothesis  properties  differences  m a r g i n a l overdominance at  between  the  structural  of  present  i n v o l v i n g the oyster,  these  traits  c o r r e l a t i o n between  examined over p h y s i o l o g i c a l  heterozygotes  unlikely  the  for  overdominance  Pacific  and  in a variety  functional  of  for a p o s i t i v e  Significant  genotypes  and  the  associations  and f i t n e s s - r e l a t e d  explanations  objective  of  i n the  kinetic  pH.  the  and h e t e r o z y g o s i t y  locus  The  largely  The  responsible  body w e i g h t  and  but  significant  adductor  of  this  muscle  at  the  Pqm-2  overdominant  o v e r d o m i n a n c e was tissues,  and was  locus enzyme  similar  in  consistently  observed three  i n p o p u l a t i o n samples  different  seasons.  exhibited but not  pathway  their  associated  that  variation  may  partitioning  affect  effect  activities  clearly observed for  of  glycogen  Pgm-2 l o c u s  evidence the  the  groups  differing The  its  i m p a c t on m a n t l e  gigas.  none  was  activity by  a  point.  the  PGM  that  of  could  concentrations  of  overdominance  glycogen  overdominance  l a r g e r body w e i g h t s of  It  activities  glycogen  expression  f a v o r i n g the  in Crassostrea  directly  between  but  between g e n o t y p e s .  c a u s e of  detected  s y n t h e s i s pathway enzymes,  and  were  synthesis  the  for  mantle,  levels.  and  account  genotypes  enzyme  glycogen  the  in their  -which  PGM a c t i v i t y  were  genotypic  PGM a c t i v i t y ,  the  glycogen  in  Pqm-2  glycogen,  glucose-6-phosphate branch  associations  Pgm-2  provided direct as  the  of  the  Pgm-2  genotype-dependent rates  at  of  tissues,  in t h e i r  Pgm-2  Non-random  adjacent  muscle  with v a r i a t i o n  suggested  functions.  concentrations  adductor  i m p a c t of  on t h e m e t a b o l i s m of  i n w h i c h PGM  different  intertidal positions  A physiological  p o l y m o r p h i s m was d e m o n s t r a t e d biochemical  f r o m two  levels,  explanation  h e t e r o z y g o t e s at  the  iv  TABLE OF CONTENTS  ABSTRACT  i i  L I S T OF TABLES  vi  L I S T OF FIGURES  .  ix  ACKNOWLEDGEMENTS CHAPTER  1:  .'  GENERAL INTRODUCTION  xi 1  The  Study Animal  8  The  S t u d y Enzyme  11  Format CHAPTER 2:  of  the  Thesis  14  BIOCHEMICAL CHARACTERIZATION OF PGM-2 GENOTYPES  INTRODUCTION  .15 .15  MATERIALS AND METHODS  21  RESULTS  33  Electrophoresis Biochemical  and Pqm-2 A l l e l e  Properties  of  Frequencies  Pqm-2 G e n o t y p e s  DISCUSSION CHAPTER 3:  33 41 71  ENVIRONMENTAL AND GENOTYPIC EFFECTS ON PGM  ACTIVITY  87  INTRODUCTION  87  MATERIALS AND METHODS  92  RESULTS  95  Effects Effect  of of  Season and I n t e r t i d a l Pqm-2 G e n o t y p e  Predicted Effects DISCUSSION  of  a Pgm-2 N u l l  Position >  99 103  Allele  115 121  CHAPTER 4:  PHYSIOLOGICAL EFFECTS OF THE PGM-2 LOCUS ON  GLYCOGEN METABOLISM  140  INTRODUCTION  140  MATERIALS AND METHODS  145  RESULTS  149  Effects Effect  of of  Season and I n t e r t i d a l  Position  153  Pqm-2 G e n o t y p e  157  DISCUSSION CHAPTER 5:  171  A C T I V I T Y STRUCTURE OF THE GLYCOGEN SYNTHESIS  PATHWAY  195  INTRODUCTION  195  MATERIALS AND METHODS  198  RESULTS  -  202  Electrophoresis  202  Correlations  203  between Pathway Enzyme A c t i v i t i e s  Pathway A c t i v i t i e s Multiple  Regression  of  Pqm-2 G e n o t y p i c G r o u p s  Analyses  DISCUSSION CHAPTER 6:  GENERAL DISCUSSION  LITERATURE CITED  206 218 223 236 ..248  vi  L I S T OF TABLES  Table  I.  Pqm-2 a l l e l e  Weinberg the  II.  Table  III.  conformity  and h e t e r o z y g o t e  Effect of  IV.  of  summary f o r temperature  Pgm-2-104  on  the  at  allozyme  ..42  Vmax(f)/Vmax(r)  f o u r Pgm-2 h o m o z y g o t e s  58  specific  activity,  PGM a c t i v i t y / g  protein  extracted/g  analyses  tissue  of  variance  tissue,  i n the  on  and  PGM  soluble  m a n t l e and a d d u c t o r  tissues  V.  97  Seasonal  activities Table  deficiencies  the  from  Table  Hardy-  ....39  F-ratios  muscle  to  sampling dates  Purification  ratios Table  expectations,  four  Table  frequencies,  VI.  specific  variation  (units/mg  in  protein)  Seasonal  variation  activities  (units/mg  of  the  mantle  seven  Pgm-2 g e n o t y p e s  in  the  protein)  specific  adductor of  seven  .104  muscle Pqm-2  genotypes Table  106  VII.  Decomposition  (units/mg protein) protein  levels  into  of  Pqm-2  enzyme  expressed  on  specific  activities  activities  and  soluble  a gram wet  tissue  basis Table  .110  VIII.  protein (mg/g  Comparison and  tissue)  possessing Table  weight  IX.  units/g of  of  enzyme  tissue)  homozygote  or l a c k i n g t h e Predicted  activities  and s o l u b l e and  reductions  protein  heterozygote  Pgm-2-100 of  (units/mg levels classes  allele enzyme  113 activities  (units/mg (mg/g null Table  and u n i t s / g  tissue) allele  X.  locus  Table  i n Pgm-2  is  of  polymorphism  XI.  locus  concentrations  XII.  on  the  glucosyl  units/g  Table  XIII.  position  117  Pgm-2 s t r u c t u r a l tightly-linked  of  132  v a r i a n c e on  activities  glycogen  i n the  mantle  tissues  151  effects mantle  of  season  glycogen  tissue)  Combined on t h e  heterozygote  of  and  intertidal  concentrations  Pgm-2  (jxmoles  homozygote  and 163  effects  of  adductor muscle units/g  tissue)  season  and i n t e r t i d a l  glycogen  concentrations  of  Pgm-2 homozygote  classes  and  and ...167  Table XIV. Glycogen concentrations  homozygotes  a  classes  (Mmoles g l u c o s y l  tissue)  assuming  two a l l e l e s  from a n a l y s e s  Combined  heterozygote  for  levels  0.044  hypothetical  and PGM s p e c i f i c  position  of  multi-allelic a  protein  classes  a frequency  the by  and a d d u c t o r m u s c l e Table  at  segregating  F-ratios  and s o l u b l e  homozygote  present  Collapse  regulatory  tissue)  specific  (Mmoles g l u c o s y l  activities  and h e t e r o z y g o t e s  (units/mg  p o s s e s s i n g or  units/g  protein) lacking  of the  Pqm-2-1 00 a l l e l e Table four  XV.  Kinetic  homozygotes  169 parameters and t h e  i n the  three  mantle  tissues  heterozygotes  for  of  the  t h e Pqm  -2-100 a l l e l e  178  T a b l e X V I . Product-moment c o r r e l a t i o n c o e f f i c i e n t s the  activities  pathway  of  enzymes  in  the  glycogen  between synthesis 204  viii  Table XVII.  F - r a t i o s from t h e  mantle  activities  enzymes  and m a n t l e  Table XVIII. PGM, the  and  high Table the  variance  on  the  synthesis  pathway  concentrations soluble  207  protein)  f o u r Pqm-2 g e n o t y p i c  of  HK,  classes  in  i n t e r t i d a l zone  and GS o f  210  (units/g  f o u r Pqm-2  soluble  protein)  genotypic  of  classes  HK,  PGM,  in  the  i n t e r t i d a l zone XX. R e s u l t s glycogen  glycogen  levels  glycogen  glycogen  212  from t h e m u l t i p l e  synthesis  Table XXI. Results the  of  glycogen  (units/g  GS o f  Table XIX. A c t i v i t i e s UDPGP,  the  glycogen  Activities  UDPGP, low  of  analyses  levels  i n the  pathway low  regression enzyme  activities  i n the  of on  i n t e r t i d a l sample  from t h e m u l t i p l e r e g r e s s i o n synthesis  analysis  pathway  enzyme  219 analysis  of  activities  on  h i g h i n t e r t i d a l sample  221  L I S T OF FIGURES  Figure  1.  under Figure  Electrophoretic (A)  2.  s t a n d a r d and Effect  of  (in  MM)  constants half)  staining  and  (B)  catalytic  temperature for  patterns  of  oyster  PGM  running conditions  on t h e  apparent  glucose-1-phosphate  glucose-1,6-diphosphate  (2;  Michaelis (1;  lower  upper  half)  of  s e v e n Pqm-2 g e n o t y p e s Figure (in  3.  Effect  MM)  of  for  .35  ...45  pH on t h e  apparent  glucose-1-phosphate  glucose-1,6-diphosphate  (2;  lower  Michaelis  constants  (1;  half)  half)  upper of  seven  and  Pqm-2  genotypes Figure  4.  49 Effect  s e v e n Pgm-2  of  temperature  genotypes  for  the  on t h e  Vmax/Km r a t i o s  forward r e a c t i o n  of  direction 54  Figure  5.  Effect  genotypes Figure  6.  of  for  the  Figure at Figure  7.  forward r e a c t i o n  four  of  seven  Pgm-2  direction (in  .....56  MM) f o r  Pqm-2 h o m o z y g o t e s e s t i m a t e d  glucosefrom  equation  the ...61  PH-dependent  activities  of  four  Pqm-2  homozygotes  20°C 8.  64 Thermal  genotypes at Figure  Vmax/Km r a t i o s  Apparent M i c h a e l i s constants  6-phosphate of Haldane  pH on t h e  9.  Effect  inactivation  plots  of  seven  Pqm-2  50°C of  Pqm-2 h o m o z y g o t e s  67 magnesium  i o n on t h e  activities  of  four 69  X  Figure  10.  Effect  apparent  of  Michaelis  phosphate  (1;  ratios  lower  Figure  temperature  (2;  11.  constants  upper h a l f ) half)  Effect  of  half) Figure  of  12.  Figure  Figure  at  and  and s o l u b l e  Vmax/K'm  rn v i v o  75 apparent  glucose-1-phosphate (2;  (1; lower  the  two  concentrations  Pqm-2 homozygote  flux  PGI/PGM a c t i v i t y  protein), protein  position PGM  (mg/g  on  activity  tissue)  in  tissues  100  mantle  and a d d u c t o r  (/nmoles g l u c o s y l  v a r i a t i o n i n the  Predicted flux  percent  intertidal  units/g  intertidal positions  Seasonal  of  77  v a r i a t i o n i n the  concentrations  glycogen  15.  and  and a d d u c t o r m u s c l e  glycogen  tissue)  glucose-1-  corresponding  estimated  jiM) f o r  (units/mg  Seasonal  14.  muscle  for  vivo  s e v e n Pqm-2 g e n o t y p e s  season  tissue),  13.  tissue) Figure  of  activity  mantle  muscle  MM)  in  seven Pqm-2 g e n o t y p e s  specific  the  estimated  and c o r r e s p o n d i n g Vmax/K'm r a t i o s  Effect  (units/g  (in  (in  and  pH on t h e  Michaelis constants upper h a l f )  of  on t h e  to ratio  154  mantle  (jxmoles  glucosyl  and h e t e r o z y g o t e  advantage  glycogen  as  of  Pgm-2 a  and a d d u c t o r units/g  classes  ...159  heterozygotes  function  of  the ....184  xi  ACKNOWLEDGEMENTS  I to  his  would l i k e oyster  to  thank  l e a s e and t o  assistance  in  Devlin,  Matsumoto,  on  E.  the  design  the  permitting welcoming  the me  members  stages  of  my  particular like  to  to  order  use  of  his  research. B.  Devlin,  I would l i k e  Operating  continued  Columbia to  group.  to  wish Suarez,  acknowledge  to  C.F.  for  their  Mommsen, B .  valuable  advice  experimental  kinetic  facilities,  data. kindly  and  for  Numerous s t u d e n t s during  C.F.  the  Schluter.  I  Wehrhahn, f o r  throughout financial Scholarship, and  my  and  various  e x p r e s s my g r a t i t u d e  Fellowships,  Wehrhahn.  access  Hochachka f o r  and D .  Postgraduate  Graduate  T.  suggestions  encouragement  a N.S.E.R.C.  grants  P.W.  thank my s u p e r v i s o r ,  Finally,  British  R.  Mesa  enzyme  laboratory  I  providing  and on the  the  to  valuable  and  of  of  offered  K.  provided  PGM,  research  for  samples.  Hall  his  advice  by  analysis  and  of  oyster  in  especially  provided  Tamm  are  into  faculty  S.  and J . G . of  and s t a t i s t i c a l thanks  MacClelland  collection  purification  Special  P.  in  would his  study.  assistance University N.S.E.R.C.  1  CHAPTER 1  GENERAL  The of  evolutionary  variation  among  variation  between  temporally  (Lewontin  all  evolutionary  existence  of  and  (1982),  (1859)  1974,  the  "populational"  p.  has,  naturally-occurring  played  genetic  always  be,  All intra-  is  a principal  occur.  concerns  the  an  agent  synthetic  the  that of  theories  A major source  relative of  the  its  that  d e p e n d on  constitute  pioneered  "typological"  with a  the  importance  of  this  of  the  life.  The c r u c i a l  role  study  has  one  of  role  this  been,  on t h e  evolutionary  but d i f f e r  and  presence  of  these  controversies  Darwinian natural process.  new will  i n how  and p r e s e n t  was  by  Darwin  rely directly  past  the  by  studies.  of  the  physiological,  evolutionary  i m p o r t a n c e of  period a significant  the  adoption  inter-populational variation,  changes  as  focus  in  to  mechanism,  i n d i v i d u a l s as  biological  variation  evolutionary  or  of  a prime reason  a  recognized  v a r i a t i o n between  characteristics  perspective  of  of  and  As a r g u e d e l o q u e n t l y  revolution  that  fundamental by  species.  into  both s p a t i a l l y  continue,  attributes  conversion  population  affecting  replacement  world-view  the  Irrespective  variation  conceptual  as  a  and s p e c i e s ,  and w i l l  biological  the  within  12).  behavioral of  involved  may be d e f i n e d  populations  change  distinctiveness Mayr  process  individuals  genetic  morphological  INTRODUCTION  Early  attributed  selection i n the to  neo-  random  2  genetic  drift  adaptive  differences  (e.g. in  writings  time  from  a  of  (e.g.  p o i n t e d out  the  Wright  pluralistic  A  over  way  to  allow  has  of  1944).  these  Gould  reflected  (1983)  same a u t h o r s  of  in  the  1986).  Only  shifted  the  work of  Sewall  recently  alternative  of  modern  G o u l d and L e w o n t i n 1979) of  at has  interpretation  "hardening  (Provine  were  n e o - D a r w i n i s t movement  to disagreements evolutionary on  termed the  that  natural  selection  has given  modes  " c l a s s i c a l " and t h e Proponents  of  function,  by r e m o v i n g d e l e t e r i o u s  mutants  contrast,  the  that  selection  is  b a l a n c e d view actively  through various  overdominance,  holds  maintain t y p e s of  frequency-dependent  t i m e and s p a c e ) .  relative  another  of  the  long-standing in natural  opposing  views  "balanced" hypotheses the  classical  serves from  a  balancing  selection, is  school  "purifying"  populations.  major r o l e  genetic  Genie h e t e r o z y g o s i t y  importance  operates  summarized t h e s e  mainly  to  its  process,  how  D o b z h a n s k y (1955)  over  selection  over  races  sentiments  occurred  consideration  population structure.  populations  geographic  adaptationist  he t e r m e d t h e trend  non-  and  Simpson  a strict  centered  populations.  contend  the  i n t e r p r e t e d as  change.  addition  what he  of  paradigm (cf.  the  d i r e c t i n g the  dispute  to  same p e r i o d  adaptationist  In  These  publications  similar  this  evolutionary  founders  that  the  of  the  then  subspecies  D o b z h a n s k y 1937;  same p a t t e r n s  in  between  how l a t e r  synthesis".  in  p r o d u c i n g what were  Robson a n d R i c h a r d s 1 9 3 6 ) .  the  this  in  of  In  natural  variation within selection variable thus  (e.g.  selection  viewed  by  the  3  "balanced"  school  as  adaptive  environmental  circumstances),  predicts  heterozygosity  to  that  population  the  (1974)  adaptive  polymorphism  on t h e  but  the  of  has  population  isozymes)  in  protein  is  (given  "classical"  the  the  current  high  levels  populations,  simply  a  of  molecular selection  level.  heated  of  and s e l e c t i v e  in  study  f o r many y e a r s  et  al.  Nei  Wills  1981;  1985).  A review  1975;  Kimura of  (see Ewens  1977;  1983;  the  Nei  evidence  that  against  various neutral predictions  Suffice  it  to  say  that  approaches  controversy,  since  robust  to  have  not  based  1983).  to  has  1983;  been  will  not  (cf.  of  Nevo  the  McDonald  b o t h n e u t r a l and  selection  of  areas Ayala 1978;  O h t a and A o k i for  be p r e s e n t e d  virtually  of  allelic  garnered  successful  for  fields  predictions  1978;  of  Neutral  (i.e.  been  account  various  i n L e w o n t i n 1974;  Koehn  indirect  Arguments  Comparisons  Gillespie and  this  has d o m i n a t e d t h e s e  discussions  by  of  the  allozymic  models  uncovered  (Kimura  genetics.  distributions  enzyme  form a c o r n e r s t o n e  discussion  evolutionary  of  sufficiently  little  of  in determining  molecular evolution  neutral  statistical  school  controversy  continuation  v a r i a t i o n w i t h i n and between s p e c i e s  1974;  specific  and c o n t r i b u t e s  s t r u c t u r e and f u n c t i o n  stimulated  and  that  of  the  purifying  various  this  the  transient  natural  now waged a t  and  quantities  is  argued  procedures,  n e u t r a l theory  theory  whereas  significance  i m p o r t a n c e of  aspects  has  observed  electrophoretic dispute,  stable  adaptedness.  Lewontin over  and  1983) in  here. or  resolving  theory any  and  are  observed  4  distribution  of  a l l e l i c polymorphism.  An a l t e r n a t i v e period  research strategy  i n v o l v e d the  allozymic  d i r e c t measurement  variation  through  and p h y s i o l o g i c a l a t t r i b u t e s their to  interactions  fitness  the  this  differences  evolutionary  blue  to  has  (cf.  (see  Mytilus  Hilbish  killifish, 1984b;  Swan  and  and P l a c e  in 1983;  1985b).  Colias Watt,  The  (Koehn,  Powers  1983),  Carter  linking  "character  states"  exposed  the  (cf.  selective  of  discipline  been  the  (Watt  1985; these  allelic  Lewontin process.  in  1981;  1983;  Hilbish,  1979,  in  the  1984a,  Watt,  isomerase Cassin  reviewed  i n Watt  studies  lie  that  1980;  r e v i e w e d by P o w e r s ,  variation  1972)  the  r e v i e w e d by Koehn  phosphoglucose  1977,  of  successfully  locus  and Powers  1982b;  of  theory,  dehydrogenase-B locus  1982a,  of  1985;  rise  Although  neutral  Siebenaller  and B l o w e r  strength  1978).  N e w e l l and Immerman  (Place  and  through  may g i v e  separate  and Koehn  on  significance  Koehn  and has  same  biochemical  which,  aminopeptidase-1  lactate  butterflies  great  mechanistic  to  the  a  the  selection the  v a l i d i t y of  1985a)  Fundulus h e t e r o c l i t u s  DiMichele  DiMichele locus  1987),  the  Koehn and  Hilbish  adaptive  1975;  into  the  edulis  1982;  the  Clarke  Watt  polymorphisms at  D e a t o n and Koehn  comparison of  to  developed  genetics  mussel,  over  natural  enzyme g e n o t y p e s  relevant  Koehn and Immerman 1981;  and  of  i n i t i a l l y with testing  approach  applied  the  of  evolved  with environmental v a r i a b l e s ,  polymorphism s t u d i e d  concerned  that  onto may  in  and  1985a, their  phenotypic in  turn  be  5  Examination phenotypic  their  (usually  to  7)  at  classes.  procedures.  Following this  organisms 1987). in  examined  by M i t t o n  These a s s o c i a t i o n s bivalves. has  been  Green  and S c o t t 1985),  1988),  and  correlated 1982;  et  nutritive  The  viability  with rates  of  (Zouros et  Koehn and S c o t t  (Hawkins, stress  (Rodhouse  is  consumption Diehl  and G a f f n e y  that  of  these  1984;  1983;  al.  Bayne and Day 1 9 8 6 ) ,  underlying cause(s)  One p o s s i b i l i t y  al.  et  1984;  and  Foltz  documented  and  Fujio  Koehn,  Diehl  al.  and  and  (Koehn  rate  1980;  Diehl  1986),  et  of  multiple-locus  Miles  Koehn and G a f f n e y  oxygen  diversity  extensively  and  of  between  c o r r e l a t e d w i t h growth  Singh  (Rodhouse  number  Zouros  organisms,  positively  1983;  fecundity  Garton,  turnover  al.  these  regression  morphological,  i n a wide  been most  Zouros,  large  1984;  of  physiological  linear  a  loci  degree  correlations  traits  scored  discrete  the  different  and G r a n t  of  and  standard  and  have  In  ( S i n g h a n d Z o u r o s 1978; 1982;  between  protocol,  and f i t n e s s - r e l a t e d  heterozygosity  series  significant  heterozygosity  (reviewed  marine  by  basic  demonstrated  physiological  a  on  consider  electrophoretic  and v a r i o u s p h e n o t y p i c  then  multiple-locus  into  to  i n d i v i d u a l s are  Relationships  are  have  polymorphisms  expanded  studies,  pooled  parameters  studies  enzyme  a s m a l l number of  and  heterozygosity  of  recently  In t h e s e  genotypes 5  impact  has  effects.  heterozygosity enzyme  the  characters  multiple-locus for  of  negatively  and  Shumway  1985),  weight  Koehn  protein  loss  under  1984).  relationships  heterozygotes at  these  enzyme  is  unknown. loci  are  6  functionally  superior  overdominant pooled  together  phenotype. are  deleterious  the  locus  in  a  homozygotes  for  within  more  loci  alleles exists  between  homozygous,  groups  Therefore,  detrimental phenotypic  homozygote  at  but  no  heterozygosity  per  se.  A  related  inbreeding,  result  Longwell  and  is this  in decreased Stiles  simply  the  heterozygote feature  of  by  overdominance). loci, are  not  the  species larval  loci  in the  marine at  in  is  expected  patterns  by  a r e c a u s e d by  v i a b i l i t y and  has  growth  been (e.g.  According  to  homozygotes  inbreeding depression. Obviously,  the  i n the  examined.  multiple-locus  enzyme  of  be  heterozygous,  of m a r i n e b i v a l v e s  et  to  w o u l d be m a n i f e s t e d  the  Beattie  If  a proportion  expected  reduced performance of  deficiencies  their  for  1987).  requires  met  segregating  al.  coefficients  certainly  are  reflected  that  correlations but  advantage  is  1973;  a manifestation  hypothesis  are  buffered  themselves,  alleles  effects  when  loci  these  but  and  these  electrophoretic  direct  hypothesis  explanation,  inbreeding is  that  which i n s e v e r a l  shown t o  this  the  classes  relationship,  that  that  these d e l e t e r i o u s the  is  The  and hence  efficient  (associative  genotypic  the  are a d d i t i v e ,  explanation  tightly-linked recessive  (overdominance).  electrophoretic  disequilibrium  present  homozygotes  each  result  by  by  linkage  at  An a l t e r n a t i v e  not c a u s e d  instead  of  effects  to  expression  of  significant  study p o p u l a t i o n . This c o r o l l a r y bivalves,  in  electrophoretic  population stuctures,  which loci  particularly  marked  a r e a common at  the  early  7  post-settlement Green  1984).  are caused  stage  A final  by t h e  non-functional studied.  deficiencies,  and 2)  contain  of  the  simultaneously  large  number  credibility selective  of  species  prove  the  theory.  hypotheses  of  considering  is  studies. also  of  valuable  overdominance, expressed  The  at  the  traits  large  Distinguishing importance  associative  loci  examined  in these  of  my  study  for  was  of  the  a the  potential  variation diversity  between for  is of  these  selective  improvement  of  greatly  if  differ  than  responsible  the  the  of  side,  would s t r a i n  genetic  would  a matter  are  this  rather  objective  is  multiple-  significance  that  vital  would  theoretical  loci  breeding programs. Breeding methodologies commercially  classes  heterozygotes.  Understanding  maintenance  in these  heterozygotes  between  functional  neutral  relevant,  heterozygote  of  On t h e  the  the  the  traits  electrophoretic  (producing  as  relationships  importance.  they  heterozygotes  null  and f i t n e s s - r e l a t e d  that  loci  b e c a u s e homozygote  c a u s e of  and  electrophoretic  1)  low a c t i v i t y  Singh is  alleles  properties  polymorphisms  involved  alternative  for  enzyme  for  null  the  of  basis  particularly  of  null  account  these  the  1984;  these patterns  at  superior  and p r a c t i c a l that  Foltz  of  alleles,  heterozygosity  demonstrating could  products)  the  a percentage  theoretical  for  presence  thus  functional  Establishing locus  and  misclassification could  two  explanation  enzyme  The  Zouros  undetected  homozygotes  for  (see  overdominance,  was  studies.  to  determine  if  evidence  8  favoring  the  overdominance h y p o t h e s i s  enzyme  locus  relationship  involved  through  physiological genotypes. the  an  to  oyster,  a  multiple-locus  examination  properties  I chose  Pacific  in  c o u l d be o b t a i n e d  of  study  of  the  an  heterozygosity biochemical  homozygous  the  for  and  and  heterozygous  phosphoglucomutase-2  locus  in  Crassostrea qigas.  THE STUDY ANIMAL  The  Japanese,  (Thunberg) western the  is  southern  where  has  1971).  This  factor has  and  its  oyster  or h i g h e r )  region  (Quayle  private  oyster  side  of  Columbia. stock,  geographic and  the  from C h i n a  Japanese  to  archipelago  the  century for commercial purposes  of  established  in  state  isolated (Quayle  c o l o n i z a t i o n of spawn  1969).  i n Nanoose 20  Originally  established  Bay, km from from  situated  on  of  r e p r o d u c t i o n of  lease the  qigas  geographic l o c a t e d on a the  Sound is  in  temperatures  Nanaimo,  Pendrell this  by C .  this  p o p u l a t i o n was  north  British  Reproduction  in  of  The p r i n c i p a l  warmer summer w a t e r  study  production  of  new h a b i t a t  experienced  The  west c o a s t  pockets  successfully.  requires  natural  this  the  the  turn  Island  the  range extends  to  i n t r o d u c e d to  Vancouver  supplementing  native  gigas  was  1969).  continuous  species  includes  than u s u a l l y  lease  Crassostrea  species  i n a b i l i t y to  Pacific  (21°C  the  oyster,  intertidal  Washington  limiting  been  the  the  become w e l l  Columbia  its  U.S.S.R.,  North America at and  Pacific  a temperate,  Pacific  (Stenzel  or  eastern British breeding  dependent  bay w i t h s e e d  on  from  9  local  suppliers  Sex  (P.  MacClelland,  determination  oysters,  is  complex,  environmental  influences  1983).  The  sexes are  prevailing  pattern  to  females  and  after  the  larvae  generally  as  they  and  begin  related  virqinica,  C.  the  and Q u a y l e  The P a c i f i c surviving  for  The  metabolic  de  is  undergo  stage  of  may  frequencies.  but  this be  adult  species,  f o u n d i n Yonge  externally, 30  they life.  and  is  switching  15 t o  which  sessile  The  genus C r a s s o s t r e a  as m a l e s  to  Buroker  hermaphroditic  r a n g i n g from  substrate  oyster  is  prolonged of  a facultative periods marine  in  days,  adhere, Excellent  the  closely  (1960),  Galtsoff  the  a c c o m p l i s h e d by 2 0 - f o l d and t h e  utilization  Virtually  pronounced  all  seasonal  anaerobe,  bivalves  ATP by s u b s t r a t e - l e v e l  Zwaan 1 9 8 3 ) .  life  and  (1969).  ability  rates  generating  i n the  1977;  but  low  other  genetic  Haley  at  in  F e r t i l i z a t i o n occurs  their  biology  as  both  separate,  change  a suitable  of  conditions  sex  a planktonic larval select  to  detected  grow o l d e r .  descriptions  oxygen.  usually  gigas,  1964;  young a n i m a l s b e g i n n i n g  metamorphose,  (1964),  (Galtsoff  of  communication).  Crassostrea responding  are  individuals  protandrous;  in  personal  complete to  novel metabolic  of  metabolism  fluctuations  with p r e v a i l i n g a b i o t i c  conditions,  and t h e  r e p r o d u c t i o n (reviewed  annual c y c l e  of  the  pathways  in C.  availability  of  basal  (reviewed  which are  of  anoxic  in their  phosphorylations  aspects  absence  tolerate  reductions  of  capable  by  gigas  integrated of  food,  by G a b b o t t  1983).  10  A  dominant  seasonal  reproduction, glycogen. species  fall  involves  The  gametogenesis 1969).  on  be  prior  differences  between  central  role  played  m e t a b o l i s m of  oysters  generally  Crassostrea  gigas  variation.  In  favoring  a  viability  has  C.  net been  gigas.  These  and F u j i n o  1978),  unidentified  by  (Gabbott  other  (1985),  were  catalase  for  (Quayle glycogen  of  Fitness-related  turn  be  levels  expressed  because  in  the  marine  of  the  energy  an a v e r a g e  19.4%.  of  on of  this  species  53%  of  and  for  the  the mean  evidence  growth  and/or  polymorphic l o c i  aminopeptidase  (Nagaya,  Nakamura and S u g i t a  (Buroker  allozymic  Circumstantial  f o r a number of  (Fujio,  invertebrates,  levels  studies  advantage  leucine  protein  summer  abilities  polymorphic  was  heterozygote  the  1975).  substantial  individual  gigas,  Pgm-2 p o l y m o r p h i s m  carbohydrate  electrophoretic  include  in  glycogen  for  and F u j i o  muscle  may  of  gland in  in  degrade g l y c o g e n .  this  suggested  the  to  different  and A u g u s t  differential  on t i s s u e  examined per  or  possesses  five  loci  the  July  of  tied  C.  early  functions  effect  found  summarized by O z a k i  heterozygosity  in late  Pqm-2 g e n o t y p e s  effects  In  m a n t l e and d i g e s t i v e  spawning  synthesize  between  season.  degraded i n the  through  these  enzyme  the  closely  degradation  varies  spawning  physiological  through  As  in  and  cycle  phosphoglucomutase  a  to  this  their  to  expressed  genotypes  synthesis  s p r i n g and i s  Since  metabolism, must  of  accumulates  and e a r l y  in marine b i v a l v e s ,  the  timing  depending  glycogen  cycle  1979),  and  in  Sasaki  1979),  an  aspartate  11  aminotransferase  (Sugita  relationships  not  However, Fujio  have  after  (1982)  to  higher  five  was  greater  populations. out  been e x t e n s i v e l y  pooling data  at  locus  exhibited  Fujio  enzyme  involved  if  growth r a t e s  loci  positive  in this  results  Pqm-2  in  C.  of  body  between  weight.  relationship;  Fujio's  gigas.  populations,  correlation  and a d u l t  o v e r d o m i n a n c e was of  Multiple-locus  studied  body w e i g h t s t h a n homozygotes  B a s e d on t h e  determine  1982).  from 20 w i l d J a p a n e s e  observed a s i g n i f i c a n t  heterozygosity Pgm-2  and  The  heterozygotes  in  18 of  (1982)  the  study,  indeed r e s p o n s i b l e  20  I  set  for  the  heterozygotes.  THE STUDY ENZYME  Phosphoglucomutase phosphate: catalyzes  the  and magnesium functional a  i n t e r c o n v e r s i o n of  rabbit  properties  muscle  enzyme's  been  Pullman"  (1954)  phosphorylated  that  inactive  complete  determined first  of  by  of  from  molecular  s e q u e n c e of Ray  et  enzyme,  glucose-1,6-diphosphate's  al.  that  561  from  daltons.  amino a c i d s  (1983). PGM may  Najjar exist  representing  f u n c t i o n was  The to  and  ( 1 9 7 2 ) . PGM  weights  respectively.  sole  (G1P) and  stuctural  62,000-67,000  demonstrated  the  its  f o u n d i n Ray and Peck  and d e p h o s p h o r y l a t e d s t a t e s ,  forms of  phosphotransferase)  glucose-1,6-diphosphate  review  exhibiting  ranging  alpha-D-glucose-1 -  glucose-1-phosphate  in presence  may be  enzyme,  organisms  recently  and  (G6P)  i o n . A comprehensive  monomeric  different  E . C . 2.7.5.1,  alpha-D-glucose-1,6-diphosphate  glucose-6-phosphate  is  (PGM,  The has and in  active  discovery convert  the  12  dephospho PGM's  into  the  reaction  phosphoenzyme  mechanism.  the  dephosphoenzyme  20  catalytic  approximates (1966)  Ray and R o s c e l l i  hence,  PGM was  surrounding  (1964a)  showed  formed  once  reaction  in  a " u n i - u n i " mechanism.  However,  Hanabusa  PGM e x t r a c t e d the  central  from b a c t e r i a  diphosphate  reaction  dissociated  complex,  p r o d u c t " and t h e  "second  Cleland  Similar patterns  (1963).  efficient  phosphates  uncertainty  the  pong" m e c h a n i s m :  less  to  r a b b i t muscle  cycles,  found that  enzyme's  of  led  substrate"  interconversion  by  PGM  phosphoglucomutase depending  involved,  and t h e  i n the  have  on  along  its  5-  et a  particular  assay  acting  as  et  al.  and  from  the  "first  1969).  conditions  by  during  6-carbon  origin,  the  proposed  described  continuum  phylogenetic  species  frequently  al.  every  displayed a "ping-  scheme  been  other  (Passonneau  functions  extremes  of  thus  this  that  the  sugar  Therefore,  between the  these  substrates  (Ray  and  Peck  1972).  The  reaction  equilibrium 1-phosphate Because  of  catalyzed  constant to  strongly  flux  its  reversibility,  PGM's  of  metabolic  activities  are  under  synthetase,  glycogen  role  conversion  is  Lacking to  any  of  for  but  regulatory  =  17.2).  in both  the  regulatory  enzymes  control:  to  whose  glycogen  and p h o s p h o f r u c t o k i n a s e . PGM's c a t a l y t i c  its  glucose-  respond e f f i c i e n t l y  closely-positioned  stringent  relevant  the  reversible,  PGM p a r t i c i p a t e s  phosphorylase,  p a r a m e t e r most  freely  (Keq = [ G 6 P ] / [ G 1 P ]  glycogen.  r a t e s d e t e r m i n e d by t h r e e  enzymic  favors  glucose-6-phosphate  s y n t h e s i s and d e g r a d a t i o n properties,  by PGM i s  function  The is  13  its in  Vmax/Km r a t i o , vivo  (see  (Hoffman  1981;  between  Pqm-2 to  Watt  from  1984).  anemone,  limited  thus  enzyme all  in  studies  organisms. of  marine  larger  and  only  al.  1985).  and has  et  for  this  al.  Pgm  enzyme  Pqm-2 l o c u s  genetic (see  characterized 1979)  The  and  the  biochemical  for  the  were  adaptive  a h i g h l y polymorphic included in v i r t u a l l y in  these  locus-specific  comparisons  detected  significant  The  in other could  in C. gigas  heterozygotes  most  in both species  genotypes  1985).  are  organisms  heterozygosity  studies,  differences  body w e i g h t s of  been  heterozygotes  between  Diehl  the  et  support  these  of  been  p o l y m o r p h i s m . PGM i s  bivalves,  two o f  biochemical at  little  In  1984;  variants  (Fucci  (Hoffman  provided  differences  ratios.  PGM have  multiple-locus  heterozygosity that  of  Vmax levels  p o l y m o r p h i c enzyme,  involving  differences  activity  significance  between Pgm g e n o t y p e s  either  homozygotes  Gaffney  isozymes  senile  detected  of  a widely  saturated  nature  functional  physiological  melanoqaster  differences  significance  steady-state  reported in a d i v e r s i t y  Allelic  Metridium  and  is  be  composite  t h r o u g h t h e s e Vmax/Km  been  Drosophila  u n l i k e l y to  its  Therefore,  g e n o t y p e s of  having  is  and v i a  1983).  Phosphoglucomutase  Gauldie  enzyme  incorporates  be e x p r e s s e d  variants  the  A t k i n s o n 1977),  simultaneously  likely  since  (e.g.  Rodhouse  significant  and  effects  of  bivalve  species  suggests  exist  between  allelic  that  may a c c o u n t  r e p o r t e d by F u j i o  for  the  (1982).  1 4  FORMAT OF THE THESIS  The t h e s i s  has been o r g a n i z e d i n t o  chapters.  Chapter  2  properties  of  Pqm-2  mantle  and  genotypes from t h e  adductor  muscle  metabolism  is  effects the  s t r u c t u r e of  the  of  synthesis  findings locus  of  of  glycogen.  the  4.  Chapter  of  in three  5  implications is  presented  Pgm-2 seasons of  on  the  glycogen  pathway t o  the  the  analyzes  Pqm-2  A general discussion  relationships  of  assessment  synthesis  effects  each c h a p t e r and t h e i r  heterozygosity  compares  activities  An  discrete  and b i o c h e m i c a l  polymorphism  Chapter  glycogen  more d e t a i l e d e x a m i n a t i o n o f the  this  of  3  positions  population. of  subject  Chapter  specific  intertidal  Nanoose Bay s t u d y  series  the p u r i f i c a t i o n  genotypes.  s a m p l e d from two  physiological  activity  seven  presents  a  of  for  the  allow a  locus the  on  major  multiple-  in Chapter  6.  15  CHAPTER 2  BIOCHEMICAL CHARACTERIZATION OF PGM-2 GENOTYPES  INTRODUCTION  Overdominance, controversial  or heterozygote  explanation  variation  in  formulation  by F i s h e r  peaked of  i n the  1951),  British After  the  the  his  genetics  al.  in  have  1974);  inherent  the  frequency  polymorphic l o c i  haploids  has  the  high  s u c h as  difficulties  e m p i r i c a l work  (e.g.  D o b z h a n s k y and L e v e n e  Lerner  of  the  (1954),  of  the  of  large  Lewontin  in  of  played  depressions p.  variation  establishing  and  by  Evidence  estimates  1974,  Levin  stable  and-  from p r e d i c t e d  Maruyama  Selander  1965).  allozymic  in  (e.g. fitness  207);  electrophoretic  and  the  experimental  role  viability  genetic  (e.g.  of  balanced polymorphisms.  Yamazaki of  and  (Ford  patterns  a variety  distributions  levels E. coli  through the  l e d by E . B . F o r d  unobserved  (e.g.  theoretical overdominance  included: discrepancies  (e.g.  genetic  of  discredited  maintaining  upon i n b r e e d i n g  asymmetrical  1976);  school  remained a  of  its  popularity  and e x a m i n a t i o n  studies  since  i d e a s of  and dominance components  et  expected  The  colleagues  provocative  overdominance  additive  (1922).  has  maintenance  populations  in natural populations,  overdominance  Mukai  and  discovery  theoretical  against  the  1940s and 1950s l a r g e l y  ecological  variation  at  natural  Dobzhansky  1948,  for  superiority,  the  alleles  1972;  Coyne  displayed 1980);  by the  multi-allelic  16  equilibria  by  overdominance the  (e.g.  Lewontin,  potential  roles  Tuljapurkar  1978);  heterogeneity  and f r e q u e n c y - d e p e n d e n t  polymorphisms  (Hedrick,  Hedrick  and,  1986);  examples  that  in  on  a  various  overdominance  traits  (reviewed  1987).  In m a r i n e b i v a l v e s ,  heterozygotes, in  the  1978;  1983;  and have  been  American o y s t e r  1982;  Zouros  Despite  relationships  remains  associative  the  1980;  Singh  al.  1983;  Foltz,  the  undetected  overdominance.  results presence  Foltz  and  discussed  1982;  Koehn  and  N e w k i r k and Z o u r o s Foltz  1986a,  u n d e r l y i n g c a u s e of  could also of  of  ( S i n g h and Z o u r o s  unknown. A l t h o u g h c o n s i s t e n t these  and  deficiencies  S i n g h and G r e e n 1984; work,  documenting  multiple-locus  studied  Miles  extensive  overdominance,  inbreeding,  related  by  fitness-related  Zouros  potentially  1984;  282)  heterozygosity  and  involving  et  p.  sparked  organisms,  Crassostrea v i r g i n i c a  and  of  against  (1983,  been  1984;  extensively  paucity case  multiple-locus  Grant  1979;  it".  associations  most  a  Kimura  has  of  maintaining Clarke  The  physiological  and  the  Z o u r o s and F o l t z  1986b).  locus  by M i t t o n  Zouros, Singh  Shumway  variety  morphological,  heterozygosity,  that  can m a i n t a i n  between  in 1976;  occurrence.  wide  correlations  Ewing  and  environmental  importantly,  overwhelming faith  of  selection  and  most  its  so  interest  studies,  significant and  appears  "only b l i n d  Renewed recent  perhaps  demonstrating  overdominance stated  Ginevan  Ginzburg  null  with  these single-  be p r o d u c e d by alleles,  or  17  Inbreeding Zouros,  studied,  the  o r i g i n a l l y d i s c o u n t e d as  S i n g h and M i l e s  inbreeding  that  was  coefficients,  was  affect  and  all  at  was  provided  (1981)  genomic  e m p h a s i z e d by Smouse Foltz  equally.  a small subset  overall  who of  (1986)  (see  segregation  of  alleles  two l o c i  providing  support  explanatory they  Zouros, power of  allele  in these  null  Zouros,  the  possessing  absence  of  and  two  of  At p r e s e n t ,  hypotheses  appear c a p a b l e of  traits,  at  least  for  accurately  conclusion  1986b) C.  between oysters.  considered  appears l i m i t e d four  the  because others  from an i m p o r t a n t t h e o r e t i c a l  homozygotes (1980)  i n the  large  sample  i m p l i e s an u n r e a l i s t i c a l l y  heterozygotes  the  thus  However,  at  the  ( P q i , G o t , Pgm, X d h ) . The n u l l  alleles  neither  Zouros  virqinica,  ( L a p - 2 ) but not  null  in  also  detected  still  functional  relationships  not  alleles  reconcile.  the  individual  (1980).  null  Pierce  that  Miles  Miles  advantage  and  and  suffers  loci  against  originally  locus  also  Singh  high selective  one  in  seven  evidence  discussion  (1986a,  the  process  hypothesis  e a r l i e r studies  explanation  limitation:  a  Singh  were o b s e r v e d a t  involved  of  for  at  a  of  from a  does  however  the  Mitton  heterozygosity,  Foltz  by  by  loci  Recently,  unlikely  Further  showed  1987). null  heterogeneity  than expected  loci  Chakraborty  heterozygosity  the  c a l c u l a t e d f o r each of  inbreeding hypothesis  reflect  because  s i g n i f i c a n t l y greater  should  (1980)  and  (1980)  an e x p l a n a t i o n by  that  over is  inbreeding  genotypes  difficult  or  null  allele  providing a general explanation heterozygosity  and  to  for  fitness-related  18  Distinguishing overdominance correlations  hypotheses  consequence  at of  deleterious  the  hybrid  1977,  p.  loci  P.  despite  9-46;  Smouse rigida  tested  or the (1986)  the  overdominance  overdominance these  to  of  by B u s h , of  hypotheses.  Any  to  at  the  a  presence  number  between between  heterosis, remains  (see  Gowen 1952;  Wright  P.  to  determining  question.  attenuata  "adaptive  and  inbreeding for  discriminate  several  Comparison by  Strauss model  i n a number of  Ledig  evidence  in  distance"  relationships  the  of  which  relationships  Smouse  cannot  dispute for  a  1987),  this  the  heterozygosity  predictions  (1987)  only  versus  the  overdominance between  genuine  overdominance.  the  o n l y way t o p r o v i d e c o n c r e t e  explanation  competing  polymorphisms  solve  progeny  p r o v i d e d by t h e s e a p p r o a c h e s or a s s o c i a t i v e  to  they  by t h e  approaches  rate  a p p l i c a t i o n of  populations  Perhaps  study  Indirect  helped  caused  are  explanations  Foltz  heterozygosity-growth not  or  Are  p r o d u c e d by  1971)? The d i s t i n c t i o n  of  1981).  i n b r e d and c r o s s b r e d 1987)  and  decades  Sedcole  s p e c i e s have  (1986, of  scored,  d i r e c t l y analogous  (Zouros  difficult.  segregating  Ohta  associative  heterozygosity  loci  alleles  and  more  overdominance  (cf.  is  vigor  c a u s e of  Pinus of  much  "dominance" and " o v e r d o m i n a n c e "  unresolved  the  enzyme  recessive  these a l t e r n a t i v e s  or  is  associative  tightly-linked  the  overdominance  involving multiple-locus  overdominance  of  between t h e  and s i m u l t a n e o u s l y  hypotheses, directly  and  is  to  produce  evidence  distinguish  study the  these  for  the  between enzyme  biochemical  and  19  physiological  support  (cf.  1975;  Clarke  research  strategy  for  these'  Koehn  1978;  that  biochemical  level  for  These d i f f e r e n c e s  confer  a  net  marginal (cf.  superior the  kinetic  Second,  the  manifested  at  the  this  must  the be  to  allelic of  the  array  compatible  with  locus  of  the  multi-allelic  equilibria  by  Ginzburg  and  Tuljapurkar  1978),  pertinent  b e c a u s e most  I  possess  have  3 to  taken  phosphoglucomutase-2  locus  oyster,  gigas.  Crassostrea  loci  this  at  this in  either  intermediacy  thus  moderate  direct  be  The  physiological of  explaining  relationship.  locus  under a  stable  maintaining (cf.  study  stable  Lewontin, is  especially  studies  involving  frequencies.  approach  (PGM; E . C . 2 . 7 . 5 . 1 ) PGM c a t a l y z e s  loci.  physiological  requirement these  by  eliminating  producing of  to 1)  produced  heterozygosity the  the  chosen  i n magnitude  some  overdominance  5 alleles  locus  be c a p a b l e  difficulties  at  h e t e r o z y g o t e s must  in question.  conditions  it  tightly-linked  of  genotypes at  enzyme  the  through  through  i n the  Given  oysters  state.  unknown  level  locus  must  overdominance  v a r i a t i o n must  the  First,  expressed  heterozygotes,  of  phenotypic  this  sufficient  superiority  fitness  equilibrium  of  terms  To s u c c e e d ,  from h e t e r o z y g o t e  genuine  properties  functional  be  at  heterozygotes  contribution  involvement  Third,  2)  are  variants  must  to  mechanistic  1985b).  differences  arising  or  attributable  i m p a c t of the  1959),  in  requirements.  between a l l e l i c  advantage  potential  effect  functional  overdominance  Wallace  Watt  must meet t h r e e  demonstrate  study.  results  the  in  with the  the  Pacific  interconversion  of  20  glucose-1-phosphate the  cofactors  functions  and g l u c o s e - 6 - p h o s p h a t e  glucose-1,6-diphosphate  metabolically  glycogen.  In  significant  the  positive  Japan.  The Pgm-2 l o c u s  weights The  weight  oyster,  a  differences  exist  locus  this  18 o f  chapter  between t h e  in C . gigas  overdominance functional  of  in  of  the  hypothesis.  differences,  the  the  seasonal cycle,  large  was  to  kinetic  Walsh,  in  v a r i a t i o n and d a i l y c h a n g e s  aerobic  and  anaerobic  Pgm-2  o r pH t h a t  allozymes  c o u l d be  heterozygotes  that  over  could provide  superior rates  of  and  the  biochemical  in both  at  the  i n s u p p o r t of  properties  of  the  of  these  seven  Pgm-2  important ranges  the  intertidal  temperature,  associated  Kinetic  a  if  mean  sampled.  presence  metabolism  for  greater  common a l l o z y m e s  with  of  the  (e.g.  of  i m p a r t i n g a net foundation  g r o w t h r e p o r t e d by F u j i o  for  of  zone  through  the  tidal  transitions  Wijsman  differences  these broad ranges  responsible  a  involved  populations  a consequence  M c D o n a l d and B o o t h 1 9 8 4 ) .  between  their  situated  and i n t r a c e l l u l a r p H , as  between  enzymes  determine  assess  fluctuations  of  observed  exhibited  g e n o t y p e s were measured o v e r p h y s i o l o g i c a l l y  experience  degradation  heterozygosity  provide evidence  To  t e m p e r a t u r e and p H . O y s t e r s  and  five  20  f o u r most  that  magnesium i o n ,  on 20 w i l d p o p u l a t i o n s a r o u n d  was was one  t h a n homozygotes  of  (1982)  between  study  presence  and  Fujio  c o r r e l a t i o n . Pgm-2 h e t e r o z y g o t e s  objective  Pqm-2  in  and  synthesis  relationship  body  this  the  Pacific  adult  in  in  i n the  may  1975; exist  temperature advantage  to  explaining  (1982).  21  MATERIALS AND METHODS  Chemicals. coupling Sigma.  All  buffers,  enzymes  for  enzymatic  (DE32,  ultrafiltration G-250  p r o v i d e d by B i o - R a d ,  and t h e  were  and from  s u p p l i e d by P h a r m a c i a ,  DEAE-  from  (CF 25)  s t a n d a r d s used  proteins, obtained  microgranular)  membrane c o n e s  and  cofactors,  assays  Sephadex G-100 and G-25 were  Cellulose  blue  used  substrates,  Whatman,  from  Amicon.  and  Coomassie  for protein determinations  electrostarch  for  were  electrophoresis  from Connaught L a b o r a t o r i e s .  Animals. the  Oysters  i n t e r t i d a l zone  Bay  on  site  of  Vancouver  were e s t a b l i s h e d situated  h i g h water the  were  at  at  for  0.5-0.8  on  Kristjansson was acid, 1979)  and  centrifuge.  Nanoose  Sampling  stations  lease;  m above mean low water  and a f t e r  gels  a small  starch  (w/v)  gel  according  A p p r o x i m a t e l y 0.5  g of  i n an e q u a l volume o f 1 mM MgC12,  centrifuged  for  piece  immediately  Horizontal  (1963).  1 mM EDTA,  on t h e  pH 7.4 5  min  S u p e r n a t a n t was a p p l i e d t o  of  at  water  (MLW),  and a  returned  to  the  methods  frozen mantle  A of  to was  -40°C.  electrophoresis  top speed  the  a low  mantle  f r o z e n at  10 mM T r i s ,  (buffer  from  in  1 . 7 - 2 . 0 m above MLW. A n i m a l s were  12%  homogenized  year p e r i o d  farm l o c a t e d  Columbia.  heights  electrophoresis,  Electrophoresis. performed  British  two t i d a l  l a b o r a t o r y on i c e ,  dissected  over a t h r e e  a private oyster  Island,  at  site  collected  was of  tissue  10 mM m a l e i c  Fucci in a  et  al.  clinical  g e l s w i t h Whatman No.  22  2 filter  paper w i c k s .  approximately  5  h  Gels  patterns, employed.  buffer  100 mM T r i s ,  gel  buffer  Under  of  and c o f a c t o r s  while  migrating  the  variation  gel,  run  were  under  the  same l e n g t h  subsequently homozygotes  for  The  the  serving  of  time  for most  the  were  electrode  pH  8.3.  The  buffer.  Under  concentrations gel  catalytically  and  active  glucose-1-phosphate,  the  presence as  L o v e r r e and  two  as  slices were  i n a water to  staining  allele  resolution  Tris-maleic acid buffer  were of  for 1976;  and  Beveridge  of  the  was  and,  same  in  the  incubated  the  50°C.  other,  The  slices  solutions  and  intensities.  300  staining  differential  hidden  described  treated  a control,  bath at  of  Catamo  (Beaumont  room t e m p e r a t u r e  common  allozymic  standard  classes  conditions,  bath at  pH-dependent  e x a m i n e d w i t h the  examine  (1978),  transferred  scored  the  by  mM MgC12.  (Trippa,  al.  manner. One s l i c e ,  immediately  fashion.  et  standard  15 min i n a w a t e r  to  for  outlined  the  electrode  and M y t i l u s e d u l i s  Following Trippa  for  and 1.5  PGM e l e c t r o m o r p h i c  1978)  of  mA)  conditions  1 mM  w i t h i n the  1983).  for  field:  were u s e d  al.  as  saturating  techniques  et  following  the  PGM a l l o z y m e s  electric  both D r o s o p h i l a melanoqaster Trippa  visually  were a d d e d t o  5 MMglucose-1,6-diphosphate,  Two  (35  5 mM EDTA,  following  t o make t h e  i n the  V  "standard" c o n d i t i o n s  d i l u t i o n of  conditions,  buffers  200  running  100 mM b o r a t e ,  "catalytic"  electrode  stained  different  was a 1 0 - f o l d  substrate  at  For improved r e s o l u t i o n  two  routinely was  run  and PGM was  Shaw a n d P r a s a d ( 1 9 7 0 ) . banding  were  treated  in  cryptic alleles system  described  this was in  23  Beaumont and B e v e r i d g e of  the  8  most  7.4  gel  electrode each  and  adjusted  to  allozymes  purified,  tissue  appropriate  homogenizer  supernatant  in  above  examined  maleic  acid,  100 mM m a l e i c  or 6.0.  Gels  and " c a t a l y t i c "  acid  run  at  conditions  before  was  s t e p s were c a r r i e d o u t  resuspended  represented  ,homozygous  for  the  Ultra-Turrax  of  buffer at  through  of  brought  A and s t i r r e d  42,000  x  to  50% s a t u r a t i o n  12,000 x  at  g  20  30 m i n . The h  wool.  and  stirring  the  The c r u d e  w i t h an  12,000 x g f o r  stirring  for  1  glass  After  75% s a t u r a t i o n  A and d i a l y z e d  same  of  for  for  filter  centrifuged to  g  a  ammonium s u l f a t e .  brought  dialyzed  Pgm-2  w i t h an  was  the  each  4  or  m i n c e d and h o m o g e n i z e d  in buffer  c h a n g e s of  for  the  ice  and a d d u c t o r  slowly  at  material  for  on  mantle  and e q u i l i b r a t e d w i t h  centrifugation  identical  individuals  3 volumes  suspension  The  pH 7.4  was  10 mM  Tris,  composed  A p p r o x i m a t e l y 70-80 g of  was  solution  supernatant  vol  the  5-8  passed  homogenate was  as  either  oysters,  genotypes,  mM  p r o c e d u r e was  homogenate was c e n t r i f u g e d  the  100  and s t a r t i n g  from  allele.  tissue  saturated  120  10 mM T r i s ,  A l l purification  4 ° C . The f o l l o w i n g  muscle  a  of  previously.  Purification.  pooled  Pqm-2  a  pH were compared under  described  at  employing  buffer,  buffer  A sample  frequent  electrophoretically pH  (1983).  ice-cold for  1  h  20 m i n . The  w i t h ammonium  sulfate  for  min,  overnight  1 h.  After  a  final  the  pellets  were  against  three  100  buffer.  sample  was a d j u s t e d  to  pH 7.6  and l o a d e d  onto  24  a D E A E - C e l l u l o s e column Tris,  5  (buffer  mM  maleic  B) a t  a flow  minimum of gradient  vol  x 30 cm) mM  of  5.6  ml/h.  3 column v o l u m e s ,  the  enzyme  the  15-25  acid,  equilibrated  0.5  (0-200  containing  (1.5  rate  mM)  of  greatest  ml)  and  NaCl  MgC12,  (200  specific  After was ml  to  5  mM  mM EDTA,  pH  7.6  washing  eluted in  activity  concentrated  0.1  with  with a  total).  were  approximately  The  column  concentrated (1.5  carried  x 60 cm)  out  were p o o l e d 7.6  and  at  of  7-9  (0-  of  ml/h (B)  (0.9  as  NaCl  before,  also contained (total  vol  7-14  the  and t h e  1 ml by c e n t r i f u g a t i o n  Sephadex  G-25 column  a  rate  of  enzyme  (0.9  8.4  The  stable,  exhibiting period.  2,000  Elution  to  to  on a v e r a g e  a  Electrophoretic  to  achieve  a  DEAE-  and e l u t e d  with a  linear  150 ml)  a  at  fractions  sample  flow  rate  elution  were  again  was c o n c e n t r a t e d  2,000 x g on  fractions  in t h i s  10% l o s s  pH  a second  20% g l y c e r o l  stored  was  fractions  e q u i l i b r a t i o n and  at  x  G-100  an  through a  in a c t i v i t y of  The  and f r o z e n  manner were  examination  A at  were p o o l e d .  (v/v)  these  to  Amicon  e q u i l i b r a t e d with buffer  m l / h and peak  PGM a l l o z y m e s  at  The p r e p a r a t i o n was p a s s e d  x 15 cm)  was b r o u g h t  -70°C.  month  vol  ml on an  adjusted  loaded onto  washed,  (total  1.5  peak  added  2 mM G 1 P . Peak ml)  A.  was  was  Fractions  a Sephadex  m l / h and t h e  was  (total  to  buffer  The sample  except  CF 25 u l t r a f i l t r a t i o n c o n e .  purified  8.4  (G1P)  x 15 c m ) ,  200 mM) of  approximately  flow  of  ml).  applied with  2 mM. The sample  column  gradient  combined  vol  rate  glucose-1-phosphate  Cellulose  buffer  was  equlibrated  a flow  (total  concentration  7.2  sample  a  linear  combined  Amicon CF 25 u l t r a f i l t r a t i o n cone by c e n t r i f u g a t i o n g.  with  at  extremely over  a  2  partially  25  purified and  preparations  showed  locus.  that  they  General  triplicate  at  (1976)  nm  on  was  a  a blank c u v e t t e .  50  PGM a c t i v i t y  mM  et  al.  1  unit  (20°C)  i n a f i n a l volume o f  the  reaction  activity convert  is  defined  1 Mmol of  Preliminary the  4  Kinetic  Pgm-2  by  as  K-2/RD  kinetic  probe  MgC12,  allozymes for  the  measuring  inserted  a coupled assay  One  of  forward r e a c t i o n  enzyme  initial  rates  in  mM  pH 7 . 0  containing 10 min and of  PGM  required  to  at 1 5 ° C .  on  each  purification (G1P  assay  0.4  unit  were c a r r i e d o u t earlier  into  medium  (G6PDH),  f o r a minimum of G1P.  and  2 mM g l u c o s e - 1 -  product per minute  following  of  at  holder  (Gl6diP),  dehydrogenase  quantity  analyses  method  circulating  cuvette  standard  mM  a d d i t i o n of  to  in  spectrophotometer.  1 m l . The a s s a y m i x t u r e ,  the  substrate  parameters  determined  The  PGM, was p r e i n c u b a t e d by t h e  by t h e  was m e a s u r e d u s i n g  glucose-6-phosphate  started  determined  a s s a y s were c o n d u c t e d  16 M M g l u c o s e - 1 , 6 - d i p h o s p h a t e  NADP,  Pgm-1  a standard.  46 t h e r m o m e t e r  3  the  e a c h p u r i f i c a t i o n s t e p on a  temperature  imidazole-HCl,  composition  by  was  UV/visible  (1967).  (G1P),  of  encoded  spectrophotometer  to a constant  phosphate  aliquot  allozymic  was c o n t r o l l e d by a Lauda  from J o s h i  an  after  gamma g l o b u l i n a s  m o n i t o r e d by a YSI model  contained  enzyme  Pye U n i c a m SP 1800  bath attached  modified  of  correct  and K i n e t i c s . A l l enzyme  Assay temperature water  UV/visible  using  the  concentration  room t e m p e r a t u r e  Enzyme A s s a y s 340  were f r e e  protein  Pye U n i c a m SP8-400 Bradford  verified  to  trials.  G6P)  triplicate  of  were at  11  26  concentrations  of  G16diP c o n c e n t r a t i o n  of  at  the  5°  the =  intervals  pH of  the  -0.017).  studied 7.5  values  over  The  a were  triplicate  pH as  the  direct  an  (1974). evaluated  at  to  linear  Currie  initial  measurements concentrations of  determined times at were  a  design  at  (cf.  For the were  to  20°C.  pH 6 . 5 ,  7.0  and  A p p a r e n t Km and Vmax  linear plot  of  E i s e n t h a l and  for  initial  the  reaction  diphosphate levels  same  was  in a  of  ranges  G1P of  cofactor rates  in  geometric (1  mM).  temperature  estimated  by  experiments  that  enabled c o n s t r u c t i o n  m i n i m i z e d the  Duggleby  of  the  and Chan  1981;  forward r e a c t i o n d i r e c t i o n ,  initial  rate  replicated  1979;  variances  of  Endrenyi  8  parameters  two d i p h o s p h a t e  G1P c o n c e n t r a t i o n taken  of  at  parameters  parameters again  s i m i l a r l y by r e p e a t i n g  each of  before  the  times  (2 mM and 20 uM) a t  M M . Kinetic  16  as  saturating  kinetic  kinetic  estimates  1982).  kinetic  plot.  optimal k i n e t i c  parameter  the  the  were m e a s u r e d o v e r and t h e  (ApKa/°C  measuring  16 uM a t  performed  5 to  parameters  of  were  vary with temperature on  Kinetic  saturating  allowing  direct  by  r a n g e of  a  30°C  rates  8 concentrations  before  These  initial  by t h e  rates  and  pH  estimated  from 0.125  Initial  of  temperature  were  series  effect  to  2 mM a t  experiments  temperature  constant  Cornish-Bowden G16diP  16 juM. T h e s e  imidazole buffer  by m e a s u r i n g  at  10 M M t o  G1P r a n g i n g from  of  a  at  each  diphosphate  for  the  initial  of  concentrations  error.  G16diP  were  measurements  For the  8  and 0.70 M M )  (16  1 mM. S e v e r a l a d d i t i o n a l  minimize experimental  G1P  concentration  cofactor rate  two  precautions  kinetic  study  27  the  4 Pqm-2 a l l o z y m e s  and  subsequently  each  of  the  most  frequent  Although  two monomers were p r e s e n t , these  purification same  was  kept  courses  of  were to  Peck  estimates,  assessed p r i o r negligible, were  to  the  the  were  Fortran  this extent  start  providing  of  the  in  not  Hoffman  at  parameters estimated  by t h e  of  linear  v a r i a t i o n of  by t h e  G1P  Since  of  the  p r o g r a m NATO w r i t t e n  kinetic  p a r a m e t e r s were  (ANOVA)  t r e a t i n g Pgm-2 g e n o t y p e  only  of  the  staining the  in  small  conversion  the  time  kinetic  represent  v a r i a b i l i t y was  was  found  to  be  daily preparations across  5  days  2.9%.  and G 1 6 d i P  method of  these  by  design  s t u d y and  less  reaction  day-to-day  activities  for  Three  heterozygotes.  Substrate  1985).  the  three  -40°C  experimental  the  at  activities  guaranteed  study.  strictly  of  A.  nature,  justified  R e p l i c a t e Km d e t e r m i n a t i o n s  exhibited a coefficient  design  1972;  by  the  kept c o n s t a n t .  Kinetic  ensure  frozen,  similar quantities  maintained  used throughout the  and  the  from n a t u r a l l y o c c u r r i n g  w i t h i n 20% t o  (Ray  and  solutions,  parameters determined single  buffer  synthetic  a condition  genotypes,  enzyme  substrate  aliquots,  in  were made by m i x i n g e q u a l  p r e p a r a t i o n s ensured that  of  stored  r a t e measurements  common Pgm-2 a l l o z y m e w i t h e a c h of  heterozygote  The  initial  equal a c t i v i t i e s  preparations  allozymes.  patterns  sequentially,  under i d e n t i c a l c o n d i t i o n s  and p H . F o r a l l  were d i l u t e d t o  heterozygote  purified  studied together  temperature  allozymes  were  biweight  from t h i s  optimal  regression  with  by C o r n i s h - B o w d e n ( 1 9 8 5 ) . The  e x a m i n e d by two-way and e i t h e r  analysis  of  variance  t e m p e r a t u r e o r pH  as  28  independent  variables.  temperature  and pH were  multiple of  the  range  Means  tests.  apparent  compared  by  Rationale  for  Km v a l u e s  individual  pH  or  covariance  (ANCOVA)  of  is  as  ordinary  least-squares  procedure.  with  This  transformation  of  the  Lineweaver-Burk plot  Hofstee  plot  v vs.  with a y - i n t e r c e p t transformations regression from t h e  Eadie-Hofstee  parameter  estimates  statistical genotypes  (-Km)  the  for  posteriori analogous  to  coefficients validity  of  slightly  on t h e  the  were data  genotypes  the  a  second  of  the  treating  changing  Differences  range  ( S o k a l and R o h l f  tests.  v/[s]  as  p.  found  to  linear  the program the  Eadie-  a regression These  for  The a b s e n c e  of  covariate  This  by  in  slope  and  the  v  as  estimates  is  a  thus  regression  irrespective the  the  Bonferroni  of  of  different  procedure  a series  507),  of  allowed  Km v a l u e s  tested  both  obtained  independence  the  line  different  errors  larger.  the  a covariate  by  biweight  parameters  between  were  1981,  in  transformation  as  time  the  to  -Km.  apparent  v/[s]  were  more r o b u s t  the  u n p l a n n e d c o m p a r i s o n of  treating  all  standard  and  linear  Pqm-2 g e n o t y p e s  multiple  for  kinetic  plot  variable.  these  of  which produces  the  comparison  dependent  analysis  1/[S])  although  each  the  estimates  of  at  by  after  vs.  identical  kinetic  by ANCOVA,  comparison  by t h e  Vmax and a s l o p e  procedures,  in  (1/v  v/[s],  yielded  outliers  the  of  statistical  Michaelis-Menten equation  from t h e  of  the  These  repeated  of  Bonferroni  First,  obtained  was  ranges  posteriori  determined  regression.  analysis  these  a  interval  were  those  over  s e v e n Pqm-2 g e n o t y p e s  follows.  Km and Vmax v a l u e s  identical  these  temperature  apparent  be  expressed  ANCOVA.  of  the  29  Kinetic  parameters  t o G1P) were e s t i m a t e d (1964b)  through the  (Haldane  1930):  where Keq i s Vmax(f)  and  and r e v e r s e and  the  app  1  reverse  procedure  constraints  reaction of  app Km (G6P)  Vmax(r)  app Km (G1P)  reaction's  reaction (G1P)  are  may  the  the  and be  and  respectively;  ([G6P]/[G1P]); of  to  the  forward  and app Km  (G6P)  Michaelis constants  glucose-1-phosphate,  rearranged  equation  . . . (1 )  maximum v e l o c i t i e s  apparent  (G6P  Roscelli  Haldane  e q u i l i b r i u m constant  directions, are  direction  Ray  imposed by t h e  Keq = Vmax(f)  glucose-6-phosphate Equation  the  by t h e  Vmax(r)  Km  for  allow  for  respectively.  estimation  of  app Km  (G6P):  app Km (G6P)  = Keq  app Km (G1P)  .  ...(2)  Vmax(f)/Vmax(r)  Therefore, different  determination Pgm-2  concentrations measurement  for  of  of  genotypes, both  their  Vmax(f)/Vmax(r) employing  reaction  directions,  ratios"  identical enables  velocities  for  under s i m i l a r  both  reaction  of  enzyme indirect  app Km's f o r G 6 P , p r o v i d i n g v a l u e s  and a p p Km (G1P) were a t t a i n e d  Maximum  the  of Keq  conditions.  directions  were  30  determined  for  temperature  the  range  imidazole-HCl either 1  f o u r Pqm-2 a l l o z y m e s of  pH  5 to  7.0  The  four  activities  in buffer  reaction  directions  temperature addition aliquots  substrate.  i n the  an  volume  equal  time  volume  from t h e  as  3,  of  the  versus  2  h  time.  a  sample  immersion  of  in  concentrations  of  was  (1974).  boiling  water  by  the  250 jul  For  assays into  into  a  each  measured  in  The G1P formed  similar  fashion  by  (1974).  Vmax was  estimated of  10 r e a c t i o n the  G1P and G6P measured  the  were  Pgm-2-100  directions  bath  for  as  the  determined  2  allozyme. with  in duplicate  as  as The  starting  were s t o p p e d min,  y-  product  tubes prepared  2 mM G1P o r G 6 P . The r e a c t i o n s a  appropriate  G6P p r o d u c e d a t  in a  e q u i l i b r i u m was a p p r o a c h e d from b o t h concentrations  of  regression  of  both  direction  Equilibrium constants  by i n c u b a t i n g  with  reverse  of  for  initiated  reaction  Bergmeyer and M i c h a l  temperature for  the  was a s s a y e d  linear  the  and  equal  t h e s e were t r a n s f e r r e d  forward  directions,  to  tubes  mixture.  by Lang and M i c h a l  reaction  50 mM  to  and 9 min i n t e r v a l s ,  The q u a n t i t y  an u n w e i g h t e d  formed/time  reaction  reaction  the  volume  were d i l u t e d  and were  6,  H C l ; for  1N NaOH.  both r e a c t i o n  intercept  above  1N  described  p r o c e d u r e of  each  of  from  reverse  For  at  bath,  forward d i r e c t i o n ,  of  interval  duplicate  the  At  in a f i n a l  equilibrated  were removed from e a c h  proceeding  similar  A. Eight r e p l i c a t e were  over  16 M M G 1 6 d i P ,  mM MgC12,  preparations  i n a s h a k i n g water  of  3  o r G6P ( r e v e r s e )  allozyme  5° intervals  The a s s a y medium c o n t a i n e d  (20°C),  4 mM G1P ( f o r w a r d )  ml.  30°C.  at  and  by the  described  31  above.  Apparent  allozymes  Michaelis  were  appropriate estimate  constants  estimated  from  Vmax(f)/Vmax(r) for  the  for  equation  ratio,  forward  G6P  for  the  four  2 by s u b s t i t u t i n g  Keq,  and  reaction  app  Km  direction  the  (G1P)  determined  previously.  Thermostability diluted MgCl2,  to 0.1  serum  equal  albumin.  pH 7.0  As  into  After  being  with  shaking  in  samples.  a set  sealed  transferred the  ice  measured 25°  of  to  an i c e  these  enzyme  decay  process.  constant,  to  interval  (1984a)  straight  i n 50 mM sodium p h o s p h a t e ,  0.5 mM  containing  kinetic  with p a r a f i l m ,  study,  kd.  (w/v)  the  studied  18 o f  the  same  the  aliquots  were  (10  x  t u b e s were  75  tubes  were  removed  1  the  experiment.  on a l l  samples  proportion to  (1985),  g e n o t y p e s was  the  treated  Least-squares  R e p l i c a t i o n of  time  of  the  After  experiment  at  Place  and  denaturation  of  the  t against  slope estimating  at  remaining  a first-order  regression  remaining at  with a negative  as  thermal  were  standard assay  control.  a  maintained  PGM a c t i v i t i e s  activity  the  over  immediately  t u b e s were  by t h e  of  and  mm).  incubated  At 5 min i n t e r v a l s  Three c o n t r o l  three  alongside  50 ul  tubes  bovine  50°C.  relative  and H a l l  activity  line  the  0.1%  bath at  bath.  duplicate  Powers  fractional  were  glass culture  bath throughout in  time  purified  For each genotype  21  i n a water  and c o n v e r t e d  each  allozymes  buffer the  30 min p e r i o d t r i p l i c a t e  in  four  p r e p a r a t i o n s were made and  homozygote  pipetted  The  activities  mM EDTA,  heterozygote four  Studies.  the  exponential  logarithm time  yielded  of a  denaturation  produced  estimates  32  of  kd f o r a l l  before,  so  genotypes the  data  between g e n o t y p e s statistically ANCOVA,  pH o p t i m a .  Initial at  to  pH u n i t  equal  appropriate replicated G6PDH  four  was  expressed at optimum  was  repeated  a second  comparisons  with  a  data),  the  comparison pH r a n g e  posteriori  equal  data  ensuring  the  relative for  paired  angular  each  Bonferroni multiple  activities  in  by  the  that  The f o u r  adjusted  20°C  to  the  measurements  were  coupling  the was  of  observed  results  activity pH  The e x p e r i m e n t  was  did  at  not  angular pooled.  interval  enzyme  the  c a r r i e d out  proportions range  at  components  proportion  on  8.0  A. A l l assay  activity  the  each  to  saturating  allozymes  genotype.  trials  transformed  the  solutions  to  f o u r g e n o t y p e s was  Magnesium I o n .  slopes  four  that  t-tests  both  of  The  and b e c a u s e  from  tested  posteriori  under  from pH 6.0  in buffer  by two-way ANOVA and a t  the  of  interval  (by  of  intervals  rate-limiting.  determined  significantly  were  these  r a t e s were measured  activities  time  constants  preparations  times a f t e r  each  from  Differences  of  At e a c h pH i n t e r v a l  not  was p o o l e d .  heterogeneity  i n 50 mM i m i d a z o l e - H C l  pH.  significantly  tests.  reaction  0.2  were d i s s o l v e d  to  range  s t a n d a r d a s s a y on  diluted  Effect  the  and p e r f o r m i n g p a i r - w i s e  conditions  differ  from b o t h t r i a l s  by e x a m i n i n g  multiple  on  d i d not  in these denaturation  Bonferroni  by t h e  that  differ  transformed Statistical the  entire  by one-way  ANOVA  and  for  tested  by  a  tests.  Pgm-2 a l l o z y m e s  50 mM i m i d a z o l e - H C l  pH 7.0  were (20°C)  diluted buffer  33  and  passed  through  a  equilibrated  with  ions.  reaction  at  Initial  1 5 ° C on f o u r  1,  3,  5,  to  the  the The  the  10  four  x  15  free  r a t e s were m e a s u r e d  by t h e  standard  at  measured.  velocities  at  was p o o l e d  for  was  each  the  relative  replicated  statistical  entire  and by one-way ANOVA f o r  Means were compared by a p o s t e r i o r i  level  Bonferroni  and  analysis. angular  range  each  0.5,  cofactor  were compared by two-way ANOVA on t h e for  assay  of  proportional activities  The e x p e r i m e n t  cm,  magnesium  magnesium c o n c e n t r a t i o n s  Reaction  proportions  concentrations,  (0.9  remove  from b o t h t r i a l s  transformed  column  buffer)  were e x p r e s s e d as  allozymes  G-25  to  mM.  maximum r a t e  data  same  replicates  and  concentration  Sephadex  of  Mg  separately.  multiple  range  tests.  RESULTS•  ELECTROPHORESIS  Two  AND PGM-2 A L L E L E  phosphoglucomutase  loci  electrophoretically  in Crassostrea  1 locus  be p o l y m o r p h i c ,  only not  appeared  a be  to  fraction reliably  of  that  scored.  exhibited  double-banded  described  for  Beardmore  1977;  conditions  this  Hoffman  homozygotes  gigas. but  Genotypes staining  1985).  in  at  a  its  Pqm-2 l o c u s  standard two-banded  was  it  could  Pqm-2  locus  similar  species  Pgm-  activity  more a n o d a l  patterns  Under  detected  The more c a t h o d a l  the  the  other  displayed  were  since  expressed at  enzyme  FREQUENCIES  (e.g.  to  those  Ward and  electrophoretic phenotype  and  34  heterozygotes  were e i t h e r  mobilities  the  of  double-banding presence  of  three-  alleles was  faster  found  carried PGM  to  (inactive)  under c a t a l y t i c  entirely  to  the  1964b),  this  became  consistent  (Figure  1B).  with  the  was  those  expected  w i t h the  added  gel  resulted  band to  in  all  react  i n the  with  the  ground t i s s u e  -40°C  behaved of  consequence procedures,  predicted  as  and t h a t  an  these d i f f e r e n t  if  of  5 MM  of  from t h e  It  is  of  not  phosphorylated  the  homogenization  they  reflect  actual  migrating alone  (not  iri  shown).  migrating  ability  of  it  Gl6diP to  the  These  staining  storage  because  stored known forms  and/or  was  slower  convert  artifact  enzyme  presumed  faster  the  and  patterns  the  G16diP  from o y s t e r s  identically.  of or  as  the  a monomeric  the  of  convert  (Ray and R o s c e l l i and  a concentration  elimination  which  known t o  ( N a j j a r and P u l l m a n 1 9 5 4 ) .  result  freshly  at  enzyme,  was  cofactor  dephosphoenzyme  state  d i d not  resolution  near  genotypes,  phosphorylated patterns  buffer  slower  electrophoresis  for  to  and  this  conversion  dephosphoenzyme  This  or absence of  eliminated  was p r o v i d e d when t h e  This  When  the  phosphoenzyme  phosphoenzyme the  presence  the  simultaneous  (active) of  on  1A).  the  running c o n d i t i o n s ,  double-banding  migrating  to  from  forms  group.  active  Evidence consistent slower  (Figure  migrating phosphorylated  charged phosphate  out  them  result  i n m o b i l i t y because of  negatively  four-banded depending  comprising  migrating dephosphorylated differed  or  f o r months whether of  PGM was  at the a  electrophoretic  vivo  proportions.  35  Figure  1. E l e c t r o p h o r e t i c s t a i n i n g p a t t e r n s o f o y s t e r PGM under (A) s t a n d a r d and (B) c a t a l y t i c r u n n i n g c o n d i t i o n s . Pgm-2 g e n o t y p e s p r e s e n t e d from l e f t t o r i g h t a r e : 9 2 / 9 2 , 927100, 9 6 / 9 6 , 96/100, 100/100, 100/104, and 1 0 4 / 1 0 4 .  37  Catalytic  running  conditions  g e n o t y p e s and a l l o w e d selected  for  No  enzyme  hidden  electromorphic sensitive Trippa,  of  class  approximately  gels. Ts  heterozygotes  of  that  the  .08)  that  this  genotypic  the  it  pH s y s t e m o f  of  be by  accurately  the  et  the  i n the  of  for  t h e Pgm  on t h e  loss  control  nor were any  oysters.  Although  w o u l d be h a r d t o  identify  by  Ts homozygotes  if  present,  influence  i n the  four  two a l l e l e s by t h e  is  kinetic  next  also  carried However,  (p < of  The v a r i a b l e to  under  detect  either  agreement pH 7.4  (Pgm-2-96  and  electrode  buffer  s t a n d a r d T r i s - b o r a t e system run at  low  electromorphic  out in  indicates  so  failed  common  this  properties  section.  (1983) most  was  conditions.  identified  (1978).  consistent  observed  T r i s - m a l e i c a c i d system at  resolve  al.  by  300  seriously  electrophoresis  more e a s i l y the  sample  common  temperature-  was o b s e r v e d ,  a b s e n c e of  presented  s t a n d a r d or c a t a l y t i c  to  in the  s u c h an a l l e l e ,  within  a  most  in D. melanogaster  resulted  Beaumont and B e v e r i d g e  authors  Pgm-2  homozygotes  the  for  Trippa  PGM a c t i v i t y  complete  class  when  of  i n d i v i d u a l s homozygous  50°C  the  and  Ts h e t e r o z y g o t e s  would not  any h e t e r o g e n e i t y  locus  demonstrated  a Ts a l l e l e  frequency  s c o r i n g of  uncovered w i t h i n  Pgm-2  as  detected  for  method,  these  the  50% o f  homozygotes  classes  was  15 min a t  No e v i d e n c e  crude  at  allele  for  the  purification.  gels containing  -2-100 a l l e l e  in  identification  L o v e r r e and Catamo (1976)  Incubation  of  unequivocal  variation  (Ts)  aided  104)  with  was  unable  that  could  at  a higher pH.  pH 6 . 0 ,  or  38  A total agreement (1979a). several  of  with  that  of  presumably (1986b).  Numerical  Their  sampling  sites  the  or  The between  11/85  3  of high)  have  No  at  effect  to  C.  the  at  to  the  2  of  were  water  distributions  50).  analysis  between  differences  were  four sampling dates,  I.  frequencies  microenvironmental conditions  significant the  100  in Table  P > .  Chi-square  the  alleles  common  these  a  Foltz  low and h i g h  w i t h 35 d f ,  heterogeneity  the  To on was two  found  between  the  pooled  on  computed i n t e r a c t i o n t e r m .  values  Statistically  the  = 34.2  by  these  most  in  and a t  virginica  been a s s i g n e d to  in  study,  segregate (1976),  indicated that  allelic  any of  the  (x  locus,  performed in t h i s  in  relative  listed  observed genotypic  8 samples,  proportions  interaction  t e r m (x  which  = 57.3  I test and  deviations  (P < . 1 0 ) .  the  were  exhibited  the  A  P < .05), largest  detected  ( 1 0 / 8 3 H i g h and heterogeneity  proportions yielded a  w i t h 35 d f ,  agreement  Hardy-Weinberg  and an a d d i t i o n a l two  nearly significant  2  in Table  significant  on t h e s e g e n o t y p i c  sample  Pqm-2  H e r s h b e r g e r and Chew  by W i l k i n s  locus  Chi-square  3/85  the  shown  frequencies  time  a  Chi-square  expectations. for  over  comparing  for  the  been  analysis  potential  heights  totals,  Pgm  table  heights.  tidal  were n o t  qigas  values  frequencies, out  at  o v e r a two y e a r p e r i o d a r e p r e s e n t e d  were homogeneous  tidal  C.  observed  contingency  carried  have  their mobilities  allele.  these  in  homologous  expressing  examine  crosses  these a l l e l e s fashion  were d e t e c t e d  r e p o r t e d by B u r o k e r ,  Although pair  Mendelian  A  8 alleles  significant  caused  by  deficiency  the of  39  Table  I. Pqm-2 a l l e l e f r e q u e n c i e s , c o n f o r m i t y t o H a r d y Weinberg e x p e c t a t i o n s , and h e t e r o z y g o t e d e f i c i e n c i e s the four sampling d a t e s .  at  Frequency at Date and T i d a l 10/83  6/84  Height  3/85  Allele  Low  High  Low  High  84  .004  .002  .003  .003  88  .004  .005  .007  92  . 133  . 125  96  .096  100  Totals  High  Low  High  Low  High  — -  .002  .002  .002  .002  .003  .006  —_  .004  .007  .006  .006  .005  .091  . 146  . 108  . 131  . 118  . 115  . 112  . 129  .075  .111  .097  .075  .097  . 100  .094  .094  .093  .596  .610  .601  .538  .652  .552  .605  .609  .617  .578  104  . 142  . 133  . 157  . 166  . 141  . 180  . 134  . 155  .141  . 163  108  .025  .050  .027  .038  .019  .032  .034  .013  .027  .027  ---  .003  .006  .005  .002  .006  .002  .002  121  100  149  160  205  222  265  695  747  9.05  10. 5  4.73  2.40  27.4***20.4**  13.0*  9.72  na  na  He  .597  .586  .593  .651  .537  .635  .591  .583  .577  .613  Ho  .579  .550  .557  .631  .454  .550  .550  .562  .528  .572  -.030  -.061  -.061  -.031  - . 155  - . 134  -.069  -.085  -.067  112 N X  1  D  na * ** ***  = P P P  not a p p l i c a b l e < .05 < .01 < .001  Low  11/85  220  - .036  41  heterozygotes the  a s m e a s u r e d by D = ( H o - H e ) / H e (where  o b s e r v e d and  values  expected  calculated  for  g e n e r a l l y much l o w e r averaging 1975)  -0.122  and - 0 . 1 7 2  Given  the  the  for for  6  homozygous  for  the  this  and  either  heterozygotes were of  the  Pgm-2  the  overdominance at  are  study  the  Pgm-2  96,  properties  to  for  the  II.  purification  The  four  examine  the  the  s t e p c a u s e d an e a r l i e r e l u t i o n  and/or  gradient,  charge  observation  in the  allozymes  from  activities  of  the  of  presumably  properties.  Hall  p u r i f i c a t i o n of  the  mussel,  three  other  between  104  of  these  alleles. three  common  alleles  expression  GENOTYPES  allozyme  starting  substrate  salt  levels  the  of  the  I,  genotypes  potential  Pgm-2-104  presence  in  by  step procedure resulted  and r e c o v e r e d 39% of saturating  Table  locus.  A p u r i f i c a t i o n summary f o r Table  1982)].  and  four  [D  (Buroker  (Fujio in  D but  locus  and h e t e r o z y g o t e s  BIOCHEMICAL PROPERTIES OF PGM-2  in  this  dominated  Pqm-2-92,  homozygotes  for  negative,  distributions  allele  biochemical  out  were a l s o  populations  locus  Pgm-2-100  and t h e  singled  Japanese frequency  at  the  samples  Washington s t a t e p o p u l a t i o n s  20  individuals  Therefore,  other  respectively).  than p r e v i o u s l y r e p o r t e d for  allelic  allele  heterozygosities,  Ho and He a r e  substrate of  because (1985)  in a  300-fold  in the  final  the  saturated  of  altered  has  edulis.  Pgm-2 a l l o z y m e s  shown  activity.  The DEAE-  enzyme binding  reported a similar  two p h o s p h o g l u c o s e  Mytilus  is  isomerase  The f i n a l were  specific  similar  to  Table  II.  Purification  summary  for  the  Pqm-2-104  allozyme.  43  Fraction  Crude Homogenate  Volume (ml)  168.0  Spec. A c t i v i t y (units/mg)  1  100  3.28  94.3  10.0  0.175  DEAECellulose  20.2  2.25  6.7  6.94  DEAESubstrate  13.2  Recovery (%)  0.0533  Ammonium Sulphate Precipitation  Sephadex G-100  Purification (fold)  15.9  42.2  90.3  130  53.2  298  39.3  44  that  shown i n T a b l e  II  f o r the  Pqm-2-104 a l l o z y m e , and a l t h o u g h  t h e s e p r e p a r a t i o n s were n o t homogeneous  (as  gels),  of  all  were  interfering and  found  w i t h the  phosphoglucose  The for  to  PGM a s s a y  Michaelis  glucose-1-phosphate over  graphically on  free  enzymes  (UDP-glucose  the  constants  (G1P)  of  and  in Figure  2.  seven  5 to  A two-way a n a l y s i s (Figure  2,  Pqm-2  of  upper h a l f )  multiple  that  significantly values  posteriori the  larger  obtained  analysis  also  (F(6,30)  =  Km v a l u e s  due  e n t i r e l y to  of  significantly  their  The  and  P < .001).  different  10,  lower  app  homozygotes,  the  revealed  (F(5,30) range  5  three  tests  the  indistinguishable.  This  of  20°C,  Pqm-2  range t e s t s  showed t h a t  genotype  comparing  this  the  result  (1A) w h i c h  was  exhibited  Km's f o r G1P t h a n t h e P g m - 2 - 9 6 / 9 6 a n d and t h e  at  Pgm-2-100/104  individual  from  its  heterozygotes  app Km (G1P) v a l u e s ,  17.6,  but  or  and  heterozygote.  temperatures  by  s i g n i f i c a n t l y l o w e r app Km's f o r G1P o f arose  =  that  were  effect  Multiple  (ANOVA)  30°C  15,  3 0 ° C were  genotypes  at  t h e P q m - 2 - 9 2 / 9 2 homozygote  - 2 - 9 2 / 9 2 genotype range.  25  comparisons  showed t h a t  measured  uncovered a s i g n i f i c a n t  app  Genotypic  Km's  than those at  at  5.21,  Pgm-2-104/104  app  Bonferroni  presented  variance  P  indicated  of  genotypes  30°C are  effect  A  capable  pyrophosphorylase  temperature exerted a h i g h l y s i g n i f i c a n t .001).  SDS  glucose-1,6-diphospate  temperature range of  t h e app Km (G1P) v a l u e s  <  by  isomerase).  apparent  (G16diP)  be  determined  as  performance over the were  expected  strictly  ANCOVA the  Pgm  15 t o  30°C  intermediate  f o r a monomer  like  in  PGM,  45  Figure  2. E f f e c t of t e m p e r a t u r e on t h e a p p a r e n t M i c h a e l i s c o n s t a n t s ( i n MM) f o r g l u c o s e - 1 - p h o s p h a t e (1; upper h a l f ) a n d g l u c o s e - 1 , 6 - d i p h o s p h a t e ( 2 ; l o w e r h a l f ) of s e v e n Pgm-2 genotypes. In e a c h g r i d t h e P g m - 2 - 1 0 0 / 1 0 0 homozygote ( c l o s e d c i r c l e ) i s compared t o a d i f f e r e n t homozygote (open c i r c l e ) and t h e c o r r e s p o n d i n g h e t e r o z y g o t e (partially closed c i r c l e ) . B a r s r e p r e s e n t ±1 s t a n d a r d e r r o r where v i s i b l e o r f a l l w i t h i n t h e p l o t t e d s y m b o l .  O  92/92  O  96/96  O  104/104  O  92/100  O  96/100  O  100/104  •  100/100  •  100/100  •  100/100  1A  28  1B  ;  1C  o  26 24  E Q_ D  22 20 18 16 i  i  i  t  i  '  2A  1.6 =1  i  1  1  1  I  1  1  2B 0,  1.4  1  1  1  1  1  2C  -1.6 •  -1.4 -  -1.2  E  1.0  Q. Q. O  0.8 H  -1.0 -0.8  •^-6  0.6 H  5  10 15 20 25 30  -  -0.6  5  10 15 20 25 30  Temperature, °C  5  10 15 20 25 30  2  -  "a? '~o to o Q. CL  o  47  and  were k i n e t i c a l l y  15°C  where  the  significantly  The  temperature Multiple  decreased and  to  range  temperature  substrate  tests  lower  differ the  92/100 30°C,  but and  but  Since varies  both  the  Pqm-2-96/100  not  the  at  5,  pH of  inversely  10 o r  the  with  glucose(Figure  2,  effect  of  P  <  (5  distinct  As  where  constants This  .001). were  to  10°C)  from  those  showed  found this  for  effect  homozygote  (2A)  f o u n d by ANCOVA t o  from t h e  Pqm-2-96/100.  76.8,  and l o w e r  .001).  heterozygote  Michaelis  cofactor  app Km ( G l 6 d i P ) v a l u e s  was  25°C  two.  t h e s e app Km v a l u e s  Pgm-2-92/92  This  except  =  the  Pqm-2-92/92  displayed  to  be  which  than  the true  homozygote homozygote.  significantly  G16diP  than  Pgm-2-  the  Pgm-2-  o c c u r r e d because  heterozygotes d i f f e r e d  to the  hold  Pqm-2-100/100  for  at a  was a g a i n a t t r i b u t a b l e  tests  lower  expressed  temperature  were  effect  range  Pqm-2-92/100  not  30°C)  P <  except  ANOVA f o u n d t h i s  that  to  = 18.0,  significantly  apparent  100/104,  that  homozygotes.  temperature  Overall,  and  significantly  other  not  (20  by  the  (F(5,30)  upper  multiple  other  for  two-way  indicated  (F(6,30)  primarily  each  did  the  GIF,  displayed  a  15°C. A significant  Pqm-2 g e n o t y p e  at  than the  increasing  significant  ranges,  measured a t  with  again  be  homogeneous o v e r  three  app Km (G1P)  from e a c h o t h e r  heterozygote  apparent M i c h a e l i s constants  half),  caused  Pgm-2-92/100  lower  1,6-diphosphate lower  indistinguishable  at  15,  25,  and  20°C.  imidazole  temperature  buffer  used  in  (Yancey and Somero  this  study  1978),  the  48  influence  of  allozymes  contained  variation  temperature  in  on t h e  a confounding  buffer  pH.  To  apparent M i c h a e l i s constants pH 6 . 5 , 3).  7.0,  pH  and 7.5  exerted  517.0,  P  <  tests  showed  that  significantly (F(6,12) <  to  Once  responsible  for  tests  more, this  the than  Pgm-2-96/96  <  .001)  kinetic  the  from  heterozygotes, significantly  only  For the  A c o m p a r i s o n of  the  each  pH  both  from  Km  (F(6,12)  was  showed the  genotypes  estimates,  lower  values  and Pqm-2-  significantly  homozygote.  For  Pqm-2-92/100 which  largely  Pgm-2-92/92  e a c h was  two,  P  Multiple  the  between t h e  (G1P)  = 61.4,  genotype. of  range  differed  app  homozygote of  =  contributed  The P q m - 2 - 1 0 0 / 1 0 0  although  tests  this  (F(2,12)  app Km ( G 1 6 d i P )  Pgm-2-104/104  different over  of  (Figure  multiple  significantly  homozygotes.  these  indistinguishable  the  effect  difference  the  at  at  app Km's f o r  G16diP  app Km (G1P) v a l u e s  9 6 / 9 6 g e n o t y p e s were i d e n t i c a l , different  pH a l o n e ,  20°C  Pqm-2 g e n o t y p e a l s o  significant  other  of  parameters,  Pgm-2-92/92  homozygotes.  three  and  expressed  Pgm-2-92/92 genotype d i s p l a y e d the  of  on t h e  and app Km ( G 1 6 d i P )  comparing the  t h e s e PGM  simultaneous  effect  effect  v a r i a t i o n w i t h pH  uncovered a s i g n i f i c a n t and  P  means  P < .05)  the  the  temperature  significant  each o t h e r .  the  = 7.14,  .001).  range  from  examine  of  of  f o r G1P and G l 6 d i P were m e a s u r e d  For both  the  functioning  effect  a constant  = 308.4,  .001).  significantly  at  a highly  b o t h G1P ( F ( 2 , 1 2 )  kinetic  the  to  again  be were  pH r a n g e .  patterns  apparent M i c h a e l i s constants  p r o d u c e d by v a r i a t i o n f o r G1P and G 1 6 d i P  i n pH on  in Figure  3  49  Figure  3. E f f e c t o f pH on t h e a p p a r e n t M i c h a e l i s c o n s t a n t s (in uM) f o r g l u c o s e - 1 - p h o s p h a t e ( 1 ; upper h a l f ) and g l u c o s e 1 , 6 - d i p h o s p h a t e (2; l o w e r h a l f ) of s e v e n Pqm-2 g e n o t y p e s . G e n o t y p i c c o m p a r i s o n s a r e shown a t t h e t o p of t h e f i g u r e . B a r s r e p r e s e n t ±1 s t a n d a r d e r r o r where v i s i b l e o r f a l l w i t h i n the p l o t t e d s y m b o l .  O  92/92...  O  96/96  O  104/104..  O  92/100  O  96/100  O  100/104  •  100/100  •  100/100  •  100/100  34  34  30  Q_  26  E  22  CL CL D  18  30  ^  26  t  22  E  18  14  14  2A  2B  2  1.4-  'ST*  1.2-  1.2  1.0-  i.o  ^.  hl.4  XI  to  & E CL • QL O  Q. CL O  0.8-  ^  to 5  0.8 ^ 8^-  °  CL CL  0.6-  0.6 I '  6.5  1  1  ' I  1  1  7.0  1  1  I  7.5  I '  6.5  1  '  1  I '  1  7.0  pH  '  1  I  7.5  I  1  6.5  1  ' 'I  7.0  7.5  °  51  shows  that  increase (G1P)  one  is  virtually  i n pH from 6 . 5  but o n l y  increase  to  a slight  7.0  caused  increase  i n pH from 7.0  to  7.5  substrate  effect  and c o f a c t o r  dissociation residue  state  on the  hypothesis  of  conditions  the  proportions Active  site  Sanger  (Hashimoto,  7.0)  described  These the  for  Figure pH of  2  moiety  3  protonated  role  further  s i d e c h a i n of  standard  present  state  forms  Handler  the the  favors  roughly  equal  (Somero  1981).  (Milstein  coli to  has  the  this  b i n d i n g of of  pH  and  catalytic histidine  form o f  phosphoenzyme  and  flounder  (Joshi  deprotonated  an e f f e c t  the  histidine  1969),  adjacent  PGM a l s o  by  for  physiological  in  and E .  This  enzyme  a  in  alpha-imidazole  from r a b b i t  and  G1P t o  the  played  Under  that  (below pH 7 . 0 ) ,  this  this  (above  pH  G16diP to  the  not  previously  enzyme.  i n the  imidazole  a  [the  is  oyster  suggests  b i n d i n g of  that  of  of  reactants  (Joshi  If  A  i n app Km ( G 1 6 d i P ) .  affinity  PGM i s o l a t e d  pH-induced effects  variation  the  possess a h i s t i d i n e  residue.  dephosphoenzyme  i n app Km  R i o and H a n d l e r 1 9 6 6 ) , all  the  a marked d e c l i n e  imidazole  these  human  Figure  and  o t h e r . An  p r o t o n a t e d and d e p r o t o n a t e d  del  favors  on  (1972)].  sequences of  of  i n app Km ( G 1 6 d i P ) .  suggestive the  imidazole  of  phosphoserine  residue  of  Reeves  1964)  present,  is  the  increase  pH  b i n d i n g of  1961),  Handler  of  image  produced only a small decrease  app Km (G1P) but a s u b s t a n t i a l antagonistic  a mirror  app Km  are capable  (G1P)  and  were a p p a r e n t l y c a u s e d buffer  decreases  0.017  of  explaining  (G16diP)  estimates  by t e m p e r a t u r e . pH  some o f  units/°C,  in  Since  the  and  was  52  initially  adjusted  to  pH  7.0  increased  from 5 t o  30°C  the  pH of  the  linearly  from  7.3  to  variation  i n app  changes 20 t o  from  30°C  these  variation with  in  20  suggest more  20°C  with  below  in  pH  an  effect  changes  7.0)  For  are  only  the  with  to  temperature  7.0,  as  to  pH d e c r e a s e d  in vivo to  were t h e  functioning  variation  in  of  from  from 5 t o 1 0 ° C  also  The  consistent  i n app Km ( G l 6 d i P )  to  the  predicted  is  pH from 7.3  below  from  temperature.  2 0 ° C were e x p e c t e d  3 0 ° C as  negligible  increases  decreases  attributable  observed  the  largely  was  decreased  the  and t h e  i n p H . The d e c l i n e s  from  the  minor changes 7.0.  These  over drop  observed  observations  these allozymes  i n t r a c e l l u l a r pH t h a n  may  to  be  changes  temperature.  Maximum v e l o c i t y  (Vmax)  estimates  regression  were s t a n d a r d i z e d t o  (to  for  correct  the  slight  preparations)  temperature  were c u r v e d s l i g h t l y as  (Ray and Peck 1985).  significant  10 t o  found  30°C.  in  Plots  biweight  activities  of  log  downward a t  previously  1972)  and t h e  Two-way - a n a l y s i s  differences  by  at 5 ° C between  and compared between g e n o t y p e s o v e r  r a n g e of  shown)  obtained  a common p r o t e i n c o n t e n t  differences  temperature  (Hoffman  6.8.  buffer  5 to  ambient  muscle  assay  temperature  of  sensitive  (not  as  range  that  allozyme  to  7.0)  alone;  20°C,  temperature,  (pH 7.2  i n app Km ( G 1 6 d i P )  temperature buffer  (G1P)  pH f e l l  be  these  from  in  (as  to  Km  10 t o  changes  appear  the  approximately  at  for sea  (Vmax)  higher  PGM i s o l a t e d anemone,  of  between g e n o t y p e s  i n the  against  temperatures from r a b b i t  Metridium  variance  the  senile  found  sensitivity  no of  53  log  (Vmax) t o  (F(6,12)  either  = 1.56,  genotypes  (F(6,12)  phosphate  =  i n the shown  homozygote  Over had  (A)  other  the  in  different  from  vivo catalytic that  divergent  from  5  forward  to  4  5,  ratios  and by  between  two-way  genotypes  P < .001)  and  were  p r o d u c e d by  the  for  respectively.  6.5  to was  but not  the  7.5  In F i g u r e  the  Pgm-2-92/92  behavior genotype  S i n c e Vmax/Km r a t i o s  most  function one  (Hoffman 1981; allozyme,  of  Pgm-2-92/92  or  also  t h a n t h e PqmPqm-2-104/104  being  again  significantly  the  Pqm-2-100/104  accurately Watt 1 9 8 3 ) ,  Pgm-2-92,  ratio  homozygote  heterozygotes  not  4  heterozygote.  greater  Pqm-2-100/100  Pgm-2-92/100  the  Pgm-2-92/100  significantly  the  by  glucose-1-  d i s p l a y e d a l a r g e r o v e r a l l Vmax/Km except  entirely  but  reflect these  consistently  in  results displays  properties.  Vmax(f)/Vmax(r) for  30°C  of  = 6.90,  that  Pqm-2-96/100  kinetic  of  caused  functions  which  the  only  Ratios  (F(6,30)  .001)  intermediate  the  heterozygote.  <  these  Pqm-2  is  a Vmax/Km r a t i o t h a t  resulted  seven  Figures  differences  F i g u r e s 2 and 3 ,  genotypes  The  the  o r pH  result  pH r a n g e of  genotypes.  P > .40)  apparent M i c h a e l i s constants in  2 - 9 6 / 9 6 homozygote  show  P  of  in  c o m p a r i s o n s of  significant  9.07,  significant  than a l l  presented  Statistical  = 1.15,  r e a c t i o n d i r e c t i o n as  with both temperature  differences  this  are  (F(5,24)  Vmax/Km r a t i o s  forward  pH  revealed  associated pH  the  and  respectively. ANOVA  P > .20).  for  temperature  temperature  are  presented  r e a c t i o n exceeded  the  the  four  in Table  reverse  by  Pgm-2 III.  a  homozygotes  The r a t e  factor  of  of  the  three,  54  Figure  4. E f f e c t o f t e m p e r a t u r e on t h e Vmax/Km r a t i o s o f seven Pqm-2 g e n o t y p e s f o r t h e f o r w a r d r e a c t i o n d i r e c t i o n . Bars r e p r e s e n t ±1 s t a n d a r d e r r o r where v i s i b l e o r f a l l w i t h i n the p l o t t e d symbol.  O  92/92...  O  9.6/96.  O  104/104  O  92/100  O  96/100  O  100/104  •  100/100  •  100/100  •  100/100  A  8-  B  -  c  7-  E X  o  :  654-  J  3-  /  2-  -/  1i 5  i i i i i 10 15 20 25 30  -  J/  :  5  -  10 15 20 25 30  Temperature, °C  ef  :  •••••i —\—V 1 1 1 5 10 15 20 25 30  cn cn  56  Figure  5. E f f e c t o f pH on t h e Vmax/Km r a t i o s o f s e v e n Pgm-2 genotypes for the forward r e a c t i o n d i r e c t i o n . Bars r e p r e s e n t ±1 s t a n d a r d e r r o r where v i s i b l e o r f a l l w i t h i n the p l o t t e d symbol.  O  92/92  O 96/96  O  O  92/100  O 96/100  O 100/104  •  100/100  •  •  100/100  104/104  1Q0/100  58  Table  III. E f f e c t of t e m p e r a t u r e o f f o u r Pgm-2 h o m o z y g o t e s .  on t h e  Vmax(f)/Vmax(r)  ratios  59  Pgm-2 Genotype Temperature  92/92  96/96  100/100  104/104  5  3.07+.16  2.99±.10  3.08±.14  2. 97±. 11  10  3.01±.08  2.92±.09  2.93±.09  3. 13±.10  15  3.12±.06  3.06±.06  3.04±.05  3. 0 7 ± . 0 7  20  3.09±.05  3.01+.05  3.10±.06  3. 0 3 ± . 0 5  25  2.84±.07  2.961.06  3.02±.06  3. 10±.07  30  2.87±.05  2.93±.05  2.83±.05  2. 7 7 ± . 0 6  60  similar  to  Roscelli  (1964b).  ratios 4.09,  that  detected P < .05),  the  higher  reported Two-way  that  multiple  ratios  homozygotes  (F(3,15)  always  formed/time highly  the Vmax(f)  linear  The  conversion as  departures  P  used  to  found to compared  .90).  of  substrate  to  of  direction  from l i n e a r i t y even  30°C. four  Pgm-2  ratios  and  lines thus the  (1964b),  always  No  82.0  product over  were  to  of  were  determination  regression  for  83.4%, from  the  confirming a assay p e r i o d .  the  slopes  negative,  though s u b s t r a t e  = by  these  averaged  f o u n d by Ray and R o s c e l l i  (F(5,15)  be c a u s e d  the  calculate  the  Vmax  The r e g r e s s i o n s  The c o e f f i c i e n t s  of  the  temperature  between  >  on  from  indicating  conversion  was  lower.  tendency  17.3±0.32. previously (1942) range.  constants  to d e c l i n e  estimated  are  time  slopes  Equilibrium  the  15°C  and V m a x ( r ) r e g r e s s i o n s  reverse  1% o r  vs.  0.17,  of  tests  observed  PGM by Ray and  variance  r e a c t i o n d i r e c t i o n approximated u n i t y ,  However, the  =  significant.  respectively. forward  were  effect  at  muscle  of  range  measured  differences  rabbit  analysis  a significant  significant  product  for  as  with  18.0±0.45, Because  estimate  of  Estimates  17.2 of  while  at  30°C  in Figure  it  was  were  reaction,  was a c c e p t e d  the  PGM  reaction  temperature.  values  this  calculated  slightly  Since  for  the  a  to  larger  be than  C o l o w i c k and S u t h e r l a n d ' s the  were c a l c u l a t e d 6.  showed  A t 5 ° C , Keq was  entire  apparent M i c h a e l i s constants  f o u r Pgm-2 homozygotes presented  the  increasing  these  reported for  for  temperature f o r G6P of  from e q u a t i o n  2  Vmax(f)/Vmax(r) r a t i o s  and of  Figure  6. A p p a r e n t M i c h a e l i s c o n s t a n t s ( i n uM) f o r g l u c o s e p h o s p h a t e of f o u r Pgm-2 h o m o z y g o t e s e s t i m a t e d from t h e Haldane e q u a t i o n .  160  H  150  H  140  8>  130  A  120  H  CD.  | CL  Q. o 110 H  100 H O 96/96 90  H  •  iqo/ioo_  • T  104/104  1  1  1  1  15  20  25  30  Temperature, °C  63  these  genotypes  values  over  patterns The  were  this  observed  ability  genotypes approach viewed there  for is  is  temperature for  to the  reverse  provisional,  constraints  pH  between  differences  that  further  6.0  to  8.0 7.2  slightly  pH  optima  humans  (Hashimoto  and et  similar  form,  proportional  (Hoffman 1 9 8 5 ) ,  al.  .001).  Multiple  of  Pgm-2-92/92  this  these  pH i n t e r v a l showed of  the  In t h e  ANOVA  tests  into  that  Pgm-2-92/92  must  be  that  through  f o u r Pgm-2  (Figure for  7),  PGM  two  the  on  the  that  but  which  isolated (Fucci  11  this  et  al.  species were  very  transformed  significant  differences  (F(3,308) the  the  s t a n d a r d p h y s i o l o g i c a l pH r a n g e of  P  <  activity Pgm-2-  D e c o m p o s i t i o n of ANOVA's a t  greater  pH r a n g e of 7.0  15.3,  exceeded  one-way  was c a u s e d by t h e  =  overall  Pgm-2-96/96.  over  from  angular  individual  homozygote  is  f l o u n d e r and s h a r k  significantly not  allozymes  bacterial  revealed that  homozygote  overall analysis  and  showed  and P q m - 2 - 1 0 4 / 1 0 4  all  (1969),  f o u r homozygotes  range  indirect  A l t h o u g h t h e s e pH p r o f i l e s  a two-way  between  100/100  1966),  activities  Pgm-2  may be c o n c l u d e d i s  D. melanoqaster  Handler  1966).  existed  the  and  Handler  (Hanabusa in  the  (Joshi  2.  Haldane r e l a t i o n s h i p .  pH  1979),  this  differentiation  at  Metridium senile  by  the  Figure  T h e r e f o r e , these values  all  (G6P)  s i m i l a r to  kinetic  r a n g e of  than  were v e r y  Km  in  exhibited optimal a c t i v i t y lower  app  app Km's f o r G1P seen  and  for  their  reaction direction  i m p o s e d by t h e  the  range  restricted.  no e v i d e n c e  Over  their  detect  severely  as  indistinguishable,  to  7.4  each  activity  6.0  to  the  6.6. four  64  Figure  7. PH-dependent a c t i v i t i e s o f f o u r Pgm-2 h o m o z y g o t e s a t 20°C. B a r s r e p r e s e n t ±1 s t a n d a r d e r r o r where v i s i b l e o r f a l l w i t h i n the p l o t t e d symbol.  Percent Activity  66  allozymes  were homogeneous.  "shoulder"  which  o c c u r r e d between 104 a l l o z y m e s  for  the  pH 7.4  between  pH 7.6  constants,  determined  for x  genotype  the  tests  was  - 2 - 1 0 4 / 1 0 4 homozygote significantly genotypes  it  more s t a b l e  heterozygotes,  the  significantly  Pgm-2-92/100  0.0212)  w h i c h , as  effect of  P <  the  .001).  kd's of  the  =  of  magnesium four  Statistical  to  decline  c o m p a r i s o n of  the  log were  significantly  The  Pgm-2-100/100  and m u l t i p l e  different  range  from t h e Pgm  and  -0.0295,  properties  genotype (kd  or  were  Pgm-2-96/96  and  intermediate  of  exhibited  =  -0.0159)  Pqm-2-96/100  a than  (kd = -  indistinguishable.  ion  on  the  Pgm-2 a l l o z y m e s  at  denaturation  time,  be  -0.0283  -0.0206) were  their  regressing  Pqm-2-92/92  Pgm-2-100/104  (kd  in  T h e s e two h o m o z y g o t e s  Each v a r i a n t d i s p l a y e d o p t i m a l a c t i v i t y tendency  by  (kd = - 0 . 0 1 4 3 ) ,  than the  expected,  of  Thermal  incubation  lower d e n a t u r a t i o n c o n s t a n t  either  velocities  against  significantly  (exhibiting Because  The  was  and P g m - 2 -  Pgm-2 g e n o t y p e s  genotype  (kd = - 0 . 0 1 7 6 ) .  respectively).  Pgm-2-100  by ANCOVA a n d f o u n d t o  stable  allozymes  7.8.  each  = 383.0,  most  i n d i c a t e d that  50°C  10)  statistically (F(6,448)  the  (Figure 8).  at  heterogeneous  Pgm-2-96  between  stabilities  activity  and  and f o r  and  each c u r v e d i s p l a y e d a  existed  temperature  compared  Pqm-2-92  and 7 . 6  Marked d i f f e r e n c e s  (fractional  Above pH 7.4  higher angular  at  initial  is  shown  3  mM  cofactor  reaction  in Figure  and  a  9.  slight  concentrations.  transformed  proportional  67  Figure  8. T h e r m a l i n a c t i v a t i o n p l o t s of seven Pgm-2 g e n o t y p e s at 5 0 ° C . B a r s r e p r e s e n t ±1 s t a n d a r d e r r o r where v i s i b l e or f a l l w i t h i n the p l o t t e d symbol.  0  5  O  92/92  O 96/96  O 104/104  O  92/100  o  96/100  O 100/104  •  100/100  •  100/100  •  10 15 20 25 30  0  5  10 15 20 25 30  Time, minutes  0  5  100/10Q  10 15 20 25 30  69  Figure  9. E f f e c t of magnesium i o n on t h e a c t i v i t i e s of f o u r Pqm-2 h o m o z y g o t e s . B a r s r e p r e s e n t ±1 s t a n d a r d e r r o r where v i s i b l e o r f a l l w i t h i n t h e p l o t t e d s y m b o l .  71  activities between  by two-way  ANOVA d e t e c t e d  Pqm-2 a l l o z y m e s  (F(3,140)  no s i g n i f i c a n t  = 1.22,  P >  differences  .20).  DISCUSSION  A selection-based multiple-locus be  heterozygosity  f o u n d e d upon t h e  the  allelic  explanation  relationships  (cf.  of  functional  differences  verification  of  requirements their of  the  on  polymorphic  kinetic  and  have  and  1978).  in  genotypes  glucose-1-phosphate  (Figures  5),  thermostabilities  2)  Pgm-2 l o c u s  (1982).  exist  differences  places  apparent  and  pH  (Figure 8).  for adult Several  are  It  between  Ginzburg  the  gigas,  four  observed  to  between for  ranges  Vmax/Km (Figure  account  described at  most  constants over  3),  to  stable  this  argument s u g g e s t  that  of  ratios 7),  must now be d e t e r m i n e d i f  sufficient  of  a  in Crassostrea  activities  body w e i g h t lines  relative  manifestation  Michaelis  (Figure  these  stringent  Lewontin,  were  these  however,  allowing  1959;  must  between  Once  heterozygotes  conditions  pH-dependent  differences  overdominance Fujio  their  traits  established,  and g l u c o s e - 1 , 6 - d i p h o s p h a t e  (Figure  4 and  biochemical  of  structural differences  Pgm-2  1978).  hypothesis  Mandel  F o r the  between  involved in  c o n c e r n i n g both the  (e.g.  Significant  temperature  been  the  common a l l o z y m e s .  loci  Koehn  properties  equilibrium  and T u l j a p u r k a r  specific  1975;  homozygotes  overdominance  associations  biochemical differences  overdominance  the  constituent  of  the  Clarke  the  and v a r i o u s p h e n o t y p i c  existence  products  for  and these  for  the  locus  by  they  are  72  not.  First,  consideration  of  the  phosphoglucomutase  suggests  differences  be as  under  may not  _in  vivo  established  that  it  u n i " and " p i n g - p o n g " k i n e t i c s phosphates extracted caused  interconverted (reviewed  by  diphosphate occurs  frequently,  p r o d u c t " as pong"  from t h e  well  as  mechanism  mechanism,  against  G1P  vicinity  of  accordance Joshi  and  an enzyme have  the  on  the  double  in  1969;  various  f o r G1P i s  b o t h G1P and G 1 6 d i P by t h e  have  sugar  variation  of  is  of if  the this  as  "first  classic  "ping-  this  variation  initial  levels of  rate  complex:  the  was  of  in  velocity  Gl6diP  parallel  in  the  lines  in  (Ray and R o s c e l l i  1964a;  H a s h i m o t o and H a n d l e r 1 9 6 6 ) . F o r  kinetics,  experimentally  PGM  acts  Despite  reciprocal plots  ping-pong  constant  This  reaction  (1963).  at  all,  specific  dissociation  p i n g - p o n g mechanism  H a n d l e r 1966, obeying  the  1972).  substrate"  Cleland  at  o r g a n i s m from w h i c h i t  Km ( G l 6 d i P ) p r o d u c e a s e r i e s with  on  glucose-1,6-diphosphate  concentration  verified  Michaelis  the  enzyme's c e n t r a l  "second  of  reaction  in  kinetic  a l o n g a c o n t i n u u m between " u n i -  by Ray and Peck  differences  observed  studies  depending  and  mechanism of  o r even e x p r e s s e d  Previous  functions  reaction  the  pronounced,  conditions.  that  catalytic  that  related  following  Ray and the to  the  Roscelli  true  or  apparent  equation:  (1964a)  "realized" Km's  for  73  K'm  (G1P) =  app Km ( G 1 P ) [ G 1 6 d i P ] app Km ( G 1 6 d i P )  where Km  K'm (G1P)  (G1P)  and  constants  1978) are  app  of  to  to as  Km  range  orders  80 of  organisms  (Ray  1969),  suggesting  that  active  phosphorylated demonstrated in  which a r e  unexpected  effect only  of  occur  if  but  state.  f o r G 1 P , app  apparent  Michaelis  and [ G l 6 d i P ]  concentrations  is  the  However, in  if  the have  from what  is  proportion  1 uM t h e  et  al.  (see  Km  1964b;  the  recent  activity  about  at  assuming  and  Peck  ratios  of  the  studies PGM  by 1984)  this  exists  can  in  the The  phosphorylated  and  with  unsaturating  realized Michaelis constants  Vmax/K'm  the  1972).  consistent an  in  saturating  PGM,  enzyme  PGM i s  al.  a deinhibitory  the  both  of  in  et  by B e i t n e r  exists  known  G16diP  entirely  i n t e r p r e t e d as  of  been  levels  Passonneau  (reviewed  of  These  for  a number of  Ray  oyster  1969).  PGM's  cofactor been  have  Haberman and N o r d e n b e r g  e x i s t s _in v i v o  concentrations  forms of  corresponding  above  enzyme  Therefore, of  the  Roscelli  detection  dephosphorylated  and  the  state  electrophoretic  concentration  and  some  dephosphorylated  suggestions.  magnitude  results  G16diP,  Gl6diP  uM ( P a s s o n n e a u  G16diP  These  are  respectively,  activations  increases  levels.  (G16diP)  from 5 /uM ( B e i t n e r ,  these  have  r e a l i z e d M i c h a e l i s constant  tissues,  h i g h as  several  + [Gl6diP]  Gl6diP.  mammalian  observed  the  f o r G1P and G l 6 d i P ,  concentration  In  is  ...(3)  these G16diP  for  7 Pgm-2 g e n o t y p e s  G1P as  74  functions equation  of  temperature  3 and a r e p r e s e n t e d  Comparison presented  of  are  have  importantly,  two-way  found  the  that  Pqm-2 g e n o t y p e reduced.  to  detect  4 and 5  by  significant either to  estimates  ratios  (Figure  between  genotypes  P  <  their tests  .05)  and  11),  effects  50%  for  the  between  1.75,  (Figure  significant  unable  ratios  to  detect  hence  10).  P  More  (F(6,12)  to  substantially  were  unable  in  their  = 0.913,  P >  .40)  >  over  .40)  Over  differences  any  the  estimates  genotypes  the  were  = 4.04,  pH  still  (F(6,12)  m a g n i t u d e was d i m i n i s h e d and a p o s t e r i o r i were  and  or  i n t h e s e m o d i f i e d K'm v a l u e s Vmax/K'm  (G1P)  app Km (G1P) and app  f o r G1P ( F ( 6 , 3 0 )  30°C  K'm  these analyses  differences  to  the  previously attributable  individually,  =  those  same amount.  eliminated  results  (F(6,30)  range o f . 5  studied  that  with  v a r i a n c e on t h e s e new  been  the  any s i g n i f i c a n t  temperature  shows  from  respectively.  parameters  approximately  of  calculated  10 and 11,  modified  analysis  had  Vmax/K'm  been  in Figures  r e a l i z e d Michaelis constants or  have  i n c r e a s e d by r o u g h l y t h e  In c o n t r a s t  (Gl6diP)  3,  reduced  Vmax/K'm r a t i o s  pH  these  i n F i g u r e s 2,  estimates  Km  and  range present  =  4.70,  P < .05),  multiple  differences  the  but  range  between  the  genotypes.  The h o m o g e n i z a t i o n of genotypes their amino  app  appears  to  (G1P) and app  acid  the  be Km  substitution(s)  a  K'm  (G1P)  consequence (Gl6diP) giving  estimates of  values. rise  to  the  of  Pgm-2  c o v a r i a t i o n of  Apparently, the  differences  the in  Figure  10. E f f e c t of t e m p e r a t u r e on t h e e s t i m a t e d in_ v i v o a p p a r e n t M i c h a e l i s c o n s t a n t s ( i n MM) f o r g l u c o s e - 1 p h o s p h a t e (1; u p p e r h a l f ) and c o r r e s p o n d i n g Vmax/K'm r a t i o s (2; l o w e r h a l f ) of s e v e n Pgm-2 g e n o t y p e s .  O  92/92  O  ?6/?6  O  104/104 _  O  92/100  O  96/100  O  100/104  •  100/100  9  100/100  1A  1B  1C  hie 14  10 £  8 Q.  he T  Q_ D  r  2B  14 12  £  10  ^  8 D  h2 1 5  1 1 1 1 1 10 15 20 25 30  -  i  5  r i r 10 15 20 25 30  Temperature, °C  "I  5  1  1  1  1  r  10 15 20 25 30  -  77  Figure  1 1 . E f f e c t of pH on t h e e s t i m a t e d in_ v i v o a p p a r e n t M i c h a e l i s c o n s t a n t s ( i n uM) f o r g l u c o s e - 1 - p h o s p h a t e (1; u p p e r h a l f ) and c o r r e s p o n d i n g Vmax/K'm r a t i o s (2; l o w e r h a l f ) o f s e v e n Pgm-2 g e n o t y p e s .  • Vmax/K'm  "T"  I cn  1  I oo  1  I  T"  r  ^  Vmax/K'm  8Z.  app K'm(G1P), fM  to  tn  oo  =2  r  O  app K'm(G1P), yUM  79  electrophoretic  mobility  binding  internal reaction  and/or  a similar 92/92 for  fashion.  This  homozygote  Upon  the  range  to  cancel  for  the  other  into  Pgm-2-92/92 genotypes.  also  exhibited  the  exception  possessing  the  that of  were  3,  such that  homozygote  the  become  pH r a n g e o f  however  of  Pqm-2-104  the  allele.  ratios  expressed  over  stressed reaction  this  here  is  for  mechanism  these to  expressed  be much l e s s  than expected  still  significant  conform  to  concentration  in  the of  other  Figure  (G1P)  2).  values  estimates from  the  parameters  (Figure of  behavior effect  3)  is  with  genotypes of  these  of  Pqm-2  the  into  important  point  consideration the  the  ping-pong  interdependence function,  the  being  of  app  kinetic  i_n v i v o between t h e s e Pgm-2 g e n o t y p e s may from t h e  differences  shown  by  the  separately.  s h o u l d be e m p h a s i z e d  h o l d under  (see  kinetic  The anomalous  PGM, and t h u s  differences  It  most  parameter  K'm  7.5  Pgm-2-  constants  indistinguishable  Km (G1P) a n d app Km ( G l 6 d i P ) on c a t a l y t i c  parameters considered  lower  the  6.5  However,  by t a k i n g of  studied  in  t h e s e K'm (G1P) e s t i m a t e s and Vmax/K'm  pH r a n g e . that  the  than  app Km (G1P) v a l u e s  g e n o t y p e s may i n p a r t e x p l a i n t h e genotype  for  smaller  these  the  f o r G1P and G l 6 d i P  apparent  temperatures  out  affect  apparent M i c h a e l i s  The c o v a r i a t i o n of  over  allozymes  pathways most  equation  each other  these  exhibited  G16diP  substitution  act  trend is  which  b o t h G1P and  genotypes over  between  vivo  these  conditions  ping-pong  G16diP i s  that  if  mechanism  in fact  kinetic oyster or  saturating.  the  differences  may  PGM d o e s  not  intracellular  However,  even  if  80  these  functional  properties either a  of  differences  Pgm-2 g e n o t y p e s  the m a n i f e s t a t i o n  stable  as  daltons  staining  m o b i l i t y on SDS g e l s  columns,  are a l l  monomeric  not  1969;  fact  the  over  overdominance  differences  in c a t a l y t i c (1959)  heterozygotes over  different  as  exceeds  of  that  must  this  of  it  of  studies.  (e.g.  Watt  properties  this  must  homozygotes  were  the and  1983), expected  were  in  examined.  If  locus  be o f  the  via  1977,  that  conditions  to  For a  Schwartz  Pgm-2 l o c u s  at  The  filtration  overdominance  "marginal", i . e . ,  the  Pgm-2 l o c u s  the  by  these  type  termed  mean p e r f o r m a n c e of only  when  averaged  in C . gigas are  dominated  96/100 and 100/104 g e n o t y p e s  a l l heterozygotes). be r e s p o n s i b l e  locus.  the  expressed  function,  that  at  Pgm-2-92/100,  74%  on g e l  to  conditions.  Heterozygotes by t h e  at  range is  1972).  PGM, i n a d d i t i o n  patterns  of  established  Peck  oyster  intermediate the  have  with these previous  Heterozygotes  strictly  observed  Wallace  of  r a n g i n g from 62,000  heteromultimers  biochemical  by  characterizations  and  of  expression  of  of  S i n g h , Hubby and L e w o n t i n 1974;  possible.  display  patterns  with  maintenance  organisms  Ray  and e l u t i o n  the  properties  Laughner  by  biochemical  compatible  or the  Molecular of  the  appear  overdominance  i n agreement  enzyme,  superior  to  not  from a v a r i e t y  (reviewed  electrophoretic  is  expressed  a monomer w i t h a m o l e c u l a r w e i g h t  67,000  its  of  do  polymorphic e q u i l i b r i u m .  phosphoglucomutase it  are  The  Therefore,  for  expression  it  is  these  overdominant e f f e c t s of  (representing heterozygotes attributed  to  m a r g i n a l o v e r d o m i n a n c e by  the  81  three  most  conditions 104  on t h e  allozymes  Because  of  2-100/100 the  less  below  of  homozygotes across  1981),  2)  or  Furthermore,  expressed  over  Powers  must  Pgm-2-100/100  average  different  b e h a v i o r of  meeting  marginal  overdominance.  of  characteristics  the  same d o e s not  allozymes. of  the  that  marginal  of  (e.g.  above  in  value  of and  properties  selective either  1)  are 96/100  values  of  variable  microenvironmental Koehn and Immerman advantages  o r pH ( e . g .  between d i f f e r e n t  Place  catalytic  1981).  Pgm-2 homozygotes  The Pgm-2-92  Pqm-  above  Pgm-2-92/100,  (e.g.  requirements  differ  from t h e  i n s u c h a m a n n e r , and  temperature  Walsh  frequent respective  and/or s t r u c t u r a l  trade-offs  for  allozyme  the  not  seem  p r o d u c t i o n of  possesses a  suite  allozyme,  Pgm-2-96  or  Pgm-2-104  a c a s e c o u l d be made f o r  the  expression  overdominance  from t h e  do  Pgm-2-100  hold for  Consequently,  through  kinetic  The o v e r a l l p e r f o r m a n c e of of  the  i n the  states  allozyme.  less  their  96 and  f u n c t i o n a l parameters  seasons,  ranges  o r 3)  capable  of  superior  arise  the  structural properties  Pgm-2-92,  these  genotype  that  physiological  1979),  of  the  Variation  could  of  the  restrictive  common Pgm-2-100  fluctuate  different  reversals  of  by r o u g h l y e q u a l amounts  100/104 h e t e r o z y g o t e s .  conditions,  places  properties  magnitude,  upon t h e  selection  or  the  most  frequencies  common homozygotes  sufficient  these  the  functional  genotype.  conferred  and  to  must d i v e r g e  that  with  heterozygotes  biochemical properties  similar  the  homozygotes  Pgm-2  relative  the  alleles,  and  common  either  by t h e  the  Pgm-2-92/100  heterozygote  but  by  82  invoking a variable selection catalytic  f u n c t i o n and  similarity the  of  Pgm-2-100  the  regime,  or  from t r a d e - o f f s  thermostability.  Pgm-2-96  allozyme  However,  and Pgm-2-104  precludes  evidence  for  reversals  Michaelis  constants  relative  to  pH  (see  seem  of  performance  over  F i g u r e s 2 and 3 ) ,  far  too  small to  differential  with  p o l y m o r p h i s m by Lewontin,  for  between  homozygotes  differentiation distinctiveness  In  may of  genotypes  be  and be  the  heterozygote  100/104  of  much  heterozygotes. weakly  Pgm-2-92  to  both  both of  and the  these  some  apparent  and  Pgm-2-104 and  fluctuations advantage. is  The also  multi-allelic analyses,  found the  that  than t h a t  with  the  variance  kinetic  fitness,  hence  in  expressed  Although  associated allozyme  compared causes  lower  is  the  numerical  (1978)  to  t h e Pgm-  temperature  a stable  their  Tuljapurkar  heterozygotes  for  these  s u c h an e q u i l i b r i u m r e q u i r e t h a t  of  relative  heterozygotes  maintenance  and  of  of  heterozygote  these  overdominance.  fitness  92/100  of  the  Ginzburg  conditions  produce a net  Pgm-2-96  ranges  magnitude  greater  Although there involving  different  the  behavior  incompatible  allozymes  f o r G1P and G 1 6 d i P of  Pgm-2-100  the  a s i m i l a r argument  2 - 9 6 / 1 0 0 and P g m - 2 - 1 0 0 / 1 0 4 h e t e r o z y g o t e s .  between  th e v  Pgm-2-  Pgm-2-96/100 conditions  the  to  and be  violated.  The allozymes  biochemical are also  distributions. (G1P)  values  The and  differences  contradictory Pqm-2-92 therefore  to  uncovered their  allozyme larger  between  observed  displayed  frequency  lower  catalytically.  these  app Km  important  83  Vmax/Km three  ratios  others.  activities to  occur  15 t o  This allozyme also  in  marine  bivalves  metabolism  1984).  Because  might  be  of  l e a d to  frequency  of  balanced  its  argument however allele  enjoy  this  catalytic  allozyme  in a l l  other  f r e q u e n c y of  overdominant -2-100/100  that  loci  genotype  Although  the  may i n f a c t  similarity  differentiation that  kinetic  of  to the  be b e s t the  intermediacy  allele  to  these somehow This  t h e Pgm-  sensitive  to  Pgm-2-100  at  explain Smouse  the  (1986)  multi-allelic  homozygote.  properties  The Pgm  that  were  the  other  homozygotes.  expressed  by t h e  Pgm-2-100/100  Pgm-2-96  at  are  to  Recently,  fittest  suited  two o t h e r  The  with  from t h e  unable  kinetic  levels  could  C. gigas.  r e m o v a l of  equally  allele.  with respect  parameter  that  examined.  common  displayed  of  allele  thermolability.  was  s h o u l d produce the  intermediate  great  most  prolonged  Pgm-2-92  these  selective  similarly  Pgm-2-100  the  generally  homozygote  is  of  conflict  greater  greater  demonstrated  advantage  indistinguishable  properties  the  in  the  M c D o n a l d and B o o t h  the  unless  allozyme  but  The b i o c h e m i c a l d a t a  has p o i n t e d o u t  is  properties  this  denaturation,  Walsh,  in populations  the  been  consequence  a selective  allele  would p r e d i c t  thermal  high  a  significantly  because  significantly  w h i c h have  1975;  fixation  advantageous by  as  p H ' s compared t o  superior properties  to its  all  exhibited  (Wijsman  these  expected  eventually  2-96  3 0 ° C and a t  u n d e r low pH c o n d i t i o n s ,  anaerobic  low  from  for  in  allele  allozymes this  vivo  locus  function,  and  the  weakens t h e produces  the  its  limited argument fittest  84  genotype. over  The P g m - 2 - 1 0 0 / 1 0 0  the  thermal  other  allozymes  maintaining instances exactly  the  observed  opposite Place  been  Merritt  reached  of  in  Hoffman 1981;  this  nature  parameters  that  these  that  (kcat).  of the  actual  in  Figures  for  not  kcat  estimates  of  kcat have  kcat,  and 5;  only  genotypes  would  these  nature  (e.g.  1987).  genotypes  reached  by  presence  of  in  enzymic  One p a r t i c u l a r l y i m p o r t a n t was  substrate  turnover  and k c a t / K m r a t i o s gone  unnoticed  concentration.  under  to  a  changed.  by  the term  However,  since  common  identical to  between  a composite  protein  conditions,  those  relative'positions be  1981,  dilemma . t h a t  this  remain s i m i l a r  the  al.  is  maintenance  undetected  standardized  must  several  of  the a  b e c a u s e Vmax i s  and enzyme were  the  measured in  et  conclusions  Pgm-2  easily  kcat/Km r a t i o s  different  for  of  Zera  the  and compared t o g e t h e r  4  inability  examined.  be  of Vmax f o r  concentration the  not  could  p r o d u c t of  Vmax  The  account  in  in  frequencies  Oakeshott  1985;  concern  Differences  genotypes  the  Hall  between  were  could  substitution  in a l l e l e  studies  differences  because  (e.g.  clearly  limitation  differences  number  to  previous  functional  property  1984a).  lower  stabilities  in a balanced polymorphism i s  important  studies  unclear  predicted  Powers  by  temperature is  superiority  significantly  played  clinal variation  that  exhibit  its  The r o l e  their  differences  1972;  An  to  and  these a l l e l e s  has  in  did  through  enzyme p o l y m o r p h i s m s  biochemical of  homozygotes  denaturation constant.  between  1982;  genotype  of  presented the  Although  curves it  is  85  possible  that  the  kcat/Km r a t i o s  104/104 homozygotes relative the  to  the  p r o b l e m of  three  most  important  in t h i s  Passonneau  1969).  demonstrated  the  for  PGM i s  and d e g r a d a t i o n  In summary, t h e the  7 Pgm-2 g e n o t y p e s  of  e x p l a i n i n g the  subject  to  is  provide  its  nonspecificity:  to of  fitness is  insight  of  of  Gabbott  and  of the  Lowry and  between Pgm as  Fisher  and  function  for  predominant cycle  role  of  glycogen  observed  between  this  at  seem c a p a b l e  locus  (1983),  by  Fujio  heterozygote  a l l polymorphic  selection. arising  complex,  i n t o how t h i s  this  is  phosphate  substrates,  s t u d y do not  be m a n i f e s t e d  differences  PGM  1975).  Ginzburg  extremely  (e.g.  by Q u i c k ,  differences  balancing  a  could exist  seasonal  examined i n t h i s  form  properties little  not  for d i f f e r e n t  in the  biochemical  expected  of  phosphoglucomutase  overdominance r e p o r t e d at  some  interpretation kinetic  of  importance  (e.g.  As shown by T u r e l l i  superiority  the  5 and 6 c a r b o n s u g a r s  plays  extent  seems u n l i k e l y and  intermolecular transfer  relative  reaction  similar  overdominance of  3 human PGM i s o z y m e s  The  9 6 / 9 6 and  remains.  p r o b a b l y l i m i t e d because  primary  (1982).  marginal  their affinities the  a  this  Therefore, differences  in  (1974).  synthesis  the  concerns  a v a r i e t y of  genotypes  oyster  study  Pgm-2-92/92, to  genotype,  property  c a t a l y z i n g the  g r o u p between  Harris  for  the  shifted  common h e t e r o z y g o t e s  c a p a b l e of  -2  be  Pqm-2-100/100  accounting  Another examined  may a l l  for  the  allozymic  loci  Although  from  iin  the  vitro  biochemical data variation  could  86  be  actively  next chapter specific at  this  m a i n t a i n e d by s e l e c t i o n will  activity locus.  examine  the  variation  in natural populations.  potential in the  contribution  expression  of  of  The  Pgm-2  overdominance  87  CHAPTER 3  ENVIRONMENTAL AND GENOTYPIC EFFECTS ON PGM A C T I V I T Y  INTRODUCTION  Many  studies  genotypes in  specific  and In  at  enzyme a c t i v i t y  several  result Lai  1976;  cases, by  the  (e.g.  cause  of  Scandalios  in  1980;  affected  by  number of  s t u d i e s have  produced  by p o l y m o r p h i s m s a t  timing  Paigen has  1986).  cause  override exist  elements  Variation  presented  the  tissue  of and at  differences kinetic  that  Baum  the  of  has  found  a  growing  (cf.  are Paigen  developmental  Laurie-Ahlberg of  gene of  regulatory enzyme  and  loci 1985;  organization the  selective  polymorphisms activity  structural differences Carlson  and are  differences  the  to  (e.g.  Estelle  factors,  interpretation  Wilson,  when  s t r u c t u r a l enzyme  levels  in steady-state  (e.g.  been  1983;  these  1982;  two  1979).  concentrations  control  v a r i a t i o n . Do  and/or  between a l l o z y m e s  that  Hay  variation  " r e g u l a t o r y " gene l o c i  these  1973;  concentrations  and g e n e t i c  expression  a dilemma f o r allozymic  procedures  enzyme  demonstrated  levels  activity  v i v o enzyme  environmental  by S c a n d a l i o s  i m p o r t a n c e of that  genetic  and/or  (reviewed  Although  both  Johnson  K i n g and M c D o n a l d  1984).  i.e.,  this  of  differences  D a n f o r t h and B e a r d m o r e  immunoelectrophoretic in  properties  have d e t e c t e d  V i g u e and  1977;  Hodgetts  1979),  biochemical loci  Hickey  from d i f f e r e n c e s  and  the  p o l y m o r p h i c enzyme  Armstrong  examined  comparing  that White  levels  may a l s o 1977)?  88  Or  are  both  relative  even  functionally  importance of  The  answers  for  the  discussion  differences of  these  these questions  (Amy)  studied  Zera,  in specific  loci  that  be  of  little  in  exist  observed  consequence  to  the  Amy-4,6  Anderson Amy), may  and  which  Santos  an i m p o r t a n t  structural attempts  locus  to  assess  polymorphism potential  must  Marked both  Therefore,  maintenance However,  the  of high  the  larger  for  McDonald,  A t k i n s o n 1977).  polymorphisms. selective  From  These  examine  high  suggest and  considerations, a specific  the  between t h e s e d i f f e r e n t  for  levels  regulatory  these of  1978  under  patterns  between  importance  simultaneously  activity  functioning  these  Michaelis  (e.g.  increased  may  activity  f o r A d h ; Hoorn and S c h a r l o o  i n t e r a c t i o n may e x i s t  interaction  1985).  1985).  substrates  (cf.  melanogaster  b o t h l i n k e d and u n l i n k e d  respective 1980  the  (Adh) and  also display  an a d d i t i o n a l a d v a n t a g e  conditions  dehydrogenase  homozygotes  in combination with t h e i r  provide  substrate that  their  the  unknown,  between t h e s e enzyme v a r i a n t s  Adh-F/F  for  is  between g e n o t y p e s a t  (Laurie-Ahlberg  in natural populations.  constants  what  largely  Hall  to  polymorphisms and  so,  Drosophila  and  are a t t r i b u t a b l e  differences  if  still  alcohol  Koehn  activity  r e g u l a t o r y polymorphisms kinetic  are  polymorphisms  in  and,  each?  intensively  alpha-amylase (see  to  significant  enzyme  existence types  and  of  genetic  extensively  studied  variation.  Regulatory  p o l y m o r p h i s m s have  been  most  89  in D. melanoqaster  (reviewed  species,  variation  genetic  appears to all  23  be t h e  lines  chromosomes  activity  studied  genetic  from  In  structure  in  to  that  variation  in  the  the  in  at  the  anemone,  Tigriopus the  of  oyster,  aminopeptidase-1  senile,  1981).  The  variants  affecting  the  in  Siebenaller allozymes since  the  similar  in  genetic  regulatory  of  is  1983), (Martin  of  to  cis-  enzyme for  locus  synthesis  s u g g e s t e d by  i n the the  in M. e d u l i s .  activities arise  from  substrate  genotypes  studied.  locus  were  loci  locus and  (Koehn  shown  differences  differences  t u r n o v e r numbers  the  by  and  Koehn  (kcat  can for  observed  between  aminopeptidase-1  the  regulatory  or d e g r a d a t i o n  Here,  in  copepod,  Pgi  1979),  edulis  these a c t i v i t y  not  variation  structural  Gpt l o c u s  virginica  measured c o n c e n t r a t i o n of  in a l l  their  or t r a n s - a c t i n g  enzyme  their  is  action  rates  (1981)  of  in  this  Mytilus  aminopeptidase-1  variation  but  a  regulatory  i n the m u s s e l ,  be e x c l u d e d a s an e x p l a n a t i o n only  variation no  in  third  and Odh (Walsh 1981) the  of  (1982)  exhibited  observed  activities  levels  48  populations  that  this  example,  al.  and  in Drosophila e x i s t s  Crassostrea  Immerman  second  ( B u r t o n and Feldman  locus  et  invertebrates,  (Hoffman 1981)  californicus  For  characterized,  specific  Metridium  exception.  o u r l i m i t e d knowledge  observed  Pgi  activity  their  and o r g a n i z a t i o n . However,  analogous  genotypes  been  light  to  In  enzyme  48  natural  marine  p o l y m o r p h i s m s have y e t  1985).  Laurie-Ahlberg from  component  levels.  surprising  by  established  extracted  significant  affecting  r u l e r a t h e r than the  enzymes  isogenic  by L a u r i e - A h l b e r g  and the  values),  enzyme  was  90  In  this  activity Pacific  chapter,  oyster,  prevailing  examination  loci  interaction  must  with  seasonal  salinity,  (Livingstone  1981;  these  the  factors  observed  to  1983).  bivalves their  involves  primary  of  the  energy  and  degraded Whyte  in the  and  glycogen exhibit  spring,  produce to  the  for  the  of  differential  important  basis  dominant and  C. gigas,  Since  activity  aminopeptidase-1  locus  of  have  of  in  been al.  In  British  synthesized  in  1969;  functions  seasonally tissues  influence  interactions  the  g l a n d and  (Quayle  of  in and or  these could  relevant  p o l y m o r p h i s m , as (Koehn,  and  marine  on PGM a c t i v i t y  in M. e d u l i s  of  glycogen,  1975).  between  this  cycle  o r more  cycles  may v a r y  factors  reflect  Chambers e t  spawning  genotype-by-environment significance  that  and d i g e s t i v e  The p o t e n t i a l  at  potential  one  phosphoglucomutase level  marine  Livingstone  (Gabbott  to  the  availability,  degradation  responses  physiological  functional  to  annual  mantle  In  enzymes  is  in  reproductive  (e.g.  glycogen  i n the  conditions.  and  annual  In r e s p o n s e  summer p r i o r  its  food  a number of  synthesis  1982).  microenvironmental  (i.e.  s t o r a g e compound  early  metabolism,  environmental  the  specific  their  G a b b o t t and Head 1980;  stored  Englar  of  in  differences  in metabolism  on a s e a s o n a l  1976;  Columbia p o p u l a t i o n s fall  1983).  activity  consider  and t h e i r  activities  variation  described.  specific  conditions  Gabbott  One  are  changes  etc.)  fluctuate  Livingstone  gigas of  of  p o l y m o r p h i c Pqm-2 l o c u s  simultaneously  environmental  temperature,  the  Crassostrea  the  polymorphic  Clarke  patterns  between g e n o t y p e s a t  bivalves,  1975;  the  Newell  found and  91  Immerman  1980;  activities (mantle  of  The  most  (1982). and  a monomeric enzyme  like  p l a y e d by f u n c t i o n a l  that  differences  mechanism  left  open  the  produce  differences  potential  different  different  genotypes.  developed  that  through  spatial  (reviewed  by H e d r i c k ,  some d e a l i n g Gillespie  applicability chapter  is  intermediate  effects  A number of  or  of  of  Langley  1974;  t h e s e models  limited, heterozygote  this  between t h e  four  confer via is  of  the  to  however,  the  1975; results  because  behavior;  marginal  the  gene  some  for role  locus,  variation  have  been  heterogeneity 1976),  variation  Gillespie presented they Pgm-2  in  advantage  Felsenstein  activity  net  PGM a c t i v i t y  environmental  Latter  a  required  models  1976;  by the  producing a heterozygote  enzyme  locus  s t r u c t u r a l enzyme  levels  the  that  regulatory  G i n e v a n and Ewing with  at  discounted  theoretical  temporal  explicitly  of  the  steady-state  are capable  and  at  if  explaining  to  that  conclusion  and  genotypes  concluded  observed  1959)  (low  winter).  between  homozygotes  Wallace  PGM. T h i s  I  and  tissues  determine  of  insufficient  over  (cf.  to  described  preceding chapter,  heterozygotes  overdominance  was  are capable  were  specific  i n two  fall  expressed  body w e i g h t  structural  of  (summer,  activities,  adult  the  i n t e r t i d a l heights  chapter  in C. gigas,  In t h e  two  seasons  this  common Pgm-2 a l l o z y m e s  advantage  but  three  of  for  Therefore,  g e n o t y p e s were m e a s u r e d  in s p e c i f i c  Pqm-2 l o c u s  kinetic  of  objective  overdominance Fujio  Pgm-2  i n each  differences the  7  and a d d u c t o r m u s c l e j , a t  h i g h water)  at  Koehn and Immerman 1 9 8 1 ) .  are  (e.g.  1977).  The  in  this  based  on  heterozygotes  92  reported  here  specific  d i s p l a y the  unusual feature  of  overdominance  for  activity.  MATERIALS AND METHODS  Animals. (early B.C.  O y s t e r s were c o l l e c t e d  N o v e m b e r ) , and w i n t e r study  site  length,  from  intertidal were  each  zone  two  (designated  t r a n s p o r t e d back t o  immediately  upon  for  in Chapter  conditions all  were  oysters.  used The  2.  they  gel For  genotypes  their  running  specific  Specific  size  from  Nanoose B a y ,  5-20  stations  ice  cm  located  from t h e i r  oysters  were  of  each  of  sample, identify oysters  prior  where,  was  -40°C.  running  Pgm-2 g e n o t y p e  s c o r e d as  to  the  was p e r f o r m e d as  were c h e c k e d a s e c o n d  conditions  in  mantle  "standard" the  shell  shells,  f r o z e n at  electrophoresis  of  Animals  in coolers  a small section  the  fall  Samples c o n s i s t e d  were e x c i s e d  i n i t i a l l y to  9 6 / 9 6 and 104/104 h o m o z y g o t e s "catalytic"  2.  June),  "low" and " h i g h " w a t e r ) .  After  Starch  (late  from t h e  l a b o r a t o r y on  electrophoresis  Electrophoresis. outlined  as  arrival,  in  sampling  the  b l o t t e d d r y , and w e i g h e d . dissected  in Chapter  ranging  of  summer  ( e a r l y March)  described  150-250 m a t u r e o y s t e r s ,  i n the  the  of  Pgm-2-92/92, time  under  d e t e r m i n a t i o n of  activities.  Activity  sample  were  classes  (12.0-23.9  Measurements•  divided into g;  Oysters  four a r b i t r a r i l y  24.0-35.9  g;  from  each  assigned  36.0-47.9  g;  seasonal  body  weight  +48.0  g).  93  Specific  activities  of  -2-100/100,  92/100,  subsample  3-6  of  these weight the  PGM  all  exactly The  nearest  the  were  was  grinder  i n 5 ml of  maleic  acid,  1  approximately  0.5  of  these  selected  1 g of left  of  tissue mantle  the  on i c e  lobe,  ice-cold mM MgC12, g of  the  was u s e d  extraction 1  of  mM  these study,  pH  PGM a c t i v i t y  activity  direction  spectrophotometer  at  supernatant  as  to  was  the most  weighed  ensure  all  that  oysters. tissue  mM T r i s ,  of  1OmM  Similarly,  the  adductor  i n 3.5  ml of  12,000 x g  for  removed f o r  the  protein.  was measured 340  and  homogenized  and s o l u b l e  Unless  from the  7.4).  at  and  4 ° C . From  in  (10  portion  and  mantle  10 ml Wheaton  buffer  EDTA,  "quick"  the  in the  was t a k e n  by hand i n a  weighed,  1 ml a l i q u o t  Phosphoglucomutase  UV/visible  for  for  blotted,  The c r u d e homogenates were c e n t r i f u g e d  reaction  for  or at  buffer.  forward  homozygotes  was d i s s e c t e d  mantle  homogenized  dissected,  of  of  genotypes.  was  measurement  on a  each  included  muscle  20 min and a  from  all  were d e t e r m i n e d  m i l l i g r a m . Extreme c a r e  same r e g i o n  tissue  selected  (and h e t e r o z y g o t e s  sample)  activities  of  were d e t e r m i n e d  rarity,  s t e p s were c a r r i e d o u t  region  the  their  (Pgm  weight.  approximately  posterior the  body  100/104)  alleles  fall  adductor muscle  specified,  to  i n the  common Pqm-2 g e n o t y p e s  randomly  Owing t o  and 104  specific  posterior  mantle  96  of  and  individuals  examined  irrespective  f o u r most  96/100  classes.  Pgm-2-92,  alleles  the  in  the  nm on a Pye U n i c a m SP  1800  described  in  at  15°C  Chapter  2.  The  94  reaction  medium c o n t a i n e d  glucose-1-phosphate,  glucose-6-phosphate  final  volume  quantity  of  1  enzyme  dehydrogenase,  m l . One u n i t  r e q u i r e d to  of  pH  glucose-6-phosphate  1  is  defined  Mmole  to  conditions.  A s s a y s were p e r f o r m e d i n t r i p l i c a t e  c r u d e homogenate  from t h e  muscle.  After  frozen  at  General  p r o t e i n was m e a s u r e d  by  the  completion  -70°C  method  UV/visible Specific  prior  of  of to  mantle  the  determination  (1976)  was e x p r e s s e d  in units  i n t h e s e c r u d e homogenates.  weight  and  the  and  each  PGM a c t i v i t y  in  the  adductor  collection,  was c o m p l e t e d  the  loss  of  any  PGM a c t i v i t y  precautions t y p i c a l day  were  room  /xl  of  adductor were  protein.  temperature  Pye Unicam a  SP8-400  standard.  PGM a c t i v i t y  tissue  p e r mg  wet  tissue  weight  content  of  were  75%  tissue  d e t e r m i n a t i o n of  w i t h i n a 4 month p e r i o d . To  day-to-day v a r i a b i l i t y in extraction eliminate  20  soluble  muscle  the  in  (Walsh,  1984).  seasonal  and  a  wet  a  these  samples  PGM a c t i v i t y / g  protein extracted/g  50%  M c D o n a l d and B o o t h  the  of  by a s s u m i n g an i n t r a c e l l u l a r w a t e r  mantle  For  a  the  u s i n g gamma g o b u l i n as  protein  calculated  on  as  under  using  of  in  glucose-1-  and 10 ul from t h e  in t r i p l i c a t e at  Bradford  soluble  minute  t h e s e enzyme a s s a y s  spectrophotometer activity  per  of  2 mM  mM NADP,  (20°C)  activity  convert  0.4  7.0  phosphate  the  3 mM MgC12,  16 uM g l u c o s e - 1 , 6 - d i p h o s p h a t e ,  1 unit  of  50 mM i m i d a z o l e - H C l ,  bias over  taken.  equal  i n the time First,  numbers  results  i n the of were  and/or assay due  frozen  the  to  the  specific minimize techniques  spontaneous  oysters,  several  12 i n d i v i d u a l s s t u d i e d  selected  from  each  on  tidal  95  height. chosen  Second, for  all  day,  genotypes  Statistical factorial  variance  H o m o g e n e i t y of  that data  of  only  the  required  normalize  variances.  posteriori  Bonferroni  any  the  that  maximum Third,  the  PGM a c t i v i t y  and  given  day.  activity  over so  for  were  same  These  measurements length  of  no s y s t e m a t i c  time  errors  results.  A n a l y s e s . The s p e c i f i c  (1981).  weight  i n the  on  days  a  same g e n o t y p e .  assayed  out  genotypes  ensuring  specific  were c a r r i e d  analysis  revealed  the  randomized the  different  thus  were  randomized a c r o s s  w o u l d be p r e s e n t  Rohlf  was  guaranteed that  suitably  four  3 i n d i v i d u a l s of  protein  precautions  and  of  each  of  i n which these genotypes  soluble  for  minimum  study  representation order  a  a c t i v i t y d a t a was a n a l y z e d by  (ANOVA)  as  variance  soluble  d e s c r i b e d in Sokal  tests  on  the  protein extracted/g  transformation  to  the  log  raw wet  data tissue  scale  Means were compared s t a t i s t i c a l l y m u l t i p l e range  and  to  using a  tests.  RESULTS  The for  specific  each  season  genotype weight  a c t i v i t y measurements  by a t h r e e - f a c t o r  and body w e i g h t was  variation for  the  the  three  found  to  class  i n PGM s p e c i f i c  fall  sample  largest  weight  independent a  activity  (F(3,144)  significant only  = 2.71,  classes  o r i g i n a l l y analyzed  ANOVA w i t h t i d a l  as  explain  were  (over  in the  P < .05). 24.0  g)  height,  Pqm-2  variables.  Body  portion adductor Here,  of  muscle  oysters  exhibited  the  in  greater  96  specific g),  activities  negligible  differences effect  eliminated  as  summarized season  a  height  genotype.  muscle  tissue  The  alone  in the  mantle.  and s o l u b l e tissue of  interaction on t h e protein  and  a highly  for  the  observed;  of  these  the  each,  tissues, two.  analyses  protein separately weight  basis. genotype,  To  levels  was  muscle,  however, factors  factors  level  of  in these  the  no and  found  effect  of  that  was  on  specific  the  activity  protein levels, the  or  relative  p e r f o r m e d on PGM a c t i v i t y b o t h on a p e r  IV shows t h a t  and  tidal  result  the  gram  significant  height-by-season  from t h e i r  phosphoglucomutase. tissues  in  determine  by e x p r e s s i n g  Table  is  season-  an e f f e c t  soluble  were  term appear l a r g e l y t o  activity  it  f o r m a n t l e and a d d u c t o r  adductor muscle,  effects  of  season,  the  significant  the m a r g i n a l l y s i g n i f i c a n t  from t h e s e  of  to  e f f e c t s . o n PGM  environmental  analyses  any  which  adductor  c o u l d be m e d i a t e d t h r o u g h d i f f e r e n c e s  combination  effects  was  significant  contribution  Due  analysis  highly significant  overall  detect  r e m a r k a b l y s i m i l a r . The o n l y d i f f e r e n c e  tissues  PGM e x t r a c t e d  some  overall  these  (12.0-23.9  PGM a c t i v i t y ,  i n t e r a c t i o n t e r m was a l s o  position  activity  exerted  on  to  groups.  mantle  In e a c h t i s s u e  were  these  The  the  o c c u r r e d between  Pgm-2  absent  in  class  unable  the  weight  IV. For both  genotype  interactions  tidal  body  lightest  were  between  factor  activity.  by-tidal  between  of  in Table  and  specific  wet  i n the  a l t h o u g h m u l t i p l e range t e s t s  significant  of  than those  influences  Although  differed significantly  soluble between  97  Table  IV. F - r a t i o s from a n a l y s e s o f v a r i a n c e on PGM s p e c i f i c a c t i v i t y , PGM a c t i v i t y / g t i s s u e , and s o l u b l e p r o t e i n e x t r a c t e d / g t i s s u e i n t h e m a n t l e and a d d u c t o r m u s c l e tissues.  98  Mantle Source of Variation  df  Specific Activity  PGM Activity  Adductor Soluble Protein  Specific Activity  PGM Activity  2  39.3***  81.7***  77.9***  Tidal Height  1  2.52  13.3***  5.48*  5.56*  6.75**  3.46  Genotype  6  11.9***  9.60***  2.62*  21.9***  12.8***  1.40  Genotype x Season  12  1.34  0.83  0.54  1.09  0.53  0.50  Genotype x Tidal Height  6  0.54  1.29  0.24  0.84  1.06  0.40  Season x Tidal Height  2  23.8***  17.8***  0.35  13.7***  5.12**  3.73*  0.74  0.80  1.14  1.20  0.52  0.37  12  Error  456  * ** ***  P < P < P <  .05 .01 .001  167.1***  Soluble Protein  Season  Genotype x Tidal Height x Season  271.3***  Muscle  23.0*"  99  seasons,  r e l a t i v e l y minor e f f e c t s  factors  and  the  their  significant  respective  effects  of  were a p p a r e n t  for  i n t e r a c t i o n terms.  these  factors  will  the  other  The s o u r c e  of  now be examined  in  detail.  EFFECTS OF SEASON AND INTERTIDAL  The activity and  influence of  12.  muscle  At a l l  found to d i s p l a y soluble in  basis, of  were  also  fall  was  i n the  with  statistical t  Second,  = 3.56, adductor  were  higher  levels  levels  the  on a s o l u b l e  fall,  First,  i n the  P <  exceeded  Z  .001,  Sokal  and  its  no d i f f e r e n c e s  level between  seasonal  tissue a  protein  important  and  variation  exhibiting  rank levels  differences  seasonal  fluctuations  significantly  these  (r  transformed Rohlf  winter  but  seasons.  =  tissue 0.43;  coefficients  1981,  concentration  i n the  greater  p e r gram  adductor muscle  these  protein  was  m a n t l e were more p r o n o u n c e d  of  muscle  muscle  PGM a c t i v i t y  PGM a c t i v i t y  i n the  g r a p h i c a l l y in  soluble  several  mantle  p r o t e i n o r wet  tissues,  observed  t h a n seen  of  specific  i n the  adductor  However, the  two t i s s u e s .  comparison  significantly there  the  a d d u c t o r m u s c l e and showed a  (r = 0.65)  yields  the  the  protein  year,  in both  on t h e  are presented  > summer. A l t h o u g h  in  protein  association weight  similar  position  and s o l u b l e  mantle.  expressed  maximal  soluble  the  significantly  > winter  o c c u r r e d between  than  and t i d a l  C. qigas  of  p r o t e i n than the  order  in  of  times  PGM a c t i v i t y ,  weight  season  phosphoglucomutase  adductor  Figure  of  POSITION  p.  589).  i n the  summer  i n the  mantle  Third,  oysters  100  Figure  12. E f f e c t of s e a s o n and i n t e r t i d a l p o s i t i o n on s p e c i f i c a c t i v i t y ( u n i t s / m g p r o t e i n ) , PGM a c t i v i t y ( u n i t s / g t i s s u e ) , and s o l u b l e p r o t e i n (mg/g t i s s u e ) i n t h e m a n t l e and a d d u c t o r m u s c l e t i s s u e s . Open c i r c l e s = l o w . water; c l o s e d c i r c l e s = h i g h water. Low w a t e r sample s i z e s : summer n=64, f a l l n=96, w i n t e r n=89. H i g h water sample s i z e s : summer n=57, f a l l n = l 0 0 , w i n t e r n=92. Bars r e p r e s e n t ±1 s t a n d a r d e r r o r where v i s i b l e o r f a l l w i t h i n the p l o t t e d symbol.  Specific Activity, units/mg  101  Specific Activity, units/mg  102  from  the  low  concentrations intertidal winter  water  in t h e i r  area  for  the  tendency  in  PGM  concentration  i n the  the  for  adductor  all  this  tissue  specific  activity  v i v o enzyme  activity,  tissues  activity  fluctuated  of  seasons tended to  specific  by  tidal  to p a r a l l e l eliminate  64%,  variation shows  high  summer and  total  protein  in  specific  than observed  the  It was  protein  the  on s p e c i f i c  only exactly  in  pattern  higher was  in  the  reversed.  influence  tissue  amount  10% i n  of  in  both weight  soluble adductor  concentrations  between  tissue  levels,  seasonal the  was  noted  was  on  on  In  relationship that  observed.  between  activities  intertidal The i n c r e a s e d  area,  any  Figure  s e a s o n and  attributable  summer and f a l l ,  and  differences  compensatory  i n t e r t i d a l zone p o s s e s s e d g r e a t e r situated  In t h e  season  the  activity  activity  of  levels.  because  interaction  PGM l e v e l s .  its  the  than produce  i n PGM s p e c i f i c  that  of  PGM a c t i v i t y / g  is not  effect  and o n l y  the  this  the  greater  p e r gram wet  on t i s s u e  winter  protein  the  variation  protein  effects  animals  with  whereas  mantle  rather  activity.  position  low  of  50% l o w e r  significant  in tissue  between t h e s e measures  also  in  in the  Because  seasonal  PGM e x t r a c t e d  These changes  12  those  covary  on s o l u b l e  muscle.  seasonal  soluble  only  was a c o n s e q u e n c e  v a r i e d by 27% i n the  in  to  the  the  not  seasonally  acted  but  was n e a r l y  protein  thus  than  tissue.  activity mantle,  greater  tissues  seasons,  12 shows t h a t  the  had  muscle.  Figure PGM  mantle  adductor muscle  of  activity  site  to  their  oysters of  in  PGM t h a n  but  in  the  PGM a c t i v i t y  of  103  oysters their in  at  lower  high  tissue  specific  season. in  the  activity  IV  for  o c c u r r e d because relative  to  on t h e  factors  between  muscle  other  tidal  two s e a s o n s .  on PGM s p e c i f i c  differences  heights  in  this  interaction  seen  protein concentration  tidal  interaction  combined w i t h  the  season-by-tidal height  a r e v e r s a l of  similar  i n the w i n t e r , accentuated  observed  adductor  of  the  site  protein levels,  The s i g n i f i c a n t  Table  effect  water  ranking  in  also  the  fall  T h i s c a n be s e e n t o have  between  these  no  environmental  activity.  EFFECT OF PGM-2 GENOTYPE  The m a n t l e and a d d u c t o r m u s c l e Pgm-2  genotypes  presented both  measured  positions  has  Pqm-2-92/92,  9 6 / 9 6 and  evident  both  in to  respective specific height  and  tissue.  probability  of  the  three  specific  This  expected 1/3.  to  Using data  heterozygote  (occurring  16  out  was of  greater  18 t i m e s  rarity  The  clear  Pqm-2  across  cases than  the  trend  than  their f o r PGM  season, activity  positions  specific  either  i n the m a n t l e ,  from of  homozygotes  the  are  heterozygotes  specific  both  seven  data  the  from b o t h t i d a l the  the  overdominance  the  exceed  of  and w i n t e r  activities  apparent  By c h a n c e a l o n e  36 c o m p a r i s o n s and i n 33 o f the  each season  a c t i v i t y was e x t r e m e l y c o n s i s t e n t  was  of  summer, f a l l ,  homozygotes.  was t h a t  greater  homozygotes.  activities  been p o o l e d owing t o  104/104  tables  exhibit  heterozygote  were  the  i n T a b l e s V and V I . W i t h i n  tidal  tended  in  specific  tidal of by  a a  there  activity  homozygote  and 17 out  of  18  104  Table V . Seasonal v a r i a t i o n i n the mantle s p e c i f i c ( u n i t s / m g p r o t e i n ) of s e v e n Pgm-2 g e n o t y p e s .  activities  105  Season Genotype  N  Summer  N  Fal 1  N  Winter  5  0 . 085+ . 0 1 5  8  0 . 128+. 011  9  0 . 085+ . 0 1 0  22  0 .101+.O06  23  0 . 111 + . 0 0 7  41  0 . 129+. 005  35  0 . 115 + . 0 0 5  99  0 .120+.003  4  0 . 082 + . 0 1 6  7  0 . 113+. 011  1 1 0 . 066 + . 0 0 9  22  0 .. 0 8 4 + . 0 0 6  96/100  25  0 . 085+, . 0 0 6  42  0 . 129+. 005  31  0 . 112+ . 0 0 5  98  0 .. 1 1 2 + . 0 0 3  100/100  29  0 . 086+ . 0 0 6  42  0 . 1 0 9 ± . 005  40  0 . 091 + . 0 0 5  111  0 .. 0 9 7 + . 0 0 3  100/104  23  0 . 100+. .007  40  0 . 1 2 8 ± . 005  40  0 . 119+, .005  103  0 . , 118+.003  104/104  12  0 . 0 7 8 + . .009  16  0 . 109+. 008  15  0 . 079+. .008  43  0. 090+.005  92/92 92/100 96/96  N  Pooled  «  F(6.77)  * ***  P < P <  .05 .001  = 2.. 4 2 *  F(6,144)  = 31 . 0 9 *  F(6,132)  = 8., 2 3 * * *  F(6,456)  =  11.9***  106  Table V I . S e a s o n a l v a r i a t i o n i n the a d d u c t o r muscle s p e c i f i c a c t i v i t i e s ( u n i t s / m g p r o t e i n ) o f s e v e n Pgm-2 g e n o t y p e s .  107  Season Genotype  N  Summer  N  Fall  N  Winter  5  0 . 127± .013  8  0 . .219 + . 0 1 3  9  0 . . 176+ . 0 1 0  22  0 . . 1 8 1 ±.007  23  0 . 1 5 5 ± .006  41  0 .. 2 3 3 ± . 0 0 6  35  0 ., 2 3 0 ± . 0 0 5  99  0 . , 2 1 4 ± . .003  4  0 . 1 3 0 ± . .014  7  0 . 219+. .014  1 1 0 . . 175+,. 0 0 9  22  0 . . 181 + .007  96/100  25  0 . 1 4 6 ± .. 0 0 6  42  0 . 236+,.006  31  0 . 226 + .006  98  0 . 2 1 0 ± .003  100/100  29  0 . 124+. . 0 0 5  42  0 . 2 0 6 ± . .006  40  0 . 191 + ., 0 0 5  11 1  0 . 1 7 9 ± , .003  100/104  23  0 . 1 5 3 ± . .006  40  0 . 238+..006  40  0 . 233+..005  103  0 . 2 1 7 ± . ,003  104/104  12  0 . 1 2 1 ± . .008  16  0 . 204+..009  15  0 . 181 + ..008  43  0 . 173+. 005  92/92 92/100 96/96  F(6.77)  * ***  P < P <  .05 .001  = 2.42*  F(6.144)  = 3.09*  F(6.132)  = 8.23***  N  Pooled  F(6,456)  =  11.9***  108  times  i n the  occurring  adductor muscle).  by c h a n c e a l o n e  Although the  three  seasons,  range t e s t s genotypes. across  were  b e h a v e d as  the  were  activity was  homozygote tendencies that  found for the  this  the  time  homogeneous  tissue.  The m a g n i t u d e o f homozygotes  both t i s s u e s . exceeded  any  groups, the  and  by  the  homozygous found  in  difference  heterozygotes activities in  Pgm-2-92/92.  the  in  except  four  that  homozygotes.  i n the  mantle when  heterozygotes.  The  elevated  24%  activities  fall  was  as  heterozygotes  homozygote's  muscle  seasons.  the  became h i g h l y  except  anomalous  not  in  was c o m b i n e d  specific  from a l l  of  p o r t i o n of  between  Pgm-2  adductor  and  The s p e c i f i c  homozygotes  .  tissues  adductor muscle  apparently  heterozygous  differences  three  results  weak a n d m u l t i p l e  unusually high level  the  nearly  between  the  Pqm-2-92/92  were d i s p l a y e d i n o t h e r  these  1 3  each genotype  were d i s t i n c t  this  because at  for  exhibiting  i n d i s t i n g u i s h a b l e from  questionable  differentiate  a l l homozygotes  b e h a v i o r of  of  to  the  group  was c a u s e d by i t s  significance  sometimes  data  mantle,  heterozygotes  exceptional  was  positions  a homogeneous  results  three  The  unable  s i g n i f i c a n t l y exceeded  Similar the  In  10"  explained a significant  effect  However, when t h e  significant.  that  the  tidal  these  a c t i v i t y observed in both  often  seasons'and  of  approximately 4 x  Pgm-2 g e n o t y p e  v a r i a t i o n in s p e c i f i c  all  it  is  The p r o b a b i l i t y  result  appears  activity and  of  no  this  similar  Therefore,  I  genotypes  represent  the  believe  a d d u c t o r muscle  specific  activity  was n e a r l y i d e n t i c a l of  Pgm-2  mantle  in  heterozygotes and  20% i n  the  109  adductor  muscle.  The  specific  decomposed  in  activities  Table  VII  to  o b s e r v e d was a t t r i b u t a b l e levels  or  effects  of  soluble  of  larger  vivo  in  heterozygotes tissue the  relative  PGM a c t i v i t y  to  differences  activities muscle,  24%  act  the  it  consistently  enzyme  was  not  activities  the  activities.  and  can  be  gram of  specific  also  exerted  soluble  protein Multiple of  these  Pgm-2-100/104  seen t h a t  in both  higher  these  than  relative  levels  measurements  between  genotypes  of  would  increasing to  and  tissues  Since these differences rather  to  adductor  adductor muscle.  heterozygotes  differences  in their  amount of  o n l y between  their  tissues, per  Pgm-2 g e n o t y p e  the  of  in magnitude  mantle  the  specific  both  similar  displayed slightly  for  found for  protein concentrations  lowering  of  activity  increased  In  the  on t h e  than homozygotes.  d i r e c t i o n of  from  but  a difference  overdominance observed  solely  enzyme As  been  overdominance  PGM a c t i v i t y  in  mantle  However,  protein  in the  specific  effect  comparing the  genotypes detected  soluble  that  larger  from t h e m a n t l e  heterozygotes  the  levels.  Surprisingly,  a marginally significant  Pgm-2-104/104.  in  o b s e r v e d between t h e s e g r o u p s  respectively).  range t e s t s  the  have  was a c o n s e q u e n c e  greater  homozygotes  (29% and  extracted  position,  a  if  concentrations.  heterozygotes  possessed  genotypes  differences  protein  Pgm-2  these  determine  to  s e a s o n and t i d a l  activities  of  the  homozygotes, must  result  i n t h e i r PGM  110  Table V I I . D e c o m p o s i t i o n of Pqm-2 s p e c i f i c a c t i v i t i e s ( u n i t s / m g p r o t e i n ) i n t o enzyme a c t i v i t i e s and s o l u b l e p r o t e i n l e v e l s e x p r e s s e d on a gram wet t i s s u e w e i g h t basis.  111  Mantle Genotype  Adductor  N  Specific' Activity  PGM« Activity  PGM' Activity  Soluble Protein  92/92  22  0 , . 1 0 1 ±. 0 0 6  2 . 4 6 ± .214  23. 7 ± 1 . 1 1  0 . , 1 8 1 ±. 0 0 7  7 . 4 0 ± . 370  40. 5+1.12  92/100  99  0 , . 1 2 0 ±. 0 0 3  3 .09+, . 100  25. 5 ± . 5 1 7  0 , .214 + . 0 0 3  8 .62+. . 173  41 . 0 ± . 5 2 6  96/96  22  o . .084+ . 0 0 6  2..07+. .214  2 3 . 2+1.11  0 . .181 + .007  7 .21 + ..370  39. 9+1.12  96/100  98  0 .. 1 1 2 ± . 0 0 3  2 9 0 ± , . 101  25. 9+.526  0 ., 2 1 0 ± . 0 0 3  8 .78+. 175  4 2 . 1+.535  100/100  11 1  0 . ,097+.. 0 0 3  2..47+, ,095  24. 9 ± . 4 8 9  0 . 1 7 9 ± . .003  7,, 3 1 ± . 164  40. 1 ± . 4 9 7  100/104  103  0 . 1 18+. .003  3., 0 7 ± . ,099  25. 9 ± . 5 1 0  0 . 2 1 7 ± . .003  8 ,. 7 9 ± . 171  41 . 2 + . 5 1 8  104/104  43  0 . 0 9 0 ± . .005  2., 0 6 ± . 153  22. 6 ± . 7 9 0  0 . 173+. .005  7.,01 + . 264  40. 6 ± . 8 0 3  11.9***  9.60***  21.9***  12.8***  F(6,456)  1  * J  * ***  un1ts/mg p r o t e i n u n i t s / g wet t i s s u e mg p r o t e 1 n / g wet t i s s u e P < .05 P < .001  Soluble Protein  2! . 6 2 *  1  Specific Activity  Muscle  1  1.40  1  1 12  Due t o  the h i g h f r e q u e n c y of  study p o p u l a t i o n , the  Pqm-2-92/100,  represent  the dominant c l a s s  next  frequent  most  three  less  96/104.  To  study  i n the  this  season,  homozygotes the  for  if  sample.  pooled  Pgm-2-100  heterozygotes  i.e.,  these  Table VIII  that  heights,  or absence  of  to h e t e r o z y g o t e s  possessing  the  Pgm-2-100  not  exhibit  this  specific  activity  each  allele  tissue,  had s p e c i f i c homozygote expressed  did  in either  the  Pqm-2-92/96,  activities classes  i n the  overdominant  and  other  effect  heterozygote less  of  differences  were o b s e r v e d between  the  amount o f  muscles.  soluble  allele  had s i g n i f i c a n t l y  both  homozygotes  to  be  for  displayed  included  for  results  from  two c l a s s e s  grouped  allele,  In  and  mantle,  the  contrast  heterozygotes  adductor  lower  group.  f o r PGM  muscle.  from the  these  absence  of  heterozygotes  l a r g e r amounts o f heterozygotes specific  Pgm-2 l o c u s .  their for  soluble lacking  the  was a  adductor Pqm-2-100  this  Heterozygotes  No  classes  protein  a c t i v i t y does  an  tissue.  genotypic  from  both level  frequent heterozygotes  any o f  In  heterozygotes  than  The  of  according  overdominance  protein extracted  overdominance for  u b i q u i t o u s at  also  r e d u c e d PGM a c t i v i t y p e r gram o f  However i n t h e  Therefore,  92/104 and  indistinguishable  significantly  in these  the  9 2 / 1 0 4 and 96/104  were  consequence  in  their  that  genotypes  formed between  Pqm-2-100 a l l e l e .  the mantle or  the  The  were  have been  the  in  locus.  Pgm-2-92/96,  they  presence  lacking  are  this  summarizes the  tidal  and h e t e r o z y g o t e s  at  heterozygotes  activity,  across  allele  9 6 / 1 0 0 a n d 100/104  heterozygotes  specific  fall  of  Pgm-2 a l l e l e s ,  determine  overdominance  to  group of  frequent  the  not  than  allele. appear  between  the  113  Table V I I I . C o m p a r i s o n o f enzyme a c t i v i t i e s ( u n i t s / m g p r o t e i n and u n i t s / g t i s s u e ) and s o l u b l e p r o t e i n l e v e l s (mg/g t i s s u e ) o f homozygote and h e t e r o z y g o t e c l a s s e s p o s s e s s i n g o r l a c k i n g t h e Pgm-2-100 a l l e l e .  1 14  Mantle Genotyplc Class  N  Specific' Activity  Adductor  PGM* Activity  Soluble Protein  3  Specific Activity  1  Muscle  PGM» Activity  Soluble' Protein  Homozygotes for 100 a l l e l e  42  0.1O9±.0O5  3.10+.163  28.4+.856  0.206±.0O6  8.83+.281  42.6+.745  Homozygotes without 100 a l l e l e  31  0.117±.005  3.00±.193  26.1+1.00  0.211+.007  8.90+.327  42.2+.863  Heterozygotes for 100 a l l e l e  123  0.1291.0O3  3.70±.095  29.2+.500  0.2361.003  9.97±.164  42.5+.435  Heterozygotes wi t h o u t 100 a l l e l e  33  0.105+.005  2.65±.187  24.9±.187  0.198±.006  8.53±.320  42.9+.850  =  7.76***  8.81***  6.71***  12.9***  7.79***  F(3,197)  units/mg protein * u n i t s / g wet t i s s u e ' mg p r o t e i n / g wet t i s s u e * * * P < .001 1  0.84  115  Pgm-2-92,  96 and 104 a l l e l e s  homozygotes rather  and  showed  behaved i n a  a  tendency  manner  analogous  to  to d i s p l a y underdominance  than overdominance.  PREDICTED EFFECTS OF A PGM-2 NULL A L L E L E  The 96/100  overdomi'nant s p e c i f i c and  100/104  undetected presence  heterozygotes of  a  null  heterozygotes,  misclassified  unknowingly  the  effect  into  of  what  four  depressing  heterozygotes  activities  allele  may be t e r m e d " n u l l  be  at  classes,  enzyme  This  central  glycogen  i n the  bivalves.  homozygotes examined viable, this The  In a c c o r d a n c e w i t h  were o b s e r v e d a t  an a p p a r e n t d e f i c i e n c y  locus  because  of  maximum f r e q u e n c y of  He)/He,  this where  heterozygosities, between  the  electrophoretically.  magnitude of  low  by  of  this  and  (see  and h i g h i n t e r t i d a l  will  He  the to  produce allele  because  prediction, i n the  of  in the  will  allele  cannot  no  1442  heterozygotes  heterozygotes  null  null  oysters  are  fully  result  at  homozygotes. exceed  the  d e f i c i e n c y as m e a s u r e d by D = (Ho are  respectively.  sampling dates  null  pooled  energy m e t a b o l i s m of  Pgm-2 l o c u s If  Null  relative  t h e i r m i s c l a s s i f i c a t i o n as  heterozygote Ho  this  the  have  o v e r d o m i n a n c e " . A Pgm-2 n u l l in C. qigas  marine  and  activities  alleles.  by  locus.  would  be l e t h a l  played  caused  this  homozygous c o n d i t i o n c o u l d role  Pqm-2-92/100,  homozygotes  homozygote  w i t h two f u n c t i o n a l  the  could  as  their  of  observed  Although  Chapter  stations  the  2),  i n the  D  values  and e x p e c t e d fluctuated  t h e i r mean l e v e l s  at  the  s t u d y p o p u l a t i o n were  -  116  0 . 0 8 5 and - 0 . 0 6 7 , required water  to  These  allele  activities  the  is  equals  heterozygotes functional  the  alleles  heterozygotes  null  allele  is  [as  0.044.  reductions  homozygote  classes  are  particularly  more  i n the  this  IX  a c t i v i t y of  of  by  is  classes.  the  that  similar  with  two  (1987)  for Lap  their  apparent i n the  the  104/104 observed  However, the than  the  of  9 6 / 9 6 and  much l o w e r a c t i v i t i e s most  null  f r e q u e n c y of  to  adductor muscle.  the  2)  the magnitude  Pgm-2-92/92,  tissue  homozygote  genotype  the  the  making  a normal  a  a n d 3)  IX shows  remarkably  100/100 homozygote e x h i b i t s once  homozygote  in Table  activity  high  differentially  shown by K a t o h and F o l t z  for  the  overdominant h e t e r o z y g o t e s ,  the  Table  for  allele  a n d PGM a c t i v i t i e s / g  in C. v i r q i n i c a ] ,  predicted  and  the  three  have 60% o f  null  values,  1)  null  site.  four  activities  a  D a r e 0.044  to a f f e c t  the  are presented  assumptions:  t h e mean o f  low w a t e r  measured f o r  t h e s e homozygotes  of  expected  predicted specific  following  The f r e q u e n c y o f  these estimates  a n d 0.034 f o r  null  specific  of  generate  site,  A  respectively.  Pgm-2-  expected,  adductor muscle  tissue.  The e x p e c t e d a consequence allele  in  the  t h e much  compared t o  be e s t i m a t e d 100/100  of  i n e q u a l i t y between  that  only  homozygotes three  the  higher  three 13% o f  rarer the  are expected  o t h e r homozygote  t h e s e homozygote c l a s s e s  frequency  of  alleles.  the  Pqm-2-100  Accordingly, scored  to  heterozygotes,  groups  the  proportion  as  it  genotypes be n u l l  is  can  Pqm-2-  of  but null  117  Table  IX. P r e d i c t e d r e d u c t i o n s of enzyme a c t i v i t i e s (units/mg and u n i t s / g t i s s u e ) and s o l u b l e p r o t e i n l e v e l s (mg/g t i s s u e ) i n Pgm-2 homozygote c l a s s e s a s s u m i n g a n u l l a l l e l e i s p r e s e n t a t a f r e q u e n c y of 0 . 0 4 4 . Soluble protein c o n c e n t r a t i o n s were c a l c u l a t e d by a s s u m i n g PGM enzyme r e p r e s e n t s (a) 1% and (b) 5% o f t h e t o t a l i n t r a c e l l u l a r protein pool.  118  Specific Genotype  A.  Observed  Activity  1  Expected  PGM A c t i v i t y ' Observed  Soluble  Expected  Observed  Protein' Expected (a) (b)  Mantle 0.117  0 . .117  3 .02  3 .02  100/100  0.0968  0 . .111  2 .47  104/104  0.0849  0 . .0998  96/96  0.0839  92/92  0 . 101  Heterozygotes  25 .8  25 .8  25 .8  2 .87  24 .9  25 .8  25 . 7  2 .06  2 .58  22. .6  25 .7  25 .5  0 . .0942  2 .07  2 .43  23. .2  25 .7  25 .4  0 .. 0 9 7 3  2 .46  2 .51  23 .7  25 .7  25 . 5  '  A d d u c t o r IM u s c l e  B.  Heterozygotes  0.214  0 . .214  a .73  8 .73  41 ,.4  41 ..4  41 .4  10O/10O  0 . 179  0 . 203  7 ,.31  8 .28  40. . 1  41 ..4  41 ..2  104/104  0 . 173  0 . 183  7 . 01  7.,47  40. 6  41 . 3  41 . 0  96/96  0.181  0 . 172  7 .,21  7..02  39. 9  41 . 3  40. 8  92/92  0.181  0 . 178  7. 40  7 ..26  40. 5  41 . 3  40. 9  units/mg protein * u n i t s / g wet t i s s u e ' mg p r o t e 1 n / g wet t i s s u e 1  119  heterozygotes 42%,  and  s h o u l d be  49%  for  respectively). homozygotes  the  were e x p e c t e d tissue  allele  homozygote  that  Table levels  the  predicted effects  IX  also  no  (a)  pool,  proportion Virtually  for  expected  null  allele  in  (b)  5%  of  1% o f  the  total  are  represent  that  present  PGM a c t i v i t y / g  overdominant  the  higher  levels  level  tissue  to 35% in  e x c e e d e d homozygotes  other which  frequency,  soluble  null  protein  allele  total  by  that  assuming  intracellular  the  true  in_ v i v o  Ottaway and Mowbray levels  1977).  are p r e d i c t e d  even a t  the  In f a c t ,  explain  patterns,  of  these  the  the  protein pool  adductor  far  too  small  to  by 29% and 24% i n t h e  in  mantle  for  in  observed account  a  PGM  muscle.  by t h e s e g e n o t y p e s .  p e r gram t i s s u e  if  5% l e v e l ,  than o b s e r v e d .  w e i g h t measurements  expressed  extracted  a  pool and,  heterozygotes are  protein  in  bracket  protein  lower  approximately  and 20% of  1960;  the  allele.  the to  for  the  actual  reductions by  and PGM  pattern,  T h e s e were c a l c u l a t e d  p r o d u c i n g no enzyme  activity  a null  expected  soluble  reductions  the  PGM  the  Czok and B u c h e r  no c h a n g e s  the  increased  1% and  of  than  this  allele's  percentages expected  (see  PGM a c c o u n t s  mantle  presents  protein product.  PGM r e p r e s e n t s protein  null  Pgm-2-100/100  heterozygotes  of  37%,  classes,  activities  larger  absence  the  i n Pgm-2 homozygotes  produces  must  of  as  specific  were s i m i l a r t o  clear  and 9 6 / 9 6  scored  significantly  The  should hold i r r e s p e c t i v e contradicts  exhibit  (approximately  92/92,  genotypes to  and t h u s  groups.  larger  Pqm-2-104/104,  Therefore,  activities/g Pqm-2-100  significantly  the The in for  The mean  heterozygotes and  adductor  1 20  muscle,  respectively.  protein  pool,  the  If  PGM r e p r e s e n t s  increased  enzyme  h e t e r o z y g o t e s can e x p l a i n  only  the  mantle  i n the  of  this  and 7% of  additional  and 34% f o r protein  the  pool,  difference  that  protein  this  are a l s o  tissue  the  variance  heterozygotes.  muscle  homozygotes,  No  to  enzyme  in  observed  in  The p r o p o r t i o n  PGM i n c r e a s e s represents  leaves a sizable  Second,  the  to  21%  5% o f  amount  the  in the  i n the  homozygotes  for  the  of  overdominant activity  this  the  of  the  should exhibit  is  expected to  three  less  Therefore,  PGM a c t i v i t y  and p r o t e i n  match any of  the  levels  predicted  larger  than  or  distributions  most  frequent  clearly  of  these  in  seen  of  a null  allele.  the  in  Visual  of  observed  i n Pgm-2 h e t e r o z y g o t e s does  effects  of  adductor  Pgm-2 a l l e l e s .  overdominance  in  and n o r m a l  d e t e c t e d no e v i d e n c e the  seen  pattern  heterozygotes be  that  inflation  mantle  a bimodal  null  effects  and PGM a c t i v i t y / g  the  normalized frequency  these d i s t r i b u t i o n s  bimodality.  of  homozygote  heterozygotes.  predicted  analyses  to  four  two a d d i t i o n a l  specific  corresponding and t h i s  of  produce  evidence  homozygotes,  expected  differences  i n h o m o z y g o t e s s h o u l d be  measurements  inspection  the  heterozygotes  from  of  was a p p a r e n t  data.  activity  expressed  adductor muscle.  still  null  them  measurements  variance  if  expected to  should d i s t i n g u i s h First,  the  intracellular  unexplained.  The p r e s e n c e of groups  4% of  the  levels  attributable  same t i s s u e s but  1% o f  this for not  121  DISCUSSION  Seasonal enzymes most  have  been  notably  Gabbott  of  a v a i l a b i l i t y of and  reproductive  the  (reviewed  changes  are  metabolism  food,  and  1978;  Phosphoglucomutase  activity  has  fluctuate  i n any s p e c i e s  the  general  pattern  that  seen  a number o f  for  Crassostrea  observed  virginica,  other i.e.,  during  the  in  n o n - r e p r o d u c t i v e phase  the  1975;  specific exhibited  activity for  by Chambers e t  The  1979).  10 out al.  (1975)  influence  specific  of has  located  enzymes  in the  lack  in  the  of  (PFK) t o low  et  al.  1980).  demonstrated  is  consistent  with  American  oyster,  in s p e c i f i c  activity  studied  by an  increase  (Chambers e t  al.  patterns  in C. v i r g i n i c a  (1979).  on enzyme  examined.  v a r y by more high  to  However,  agrees w i t h the  phosphoglucose  and  annual  followed  13 enzymes  r a r e l y been  (i.e.  a r e l a t i o n s h i p between PGM  also  and M a r t i n  reflect  their  and w i n t e r )  of  that  the  of  been  study  (fall  to  marine m o l l u s c .  (summer)  1981;  conditions  Zandee  of  bivalves,  related  state  not  tidal position  activities  phosphofructokinase oysters  of  Livingstone  in this  and body w e i g h t  in marine b i v a l v e s the  The  by  a reduction  reproductive period  Martin  marine  abiotic  the  of many  of  in these organisms  (Widdows  seasonally  activities  directly  prevailing  salinity),  cycle  specific  in a v a r i e t y  edulis  These  patterns  temperature  in  documented  Mytilus  1983).  seasonal the  variation  activity  Martin  (1979)  isomerase  than  intertidal  levels found  (PGI) and  2-fold areas,  between but  in  122  opposite  directions;  decreased position  as  activities  in  the  function  on PGM a c t i v i t y  pronounced,  in  a  PGI a c t i v i t y  but of  of  increased  tidal  in the  but  height.  present  PFK  The e f f e c t  study  was  showed an i n t e r a c t i o n w i t h s e a s o n ;  oysters  i n the  low  h i g h i n t e r t i d a l zone  activity of  tidal  much the  less  specific  i n t e r t i d a l area exceeded  those  i n b o t h J u n e and November, but  not  March.  The  causative  variation tidal  in  PGM a c t i v i t y  position  hand,  the  enzyme  levels  factors  and s e a s o n  patterns  and t h e  are consistent  i n response  1974).  However,  the  amount  of  a b i l i t y of  related  to  Fernandez  oyster  its  enzymes  On  synthesize  glycogen 1972;  i_n Its  catalyzes vivo  in  relative  u n c e r t a i n because  a  the  related  in Chapter  4).  to  Since  (L-Fando,  Garcia-  1976),  annual c y c l e  reversible  glycogen  has  in  inversely  importance in the  u t i l i z i n g amyloglucosidase  as  is  Goromosova  an a l t e r n a t i v e  in  and  i n PGM a c t i v i t y  glycogen  store  freely  both  one  Hazel  of  these  glycogen  and d e g r a d a t i o n . A f u r t h e r c o m p l i c a t i o n a r i s e s  functions  glycogen  to  (e.g.  inversely  (presented  could simply r e f l e c t  glycogenolysis. still  tissue  R-Candela  phosphoglucomutase thus  interpret.  o b s e r v e d changes  present  between  temperature c o n d i t i o n s  glycolytic  existing  and  fluctuations synthesis  glycogen  seasonal  interaction  to  b o t h m a n t l e and a d d u c t o r m u s c l e were a l s o the  the  w i t h compensatory changes  to d i f f e r e n t  the  for  observed  are d i f f i c u l t  documented f o r a v a r i e t y of Prosser  responsible  because  r e a c t i o n and  synthesis latter  and  process  d e g r a d a t i v e pathway been  detected  in  is  from M.  123  edulis is  (Alemany and R o s e l l - P e r e z  possible  glycogen  that  is  consequence  of  time  separate factors  its  relative  observed  the  may be  when  purposes,  at  not  appear  seasonal  of  a  warmer p r e v a i l i n g t e m p e r a t u r e s does  instead  is  a  it  role  in June,  it  being  observed  possible between  changes  these environmental  (e.g.  although  dominance  al.  K i n g and  in  When  to  these  in  been  and  McDonald  1987).  to  (1984)  parental  also  PGM  interspecific  hybrids  than e x h i b i t e d  by e i t h e r  reported  and t h e n  between  only  in  greater  invariably  (e.g.  in  1975), Gibson  for  et  enzyme  exceptional increased  F1  hybrids  D i c k i n s o n , Rowan and ADH  D. melanogaster  parental species,  specific  Harris  in  is  different  r e p o r t e d an  tissue  known  allele  almost  1974;  inbred l i n e s . a  the  two  differ is  on PGM  overdominance  for  Whaley (1952)  observed  the  Overdominance  i n maize m e r i s t e m a t i c  their  of  Pgm-2-100  Langley  sometimes been  For example,  activity  the  found to  r a r e l y been d e s c r i b e d ,  circumstances.  gigas,  intermediacy  Gillespie  factors  framework  homozygotes  have  has  C.  possessing  heterozygote  has  of  changes  alleles  observed  Brennen  observed  gametogenic  i n t e r p r e t e d w i t h i n the  unusual.  activity,  relative  the  in heterozygotes  electrophoretic  catalase  Therefore,  i m p o r t a n c e and i n t e r a c t i o n  effects  metabolic  extremely  1986;  the  1981).  activity.  Although  seasonal  to  At p r e s e n t  in explaining  activity  for  diminished catabolic  year.  the  specific  activity  degraded  response  of  Zaba  r e d u c e d PGM a c t i v i t y  being  compensatory this  the  1973;  activity and D .  although  this  in  simulans effect  124  may  have  resulted  populations activity shown  a clear-cut  has  to  not  freshwater  the  sand s h i n e r ,  ratios  sucker,  Watt for  of  temperature  In none of  has  different  seasons,  insensitivity  Before it  is  to  through important  factor  influence  of  the  tissue  mantle  an  M.  males  all  and  Clarke  causes  examined  significantly  of  the  (1969)  and  P e r e z and M e r r i t t  in C o l i a s  the  1983)  or o n l y  shown  specific  that  the  levels  activity  present  at  in the  study  appears  reproducibility  across  and  this  by  in this  tissues,  year  and  its  overdominance, that  systematic study  was  have of  the  arose  1981;  One  potential  activity.  been  soluble  (Livingstone  it  errors.  on PGM s p e c i f i c  levels  i n the  Therefore,  apparent  females  greater  of  it  possibility  or  edulis,  times  and Immerman 1 9 8 1 ) .  both  factors.  factors  possess at  for  i n d i v i d u a l ' s sex of  Koehn  in  heterozygotes  positions  discount  not  locus  r e p o r t e d o v e r d o m i n a n t Vmax/Km  expression,  tidal  uncontrolled  by  by K o e h n ,  increased  environmental  to  at  an e s t e r a s e  r e p o r t e d i n the  of  examining the  necessary  homozygotes  enzyme a c t i v i t y  C. gigas  clarity  been  from  The o v e r d o m i n a n c e  in i t s .  than  enzyme  have  was  heterozygotes.  unique  for  these examples  resulted  in  In n a t u r a l  Heterozygotes  isomerase  overdominance  locus  at  Notropis stramineus 1983)  size.  overdominance  Catastomus c l a r k i i  (1977,  Pgm-2  of  activities  some p h o s p h o g l u c o s e  butterflies.  increased  demonstrated.  greater  ranges  the  from t h e i r  example  been  display  intermediate  (1971).  solely  found  protein  In to  than  Livingstone  p r e - r e p r o d u c t i v e p e r i o d (Koehn if  both  sexes  produce  equal  125  quantities specific  of  between  sex  somehow about  the  (Haley  sexes  i n the  for  and  females  solely  the  the  males.  and  the  requirement  appears  overdominance arise  of  of  adjacent  their  to  in tissue  rather  from  levels.  An i d e n t i c a l p a t t e r n was  muscle  tissue  to d i f f e r  the  cathodal  Pqm-1  phosphoglucomutase reasons  why t h e  effect  on  activity majority  of  locus  locus  results.  to  is  known  to  oysters  change  activity  also  sex  and  the this  was  shown not  iji v i v o enzyme noted  the to but  activity  in  the  adductor  not  been  observed  sexes.  the  to  the  c o u l d not First,  throughout the  compared  is  Furthermore,  contribution measured  i n t h e s e crude homogenates.  Pgm-1  these  in both t i s s u e s  met.  in  A n o t h e r c o m p l i c a t i n g f a c t o r was more  allele  protein concentrations,  differences  two  what  if  (Buroker 1983),  where p r o t e i n c o n c e n t r a t i o n has  between  also  only  ( H o a g l a n d 1978)  be  f o r m a n t l e PGM s p e c i f i c  genotypic  may  sex-related  Pgm-2-100  ability  individuals  unlikely  through d i f f e r e n c e s  these  d e t e r m i n a t i o n mechanisms of  because  sex  these  From  greater  of  patterns  However,  d e p e n d i n g upon e n v i r o n m e n t a l c o n d i t i o n s size  exhibit  because  Similar  possessing  sex  will  observed overdominance  d i r e c t i o n of  multiple-locus  1978),  males  in C. gigas.  heterozygotes  biased  enzyme,  protein levels.  can account  of  the  than  in soluble  differences the  certain  activities  differences exist  a  the  this  enzyme m e a s u r e d i n t h e s e  activity  There are  of  several  had  locus  remained u n s c o r a b l e  locus. crude  its  a  the  have  s t u d y due t o  Pgm-2  of  significant  low  level  Therefore, tissue  the  of vast  homogenates  126  must  have  locus  could explain  complete Pgm l o c i  were  Pgm-1  was  and 3)  loci  could  The  heterozygote. of  an  However,  to  produce  specific  (30%  p r o p o r t i o n of  more)  absolutely  no  evidence  electrophoretic  It  is  assay  protein. been  the were  The  similar  to  either of  account  Day-to-day selection  assayed  for  spontaneous for  all  of  l i n k e d to  20-25%  the  linked  the  frequent and  the  both Pgm-2  enzyme  differences separate  the Pgm-  at  larger  must  Pgm-1  for a  PGM a c t i v i t y .  and  sizable T h e r e was  from  the  o y s t e r PGM.  for  this  errors  o v e r d o m i n a n c e as  in extraction were  enzyme  activity  PGM a c t i v i t y  genotypes because  these  by  order  and  in  soluble  should also  measurements  a  and/or  minimized  Pgm-2 g e n o t y p e s and t h e  of  in  example,  less  requirement  errors  both loss  As an  recessives,  must a c c o u n t  total  l o n g term s y s t e m a t i c  techniques.  which they  the  of  both  dominance at  substantial  locus  patterns  difficult  of  randomizing  for  staining  also  consequence  Pgm-1  two  overdominant  the  activities  and t h e  2)  in  differed  the  three  variant  apparently  Pgm-2 g e n o t y p e s , or  exhibiting  Pgm-1  existed  t h e s e dominance e f f e c t s  Pgm-2 h e t e r o z y g o t e s ,  between t h e  at  relationships.  f o r a Pgm-1  produce  that  present  low a c t i v i t y  of  it  the  Pgm-2 l o c u s ,  variants  alleles  allele  pooling  Second,  1)  w i t h the  allelic  the  if  i n d i s e q u i l i b r i u m w i t h the  pattern existed  allele.  exist  for  which behaved as  2-100  only  r e v e r s i n g dominance  Pgm-2 a l l e l e s ,  activities  results  a high a c t i v i t y  locus  Pgm-2 l o c u s .  disequilibrium  activity,  that  opposite  these  segregating  exhibited  suppose  p r o d u c e d by t h e  linkage  specific loci  been  have were  1 27  completed the  within  rates  of  identical  p e r i o d s of  i_n v i v o d e g r a d a t i o n have  heterozygotes  would  than overdominant s p e c i f i c  vitro  stabilities because  and 96 a l l e l e s , alleles their  may  and  specific  be  104  t o have  than genotypes  of the  of  existence  potential  to  The l a c k  of  rate  exclusively 1986b) loci  has in  a null  Zouros,  in  on  C.  C.  virginica,  (1980).  The  activities  predicted and  soluble  of  the  the  these  grounds. null  (1978)  effects protein  one  that  the  i m p a c t on  the  to  levels  the  9 6 / 1 0 0 and  for  growth alleles,  Foltz  (1986a,  segregating  at  several  (Lap-2) p r e v i o u s l y Singh  allele of  for  the  by n u l l  However,  a null  has  (1980) o r i g i n a l l y  caused  locus  have  errors.  Pgm-2 l o c u s  and Z o u r o s , of  Pgmthe  Pgm-2-92/100,  alleles  of  in  overdominance  was  including  s c o r e d by S i n g h and Z o u r o s  the  In  Pgm-2-92  proportion  powerful explanation  virginica  recently detected  at  in  2.  for  unlikely  S i n g h and M i l e s  that  theoretical  the  suggests  or s y s t e m a t i c  allele  activities  possibility  observed  classes  observed overdominance i s  enzyme  the  a greater  enzyme a c t i v i t y has had l i t t l e  of  the  a clear distinction  these genotypic  100/104 h e t e r o z y g o t e s . discounted  on  c o n t a i n i n g t h e more s t a b l e  provide a simple yet  overdominant  based  heterozygotes  lost  been c a u s e d by u n c o n t r o l l e d f a c t o r s  The  results,  observed in Chapter  and  in  intermediate  t h e marked t h e r m o l a b i l i t i e s o f  alleles.  loss  Hence,  these allozymes  expected  activities  spontaneous results.  of  differences  to d i s p l a y  activities,  b o t h homozygotes  activities  2-100  of  If  influenced these  have been e x p e c t e d  rather  addition,  time.  the  and  Miles  on t h e  enzyme  four  Pgm-2  128  homozygote patterns  classes  expressed  .frequency  shown i n T a b l e by t h e s e  differences  population,  genotypes.  between  marked  dichotomy  reductions  in  enzyme  activities  between  Pqm-2-100/100  and t h e  homozygote for  far  than  greater  classes.  soluble  expected  i n homozygotes  protein  product,  exhibited directly  by t h e  or  that  unidentified  Perhaps  the  the  influence  of  overdominance  is  p r o v i d e d by t h e  heterozygote  groups examined i n the Pqm-2-92,  overdominance  for  specific  weight  basis,  the mantle or a d d u c t o r  explanation functional  is  correct,  alleles,  protein  these  are expected  levels  that  null  9 6 / 9 6 and this  in  a null may  distinction the  slight  heterozygotes 1)  the  of  These  even  discrepancies and a  further  number  the  (Table  null  allele  or s o l u b l e If  the  VIII).  activity,  PGM  the  Here, d i d not  contents  null  allele  possessing  t o d i s p l a y PGM a c t i v i t i e s identical  with  two  activity  protein  heterozygotes,  were  of  Pqm-2 l o c u s .  96 and 104 a l l e l e s  muscle.  no  PGM a c t i v i t y  allele  against  fall  was  reductions  producing  involve the  allele 104/104  r e l a t i v e performance of  the  either  soluble  evidence  between  on a t i s s u e  the  for  allele  in a d d i t i o n to  explanation  in  by  overdominant h e t e r o z y g o t e s .  strongest  expressed  the  increased levels  the  exhibit  between  be a c c o u n t e d f o r by  2)  enzyme l o c i  heterozygotes  expected  Furthermore,  from a n u l l  c o n t r a d i c t the  suggest  study  protein levels  could  large  the  Pqm-2-92/92,  between  the  in  caused  g r o u p s . T h e r e was no e v i d e n c e  of  the  allozymes  is  homozygote  overdominance  incompatible with  Because  these  a  the  IX a r e  the  two and three  129  heterozygotes  for  the  indistinguishable alleles. the  of  heterozygosity the  is  a  of  r e q u i r e d ; the 96,  well  null se  as  (cf.  Instead,  In  for  these  Lerner  they  were  three  rarer  Pgm-2  for  the  results  1954)  is  enzyme  a particular allele  104 a l l e l e s  fact,  c o n t r a d i c t i n g a key p r e d i c t i o n  allele,  Pgm-2-100  or  allele.  homozygotes  overdominance  in C. gigas.  Pgm-2-92,  as  per  expression  locus  from  Therefore,  effects  Pgm-2-100  must  before  not  show  that  sufficient  activity  at  allelic  the  for Pgm-2  configuration  be p a i r e d w i t h e i t h e r  the  of  overdominant  effects  the are  manifested.  An  alternative  explanation  for  these  associated  with  the  regulatory  locus  that  hypothesis,  capable  results,  that  produces  in  heterozygotes  action  of  this  regulatory  it  putative could  control  translation,  or  involved,  its  example.  The  (i.e.  1983)  and  1983)  variants  intermediate  et  cis-acting  unlike  pattern  al.  the  a  any that  is  Doane e t  (e.g.  Dickinson  activities  of in  levels  unknown,  al.  1975;  of  processing, element  is  characterized from s t u d i e s  both  1983;  of  but  levels  mRNA  that  or  The mode of  known  emerged  to  overdominant  regulatory  has  1980;  rates  is  previously  in eukaryotes  affecting  enzyme  locus of  If  an  homozygotes.  transcription,  is  general  to  any  degradation).  behavior  Scandalios  at  is  better  linked  steady-state  regulatory  act  r e g u l a t o r y polymorphisms (e.g.  greater  relative  providing a  tightly  Pgm-2 s t r u c t u r a l l o c u s ,  enzyme  conceivably  is  of  trans-acting  Lusis  Shaffer  et  and  transcription heterozygotes,  on  al.  Bewley produce  while  those  130  acting  post-translationally  dominant/recessive and S c a n d a l i o s  tend  fashion  1980;  to  (e.g.  be  inherited  R e c h c i g l and H e s t o n  K i n g and M c D o n a l d  1983,  1987;  in  a  1967;  Lai  Gibson  et  al.  1986).  An o v e r d o m i n a n t its  effects  increased  at  effects  are  and  Day  regulatory rates  of  levels is  PGM t u r n o v e r ,  of  enzyme  unlikely  on PGM a l o n e , should also  This provide  in  a  efficiency" used  allele, alone,  between  rates  the  in. v i v o  levels  that  enzyme  in  relative  of  level  will  loci.  its  the  leads  However,  other  Bayne  putative  to  decreased  steady-state  homozygotes.  have  of  pleiotropic  greater to  of  heterozygosity  that  condition  such a protease  the  r e p o r t e d by H a w k i n s ,  resulting  in heterozygotes that  and  tissues  The  explainable  suggests that  possibility  heterozygous thus  that  multiple-locus  recently  the  i n the  above  unidentified  exert  or d e g r a d a t i o n .  observed  transcriptional  offers  locus  could theoretically  synthesis  levels  on o t h e r  turnover  (1986)  enzyme  enzyme  is  exerted  locus  Pgm-2-100  negative association protein  ATP  the  control  and  it  for  of  protein  phosphoglucomutase  regulatory  the  level  soluble  heterozygotes by  the  regulatory  act  enzyme  Since  specifically substrates  increase.  explanation general of for  is  intuitively  explanation  heterozygotes polypeptide  for  (through  synthesis)  a n d • s u p p o r t e d by a number of  recent  a p p e a l i n g because  the the  increased  it  "metabolic  reduced expenditure  s u g g e s t e d by B e r g e r studies  (e.g.  can  of  (1976)  Koehn  and  ^  131  Shumway  1982;  Garton  from p r e v i o u s l y overdominant display  unique  multimer.  the  heterozygote  of  transcription  of  explanation. the  the  Pgm-2  system i n t o results  the  rates  for  somehow These  gene p r o d u c t  product  functional  and  this  condition.  diffusible  gene  hypothetical  of  several  First,  produced  is by  a a  properties  ( p r o d u c i n g an  in  case  the  regulatory  of  function  PGM a c t i v i t y  it  is of  structural  locus  a two a l l e l e  these  capable  a stable by  if  would  locus  According complete  three  genotype at  it  a is  result.  must  to  this  model,  Associated  w i t h the  await  designated an  as  it  is  96 and  phenotype  allele  explanation  equilibrium the  possible  to  the  manifested  a  X). in  allele. different  A and B in  10  their  exists  structural is  the  Table  "A"  104 a l l e l e s for  on  g r o u p the  (see  variant  at  multi-allelic  a c c o r d i n g to  locus  Pqm-2-100  "B". Heterozygotes  overdominant  p r o v i d i n g an  classes  genuine  null  p o l y m o r p h i s m . Based  regulatory  Pqm-2-92,  the  collapsing  regulatory  d i s e q u i l i b r i u m w i t h the  a c a s e of  over  of  phenotypic  this  as  polymorphic  regulatory  summarized i n T a b l e V I I I into  results  advantages  maintenance  hypothesized  produce  if  overdominance  this  has  Pgm-2 g e n o t y p e s  allele  be t r a n s - a c t i n g  or d i m i n i s h e d p r o t e o l y t i c  interpretation  overdominance  the  polymorphisms,  study.  The  for  1984). E x t r a p o l a t i n g  heterozygous  transcription  then  The e x i s t e n c e  met  altered  factor  protease),  further  be  in  heteromultimeric  has  enhancement  a  properties  al.  regulatory  i n C . g i g a s must  could  If  Garton et  characterized  locus  requirements  1984;  the  alleles Pgm-2-  132  Table X. C o l l a p s e o f t h e m u l t i - a l l e l i c Pqm-2 s t r u c t u r a l l o c u s p o l y m o r p h i s m by a h y p o t h e t i c a l t i g h t l y - l i n k e d r e g u l a t o r y l o c u s s e g r e g a t i n g f o r two a l l e l e s .  133  Phenotypic Class  Regulatory locus Genotype  Structural locus Genotype(s)  1  "A/A"  2  "A/B"  92/100  96/100  100/104  3  "B/B"  92/92 92/96  96/96 92/104  104/104 96/104  100/100  134  92/100,  9 6 / 1 0 0 and 100/104  allele  produce  comprising 96  and  six  genotypes.  phenotypically  b o t h homozygotes  104  alleles.  This  different  eliminates  difficulties  polymorphisms  G i n z b u r g and T u l j a p u r k a r it  is  difficult  to  structural associated  by  For the  genotypes presence  a  The  a null  allele  Pgm-2-92,  genotypes  (e.g.  this  a problem  i n t e r p r e t a t i o n , however,  be e a s i l y  b r o k e n down by i n t r a g e n i c " r e c o m b i n a t i o n between  in D. melanogaster are  expected  the  fast  within  the  frequency and c l o s e l y 1979a, at  least  to account within  the  is  some o f  30 m i l l i o n this  of  the  (cf.  (1986). of  because  at  the  over the  it  could  detected,  t h e Adh l o c u s  C. gigas.  crossovers This  becomes  age  similarity  of  this  of  allele  congeneric Crassostrea  species  Buroker,  (Stenzel  disequilibrium  as  Similar  Hershberger  w h i c h a r e known t o have been years  with a  disequilibrium  p o t e n t i a l l y great  by  between  r e l a t e d genera  for  al.  suggested  distributions  1979b),  and slow a l l e l e s  Pgm-2 l o c u s  even more p r o b a b l e i n l i g h t polymorphism that  hypothesized  by A q u a d r o e t  explanation,  species).  presents  example,  Lewontin,  advantage  of  for  stable  heterozygote  persistence in  and  ( r e q u i r e d to maintain  i n p o p u l a t i o n s of  this  similarities  allele  selective  w i t h two f u n c t i o n a l a l l e l e s of  the  with maintaining  null  B  genotypes  locus  how a Pgm-2 n u l l  r e d u c e d enzyme a c t i v i t y c a n e n j o y  the  Pgm-2  for  overdominance  1978).  see  for  model c a n e x p l a i n t h e  the  multi-allelic  equivalent  and h e t e r o z y g o t e s  o b s e r v e d between the  Homozygotes  is  1971).  and  isolated  Perhaps the  Pgm-2  for  o n l y way  f o r an i n v e r s i o n t o  chromosomal r e g i o n e n c o m p a s s i n g t h e  Chew  exist  structural  1 35  locus less  that  includes  frequent  The  Instead, alleles  is  heterozygous the  consistent in  the  and  control  the  Pqm-2-92,  to  the  their state  resulting  relative  dynamics of  the  to  the  areas  Japan,  range, of  the  most  5.4%.  adjacent  In  mobility  variation appears erratic  is  of  from  large  patterns  alleles  type  too  to  3,  to  in  of of  of  the  be c a u s e d by  and  genotype  that  reconcile  equivalent Pgm-2-100  balancing as  selection,  allele  fell  that  the  within a limited  of  of  variation  alleles  in  r a t h e r d r a m a t i c a l l y , each to  coefficients of  this  by s a m p l i n g e r r o r frequencies  with  unless  i n p o p u l a t i o n s of  For  from 5 g e o g r a p h i c  The e x t e n t  p r e d i c t e d by t h e m o d e l .  is  alleles  oyster.  observed  rise  the  frequent  homozygous  Pacific  frequencies  giving  are  interpretation  C. qigas  fluctuated  the  achieved.  t h e s e Pgm-2  (1985)  the  that  less  the  of  they  are  Since  the  C. gigas  is  of  variation  alone. these  maintenance  m u l t i - a l l e l i c p o l y m o r p h i s m by o v e r d o m i n a n c e , of  three  alleles  0.596 and a c o e f f i c i e n t  29%-49%.  displayed  difficult  these  common Pgm-2 a l l e l e  classes  ranging  far  the  state  Pgm-2-100/100  Fujio  contrast,  by more t h a n a f a c t o r  stable  and  e x h i b i t i n g a mean of  only  of  frequencies  i n 23 p o p u l a t i o n s a m p l e s  of  96 and 104  fitnesses  and c u l t u r e d p o p u l a t i o n s  frequency  or  predicts  polymorphism. This  observed  Ozaki  also  equilibrium  example, of  allele  model  combined f r e q u e n c y  w i t h the  natural  of  consequence  it  Pqm-2-100  overdominant  frequencies  little  the  alleles.  proposed  individual of  either  The rarer of  o r any  a  other  functionally  f r e q u e n c y of always  the  greater  136  than  0.5  Fujio  1985),  the  that  of  exceed less  (Buroker,  frequent  activity mantle  that  are greater  also  it  sufficient  detected to  overdominant  and must  possessing  these  with  enzyme,  supported in  homozygote  displays  on a t i s s u e  these other  Ozaki  genotype  correlated  protein levels  differences this  at  96/100  these  the PGM  weight  genotypes  locus  the  kinetic  these  Pgm-2  for-  the  locus  basis  by  14%  enzyme  of  suggest  are expressed;  these genotypes capacities  demonstration  by  Fujio  that  is  the  the may of  other provide  differences  of  if  the  the  selective  biochemical importance  these effects at  entirely,  Pgm-2-92/100, i m p a r t s upon greater  listed  flux  in Table X.  basis of  the  observed  not  automatically  Pgm-2 g e n o t y p e s the  of  ( 1 9 8 2 ) . The  selective  may be l a r g e l y ,  not  effects  through  allelic  heterozygotes  were  that  it  in  structural  allozymes  l a r g e r Vmax/Km r a t i o s a n d , h e n c e ,  than turn  and  2,  possessing  The i n c r e a s e d enzyme a c t i v i t i e s 100/104  Chapter  heterozygotes  levels  the  locus  by an o v e r d o m i n a n t  phenotypic  reported  activities  strongly  results  b i o c h e m i c a l d a t a of  that  i n enzyme a c t i v i t y  and  in  this  specific  structural  neutral.  of  between  account  Pqm-2-100 a l l e l e  This  is  prediction is  t h a n t h e mean of  concluded  heterozygosity  the  this  s u p p o r t e d by t h e  was  differences  of  and h e t e r o z y g o t e s  Pgm-2-100/100  soluble  1979a;  Pqm-2-100/100  fitness  locus,  interpretation  is  which  the  1975,  respectively.  The model  If  where t h e  and  of  homozygotes  this  tissue  and 8%,  fitness  alleles.  at  activities  H e r s h b e r g e r a n d Chew  for  this  the enzyme  1 37  polymorphism through i t s subsequent  i m p a c t on f i t n e s s  These growing  results  number  Watt  As  (1977,  it  by M i t t o n  1983),  overdominance and  is  has  the  implicated  several  studies  reported  for it been  first  with  on  related  the  important  is  Grant Pgi  of  detected time  associations  the  at  it  has  explanations Pgm-2 l o c u s tightly  are  linked  d i s e q u i l i b r i u m w i t h the  Another the  with  most  studies  that  all  it  block  structural  properties  it  has  one  forward  though  or  of  examining the  heterozygosity, homozygotes  least  different  phenotypic  been  and h e t e r o z y g o t e s  Foltz by  previously  multiple-locus  locus,  the  between t h e  to  various  explain  may have of  present  these  overdominance  been  genes,  at  the  caused  by a  in  complete  locus.  i m p o r t a n t r a m i f i c a t i o n of  distinctive  one  o v e r d o m i n a n c e was m a n i f e s t e d  even  gene,  and  cases  i n b r e e d i n g and a s s o c i a t i v e  untenable:  in C. gigas  Zouros  involved  in d i s t i n g u i s h i n g  Both the  phenotypic-level  locus,  successful  associations.  between  a p o l y m o r p h i c enzyme  study  put  the  which  relationship  hypotheses  to  in  for at  alternative  of  few  Therefore,  been  and  in C o l i a s b u t t e r f l i e s  heterozygosity. has  implications  1984;  locus  one  metabolism  traits.  and a v a r i e t y  and  a  glycogen  documenting  heterozygosity  (reviewed  1987).  have  of  multiple-locus traits  influence  common together,  these  results  arises  Pgm-2 h e t e r o z y g o t e s . effects  of  practise thus  genotypes combined w i t h i n these  from In  multiple-locus to  pool  all  i m p l i c i t l y assuming  groups are  equivalent.  138  The a b s e n c e  of  the  Pgm-2-92,  that  a  represent  of  crosses  and 104 a l l e l e s  amount  in  resulted  several  collected  (discussed  by  discrepancies number  of  1984),  the  some l o c i  have  (1986)  and  is  rate  Foltz  possible:  of  interpreting  study  genotypes locus.  laboratory  observed  species  Although  these  for a  large  G a f f n e y and  Scott  suggest  that  "adaptive  states.  heterozygous  groups  studies provides  an  distance"  model  of  causes  of  accurately  the  the  demonstrated  this  adductor  at  the  has  expressed  Pgm-2 l o c u s  overdominant e f f e c t muscle  tissues,  by t h e  was  similar was  that  three  in Crassostrea  and  at The.  heterozygosity.  is  an  overdominant e f f e c t s  t y p e s of  study  in  same  involving multiple-locus  present  the  occasionally  the  heterozygous  more  by  between m u l t i p l e -  of  (e.g.  the  suggests  this  requirement  disparate  in these  at  was  1987).  present  f o r a d o p t i n g the  these  have  that  genomes  f o r enzyme a c t i v i t y  of  parents  particular  scored  heterozygotes  The m a g n i t u d e mantle  the  on  in  summary,  since  an a s s o c i a t i o n  parental  study  marine b i v a l v e s ,  e x p l a i n e d by t h e  from  locus  reason  overdominance common  of  confounding e f f e c t s  these c o r r e l a t i o n s  In  been  may depend  additional  of  between  introduced  heterozygotes  growth  and  explanation  an enzyme  Smouse  Zouros  results  potentially  be  from n a t u r a l p o p u l a t i o n s  differing  additional  species  and  may  all  e l i m i n a t i o n of  heterozygosity  heterozygotes  equivalency,  l i m i t e d numbers  i n the  in  found i n t h i s  error  heterozygote  between  progeny  of  a p p r o x i m a t e l y 24% o f  Furthermore,  at  96  sizable  assumption  locus  an o v e r d o m i n a n t e f f e c t  most  gigas. in  unaffected  the by  139  environmental or  an  these via  factors.  undetected findings.  a  locus,  null  that  alleles  relative  the  locus  to  collapsing  of  the  the  between  overdominance  is  Pqm-2 s t r u c t u r a l  levels  of  This an  the  importance  of  i n v o l v e a d e m o n s t r a t i o n of  its  system  regulatory locus.  pathway  for  PGM a c t i v i t y  interpretation  array  of  different  frequencies  and p r o v i d e an  multi-allelic  the  physiological  for  account  the  to  variables,  of  these  explanation  Pqm-2 p o l y m o r p h i s m i n a b a l a n c e d s t a t e  this  the m e t a b o l i c  this  l i n k e d to  account  polymorphism at selective  for  homozygotes.  similarities genotypes,  are unable  steady-state  in natural populations,  maintenance  glycogen  allele  tightly  produces greater  explain  e r r o r s , unexamined  explanation  regulatory locus,  structural  the  Pqm-2  The b e s t  in heterozygotes can  Systematic  the  into  Definitive  concentrations  of is  impact  on  PGM a c t i v i t y the  two  by  allele  evidence  for  o b s e r v e d o v e r d o m i n a n c e must glycogen  i n w h i c h PGM f u n c t i o n s .  effects  a  for  subject  of  metabolism,  E x a m i n a t i o n of  variation Chapter  4.  on  the  tissue  140  CHAPTER 4  PHYSIOLOGICAL  EFFECTS OF THE PGM-2 LOCUS ON GLYCOGEN METABOLISM  INTRODUCTION  A large  body of  studies  the  and  properties  differences  in  allozymes  p o l y m o r p h i c enzyme  at  structural  have d o c u m e n t e d  review  of  better  characterized  McDonald Hall  this  literature  (1983),  (1985),  which the  Koehn,  between  (1985b). allozymes  importance  natural  p o p u l a t i o n s must  (1975)  and  allozymic  Koehn  variation  physiological of  the  studies  have  significance 1981;  Hall  of  of  that  the  fall only  have  detection  between  comprehensive but  been  (1983),  of  some of  the  discussed Zera, of  by  Koehn and  biochemical  p r o v i d e s o n l y the  f o u n d a t i o n upon  functional,  ultimately  type  be b a s e d .  imparts  single  been d o n e ,  The  this  genotypes.  on a l l o z y m e s  consequence,  the  (1978),  processes  enzyme  has n o t  A  Z e r a and H a l l  d e m o n s t r a t i o n of  selective,  loci.  polymorphisms  and Watt  differences  functional  existence  it  As  is  and  of  genetic  pointed  essential  significant  variation  out to  the  by  in  Clarke  show t h a t  this  effects  on  relevant  d i f f e r e n t i a l l y affect  the  fitnesses  The  short  vast of  achieving this  been a b l e  kinetic  data  attempt  to  majority  to  (e.g.  of  biochemical  end a n d , as  a  speculate  on t h e  adaptive  Merritt  1972;  Hoffman  1985).  Studies  that  demonstrate  the  physiological  141  consequences formidable (1984)  of  difficulties.  have a d d r e s s e d  extrapolating to  in  vivo  studies,  the  variation  encounter  Zera,  Hall  and Koehn  (1985),  the  problems  that  some  of  significance  function.  and  analytical  robust  kinetic  on  the  catalytic  of  the  is  for  identifying  essential  parameters 1985a).  that  enzyme  might  Dykhuizen,  additional  complexity  of  kinetic  the  allozymes,  Dean  obstacles  inherent  and t h e  under  study.  organism's  ecology  concerning  the  be  of  the  catalytic  Hartl  in these  metabolic  unknown e f f e c t s Finally,  enough must  to  reasonable  allow  selective  polymorphism under n a t u r a l  The  ability  allozymic theoretical  loci  variation  to  measure has  difficulties.  electrophoretically  also A  be  assay and  knowledge  is  metabolic  This  information  and/or  regulatory (Watt  have  the  1983,  discussed  arise  from  the  small  magnitude  present  between  tightly  l i n k e d to  known about  assumptions  agent(s)  purified  designs  that  usually of  these  and  (1987)  phenotypes,  differentiation  in  highly  importance  studies  from  differences  realistic  study.  selective  and  faced  potential  of  background  for  and E a n e s  unavoidable use  of  arise  kinetic  properties  selected  of  number  experimental  Sufficient  function(s)  studied  are  a  physiologically  methodologies.  required  on t h e  j_n v i t r o  Such l i m i t a t i o n s  preparations,  conditions,  one  of  but may be m i n i m i z e d t h r o u g h the  allozyme  also  enzyme  that  the  the  study  be  made  to  c o u l d be  acting  conditions.  the met large  do n o t  physiological with  some  proportion  function  effects  of  interesting of  the  enzymes  independently,  but  142  catalyze  reactions  that  pathways.  Hence,  variation  must be m a n i f e s t e d  in which  they  Extending Kacser  function  the  have  between  basic  enzymic  action  of  onto  Dykhuizen  electrophoretic differences,  of  in  adaptation  in general and  Koehn  (e.g. 1987).  DiMichele 1983;  Middleton The  of  Hartl,  activity  for  have  this  entire to  a concave  selection.  and  of  through  many enzymes  relationship,  predicts  Dean  enzymes  that  of  (cf.  relationship  many  this  pathway  c o n t r o l theory  activity  which u s u a l l y  enzyme  Dykhuizen  activities  (1985)  the  argued  Therefore,  this  visible  metabolic  in c a t a l y t i c  flux.  at  the have  which  would r e s u l t  saturation that  the  exhibit  in  theory  of  m a j o r i t y of  minor  kinetic  neutral.  has  stimulated implications  (e.g. Place  has c o n t r a d i c t e d i t s  and  of  of  biochemical  considerable for  the  discussion  physiological  a l l o z y m i c v a r i a t i o n i n p a r t i c u l a r , and b i o c h e m i c a l  Burton  1981;  being  p l a t e a u of  important  of  level  of  the  selectively  theory  its  the  impact  existence  They  and Dean  relevance  Clegg  and  variants,  are  Control because  the  changes  changes  the  1981),  natural selection,  negligible Hartl,  1979,  metabolism.  substantial  1985b;  premises  flux  pushed w e l l  at  before  predicted  intermediary  point  physiological  and B u r n s 1973,  (1985)  been  the  a r e embedded w i t h i n c o m p l e x  Koehn, 1987;  predictions and Powers  Burton  and  and  Kacser  elegant  series  Zera  Pogson  and  Hall  1988).  i n some c a s e s 1982a,  1983; of  Barnes  competition  Watt  E x p e r i m e n t a l work (e.g.  1982b;  Feldman 1983),  1983;  C a v e n e r and  Hilbish, but  and  not  Deaton  in  others  Laurie-Ahlberg  experiments  by  Hartl  143  and c o - w o r k e r s allelic  substitutions  largely  between s t r a i n s  standard  Hartl  genetic  de  locus  effects  at  all  these  of  is  allelic  variation  studies  are c l e a r l y  consequences metabolic  of  has  most  of  of  this  relevant,  this  variation  influence  the  is  on f i t n e s s of  assess  lacZ  and  the  1980;  these  zwf  (Dean, The  significant Hartl  (i.e.,  gnd  1987).  measured  1987).  the  effects  growth  rates),  experiments  fluxes. both  enzyme  if  More e m p i r i c a l  the  physiological  general  validity  to  glycogen.  be s t r i c t l y locus  Pgm-2 l o c u s  To  activity  properties  isomerase  activities  demonstrating  i n enzyme  metabolism of  believed  for  the  polymorphism.  regulatory  phosphoglucose  or  exerted  experiments  overdominant  significance  function  variation  v a r i a t i o n and t h e  potential  any  1984),  metabolic  the  possess  at  ( D y k h u i z e n and H a r t l  common h e t e r o z y g o t e s a t  provided  alleles  were  loci  to  have  of  theory.  The e x p r e s s i o n three  different  known i n any  needed  loci  differences  ( D y k h u i z e n , Dean  affected  single  Hartl  alleles  allozymic  control  fitness  D y k h u i z e n , Dean and H a r t l  lacY  not  by  1983),  these chemostat  it  differ  ( D y k h u i z e n and H a r t l  allelic  polymorphic  and t h e r e f o r e  pgi and  1986;  was  No  possessing  that  electrophoretic  backgrounds)  which  on f i t n e s s  However, of  theory.  Framond  D y k h u i z e n and H a r t l  strains  different  and D y k h u i z e n 1 9 8 1 ) ,  (Dykhuizen,  only  at  supported c o n t r o l  detected (on  utilizing E. coli  by  be  the  gigas  adaptive  now be shown  Since  PGM i s  (Ray  and  Watt  in C.  the  physiologically  must  catalytic.  by  not  Peck  known  to  1972),  its  As a r g u e d  (1977,  to  for  1983),  the the  1 44  physiological expressed differ  effects  by d i f f e r e n c e s  because  activities  Pgm-2  3); and  may be of  Michaelis  activity)  is its  importance,  probably  composite  differences  that  activity  the  nature  may  more  are  measured  (as  (Chapter  between a l l o z y m e s d e t e r m i n e d by  important  that  specific  in vivo  between g e n o t y p e s  simultaneously  for  glucose-6-phosphate  (Vmax = k c a t [ e n z y m e ] )  exist  levels  Vmax  enzyme  between a l l o z y m e s  could exist  be  reaction  specific  for  to  ratios  for  constants  estimated  differences  selective  their  likely These  genotypes  in  o n l y minor d i f f e r e n t i a t i o n  A l t h o u g h kcat  enzyme  Vmax/Km r a t i o s .  variation  (Chapter  exhibited  because  between  of  glucose-1-phosphate  Pgm-2 g e n o t y p e s a r e most  in their  significantly  directions  2).  of  parameter  ensures in  that  steady-state  incorporated  (Hoffman  1981).  Examination of on  glycogen  advantages pathway  of  over  weights  glucose  at  important  quantified on pathway  the  studies  1969). are  b e c a u s e of of  the  function  of  this is  the  thus  the  may a l s o  the  Pgm-2  oyster  has  a number of  nature.  up t o  role  Because effect  of  25% of  synthetic four  s t o r a g e compound their  effects to  The  locus  involving only  major energy  likely  central  of  short,  Demonstrable  oysters.  end p r o d u c t ,  effects  Pacific  glycogen  representing  metabolism  metabolism  to  in  Glycogen is  times  (Quayle  glycogen  energy  previous  reactions.  oysters,  physiological  metabolism  from  enzymic  the  be  of  total  this  locus  variation  on  physiologically  p l a y e d by g l y c o g e n glycogen  body  is  an  in  the  easily  i n PGM a c t i v i t y  be more r e a d i l y m e a s u r a b l e  than  if  1 45  it  catalyzed  pathway  a  reaction  ( s u c h as  in  a  glycolysis).  more complex e n e r g y - p r o d u c i n g  Seasonal  synthesis  and d e g r a d a t i o n a r e w e l l  (Gabbott  1975;  (over  periods  capacity  of  to  Since glycogen  cumulative  Pgm-2  time  levels  glycogen  on  bivalves  change  genotypes  effects  w h i c h may be more e a s i l y  o p e r a t e d on a much s h o r t e r  of  documented i n m a r i n e  weeks and m o n t h s ) , exert  concentrations cycle  Bayne 1 9 7 6 ) .  patterns  have  tissue  observed  slowly  than  the  glycogen if  the  period.  MATERIALS AND METHODS  Chemicals. used  Buffers,  for  the  glycogen  Amyloglucosidase electrostarch  Animals. oysters  substrates,  was  provided  that  Pgm-2 g e n o t y p e s  electrophoresis.  Details  found  2.  in Chapter  Glycogen  Assays.  activity  experimental  the  t r a n s p o r t a t i o n and s t o r a g e  were  identified  electrophoretic  design  tissues  measurements i n v o l v e d the  Sigma.  B o e h r i n g e r Mannheim and  Glycogen concentrations  m a n t l e and a d d u c t o r m u s c l e specific  from  obtained  d e s c r i b e d in Chapter  Electrophoresis.  of  were  the  from Connaught L a b o r a t o r i e s .  collections,  i d e n t i c a l to  and s t a n d a r d s  by  for e l e c t r o p h o r e s i s  The s e a s o n a l was  assays  c o u p l i n g enzymes  of  all  by  of  3.  starch  p r o c e d u r e can  gel be  were d e t e r m i n e d i n  the  oysters  for  described  in  comparison of  selected Chapter  glycogen  3.  The  levels  1 46  in animals g; in  from  36.0-47.9  f o u r body w e i g h t  g;  48.0  g+),  seasons  (summer,  genotypic  classes.  Glycogen  individuals  present.  Hence,  specific  activity  the  mantle the  adjacent  activity,  thus  represented Tissues (PCA)  to  grinder.  half  to  A  the  Glycogen modified  1  ml  of  to  of  ml  carbonate  was  the  amyloglucosidase  tissue  2 ml of for  aliquot tube  pH 4 . 8  of the  chosen  s  was  tissue  from b o t h  the  weighed  to  and  removed  measurement  of  -70°C  PGM  study.  perchloric  to for  in  dissected  for enzymatic  transferred  was  studied  muscle  N  the  3.  mantle  0.6  some  than  w i t h an U l t r a - T u r r a x an  acid tissue  Eppendorf  up t o  3 months  amyloglucosidase  method  glycogen.  by  the  and Decker  (1974).  Duplicate  from b o t h t i s s u e s  sodium a c e t a t e , containing  (lyophilized  in Chapter  adductor  30  in  lower  blotted,  ice-cold  from K e p p l e r  mM  measured  was d i s s e c t e d  and f r o z e n a t  assayed  be  a c o m p a r a b l e r e g i o n was  tissue  and h i g h ) ,  c o m p r i s i n g 7 Pgm-2  slightly  for  the the  (low  24.0-35.9  l i m i t e d amount of  section  PCA homogenates  200  buffer,  were  taken  d e t e r m i n a t i o n of  slightly  M1 s a m p l e s 1  of  homogenized  microcentrifugation prior  that  were added t o and  the  presented  The  Similarly, one  not  adductor muscle,  ensuring that  oysters.  of  sizes  g of  milligram.  directly  all  0.4  and w i n t e r )  could  measurements  and p o s t e r i o r  nearest  fall  because sample  Approximately  ( 1 2 . 0 - 2 3 . 9 g;  from two t i d a l h e i g h t s  three  smaller  classes  were  50 mM p o t a s s i u m  approximately  6  from A s p e r g i l l u s n i g e r )  100  added  hydrogen units  in  of  10 x 75  147  mm  polycarbonate  plastic 40°C. 500 in  caps  culture  and i n c u b a t e d f o r  The h y d r o l y s i s of  Ail 0 . 6 N PCA and t h e a clinical  were  at  The  by t h e  at  340  Assays  hydrosylates  on  spun f o r  bath  5 min a t  of  the  with at  a d d i t i o n of top  speed  supernatants  microcentrifugation  Unicam  reaction  tubes  and  medium  in  tissues.  pH 7 . 5  of  hexokinase  these  then  a second absorbance absorbance free  75% i n t h e  volume of 1 M1  of  the  addition,  the  for  r e a d i n g was t a k e n . the  cuvettes  were  The  at 1.5  again  completion  present  as  i n the  (>  difference  the  Mmoles  hydrolyzed  mean o f  50%  in  the  glucosyl  adductor  sample.  four assays  by a s s u m i n g an i n t r a c e l l u l a r w a t e r m a n t l e and  units  equilibrate  reading taken.  represented  and were e x p r e s s e d  weight  added,  mM  r e a d i n g s was s t o i c h i o m e t r i c a l l y e q u a l  glucose  concentrations  individual  absorbance  300  0.8  20  r e a c t i o n was a l l o w e d t o p r o c e e d t o  amount o f  Glycogen  was  on  sample  were m i x e d t h o r o u g h l y and a l l o w e d t o an i n i t i a l  NADP,  in a final  duplicate After  UV/visible  contained  mM  room  dehydrogenase  SP8-400  1 mM A T P , 0 . 5  dehydrogenase,  both  between  of  The  from  before  tissue  water  s t o p p e d by t h e  M1 sample  Pye  performed  15 min)  the  a  were  m i x e d and t h e  to  sealed  was q u a n t i f i e d e n z y m a t i c a l l y a t  4 mM MgS04,  10 min b e f o r e  units  were  hexokinase/glucose-6-phosphate  nm  glucose-6-phosphate  cuvettes  was  were  A 500  l i b e r a t e d glucose  triethanolamine,  least  samples  Eppendorf  spectrophotometer.  ml.  tubes  -70°C.  temperature system  into  The  2 h in a shaking  glycogen  centrifuge.  pipetted  frozen  tubes.  units/g  per wet  concentration muscle  tissue  148  (Walsh,  M c D o n a l d and B o o t h 1 9 8 4 ) .  the  free  glucose  its  c o n c e n t r a t i o n was  present  i n the  C o r r e c t i o n s were  original  f o u n d t o be t o o  estimates.  To a s s e s s  the  enzymatic  hydrolysis  dissolving  100 mg o y s t e r  efficiency  of  and  stock  were h y d r o l y z e d a l o n g s i d e  and a s s a y e d  because glycogen  repeatability  (Type I I )  buffer,  each season  the  for  of  the  s t a n d a r d s were p r e p a r e d by  imidazole-HCl solution  pH 7.0  PCA homogenates  low t o a f f e c t  glycogen, glycogen  n o t made  (20°C).  in d u p l i c a t e  in  Fifty  nl  the  for  10 ml  of  50  aliquots  tissue  glucose  mM  of  this  samples  from  as  described  previously.  The of  glycogen  each  seasonal  degradation  of  determined, influence 3,  the  the  time  measurements  collection. glycogen  several  of  this  selection  of  genotypes  taken to p r o c e s s for  completely  r a n d o m i z e d , as  errors  the  glycogen  was  Statistical  were  in  on t h e  genotypes  day  the  enzymatic  out. kept  Analyses. on t h e  similar.  order it  in  The of  not  minimize  the  Chapter  which  that  selection  glycogen  was e x p e c t e d  a minimum and t h e  was  of also  the  glucose  that  day-to-  spontaneous  loss  of  genotypes.  The e f f e c t s glycogen  was  r a n d o m i z e d e a c h day s u c h  Therefore, to  to  months  spontaneous  As d e s c r i b e d i n  hydrolysis  was t h e  six  oysters  taken  results. was  the  frozen  were  them was  s i m i l a r for a l l  Pgm-2 g e n o t y p e  Although  precautions  factor  a s s a y s were c a r r i e d  were c o m p l e t e d w i t h i n  of  season,  tidal  c o n c e n t r a t i o n of  s t u d i e d was examined by a n a l y s i s  of  variance  the  position,  and  two  tissues  (ANOVA) a s  outlined  149  in  Sokal  and Rohlf  Bonferroni  (1981).  m u l t i p l e range  Means were compared by a p o s t e r i o r i  tests.  RESULTS  C o m p a r i s o n of amyloglucosidase assays  85.9%,  statistical  = 4.82,  showed  hydrolysis  i n the  concentrations  and  be  87.4%,  a  a consequence  winter  Similar reported  Pgm-2  again  relative the  the  on t h e  analysis 3,  the  tissues  ANOVA t r e a t i n g  genotype  as  a significant  adductor muscle  of  and  h y d r o l y s i s of  percentages of  season  multiple  range  higher e f f i c i e n c y Tissue  sample w i l l  winter,  but  glycogen  therefore  this  of  be  w o u l d have  results.  of  the  PGM s p e c i f i c data'for  activity  both the  tidal  position,  on  p o r t i o n of  body w e i g h t  variables. tissue the  from t h e  data  m a n t l e and  o r i g i n a l l y a n a l y z e d i n each  independent  tissue  samples However,  effect  the- f a l l .  the  with  glycogen  winter  that  the  fall  glycogen  were  of  to  f o u n d t o have a m i n o r e f f e c t  explaining in  muscle  a 3-factor  and  the  in Chapter  adductor by  to  effect  the  respectively.  difference  determined for  a negligible  of  produced a s i g n i f i c a n t  u n d e r e s t i m a t e d by 3% c o m p a r e d t o had  fall,  standards  these angular transformed  P < .05),  to  glycogen  The e f f i c i e n c y  summer,  84.8%,  ANOVA s t i l l  the  consistency  study.  comparison of  one-way  tests  the  the  i n c l u d e d w i t h the  averaged  (F(2,95)  h y d r o l y s i s of  confirmed  throughout  standards  by  the  season class,  Body w e i g h t  glycogen  was  levels,  observed v a r i a t i o n only  fall  sample  (F(3,144)  =  150  5.99,  P  <  and t i s s u e PGM  .001).  i n w h i c h body w e i g h t  specific  between  than  activity.  in  the  levels  smallest  of  this  measured i n a l l  detected weight  classes  classes.  body  glycogen  data;  the  activity  levels  association mantle, tissue  the  as  weight  was e x c l u d e d as  specific  Table  activity  Season on  and  tidal  glycogen  adductor muscle  tissues  produced  from C h a p t e r  position levels of  highly  muscles  range  tests two  specific  between  these  between  to  that  for  lower A  similar in  the  measured i n  this  were  not  minor i m p o r t a n c e ,  overall analysis.  with  the  the  enzyme  was e v i d e n t  classes  its  four  specific  seen  groups.  activities  results  body  This from  is the  3 for comparative purposes.  exerted  measured C.  possessed  adductor muscle  weight  together  data,  g r o u p and t h e  exhibited  i n the  observed  24.0-35.9  three  of  on  i n the  and glycogen  Because  g)  Multiple  this  opposite  between  XI  g.  relationship  specific  a factor  data  the  other  body w e i g h t  effect  adductor  from t h o s e  oysters  the  the  their  season  glycogen  (12.0-23.9  between  was  smallest  with  the  differences  the  significant.  in  For  class  but n o t  similar  differences  statistically  presented  g,  only  r e l a t i o n s h i p was  24.0  difference  the  a significant  a n i m a l s above  than  between  but  weight  However,  and  factors  inverse  comparison of  revealed  activity  effects  exerted  in  36.0  Statistical  activities weight  above  was a l s o  carbohydrate  a significant  g range.  weight  An  this  t h e s e v a r i a b l e s and body s i z e .  oysters higher  Interestingly,  gigas.  significant  highly  significant  in  b o t h t h e m a n t l e and  In  combination,  season-by-tidal  these height  151  Table X I . F - r a t i o s from a n a l y s e s of c o n c e n t r a t i o n s and PGM s p e c i f i c and a d d u c t o r m u s c l e t i s s u e s .  v a r i a n c e on g l y c o g e n a c t i v i t i e s i n the mantle  152  Glycogen  Content  Specific  Activity  Adductor Muscle  Mantle  Adductor Muscle  1  Source of Variation  df  Mantle  Season  2  128.2***  449.7***  39.3***  Tidal Height  1  88.2***  166.2***  2.52  5.56*  Genotype  6  1.13  1.38  11.9***  21.9***  Genotype x Season  2  3.52***  1.70  1.34  1.09  Genotype x Tidal Height  1 .55  1.15  0.54  0.84  Season x Tidal Height  17.4***  23.8***  13.7***  1.44  0.73  1 .20  448  456  456  Genotype x Tidal Height x Season  12  Error  * P < .05 * * * P < .001 from C h a p t e r 3 1  3.06***  451  111.9***  271.3***  153  interaction activity.  terms, Despite  differences First,  as  can  these  these  (as  repeated  of  but not  tissues.  genotype  the  but  were  tissues  produced  the  adductor muscle  results accounted  on  effects  adductor  muscle  the  of  of  observed specific  i n the  glycogen  effect  highly  in of  data.  results  data:  mantle  the  Pqm-2 not  significant height results,  Fourth,  for  in  was  mantle glycogen  activity  the  the  two  specific  genotype-by-environment  were  s e a s o n and t i d a l  13.  not  present  in  the  i n these  four-fold  Consequently,  > winter.  significantly  concentrations  to  tissues  POSITION  position  In e a c h s e a s o n ,  three-  muscle.  summer > f a l l  possessed  the  activity  Third,  identical  glycogen  consistently  displayed  highly  genotype-by-season-by-tidal  i n the  in Figure  adductor  a  tissue.  The  were  for  analyses.  observed  specific  data.  important  these  in  EFFECTS OF SEASON AND INTERTIDAL  graphically  of  highly significant  glycogen  observed  on PGM s p e c i f i c  several  variation  factor)  nearly  b u t not  interactions  the  from t h e  effects  enzyme a c t i v i t i e s ,  and  terms were  absent  activity  the  glycogen  genotype-by-season, interaction  alone the  Second,  a separate  in  their  similarities,  position  amount  concentrations,  for  be s e e n between t h e  intertidal  significant  observed  were  are  m a n t l e and presented  mantle glycogen  greater the  seasonal  i n the  quantities  levels  than o b s e r v e d  similar, exhibiting  O y s t e r s lower greater  on t h e  patterns rank  orders  intertidal of  in  glycogen  zone than  154  Figure  13. S e a s o n a l v a r i a t i o n i n t h e m a n t l e and a d d u c t o r m u s c l e g l y c o g e n c o n c e n t r a t i o n s (jumoles g l u c o s y l u n i t s / g t i s s u e ) a t t h e two i n t e r t i d a l p o s i t i o n s . Open c i r c l e s = l o w water; c l o s e d c i r c l e s = h i g h water. Mantle sample s i z e s : summer, low=61 and h i g h = 5 7 ; f a l l , low=96 and h i g h = l 0 0 ; w i n t e r , low=88 and h i g h = 9 l . M u s c l e sample s i z e s : summer, low=58 and h i g h = 5 7 ; f a l l and w i n t e r a r e t h e same a s t h e m a n t l e . B a r s r e p r e s e n t ±1 s t a n d a r d e r r o r where v i s i b l e o r f a l l w i t h i n t h e p l o t t e d s y m b o l .  A. Mantle  B. Adductor Muscle  156  animals  situated  relationship accounting  i n the  held for  in both t i s s u e s  the  significant  T a b l e X I . As e v i d e n t season-by-tidal were p r o d u c e d glycogen in  the  from  height by  levels  i n the  more  comparable  differences  the  from  all  three  thus  of  13,  tidal  the  at  fall  the  position  the  fall  21,  area  Here,  In  intertidal. and w i n t e r  oysters  low  the  summer,  tissue  positions  the  summer,  glycogen zone,  than  whereas  were 25 and 22%,  were  observed  i n the  and 14% more g l y c o g e n in  in  intertidal  and w i n t e r .  in  i n T a b l e XI  difference two  seen  significant  terms o b s e r v e d  high  i n the  tissue.  p o s s e s s e d 92, intertidal  seasons,  highly  Even more d r a m a t i c d i f f e r e n c e s  adductor muscle  high  This  i n t e r t i d a l had 53% more m a n t l e  sampled  region  i n t e r t i d a l area.  pronounced  between o y s t e r s  individuals  respectively.  high  effect  interaction  the  low  in  Figure  summer compared t o  oysters  the  more e x p o s e d ,  than  fall,  in  intertidal  those and  in  the  winter,  respectively.  The height  source on  of  tissue  interaction  specific  activity  the  shifts heights the  in d i f f e r e n t  low  water  activities winter  this  than  between  levels  data,  had  In t h e  differs  these  in oysters  the  reversed.  The  the  on  PGM  in Chapter  at  was c a u s e d the  summer and f a l l ,  two  site,  nature  3. by  tidal  oysters  greater  h i g h water  tidal  from  factors  interaction  significantly  i n d i v i d u a l s at was  the  measured  seasons.  station  pattern  s e a s o n and  i n T a b l e XI and d i s c u s s e d  activity  enzyme  between  concentrations  observed  shown  specific  i n the  interaction  glycogen  similar  For  this  at  specific but of  in  the  these  157  interactions glycogen causal  glycogen at  the  between t h e  fall  by  and w i n t e r  were  suggest  is  not  the  in  a  not  these  for  the  concentrations  seasons.  these  the  summer  the  same  additive of  the  tidal  positions  the  glycogen  in  These  i n PGM a c t i v i t y height  over  reversals  these  reflected  season-by-tidal of  at  the  strictly the  the  non-additive  However, a  in  into  Between  markedly  in  oysters  variation  responsible glycogen  in  position  insights  In a d d i t i o n ,  exhibited  measured  it  offer  increased  heights.  concentrations that  changed  activity  these t i d a l  activities  data  intertidal positions.  PGM s p e c i f i c  manner a t  -intertidal  between t h e s e m e a s u r e s .  levels  two  and  activity  relationship  fashion  PGM  season  and s p e c i f i c  and f a l l ,  period  between  discrepancies  per  se  that  interactions  seen  is in  tissues.  EFFECT OF PGM-2 GENOTYPE  The m a j o r f i n d i n g overdominance Crassostrea  for gigas.  heterozygotes  of  Chapter  enzyme The  3  activity  at  Pgm-2-92/100,  possessed greater 96/96,  homozygotes  and h e t e r o z y g o t e s  behaved  exhibiting  similar  in  in  the  allowed  and  patterns  in heterozygotes  mantle  comparisons  i n PGM a c t i v i t y  both  of  the  the the  expression Pgm-2  96/100,  as  locus  and  activities  104/104  exceeded  and a d d u c t o r m u s c l e ,  a priori  differences  100/100,  specific  -2-92/92,  activities  was  t h a n t h e Pqm  homogeneous  homozygotes  studied:  enzyme  by 24% and This  associations  between  glycogen  These groups,  respectively.  and t i s s u e  in  100/104  homozygotes.  tissues  of  20%  knowledge these  concentrations.  158  Consequently, genotypes three for  genotypic  the  these analyses  tissue.  and f o r  second  in  homozygote  detected order  enzyme  the  were  activities  14A),  summer  between  allelic  positions  (F(2,95)  significant present In t h e groups  significant  (F(2,172)  possessed  =  three  seasons. did  when  higher glycogen  highly  a  observed  significant  not  P  >  that  differences P  levels  will  than the  which  the  Pgm-2  across  tidal  mantle  (Figure  significantly the  two a  tidal highly  interaction  be d i s c u s s e d  existed  < .001).  genotype-  (However,  height  the Pgm-2  14,  of  differ  .30).  the  Figure  over  with  between  In t h e  averaged  P < .01)  12.0,  levels  pooled  class-by-tidal  = 6.31,  explain  associated  in  classes,  1.12,  allelic  (F(2,95) fall,  =  d i d not  concentrations  levels  classes  lost  classes.  g r o u p s . The s o u r c e o f  heterozygote  glycogen  less  information is  expressed  illustrated  the  three  Pgm-2 g e n o t y p e  directly  glycogen  of  heterozygotes  the  these  into  height  the  each  pooled  genotype-by-season-by-tidal  presents  in  Pqm-2  season,  is  heights,  mantle,  7  and  interaction  and  into  Pgm-2 g e n o t y p e  by-season  mean  for  little  between  and h e t e r o z y g o t e  homozygote  homozygotes,  be s e e n ,  in  interactions  differences  the  v a r i a t i o n in glycogen  However  were  Both  the  1)  same g e n o t y p e s  by p o o l i n g g e n o t y p e s  amount of  the  the  on  and h o m o z y g o t e s  As w i l l  interactions  term.  2)  overall analysis,  significant  performed  (Pgm-2-100/100  allele,  alleles).  either  and  groups  Pgm-2-100  In t h e  in  were  separately,  frequent in  analyses  between  The Pgm-2  was  later.) allelic  heterozygotes  two homozygote  groups,  159  Figure  14. S e a s o n a l v a r i a t i o n i n t h e m a n t l e and a d d u c t o r m u s c l e g l y c o g e n c o n c e n t r a t i o n s (Mmoles g l u c o s y l u n i t s / g t i s s u e ) of Pgm-2 homozygote a n d h e t e r o z y g o t e c l a s s e s . Open c i r c l e s = P q m - 2 - 1 0 0 / 1 0 0 h o m o z y g o t e s (summer.n=29, fall n=42 w i n t e r n = 3 8 ) . Closed circles=heterozygotes f o r the Pgm-2-100 a l l e l e (summer n = 7 l , f a l l n=123, w i n t e r n = l 0 8 ) . Open s q u a r e s = h o m o z y g o t e s f o r t h e P g m - 2 - 9 2 , 9 6 , and 104 a l l e l e s (summer n = l 8 , f a l l n = 3 l , w i n t e r n = 3 3 ) . B a r s r e p r e s e n t ±1 s t a n d a r d e r r o r where v i s i b l e o r f a l l w i t h i n the p l o t t e d symbol. f  \  A. Mantle  B. Adductor Muscle  161  but were only  f o u n d by m u l t i p l e from  in  Pgm-2-100/100  also  explained  mantle  glycogen  (F(2,155)  = 10.2,  that  i n the  seen  tests  the  classification variation  range  significantly  P < .001), fall.  more  significantly The  significant  the  situation  in was  homozygotes  glycogen  than  allelic  amount  concentrations  Pgm-2  mantle  differ  genotypes.  a  but  Both  to  of  the  the  winter  reversed now  the  from  displayed  thr^e  Pgm-2  heterozygotes.  Genotypes similarly height  pooled  i n both the  into fall  interactions  comparisons  of  relative  their  season.  to  In t h e  the  were  respective  in  5  of  their  mantle  respective  glycogen  heterozygotes significant  levels  levels  over  the  of  Pgm-2  6 comparisons. lower  homozygotes. observed  genotype-by-season  higher In a l l  unusual  between was  six  heterozygotes in  each  glycogen six  quantities  The  and w i n t e r  analyses,  available  displayed  possessed  fall  both  were  behaved  genotype-by-tidal  in  homozygotes  the  classes  Because  absent  heterozygotes  comparisons heterozygotes than  allelic  and w i n t e r .  glycogen  fall,  concentrations  these  winter  glycogen  reversals  homozygotes responsible  i n t e r a c t i o n term  seen  in and  for  in  the  Table  XI.  Seasonal concentrations The  changes of  significant  mantle  levels  was  in  these a l l e l i c  the  groups are  genotype-by-tidal not  repeated  adductor  for  height the  shown  muscle  in Figure  interaction  adductor  glycogen  muscle  14B.  i n summer tissue,  162  and,  i n both the  possessed  identical  significant the  winter  mantle  (F(2,155)  tissue  genotypes. in  the  their  these  for  the  As  classes  in the  was  for  The  glycogen  in  for  classes  the  the  possessed but  Pgm-2-100/100 at  i n 5 of  both  the  6  tidal  possible  glycogen  differentiation  levels between  was m a r g i n a l l y below  genotype-by-season data  classes  seen  patterns  that  winter  to produce a s i g n i f i c a n t adductor muscle  allelic  d i s p l a y e d lower  homozygotes.  However,  than h e t e r o z y g o t e s ,  only  revealed  heterozygotes  glycogen.  between  genotypic  Pgm-2 h e t e r o z y g o t e s  allelic  required  winter  and  b o t h homozygote  significant  respective  muscle  adductor muscles  An e x a m i n a t i o n of  comparisons, than  was  of  P < .001).  season,  in their  homozygotes  were p r e s e n t  = 8.13,  in this  difference  heights  quantities  differences  more g l y c o g e n the  summer and f a l l ,  (F(6,448)  that  interaction  =  1.70,  P  <  .10).  The m a n t l e  glycogen  heterozygotes  are  positions  Table  in  significant Significant  intertidal  site  glycogen  to  i n the  than the  heterozygotes  were  in a l l  intertidal  homozygote  XII  differences heights  low  presented  of  for  both  illustrate  the  source  height  o b s e r v e d between  seasons w i t h the  summer. In t h i s  of  possessed but  larger  At the  exception  classes of  these d i f f e r e n c e s  the  homozygotes  h i g h water  glycogen  the  interaction.  allelic  season,  and  intertidal  zone d i s p l a y e d h i g h e r q u a n t i t i e s  heterozygotes.  groups,  Pgm-2 homozygotes  separately  genotype-by-season-by-tidal  both t i d a l  the  concentrations  site  of  at  high in  mantle  however,  levels  than  were  significant  not  both  163  Table X I I . Combined e f f e c t s o f s e a s o n and i n t e r t i d a l p o s i t i o n on t h e m a n t l e g l y c o g e n c o n c e n t r a t i o n s (/xmoles g l u c o s y l u n i t s / g t i s s u e ) o f Pgm-2 homozygote and h e t e r o z y g o t e classes.  Glycogen C o n c e n t r a t i o n ' Season  Summer  Fall  Winter  Tidal Position  N  Low  15  High  N  Heterozygotes f o r 100  N  Homozygotes without 100  396.9±19.S  38  301.4±12.3  8  375.4+26.8  F(2.49) = 8.85***  14  189.1±24.3  33  236.5115.3  10  199.3±27.6  F(2.47) = 1.62  Low  22  171.4+16.0  60  220.5+9.7  14  195.2±20.0  F(2,84) = 3.76*  High  20  122.0±12.0  63  1B3.1+6.7  17  148.7+13.1  F(2,88) = 10.9***  Low  19  179.6+13.0  50  157.7+8.0  19  192.8+13.0  F(2.76) = 3.11*  High  19  174.2±10.4  58  124.2±5.9  14  155.7±12.2  F(2,79) = 9.78***  pmoles g l u c o s y l * P < .05 *** P < .001 1  Homozygotes f o r 100  u n i t s / g wet t i s s u e  165  (F(2,47)  =  1.62,  differences similar the  >  expressed  a t both  tidal  difference  high  P  .20). In between  greater  second-order seasonal  mantle  differ  genotypic  classes  winter  classes  the were  summer,  was more e x t r e m e a t t h e  the s e a s o n - b y - t i d a l  interaction  fluctuations  levels  at  these  of glycogen  fashion, differing  summer,  fall,  and w i n t e r ,  second-order  differences  these by  between  tidal  reversal  between t h e s e  responsible  by o n l y  positions  f o r the  27,  respectively. was  Pgm-2 in  significant these  averaged  in  and  27%  i n the  component  entirely  by t h e  The d i f f e r e n t i a l  and  heterozygotes with  the net  a n d w i n t e r , was t h u s  genotype-by-season-by-tidal  c o n c e n t r a t i o n s o f homozygotes when  and w i n t e r ,  The g e n o t y p i c  the f a l l  found  i n a l i n e a r and  t h e summer, combined  glycogen  identical  20,  homozygotes  groups over  Despite  fall,  were  concentrations  classes.  interaction.  positions.  positions  caused  allelic  of the  o f t h e two homozygote  glycogen  height  nearly  consequence  heights declined  interaction  between  displayed  mantle  component  concentrations i n the  t h e average  tidal  parallel  patterns  a  a t t h e two i n t e r t i d a l  In c o n t r a s t ,  heterozygotes  this  was  height  by 101, 35, a n d 12% i n t h e summer,  respectively.  of  and  but i n c o n t r a s t t o the  between a l l e l i c  Pgm-2 h o m o z y g o t e s . T a k i n g  groups, to  fall  intertidal.  this  four  these  positions,  T a b l e X I I shows t h a t of  the  over  differences, and  the  heterozygotes  a l l seasons  mantle were  and t i d a l  166  Table XIII glycogen two  levels  presents of  intertidal  between  (F(2,79) average was  =  genotypic  7.58,  evident  8%)  1.51,  allelic  at  and  thus  low  Because  slightly  In activity  of  it  was e x p r e s s e d  was only  were  96 a n d 104 a l l e l e s  significantly  indistinguishable  lower  = 1.38,  shown t h a t  specific activity  activities  are  measurements,  trend this  heterozygotes (F(2,76)  3.  =  homozygotes heterozygotes  Pgm-2 these  genotypes differences  overdominance for possessing  between  the  less  enzyme  the  most  frequent  activities  that  overdominant heterozygotes  presented,  from C h a p t e r  similar  on  0.20).  from b o t h homozygote c l a s s e s .  classes  possessing  than  displayed specific  mantle and a d d u c t o r muscle g l y c o g e n genotypic  P >  in heterozygotes  than the  area  results,  but s i n c e  Heterozygotes  intertidal  significant  summer and f a l l ,  Chapter  were  A  levels  in  differences  exceeding  glycogen  (F(6,448)  were  but the magnitude of  winter  seasons,  the  position  homozygotes  site  these  significant  3  tidal  high  statistically  three  common Pgm-2-100 a l l e l e . Pgm-2-92,  the  w i t h the  i n d i s t i n g u i s h a b l e i n the  were n o t  either  at  differences  significant  in  water  higher the  heterozygotes  than h e t e r o z y g o t e s .  was n o t  when a v e r a g e d o v e r were  groups  i n adductor muscle  significant  winter,  P < .001),  the  and  was d i m i n i s h e d (homozygotes  P > .20).  exhibited  No  changes  c l a s s e s at  In t h e  23% more g l y c o g e n  difference by  Pgm-2 h o m o z y g o t e s  summer o r f a l l .  present  seasonal  locations.  o b s e r v e d between the  the  In T a b l e X I V t h e  concentrations together As f o u n d  and  with for  of  these their  the  a c l e a r d i s t i n c t i o n was o b s e r v e d  same Pgm-2  specific between  167  Table XIII. Combined e f f e c t s of s e a s o n and i n t e r t i d a l p o s i t i o n on t h e a d d u c t o r m u s c l e g l y c o g e n c o n c e n t r a t i o n s (nmoles g l u c o s y l u n i t s / g t i s s u e ) of Pgm-2 homozygote and heterozygote classes.  Glycogen C o n c e n t r a t i o n ' Season  Summer  Fal 1  Winter  Tidal Position  N  Low  15  119.0±9.1  35  127.6+6.O  8  141 .1±12.5  F(2.46) = 1.16  High  14  60.6+6.1  33  66.3+3.8  10  74.316.9  F(2.47) = 0.95  Low  22  47.7+2.9  60  50.6+1.7  14  51.013.6  F(2.84) = 0.62  High  20  39.012.4  63  40.811.4  17  46.412.6  F(2.88) = 2.41  Low  19  44.212.0  50  40.411.2  19  43.412.0  F(2.76) = 1.51  High  19  43.312.2  58  34.0+1.2  14  39.912.6  F(2.79) = 7.58***  ' pmoles g l u c o s y l *** P < .001  Homozygotes f o r 100  u n i t s / g wet t i s s u e  Heterozygotes f o r 100  Homozygotes without 100  169  Table XIV. G l y c o g e n c o n c e n t r a t i o n s (Mmoles g l u c o s y l u n i t s / g t i s s u e ) and s p e c i f i c a c t i v i t i e s ( u n i t s / m g p r o t e i n ) o f h o m o z y g o t e s and h e t e r o z y g o t e s p o s s e s s i n g o r l a c k i n g t h e Pgm-2-100 a l l e l e .  170  Adductor  Mant1e  Muscle  N  Specific ' Activity  Glycogen' Content  Specific ' Activity  Homozygotes for 100 a l l e l e  42  0.109±.005  147.9110.0  0.2061.006  43.511.8  Homozygotes wlthout 100 a l l e l e  31  0.1171.005  169.7111.6  0.2111.007  - 48.512.1  Heterozygotes for 100 a l l e l e  123  0.1291.003  201.315.8  0.2361.003  45.6+1.1  Heterozygotes wlthout 100 a l l e l e  33  0.1051.005  148.7111.3  0.1981.006  45.112.1  Genotypic Class  F(3,197)  1  7.76***  10.5*  units/mg p r o t e i n ' from Chapter 3 ' pinoles g l u c o s y l u n i t s / g wet t i s s u e *** P < .001 1  1  12.9***  Glycogen' Content  1.21  171  these  heterozygote  Pgm-2-92/96,  92/104,  concentrations other  groups  and 96/104 h e t e r o z y g o t e s  that  were  heterozygote  homozygote  groups.  existed  between  classes  and t h e i r  concentrations despite  in t h e i r mantle glycogen  again  class  and  In t h i s the  tissue  specific  glycogen  did  not  those observed  in the  a strong p o s i t i v e activities  differ in  from  of  Adductor  The  glycogen  lower  different  stores.  showing d i f f e r e n c e s  exhibited  significantly not  levels.  than the  the two  relationship  these  genotypic  muscle  glycogen  between  these  genotypic  enzyme  activities  groups,  similar  to  mantle.  DISCUSSION  The  physiological  relevance  of  naturally occurring  enzyme  p o l y m o r p h i s m s can o n l y be a s s e s s e d t h r o u g h an e x a m i n a t i o n of effects  of  pathways  this  variation  i n which these  Crassostrea  gigas,  demonstration  of  metabolism. synthesis their  enzymes this  i m p a c t of  marine  1983).  changes  this  bivalves,  i n the  and s u b s e q u e n t  1986a).  function.  is  (Bayne  activities  Pgm-2 l o c u s  1981;  on  into  variation  Zandee e t  this  cycle  enzymes  these  in  Zaba  in glycogen  1981; levels  a  the  al.  to  1980;  involves  involved in  glycogen  in  glycogen  f u n c t i o n a l l y coupled  of of  biochemical  translates  1976;  m o b i l i z a t i o n of  changes  the  For the  seasonal  glycogen  (Livingstone  Because  of  polymorphism  One i m p o r t a n t component  gametogenesis  Whittle  output  requirement  and d e g r a d a t i o n of  term  storage for  the  annual reproductive c y c l e  Gabbott long  In  the  on  the  the  reserves  G a b b o t t and take  place  172  rather  slowly,  differing  enzyme  existing albeit  examination  glycogen  indirect,  intertidal with  activities  tissue  polymorphism.  of  The  important  seasonal  study for  are  in  British  in  mantle  one of  the  the  food  that  two  winter. levels  personal  tissues  primary  intertidal  glycogen  zone  provide of  content  found  with those this  storage  1969;  and  some  allozymic  glycogen  position  levels  winter  also  glycogen consistently  of  in  the  the  present  (Quayle  1969;  in glycogen  levels  for  et  this  al.  since  It  those  (P.  concentrations  for  t i m e of  levels:  a  displayed  could  year.  highly  oysters  natural  MacClelland,  glycogen  exerted  was  measured  sample was t a k e n when u n u s u a l l y low  the  carbohydrate  1982).  would exceed  this  mantle  reported previously  species  sites  Bayne  t h e i r mean l e v e l s  tissue  this  season  examined was e x p e c t e d  c o m m u n i c a t i o n ) , and t h u s  on  of  interactions  study  influence  C. gigas  i n Nanoose Bay were  Intertidal effect  of  However, the  have been below  of  their  The marked d i f f e r e n c e  (Eble  fall  and  their  an e v a l u a t i o n ,  effects ,  this  the  i n the  tissues  the  enables  and  the  flux.  1982).  in marine b i v a l v e s  in  in  C o l u m b i a p o p u l a t i o n s of  observed  unexpected  observed  between  genotypes  effects  levels  g e n e r a l agreement  Whyte and E n g l a r  is  Pgm-2  significant  changes  and a d d u c t o r m u s c l e  associations  physiological  concerning  on m e t a b o l i c  The  of  on g l y c o g e n  insights  variation  the  genotype  the  concentrations  highly  position  Pgm-2  of  from  significant the  significantly  low larger  173  quantities exposed  of  glycogen  high for  intertidal  zone  compared t o  animals  their  reserves. switch Mustafa  durations  would in  Zwaan  1983),  oysters  the  availability  of  extreme  at  tides  primary  to  of  mantle  were  this  these  in  the  for  demands  of  early  of  chapter  exposure  would  directly glycogen oysters  (Hochachka and  and p e r s i s t  limit  Hypoxic  for  longer  intertidal  the  zone.  metabolism  (de  reserves  capability  The i n t e r a c t i o n  could  high  aerial  on t h e s e e n e r g y  by  the  in of  observed  i n p a r t be e x p l a i n e d  caused  by  increased  summer c o u p l e d w i t h  the  more  year.  season,  intertidal  to  highly  exert  tissues.  on t i s s u e  data.  and  effects  measured  in  E x a m i n a t i o n of  glycogen  First,  position,  significant  showed a number of  activity  the  1980).  i n the  phosphoglucomutase  same f a c t o r s  enzyme  Zwaan  anaerobic  glycogen.  i n the  in  exposure  production de  effects  adductor muscle  present  from t h e  shown  aerial  higher  position  time  3,  activity and  of  and  fuel  store  these  food  Chapter  specific  energy  situated  increased  area  accentuation  genotype  of  more f r e q u e n t l y  s e a s o n and t i d a l  In  of  This  more  probably  and h e n c e a s s i m i l a t e  i n t e r t i d a l a r e a would f u r t h e r in this  between  the  the  feed  Holwerda  occur  is  intertidal.  of  the  are  oysters  periods  periods  pathways  individuals  glycogen  to  from  factors  First,  longer  low  during  Zandee,  Since  high  i n the  sampled  Two  observations.  opportunities  1972;  area.  experience  anaerobic  conditions  the  these  Second,  to  animals  intertidal  responsible  reduce  than  glycogen  levels  on  both the  concentrations important  Pgm-2 the the  effects reported  differences in  oysters  174  sampled  from  additive  fashion  PGM  the  activity  additive. net  winter  intertidal  between t h e  observed  Second,  ranking  two  had  no  significant  data.  These  muscle  despite  w i t h the  mantle.  observed  for  the  glycogen  This  in  and  is  equilibrium  vivo  is  to  transmit  dictated catalyze Start  repeated its  effects  a reversal the  fall  in and  glycogen  glycogen  data  interactions  specific  activity  in  adductor  the  enzyme a c t i v i t y suggest cannot  of  in  strictly  tissue  the  activity  is not  these  data  that  be  the  directly  factors  (e.g.  flux  reactions  entirely  to  a freely  a t t r i b u t e d to the  not  known  in other  by enzymes  1973;  glycogen,  PGM  catalyzes as  role  not  of  changes  on  concentrations.  marine b i v a l v e s ,  metabolic  mantle  analysis  significant  Phosphoglucomutase  ^n  the  these d i f f e r e n c e s  conclusion  properties  on  s i m i l a r i t y of  Together,  over  effect  of  were  great  variation  responsible tissue  the  exhibited  positions  in a non-  was  genotype-by-environment  i n the  interactions  seasons  activity  analysis  observed  these  apparent  produced h i g h l y not  when t h e  tidal  Third,  were  summer and f a l l  PGM s p e c i f i c  concentrations.  that  shifted  between  between t h e s e  that  heights  possess  reversible  organisms, Ebberink  these p i v o t a l  regulatory  reaction  far  that  to  and  Zwaan 1 9 8 0 ) . The  de  operate  through  biochemical flux  near  l i k e PGM pathways  capacities  that  from e q u i l i b r i u m (Newsholme  F o r the  reactions  synthesis are  in  appears  w i t h c o m p a r a t i v e l y low  A t k i n s o n 1977).  any  near-equilibrium reactions  rates  displaced  unexpected.  and d e g r a d a t i o n  catalyzed  by  and of  glycogen  175  synthetase  and  mammalian  glycogen  tissues,  coordinately  the  regulated  phosphorylase,  activities  i n an a n t a g o n i s t i c  phosphorylation/dephosphorylation kinases Cohen  and  1986)  general  phosphatases i n response  (see  Stalmans  1976;  regulation glycogen  of  glycogen  glycogen  Although bivalves  regulatory  synthetase Whittle  not  the  exhibits  In  Gabbott  parallels  glycogenolytic  the  and is  the  extensively  Whittle  throughout the  direction,  both glycogen  Zaba  of  about  the  synthetase  and  (1981)  marine  serve  similar  the  form  blue of  mussel  glycogen  ( C o o k , G a b b o t t and and and  its  steady  early  summer  proceeding  observed  p h o s p h o r y l a s e and  that  in the  amyloglucosidase  was b e i n g  These o b s e r v a t i o n s , the  in  glycogen  For flux  t i m e when g l y c o g e n  gametogenesis.  near-equilibrium status  deposition.  1980;  studied,  1986a), spring  different  respectively.  of  variation  and  "flux-generating"  undoubtedly tissue  1986;  Whitton  metabolism  dephosphorylated  glycogen  specific  Roach  known  classic  are  hormones,  glycogen  the mantle  and  i n c r e a s e d over a p e r i o d of for  what  marked s e a s o n a l  directly  degraded  Hems  glycogen  as  active  in a c t i v i t y  of  From  phosphorylase  elevation  activities  1983;  of  by  1986;  In  reversible  t h r o u g h a number of  represent  been  functions.  1979;  by  i n Madsen  metabolism,  control  and  edulis,  fashion  s y n t h e s i s and g l y c o g e n o l y s i s ,  the  has  synthetase  the  1976).  phosphorylase  s t e p s of  Mytilus  by Cohen  Hers  enzymes  neuronal s t i m u l a t i o n ,  condition  reviews  these  events c a t a l y z e d  (reviewed  to  physiological  effectors  of  respectively.  rapidly  together  phosphoglucomutase  with  reaction,  176  would  predict  that  Pqm-2 h e t e r o z y g o t e s if  any,  this  the  marginally greater  relative  i m p a c t on t i s s u e  t o homozygotes  glycogen  the  results  of  p r e d i c t i o n for  the  mantle  but  C l a s s i f i c a t i o n of  or h e t e r o z y g o t e s variation  in  comparisons  shown  glycogen  a d d u c t o r m u s c l e was weak,  It  activity  o n l y one of  seems r e a s o n a b l e  annual the  cycle  of  system. however,  t o assume  the the  influenced  that  glycogen  combined  activity  rates  d i c t a t e d by t h e s e  impact  of  reversals  between  (i.e.  and w i n t e r )  seasonal limited  of  these a c t i v i t y  fall  season  the  of  the  of  concentrations a  comparisons  significant (Table X I I I ) .  control  is  eight  association  for  overall  observed  of  of  the  mediated through  activities  of  by t h e s e  glycogen  regulatory appears  enzymes, to  genotypes  to  was  the  respond to  the  complex,  and h e t e r o z y g o t e s  The a p p a r e n t exhibiting  between  intertidal positions  Since  glycogen  was  its  annual c y c l e ,  it  have of  r e g u l a t o r y enzymes.  and between  stages  the  homozygotes  the  seven  muscle  T h i s may be a c o n s e q u e n c e  Pgm-2  homozygotes  contradict  phosphorylase/amyloglucosidase  differences  (summer).  number of  in  variation s t i l l  metabolism.  abilities  flux  the  of  either  amount of  and d e g r a d a t i o n  differential of  as  accounting  c o n t r o l exerted  PGM s p e c i f i c  little,  adductor  and g l y c o g e n  same e i g h t  regulation  and  Despite  the  synthesis  differential  synthetase  differences  study  the  i n T a b l e X I I . In c o n t r a s t ,  in  in  not  levels  specific  result  present  Pgm-2 g e n o t y p e s  these the  the  explained a significant  mantle  s h o u l d have  of  concentrations.  Surprisingly,  tissue.  enzyme a c t i v i t i e s  seasons within a  measured is  not  at  a  possible  177  to  determine  from  differentially  the  affects  if  glycogen  or  represents  Of  these a l t e r n a t i v e s ,  enzyme  outset  the  synthesis  a fortuitous association  activity  on  a direct the  rate  through a p a r t i t i o n i n g e f f e c t  v a r i a t i o n i n PGM a c t i v i t y and/or  through a linkage  influence of  at  of  glycogen the  glycogenolysis,  this  effect.  variation  synthesis  is  in  expected  glucose-6-phosphate  branch  point.  Net glycogen  flux  formation is  forward equation  Vnet  through  and  the  d e t e r m i n e d by t h e  reverse  (Savageau  PGM r e a c t i o n  reaction  i n the  opposing  directions  by  [G-6-P]  1  +  -  VmaxCr)/Km(r)  [G-6-P]  and Km(f) and Km(r) a r e  velocities  constants  in  the  Michaelis  directions,  6-phosphate,  and  respectively. tissue  heterozygotes  for  XV.  velocities  Maximum  the  mean enzyme a c t i v i t i e s  for  for  the  following  [G-1-P]  [G-6-P]  glucose-1-phosphate  the  allele  the  these  four are  reverse  these genotypic  the  ..(4)  maximum  f o r w a r d and  and  E s t i m a t e s of  Pgm-2-100  of  for  [G-1-P]  c o n c e n t r a t i o n s of  mantle  the  Km(r)  where V m a x ( f ) and V m a x ( r ) ,  intracellular  the  of  + [G-1-P]  Km(f)  reaction  rates  1976):  = VmaxCf)/Km(f)  and  d i r e c t i o n of  reverse  are and  kinetic  the  glucose-  parameters  homozygotes presented  and in  the Table  reaction represent classes  from  the  Chapter  Table  XV. K i n e t i c p a r a m e t e r s i n t h e m a n t l e t i s s u e s of t h e f o u r h o m o z y g o t e s and t h e t h r e e h e t e r o z y g o t e s f o r t h e Pgm-2-100  i  179  Parameter  Vmax(r)  2.47  21 .2  21.5  1.01  0.823  121.9  123.2  Vmax(r)/Km(r)  0.142  0.115  Vmax(f)/Km(f)  0.00829  0.00668  Km(f)  3  1  2  Vmax(f)  1  Homozygotes  3.02  Km(r)  2  Heterozygotes  1  3  u n i t s / g wet t i s s u e Mmoles g l u c o s e - 1 - p h o s p h a t e Mmoles g l u c o s e - 6 - p h o s p h a t e  180  3.  Similarly,  represent  the  temperature parameters from t h e (as  for  written  5 to  forward  above)  expected  to  involve  1976).  assuming of  for  e q u i l i b r i u m Vnet of  that  than  It  might  be  in heterozygotes  simultaneously conditions, numerical,  expected  reverse  negating  solution  of  any  equation  advantage  of  irrespective and for  [G-1-P]  heterozygotes the  magnitude  are  300  and  estimated  for  relationship proceeding results  are  homozygotes  i n the  these  the  two  substrates  and  Crabtree reaction  advantage.  the  the  flux  if  [G-6-P]  calculated 24%  Vnet  larger An  holds  than  identical for  Surprisingly,  concentrations,  of  conditions  For example,  direction.  However,  that these  groups  the  concentrations  V n e t shows  is  by  flux-generating  Mmoles/min/ml).  by s u b s t r a t e  glycogen  forward  under  under  thus  directions  counterbalanced  4 with various  1964b;  are  towards  larger  catalytic  genotypic  glycogenolytic  unaffected  the  Vnet.  Vmax(f)  homozygotes.  a flux  umoles/min/ml) (0.219  reaction four  10 MM, r e s p e c t i v e l y ,  (0.271  between  of  estimated  allele  (Newsholme  persists  of  heterozygotes  the  rate  G - 1 - P and G - 6 - P p r o d u c i n g a p o s i t i v e  were  the  Kinetic  Vmax(r) exceeds  both  would be e x a c t l y  greater  thus  that  over  i n C h a p t e r 2.  or both of  equilibrium concentrations  reaction  (Ray and R o s c e l l i  zero,  one  groups  Pgm-2-100  for  is  reverse  direction  the  24% l a r g e r  a displacement  the  these  three  p o s s e s s Vmax/Km r a t i o s  at  from t h e i r  by  reaction  by a f a c t o r  are approximately  for  30° presented  Heterozygotes  Since  rate  of  the  constants  expressed  Haldane e q u a t i o n ,  2).  must  means  range  Chapter  that  Michaelis  flux these  although  the  181  realized  Vnet  displacement  from  The f l u x expressed an  is  vivo  intermediate  from  synthesis  of  of  has  the  of  to  in  the the  operates  pathway  this  mechanism both  glucose,  mechanism w i l l  G - 6 - P between  metabolite. (by n e t  pull  for  maintained partitioning is is  the  into  of  is  periods  u n a v o i d a b l e . An e s t i m a t e flux  point.  The  by t h e  injection  reductions  in  of the  glucose-6-phosphate  Hue and H e r s  1974).  In  also  displacements  the  PGM When  create and  pathway  UDP-glucose  driven  create  by  enzymes the  basal  that  for  the  utilize  this  G - 6 - P branch isomerase  and g l y c o g e n  the  of  on m a n t l e  this  tissue  (PGI)  must  formation,  glycolysis  point  from G - 6 - P  metabolism  glycogen  extent  exogenous  competition  fructose-6-phosphate  Since  studies  of  must  of  of  synthesis  is  mechanism. A c t i v a t i o n  phosphoglucose  f o r m a t i o n of  G - 6 - P between  p r o v i d e d by t h e  branch  PGM  of  steps.  glycolysis.  during  be  concentrations  PGM and o t h e r  activity criteria)  entry  1967;  The p r e d o m i n a n t c o m p e t i t o r a t  which c a t a l y z e s  in the  significant  and H e r s the  at  not  However,  intermediates  reaction  this  step  by a " p u l l "  pyrophosphorylase  available  first  i n mammalian l i v e r  (De Wulf  equilibrium  pathway.  glucose-6-phosphate  the  might  enzyme c a t a l y z e d a r e a c t i o n o c c u p y i n g  lowering  intermediates, from  heterozygotes  been o b s e r v e d t o c a u s e  UDP-glucose  addition  the  synthetase  concentrations and  Pgm-2  catalyzing  glycogen  glycogen  glucose  if  of  position  for  glycogen  their  equilibrium.  advantage  ijn  responsible  d i r e c t l y d e t e r m i n e d by t h e m a g n i t u d e of  be  some  synthesis  partitioning slices  from M .  182  edulis  by Zaba and D a v i e s  Davies  (1981).  These  glucose  label  appeared  in organic  was  studied  LaPorte,  and amino  of  into  flux  at  by K a c s e r  other.  will  c a u s e an  competing  enzymes can p r o d u c e n e g a t i v e of  This  theory  hyper-sensitivity  to  parameters  specific  Koshland branch PC,  of  the  1984). point  defined  remainder  of  points  flux  control  has  by t h e  for  increased  decline  in  flux  between  coefficients,  1983),  involved at  an  relationship  depending  enzymes  may be q u a n t i f i e d  by L a P o r t e ,  [14C]-  and e m p i r i c a l l y by  identical  (Kacser  regulation  The d i v i s i o n  the  branch  (1983)  antagonistic  control  the  and  When two enzymes compete  the  language  glycogen;  under s t e a d y - s t a t e c o n d i t i o n s ,  t h r o u g h one  Gabbott  40-60% of  metabolic  through  the  Zaba,  acids.  W a l s h and K o s h l a n d ( 1 9 8 4 ) .  flux  and  found that  incorporated  theoretically  a common s u b s t r a t e rate  1981)  experiments  The p a r t i t i o n i n g of been  (1980,  on  or u l t r a the  in or  kinetic  ( L a P o r t e , W a l s h and  the  glucose-6-phosphate  partitioning  coefficient,  W a l s h and K o s h l a n d ( 1 9 8 4 ) : n -1  PC =  where  PC q u a n t i f i e s  glycogen,  Vmax(l)  parameters  for  Vmax(2)  and  intracellular  the Km(2)  Vmax(2)  (Km(1)  + [G-6-P])  Vmaxd)  (Km(2)  +  the  proportion  and  Km(1)  forward are  concentration  of  of  flux  represent  same  (5)  [G-6-P])  reaction  the  + 1  for  directed  the  (to  towards  standard  glycogen)  kinetic of  P G I , and [ G - 6 - P ]  glucose-6-phosphate.  PGM, is  the  Inspection  183  of  equation  unaffected  5 by  Furthermore, species  is  Chapter  shows the  since  very 2  that  partitioning  concentration the  Km f o r  similar  (Martin  approximated  the  to  G - 6 - P of  that  1979;  Hall  PGI  Specific  the  point  occurring  for  1985),  C.  bivalve  qigas  equation  5  PGM  in  may  be  Activity + 1  PGM S p e c i f i c  branch  glucose-6-phosphate.  PGI from s e v e r a l  estimated  is  as:  PC =  Therefore,  of  coefficient  division when  is  of  both  largely  Activity  flux  at  glycolysis  determined  . . . (6)  the  glucose-6-phosphate  and g l y c o g e n  by t h e  synthesis  PGI/PGM s p e c i f i c  are  activity  ratio.  The  expected  flux  towards  difference  between  function  of  the  15.  equal a c t i v i t i e s  flux  At is  larger  increases, declines  activities  only  their  of  12 PGI  glycogen,  As of  advantage  net  to  the  of  illustrated  the  PGM was  enzymes,  however,  ratio  7.4  and 8.4  the  flux  for  despite  their ratio  glycogen  asymptotically  In f r e s h l y of  a  50% of  towards  heterozygotes  as  Figure  activity  proceeding  mean  in  the  PGI/PGM  advantage.  flux  homozygotes  t h a n homozygotes  flux  catalytic  oysters,  is  resulting  and  these competing  ratios.  flux  ratio  11% l a r g e r  proportion  and t h e  from  of  towards  Vmax/Km the  approaches tissues  is  and t h e  heterozygotes  PGI/PGM a c t i v i t y  directed  heterozygotes 24%  Pgm-2  glycogen  the  i n the  ground specific  mantle  and  184  Figure  15. P r e d i c t e d f l u x a d v a n t a g e o f Pgm-2 h e t e r o z y g o t e s and p e r c e n t f l u x t o g l y c o g e n a s a f u n c t i o n of t h e PGI/PGM activity ratio. Open c i r c l e s = p e r c e n t f l u x e x c e s s of h e t e r o z y g o t e s over homozygotes; c l o s e d c i r c l e s = p e r c e n t flux to glycogen.  Heterozygote/Homozygote Flux, %  Percent Flux to Glycogen  186  adductor muscle, reported edulis  i n the  are  Pgm-2  at  the  hold  synthesis  flux  advantage  The o v e r a l l  rate  availability  of  However, PGM  a displacement  is  differences  in  homozygotes  by  in  1986).  to  larger  are expected  of  the  The  i m p a c t of  less  certain.  rates  this  catabolic  could  partitioning  These  patterns  overall  rate  of  be  precursors  be m a i n t a i n e d .  determined the  by  crucial  decarboxylase  to  glycogen  is  still the  net  required flux  levels  observed be  at  i n Pqm-2  homozygotes.  part  and  synthetase.  exceed  in  the  The  between  explained  by  synthesis.  factors  process  magnitude  synthetase.  pyruvate  PGM a c t i v i t y  Three  this  precursor involved,  glycogen  of  in  could also  to  scallop,  exhibit  from g l u c o n e o g e n i c  expected  mantle  of  branch p o i n t .  from e q u i l i b r i u m  still  bay  similar  because  addition  and h e t e r o z y g o t e s  these d i f f e r e n t i a l  PGM on t h i s  Ellington  and u n d e r t h e s e c o n d i t i o n s  heterozygotes  is  catalyzed  bisphosphatase  step  and  would now  specific  Mytilus  (Chih  heterozygotes  synthesis  the  reactions  fructose  the  of  of  mussel,  those  the  irrespective  synthesized  the  s i m i l a r to  and  d i c t a t e d by g l y c o g e n  is  are  1980)  excess,  glucose-6-phosphate to  of  than homozygotes,  activity  When g l y c o g e n  bypass  Zwaan  heterozygotes  enzyme  expected  glycogen  the  de  towards glycogen  their  effect  and  ratios  tissues  irradians concentricus  Therefore,  to  These  adductor muscle  (Ebberink  Argopecten  fluxes  respectively.  are  v a r i a t i o n on  suggesting 1)  the  glycogenolysis  a limited effect  reaction  mechanism  of of  187  glycogen  phosphorylase,  mobilization, pathway. differs  and 3)  The  i n the  of  the  high  relative  ensure  that  glycogen  the  the  reaction  variations affect  the  accordance glycogen  their  (Cohen  expected  Several  to  experiments  prediction. mass a c t i o n  of  to  reversible  proceed  inorganic  almost  breakdown) phosphate  compared to  moieties by  the  and  present  to  PGM  PGM w o u l d  liberated into  excess  uni-directional  between  that  from  glucose-6-  catalytic  suggests  and  flow  the  genotypes  at of  minor  would  not  rate.  a is of  requirements  controlling  the  cascade  for  the  in  (MAR)  for  14  in  rapid mobilization  regulatory  have  reactions,  borne  Zwaan (1980)  glycolytic  enzymes  of  position  i m p a c t on g l y c o g e n o l y t i c  and de  cell  architecture  an i n t e r m e d i a t e  with marine b i v a l v e s Ebberink  activation  known, and d i f f e r e n t  and a d d i t i o n a l a limited  the  well  Situated  For example, ratios  phosphorylase  T h i s h i g h molar : r a t i o  favors  glycogen  1978).  have  glycogen  degradative  glycogen  The l a r g e  r e g u l a t o r y cascade  between p h o s p h o r y l a s e is  glycolysis.  modifications  with  (i.e.  converted  phosphorylase  exhibit  believed  phosphorylase  strongly  degradative  glycogen  types  into  activity  The e l e g a n t of  1976).  from  in  is  (Stalmans  rapidly  and fed  units  glycogen  glucose-1-phosphate  phosphate  glucosyl  yet  ratio  of  of  in being p o t e n t i a l l y  molar  activity  w o u l d be  PGM  synthetase  aspects  an a l t e r n a t i v e  by  forward d i r e c t i o n  glucose-1-phosphate low  of  catalyzed  and Madsen 1 9 8 0 ) ,  exclusively because  regulatory  existence  reaction  from g l y c o g e n  (Fletterick  the  the  2)  out  PGM rate. this  monitored in  the  188  posterior  adductor  anaerobiosis  hours of  pyruvate for  valve  kinase  the  PGM  aerial  as  substrates.  Since  the  similar  to  G - 6 - P w o u l d not anoxic  In also  This  converted  into  of  second  turn  the  first  control  to  ratio  throughout  concentrations in  of  the both  oysters  differential  interconversion  affect  the  The Keq/MAR  unity  of  glycogenolytic  phosphorolytic  by a m y l o g l u c o s i d a s e  could  be  exported  is  abilities G-1-P  rates  and under  route  i n marine  f o r many y e a r s  (e.g.  Alemany  glycogen  determined.  i n the  the  both  of  the  effect  The o b s e r v a t i o n mantle  from  the  vesicular  The  and  of of  is  or  has  However,  have  yet  glycosyl  to  units  essential  M. e d u l i s  the  hydrolytic  for  v a r i a t i o n on  membrane-enclosed of  cell  existence  and  PGM a c t i v i t y  cells  free  Rosell-Perez  1981).  p r o p o r t i o n of routes  may  invertebrates  i n marine b i v a l v e s  degradative  potential  produce  phosphorolytic  mobilization  Knowledge  through  glycogenolysis.  of  Zaba  glycogen  to  by h e x o k i n a s e .  Tazawa and Yasumasu 1978;  the  pathway,  degradative  of  particles  the  glucose-6-phosphate  pathways  assessing  to  in  glycolysis  in mussels,  the  contributions  channeled  of  to  identified  during  shift  i n the  catalyze  relative  be  They  prolonged anoxia.  regulation  to  in  recognized Hino,  a 24 h p e r i o d o f  conditions.  glucose.  1973;  a  increases  be h y d r o l y z e d d i r e c t l y  been  noted  close  that  over  exposure.  remained  be e x p e c t e d  addition  this  but  8 h of  large  M. e d u l i s  major c o n t r o l p o i n t  closure,  Pgm-2 g e n o t y p e s t o  similar  the  reaction  despite  of  by  after  experiment  probably  of  induced  phosphofructokinase few  muscle  glycogen  by Bayne  et  189  al.  (1982)  would  mechanism.  If  frequently,  the  an i m p o r t a n t r o l e  phosphorolytic  PGM  glycogenolysis function  suggest  that  must  exert  is  great  too  and r e g u l a t i o n of  glycogen  Although phosphoglucomutase glycogenolysis  under  d e s c r i b e d where  it  example,  Chih  contractions scallop,  could  not  some  the  posterior  adductor  sylvaticus gairdneri  25.  effect  Circumstantial mobilization  to  on  evidence  has a l s o (Leigh  Under the  175 t o  suggesting  been  Brown  noted  of  tissues  and  glycogenolysis. glycogen  is  proximity  to  the  stored the  These  concentrated  In  cells  i n the  For  the  after  of  the  of  of  that  for  PGM c o u l d  c y t o p l a s m as  small  of  PGM exert  degradation.  PGM on  glycogen  f i e l d mouse,  Apodemus  rainbow t r o u t ,  glycogen  of  cells  large  bay  Keq/MAR r a t i o  Salmo  stored  marine  that  g l a n d and d e v e l o p i n g  contain  80  1983).  tissues  vesicular  digestive  control.  a mechanism by w h i c h PGM c o u l d mantle  in  been  glycogen  the  high concentrations  suggests  known  have  muscle  an e f f e c t  ( A l l e n d o r f , L e a r y and Knudsen  The e x t r e m e l y  on  influence  that  27 w h i l e  r e p o r t e d i n the 1977)  of  these c o n d i t i o n s rate  to  situations  degree  (1986)  from  less  effect  believed  Ellington  fell  used  phosphorylase.  and  from 3 . 5  significant  1983).  exert  is  be r e c o n c i l e d w i t h t h e  standard conditions,  phosphorylase  increased  oyster  is  the h y d r o l y t i c  significant  Argopecten i r r a d i a n s concentricus  glycogen  a  of  pathway a  to  for  lie gonad  quantities granules  of or  in  affect  bivalves, in  close  (Gabbott glycogen large  /3-  190  particles  (Eble  degradation studied.  It  glycogen 1969)  of  to  glycogen is  could  these  between  release  influence  rates  these  the  respond to  flux  possibly glycogen  of  activity  possessing glycogen Pgm-2-92,  occur,  affects  of  the  differences  the  mantle  dictated  rapidly  necessary  season,  by  this  the  rates  104  have  of  (Eble  result  PGM  reaction.  in  activities  the  potential  genotype-by-environment glycogen of  fall  had  alleles.  could  Pgm-2 g e n o t y p e s synthetase,  after  r e o r g a n i z a t i o n of  to and  mantle  completion  tissue  (Quayle  was o b s e r v e d  higher  These  activity  the  be  between  (Table X I V ) . Heterozygotes  or h e t e r o z y g o t e s  glycogen  data  In C r a s s o s t r e a q i g a s ,  glycogen  enzyme of  the  glycogen  in the  Pqm-2-100 a l l e l e  if  been  that  i n enzyme  a strong association  mantle  or  not  B-particles  G-1-P  and h e t e r o z y g o t e s  t h a n homozygotes 96,  amounts  has  the  high concentrations  with these  phosphorylase.  and  the  the  cells  from e q u i l i b r i u m a t  for  rates  s p a w n i n g and t h e  expected  that  Unfortunately,  glycogen breakdown.  synthesized  In t h i s  1982).  differential capabilities  glycogen is  al.  considerations,  observed  by  1969).  large  displacements  interactions  PGM  conceivable  Pgm-2 h o m o z y g o t e s  caused  et  in bivalve vesicular  displacements  From  of  Bayne  phosphorylase associated  substantial If  1969;  levels  for  the  results  variation  synthesis  between  of  less  mantle frequent  are exactly  as  differentially these  genotypic  classes.  When t h e  summer  sample  was  taken,  considerable  gonadal  191  development  was  probably  on t h e  between  the  (Gabbott  1975;  higher the  observed decline  low  of  A  clear  These p a t t e r n s levels  rates  asynchronous .  for  timing  N e i t h e r of  and f u r t h e r s t u d i e s genotypes  these  the  gonadal  to  gametogenesis had  heterozygotes  results  in  high i n t e r t i d a l is  not  possible.  differences  in  glycogen  onset  of  or  glycogenolysis,  perhaps  development  m e t a b o l i s m of  required  were  Pgm-2 h o m o z y g o t e s  i n the  these p o s s i b i l i t i e s  on t h e  are  levels  levels  relationship  and  than  degradation, of  strong  season  from  before  of  the  glycogen  lower  explanation  existed  differential  genotypes.  but  glycogen  reserves  In t h i s  c o u l d have a r i s e n  that  of  these  mantle  i n t e r t i d a l area,  region.  Pgm-2  of  1976).  concentrations  therefore,  because  depletion Bayne  and,  to  in  different  are mutually glycogen  determine  exclusive  in  which,  the  different if  any,  is  correct.  In t h e of  mantle  locations. in  winter,  the  Pgm-2 homozygotes  glycogen The same  than  heterozygotes  r e l a t i o n s h i p between  adductor  muscle  only  in  previously,  this  sample was  extremely  expected  levels  population growth" have been  poor,  at  (cf.  this  of  the  tissue,  significant  were  had h i g h e r  a factor glycogen  t i m e was  Bayne  high  and  at  concentrations both  genotypes  but  the  intertidal  intertidal was  differences area. food  that  the  might e x p l a i n at  experiencing Newell  1983),  this  glycogen  u t i l i z e d t o meet b a s a l m e t a b o l i c  stated  conditions lower  time.  negative  were  As  t a k e n when n a t u r a l  observed  evident  than If  "scope  reserves  requirements,  the for  might given  192  t h e dormant s t a t e  of  the  reproductive cycle.  these  results  is  that  energy  reserves  at  a faster  Gaffney  (1984)  heterozygosity American authors  s t u d i e d the and  rate  r e l a t i o n s h i p between  starvation-induced C.  virginica,  found t h a t  the  rate  more v i a b l e  If  of  weight  over  weight  i n d i v i d u a l s , thus over p e r i o d s of  were d e p l e t i n g  than homozygotes.  oyster,  heterozygous  a  loss  suggesting  lower,  levels  under  these  these  results  Gaffney  adverse are  (1984)  environmental  questionable  were  able  to  These  that  were  or m a n i p u l a t e d i n t h i s  known  and G a f f n e y between after  the  that  any  did  not  carbohydrate levels  metabolism assignment different  (1984)  starvation period. effect  It  Pgm  than  the  heterozygosity  Marked d i f f e r e n c e s  classes  are  homozygotes.  Pacific  oyster,  higher, to  rather  homozygotes Comparison  since  Rodhouse  None  of  of and  these  factors  Furthermore,  Rodhouse  significant  relationship  either  before  or  s h o u l d be p o i n t e d o u t ,  however,  by  glycogen  the  Pqm  locus  s t u d y may have been o b s c u r e d by homozygotes  more  food a v a i l a b i l i t y , water  study. a  in  heterozygotes  and h e t e r o z y g o s i t y  contributed  in their of  find  lower  conditions.  starvation period.  and  42 day p e r i o d .  compared  control  and t h e  loss  was  however,  temperature,  Rodhouse  multiple-locus  to e x h i b i t  of mantle glycogen  these  the  nutritive stress  w o u l d have been e x p e c t e d  for  in  these c o n c l u s i o n s a r e a p p l i c a b l e to  heterozygotes than  Pgm-2 h e t e r o z y g o t e s  One e x p l a n a t i o n  and  heterozygotes  on the  random  across  the  examined.  were o b s e r v e d between  the  effects  of Pgm  193  -2  genotype  glycogen great  data.  analyses  These  of  the mantle  results  were  s i m i l a r i t y shown by t h e s e  activity  measurements  glycogen Pgm-2  levels.  locus  mantle, of  i n the  glycogen  the  an  metabolism d i f f e r s  important aspects.  form o f  glycogen  glycogen  itself  inhibition  prevents  fibres  that  Extrapolating difficult  (Hers,  excessive  could these  because  their  adductor rates  Deposition different would  of rates,  but no n e t  be e x p e c t e d  if  is  to  at  the  liver,  the  Stalmans of  glycogen  in  contractile  may  function. is  have  not  exists  in  be  in  expressed.  m u s c l e may o c c u r  between is  muscle  Pqm-2 g e n o t y p e s  not  adductor  This  systems  mechanism  between  synthesis  in  muscle  1970).  r e g u l a t o r y enzymes  differences  the  i n h i b i t e d d i r e c t l y by  a similar  the  further  their  regulation  molluscan  synthesis in  of  l i v e r and m u s c l e  the  with  differences  glycogen glycogen  to  deposition  if  the  specific  variation  the  and  homologous  muscle,  of  Wulf  differences  been c h a r a c t e r i z e d . H o w e v e r , oyster  between  interfere  the  patterns  In mammals, t h e  phosphatase De  PGM  of  on g l y c o g e n m e t a b o l i s m i n  In c o n t r a s t  synthetase  muscle  in l i g h t  their  enzyme a c t i v i t y  adductor muscle.  several  in  seasonal  effect  adductor  surprising  tissues  the  Apparently,  exerted  but not  and  and  Pgm-2  at  genotypes  p r e v e n t e d beyond  this  limit.  Fewer c o n s t r a i n t s may a p p l y t o glycogen  stored  exist  the  but  for their  in  the  storage  mantle  the  mantle t i s s u e .  capacities  of  proportions are  absolute Upper  individual  quantity  limits  obviously  vesicular  known t o v a r y  of  cells,  seasonally  and  1 94  between d i f f e r e n t  populations  Moore  1982).  and  Bayne  accumulation adductor  of  enzyme a c t i v i t y could  be  i n the  tissue.  even  the  s h o u l d be  assumption  m e t a b o l i s m were genetic  the  the  that  the  with adjacent  lead  to  Pqm-2 l o c u s .  assumption  by  enzymic  erroneous The n e x t  of  respect  kinetically-linked  reactions  chapter  reactions  tests  activity of  the  restrictive  the  of  of  with  the  rates  flux still  rely heavily on  this  on  glycogen  r e m a i n d e r of  the  PGM a c t i v i t y  glycogen  metabolism effects  validity  relationships  glycogen  of  synthetase.  c o n c e r n i n g the the  the  effect  would  Pgm-2 l o c u s to  (Lowe,  metabolic  glycogen  the  conclusions  examining the  less  on  overall  like  year  comparison  t h e s e arguments  effects  randomized with  in  of  the  Pqm-2 l o c u s  though the  that  for  b a c k g r o u n d . Non-random a s s o c i a t i o n s  variation could  stressed  time  these c o n d i t i o n s ,  be d e t e r m i n e d by r e g u l a t o r y enzymes  It  same  mantle  Under  v a r i a t i o n at  manifested,  the  T h i s may a l l o w  glycogen  muscle  at  of  between  synthesis  of this the  pathway.  195  CHAPTER 5  ACTIVITY  STRUCTURE OP THE GLYCOGEN SYNTHESIS PATHWAY  INTRODUCTION  Attributing at  a  single  encounters  a physiological effect  enzyme  locus  difficulties  detection  (e.g.  Dykhuizen,  populations,  a major  genetic  background.  Marine  substantial  levels  Hershberger  a n d Chew  for  present  Although gene  that  number o f  has  revealed between  that  Ozaki  and F u j i o  are  vitro  specific 1988).  of  considerable alleles  sometimes  exert  activities  Each of  these  as  et  (e.g.  (Buroker, and thus  1982;  the  might  also  Wilton  also  may  exist  flanking  regions  Aquadro et  1986; the  al.  have  differences  factors complicates  on  et  with genotypic al.  be  on a  by s t u d i e s  variation  and 3'  one  bivalves,  disequilibrium 5'  the  segregating  marine  suggested  sequence  and t h e i r  the  pleiotropic effects  al.  linkage  associated  possess  in a d d i t i o n to  1979)  DNA  C. gigas  be  (cf.  Paigen  studying of  l i k e l y to loci  its  effects  1985),  in  to  In  heterozygosity  pathway e n z y m e s ,  studies  such as  unquantified  potential  variation  with  1987).  involves  bivalves  enzyme  yet  concerned  and H a r t l  concern  (Laurie-Ahlberg  structural  that  al.  the  different  Molecular  at  variation  melanogaster  1982).  Dean  protein  1979a;  variation  study.  regulatory  D.  those  e n e r g y - p r o d u c i n g pathways a r e  genetic  under  of  allozymic  a complex m u l t i - e n z y m e pathway  beyond  natural  central  in  to  in  Langley  assignment  in et of  196  physiological enzyme  effects  to  of  this  confounded  by  the  metabolism  of  lipid,  accumulation Pieters  et  seasonal  al.  the  well  1979;  basis,  has  polymorphic  a  specific  and/or  do  studied  not to  of  architecture  could  the  through the  creation  al.  1981)  1980,  introduce  single  or  of  isogenic by  allelic  to  the  study  of  3  in  1975;  Livingstone PGM  on  specific  a  to  seasonal  involve  detection in  of  activity  enzymes have  affect  specific  (e.g.  have  pathway's  these  and i n some  vary  the  the  1983),  Although to  and i n  the  its  levels  potential  regulatory  the  the  impact  control of  the  seasons.  genetic  D y k h u i z e n and H a r t l  amenable  the  differentially  laboratory,  enzymes  Alterations  p o l y m o r p h i s m between d i f f e r e n t  In  Gabbott  complicate  of  their  Associated with  necessarily  pathway  of  Gabbott  changes  1983).  in Chapter  properties  the  in turn  Clarke  consequences.  kinetic  affect  and  (e.g.  1980).  of  further  carbohydrate,  1981;  properties  shown  locus  and  al.  are  seasonality  tissue-specific  changes  physiological  1980;  at  bivalves  end-products  Livingstone  been  marine  documented  Zandee e t  kinetic  these  in  protein,  are  Livingstone  activity  nature  anaerobic  (e.g.  instances 1975;  of  cycles  activities  that  differences  locus.  Studies  to  allozymic  the  b a c k g r o u n d s may be lines use  of  substitutions 1981).  (e.g.  Laurie-Ahlberg  genetic (e.g.  Neither  of  standardized  transduction  Hartl these  natural populations,  and  et to  and D y k h u i z e n techniques thus  it  are is  197  necessary  to  variation  in  alleviate  bivalves,  their  contributed the  adaptive  annual cycles  between  patterns  differences  3,  the  in  their  variation  and  synthesis  pathway  enzymes  genotypic  classes  i n the  reserves 1969), of  the  due t o  potential  thus  to  be  complex  basis  were  activity  additional  were  if  the  loci,  the  studied  to  associated  documented  in  particularly be  existence  of  shown  season-dependent  o b s e r v e d were d i r e c t l y  levels  marine  understood.  concentrations a  help  enzyme p o l y m o r p h i s m s may  tissue-specific  effects,  fall  to  in  discounted. of  genetic  remaining  glycogen  in  different  Pgm-2  sample.  In t h i s  season,  glycogen  accumulated i n the  mantle  tissue  (Quayle  a l l o w i n g a more t h o r o u g h e x a m i n a t i o n of  Pgm-2 l o c u s the  in  of  activity  are rapidly  is  genetic  background e f f e c t s .  r e l a t e d w i t h PGM, c o u l d n o t  To examine  the  of  of  studied  metabolism  PGM s p e c i f i c  contribution  metabolically  of  and  glycogen  Pgm-2 g e n o t y p e s  A l t h o u g h the  the  Chapter  mantle  presence  by g e n e t i c  significance  4,  the pathway  physiological effects  Chapter  fashion.  those  metabolic  be s t u d i e d on a s e a s o n -  In  with  simultaneously  entire  due t o  the  full  differ  the  problems  Furthermore,  have t o  monitor  on g l y c o g e n  synthesis  c o n c u r r e n t o p e r a t i o n of  the  without  the  effects  complications  glycogenolytic  pathway.  198  MATERIALS AND METHODS  Chemicals. used  for  from by  Buffers,  the  electrophoresis  Sigma.  and  The e l e c t r o s t a r c h  Oysters  population  in  and  of  storage  were the  enzyme for  determined In t h e  activity  fall  For  Ridgway,  has  and c o u p l i n g  assays  enzymes  were  electrophoresis  subsample  of  and g l y c o g e n  obtained  was  supplied  loci.  100 mM T r i s - H C l chloride,  buffer,  90  mg  25 mg NADP,  0.7  phosphoglucomutase,  dehydrogenase,  2 mg MTT, and  activity  was  outlined  mg  examined  Chapter  the  enzyme  analysis  and UDP-  buffer  system was  30 min  containing 65  PMS.  of  stained  mg  120  in mg  sodium  glucose-1,6-diphosphate, units  was  pyrophosphorylase  approximately 7.5  was  electrophoretically  UDP-glucose, mg  for  UDPGP a c t i v i t y for  3.  in  hexokinase  Li-OH  pH  40 1  the  resolved  slice  study  genotype  electrophoretic  (1970).  incubating a gel  as  UDP-glucose  was  Bay  transportation  Pgm-2  selected  Tris-citrate,  S h e r b u r n e and L e w i s  pyrophosphate,  sample,  genotypes at  activity  Nanoose  i n C h a p t e r s 2 and  electrophoresis  pyrophosphorylase  100 ml o f  2.7.1.1)  entire  determine  by  units  described  measurements,  discontinuous  the  The c o l l e c t i o n ,  individuals  visually  magnesium  from  1985. been  the  (UDPGP; E . C . 2 . 7 . 7 . 9 ) the  of  by s t a r c h g e l  a l s o performed to glucose  collected  animals  Electrophoresis.  by  cofactors,  Connaught L a b o r a t o r i e s .  Animals.  2.  substrates,  80  glucose-6-phosphate Hexokinase  (HK; E . C .  electrophoretically  by  the  199  standard Tris-borate-EDTA Chapter  2,  Enzyme  and s t a i n e d  Assays.  from t h e  were  low These  classes  that  body w e i g h t crude  in  3,  the  a  of  or  the  were  buffer.  seven  hexokinase, and  229  glycogen  oysters  high  found to  intertidal  Pgm-2;  to  that  genotypic  have  assigned the  outlined  in  5 mM d i t h i o t h r e i t o l (DTT) of  DTT  stabilized  pyrophosphorylase no a d v e r s e  SP  were m e a s u r e d  1800  UV/visible  was m a i n t a i n e d a t  water  Phosphoglucomutase  standard  assay  described  i n C h a p t e r 2.  quantity  of to  of  (108  P r e p a r a t i o n s of  identical  Addition  temperature  conditions.  the  i n C h a p t e r 3.  inclusion  activities  Unicam  phosphate  from  (1970).  of  of  in  and  effects  the  glycogen on  either  phosphoglucomutase.  enzyme  bath.  121  UDP-glucose  but was  Pye  activities  represented  described  for  extraction  All  Shaw and P r a s a d  i n a subsample  site,  homogenates  except  hexokinase  described  were r a n d o m l y p i c k e d from f o u r a r b i t r a r i l y  activities  on  determined  classes  synthetase,  system  UDP-glucose pyrophosphorylase,  individuals  tissue  Chapter  buffer  mantle  intertidal  site).  8.3  a c c o r d i n g to  The  phosphoglucomutase, synthetase  pH  on  enzyme  20  at  340 nm  spectrophotometer.  Assay  15 C by a Lauda K - 2 / R D c i r c u l a t i n g activity  yl aliquots One u n i t  required  in t r i p l i c a t e  of to  glucose-6-phosphate  of  was  determined  the  per  the  c r u d e homogenates  activity convert  by  is  defined  1 /xmole o f minute  as  as the  glucose-1-  under  these  200  Hexokinase Ml  samples  of  was a s s a y e d  a c c o r d i n g to  the  homogenates.  crude  contained  50  MgC12,  mM A T P , 0.4  0.6  phosphate unit  of  C.  mM  dehydrogenase  activity  convert 15  mM t r . i e t h a n o l a m i n e  1 Mmole of  glucose  UDP-glucose  and  (G6PDH) to to  the  amount o f  by  extracts.  The a s s a y m i x t u r e c o n t a i n e d  MgC12,  1.5  (1974)  on 20 ul  NADP,  16.nM g l u c o s e - 1 , 6 - d i p h o s p h a t e ,  unit  of  for  of  G6PDH i n a f i n a l  UDPGP i s  UDP-glucose  the to  Glycogen determined Rottenberg  amount o f  by  the  (1973)  hydrazine-HCl,  10  glycogen  10 u n i t s  bovine  serum  of  B.C.  of  of  lactate  albumin  in  assayed  synthetase  was  not  phosphate,  thus  only  the  a  as  of  the  tissue 7.5,  PGM,  5  0.4 mM  and  one  1 nmole  of  15 C .  Passoneau  and  NADH,  the  of  c r u d e homogenates. 50 mM K C 1 ,  The  10  mM  10 mM U D P - g l u c o s e , 1  20  volume  the of  measured  assay the  final  activity  at  was  dehydrogenase,  in  per minute  activity  5 mM MgC12, mM  to  2.4.1.11)  mM s o d i u m a m o b a r b i t a l ,  0.15  needed  convert  per minute at  one-step  (Type I I ) ,  1 m l . One  1 m l . The u n i t , d e f i n i t i o n  50 mM T r i s - H C l pH 7 . 5 ,  phospho(enol)pyruvate, kinase,  (GS;  of  1 mM U D P - g l u c o s e , of  5 mM  glucose-6-  enzyme  aliquots  r e q u i r e d to  on 50 jul s a m p l e s  a s s a y medium c o n t a i n e d  mM o y s t e r  enzyme  direct  volume  also  5 units  volume  glucose-1-phosphate  synthetase  of  60  medium  50 mM T r i s - H C l pH  mM sodium p y r o p h o s p h a t e ,  on  mM D - g l u c o s e ,  glucose-6-phosphate was  (1974)  reaction  unit  in a t o t a l  outlined  mM  200  one  pyrophosphorylase  Bergmeyer  The  pH 7 . 5 ,  NADP,  corresponds  Bergmeyer  mM  Na-EDTA,  units  of  and of  presence  10  1 mM  pyruvate  0.02%  (w/v)  1 m l . Glycogen of  glucose-6-  dephosphorylated  I  form  201  was  quantified.  quantity  of  that  Because of  homogenates,  Similar  enzymes  concurrently activity Chapter  loss  significant  above  of  the  3,  not  the  crude  glycogen  synthesis  selection  the of  fall  Pgm-2  of  t e c h n i q u e and s y s t e m a t i c  enzyme a c t i v i t y w o u l d be  Glycogen  Assays.  concentrations previously  Statistical  of  Pgm-2  examined  product-moment expectations.  Activity by  correlation To  were  2 and 3 .  muscle  PGM  assayed specific  As d e s c r i b e d i n  genotypes  of  genotypes  d e s c r i b e d i n Chapter  were  homogenates  was  randomized  day-to-day  due t o  the  variation  spontaneous  minimized.  Determination  Analyses.  other  sample.  errors  UDP-  the  pathway were  e x p e r i m e n t a l days thus e n s u r i n g t h a t  the  cuvettes  except for  of  the  the a m o b a r b i t a l ,  required  the  above  NADH by  components  w i t h t h e m a n t l e and a d d u c t o r  the  1 Mmole  c o r r e c t i o n s by b l a n k  were in  of  d e s c r i b e d in Chapters  for  d e f i n e d as  o x i d a t i o n of  assay  corrections  measurements  assay  enzymes  the  is  t r a n s f e r the a d d i t i o n  i n the p r e s e n c e  i n t r i p l i c a t e as  The  activity  g l y c o g e n per minute under the  Soluble protein levels  measured  in  to  GS  measurements n e c e s s i t a t e d  glucose.  across  the  even  c o n t a i n e d a l l of  assays.  of  onto the primer  conditions.  activity  unit  enzyme r e q u i r e d  UDP-glucose  crude  One  the in  this  mantle  glycogen  sample have  been  4.  relationships  testing  the  significance  coefficients  examine t h e e f f e c t s  of  between  pathway of  their  from  random  Pgm-2 l o c u s  on pathway  202  activity -2 the  structure,  allelic  classes  Pgm-2-100  the  sample was p a r t i t i o n e d i n t o  d e s c r i b e d i n Chapter  allele,  heterozygotes  allele,  homozygotes  alleles,  and h e t e r o z y g o t e s  alleles).  Three-way  activities  of  treating  for  the  less  HK, PGM, UDPGP,  as  independent  genotype  class  demonstrate  i n the  an  variables.  analyses  association  these  sequential  variation  in  the  activities  regression  analysis.  its  partial  squares  to  a t t r i b u t a b l e to  the  significance  coefficient  partial each  F-tests  factor  to  its  on t h e  i n the  locus  of  Pgm-2  o r GS d a t a  thus  with  the  The e f f e c t s  UDPGP,  importance of  the  levels  effects  locus  PGM,  104  frequent  and Pqm-2  reactions.  HK,  and  each of  of  and GS on  was examined by m u l t i p l e  The r e l a t i v e  regression  procedure analogous  of  class,  Pgm-2  96,  less  HK, UDPGP,  the  Pgm-2-100  glycogen  Significant  the  for  p e r f o r m e d on  mantle  enzymic  levels  enzyme was a s s e s s e d by t e s t i n g of  of of  of  mantle glycogen  three  body w e i g h t  activities  existing  and  the  Pgm-2-92,  v a r i a n c e were  GS,  intertidal position,  genotype  of  these  f o u r Pgm  homozygotes  possessing  frequent  between  analyses  3 (i.e.,  the  linear  individual  the  t-ratio  standard e r r o r , e x p l a i n e d sums  multiple  a of  regression.  RESULTS  ELECTROPHORESIS  The  discontinuous  electrophoretic distinct  Ridgway b u f f e r  r e s o l u t i o n of  z o n e s of  activity  system  produced  excellent  U D P - g l u c o s e p y r o p h o s p h o r y l a s e . Two  were v i s i b l e ,  suggesting  the  presence  203  of  two d i s t i n c t  selected showed Using  for  locus  of  a  faint  of  genetic  for  individuals  yielding  observed at  either  the  Product-moment of in  the  of  good  HK  XVI.  correlations and  UDPGP,  cathodal also  pattern.  c o u l d not  heterozygosity  two  were  found  in those positive  with  locations,  observed  be 62 was  situated  relationship  are highly  between  the  UDPGP a n d G S , but no  samples.  in  mantle  PGM a n d UDPGP. Weak  between  PGM i n  the  enzymes  the a c t i v i t i e s  intertidal  correlated  between  pathway  o b s e r v e d between  were  weak  no  and between  differences  but not  locus  At both i n t e r t i d a l  Two  the  also  However i n a sample o f  synthesis  between  a  more  The a n o d a l r e g i o n  resolution,  were e v i d e n t  Second,  The  either  sample.  relationships  intertidal  hexokinase  BETWEEN PATHWAY ENZYME A C T I V I T I E S  c o r r e l a t i o n s were  significantly  loci.  band.  v a r i a t i o n at  entire  Table  activities  of  system,  low a c t i v i t y o f HK, d e t e r m i n a t i o n of  glycogen  positive  matrices  measurements  were c o m p l e t e l y m o n o m o r p h i c .  correlation coefficients  significant  notable  individuals  locus.  CORRELATIONS  activities  229  glycogen  buffer  single  extremely  determined  presented  and  the  but was c h a r a c t e r i z e d by a t w o - b a n d e d  the  existence  positive  activity  be e n c o d e d by two s e p a r a t e  faintly,  Because  enzyme  E x a m i n a t i o n of  both presumptive l o c i  produced  stained  loci.  T r i s - b o r a t e - E D T A pH 8.3  appeared to  the  the  that the  genetic  of  GS.  the c o r r e l a t i o n First,  oysters the  HK and  high  existed  HK  from  the  was low  intertidal. between  the  204  Table XVI. P r o d u c t - m o m e n t c o r r e l a t i o n c o e f f i c i e n t s between t h e a c t i v i t i e s of enzymes i n t h e g l y c o g e n s y n t h e s i s p a t h w a y . V a l u e s above t h e d i a g o n a l a r e f o r t h e low i n t e r t i d a l sample ( n = l 0 8 ) ; t h o s e below f o r t h e h i g h i n t e r t i d a l sample (n=121).  205  Enzyme Enzyme  HK  PGM  UDPGP  GS  HK  1 .000  0.316**  0.463***  0.084  PGM  0.135  1.000  0.512***  0.105  UDPGP  0.491***  0.419***  1 .000  0.137  GS  0.006  0.134  1 .000  * * P < .01 * * * P < .001  -0.146  206  activities high was  of  PGM and GS i n  i n t e r t i d a l the negative. of  low  intertidal,  c o r r e l a t i o n between  Statistical  coefficients h i g h and low  the  the  t h e s e enzyme  comparison  of  the  HK-PGM and PGM-GS enzyme  intertidal  zones d i d  not  whereas  the  activities  Z-transformed  pairs  detect  in  between  any  the  significant  differences.  PATHWAY A C T I V I T I E S OF PGM-2 GENOTYPIC GROUPS  The levels  results  and  the  of  the  three-factor  activities  summarized  in  Table  significant  p r o p o r t i o n of  dependent  variables.  analyses:  oysters  significantly greater  activities  from t h e  observed  of  more e x p o s e d  magnitude  of for  intertidal  the  32%.  In c o n t r a s t ,  22%  more  activity,  F  glycogen site  HK  XVII.  A  in  larger  of  HK,  the  variance  consistent low  activity,  the  zone.  a separate  factor  its  that  at  i n the  low  15%  glycogen  than  No s i g n i f i c a n t were p r e s e n t  most  for  and  all  high  those  i n any of  of the  all  sampled the  difference  was  at  area  the site  low by  displayed  21% more UDPGP  i n d i v i d u a l s higher  effects  in  from  intertidal  intertidal  five  significantly  mean a c t i v i t y the  a  possessed  As e v i d e n t  extreme  are  explained  region  more PGM a c t i v i t y ,  and 24% more g l y c o g e n  intertidal  of  GS  p a t t e r n was o b s e r v e d  intertidal area.  synthetase:  oysters  position observed  glycogen  and  f o u r enzymes c o m p a r e d t o  values,  exceeded  UDPGP,  intertidal  quantities  high  PGM,  Intertidal  the  all  ANOVA's on m a n t l e  body w e i g h t analyses.  in class  the as  207  Table XVII. F - r a t i o s from t h e a n a l y s e s o f v a r i a n c e on t h e m a n t l e a c t i v i t i e s of t h e g l y c o g e n s y n t h e s i s pathway enzymes and m a n t l e g l y c o g e n c o n c e n t r a t i o n s .  208  Source of Variation  Dependent V a r i a b l e df  HK  PGM  UDPGP  GS  Glycogen  Tidal Height  19.1***  15.0***  14.8***  67.7***  19.2***  Body Weight  0.79  1 .88  1 . 14  1.51  2.06  Allelic Class  2.40  7.76***  1.70  3.25*  10.5***  Tidal Height x Body Weight  1.48  0.75  0.36  3.67*  1.21  Tidal Height x Allelic Class  0.94  0.52  2.27  4.90**  0.61  Body Weight x Allelic Class  0.59  0.22  0.31  1.83  0.78  Tidal Height x Body Weight x Alleiic Class  1.95*  1.72  2.14*.  0.67  1.06  Error  * P < .05 ** P < .01 *** P < .001  197  209  Highly activities classes. these XIV  significant  specific  genotypic  significantly homozygotes  activities  4.  greater  and t h e  specific  d i d not d i f f e r  for  less  frequent  genotypic  concentrations.  activities only  of  glycogen  the  larger  96,  unable, the  The  for  the  A  alleles  posteriori  analysis  low  in  allele  to d e t e c t  had  96,  from t h e  and  104  homozygotes between  i n t h e i r mantle  glycogen  this  between  classes.  the  study  however,  HK, UDPGP,  T a b l e XVII  Homozygotes  h a d GS a c t i v i t i e s homozygotes  (7.31  showed  Bonferroni  Table  Pgm-2-100/100  Pgm-2-92,  in  groups  of  a n d GS  shows  between  for  that  that  t h e Pgm  were  units/g  14.4%  vs.  6.39  intermediate  m u l t i p l e range t e s t s  any s i g n i f i c a n t  differences  GS were  between  groups.  class  the  levels  differences  overall analysis.  Pgm-2-100/100  general  genotypic  present  104  however,  allelic  overall  and  PGM  allelic  activity differed significantly  The two h e t e r o z y g o u s  activities.  Pgm-2  than the  the  interest  Pgm-2 a l l e l i c  i n the  than the  units/g).  four  Pgm-2-100  Identical  might e x i s t  synthetase  Pgm-2 g e n o t y p e s -2-92,  that  for  were o b s e r v e d  Of p a r t i c u l a r  differences  the  significantly  alleles.  classes  for  activities  heterozygotes  but  were  the  the  and m a n t l e g l y c o g e n  Heterozygotes  alleles,  these  of  between  g r o u p s have been p r e s e n t e d p r e v i o u s l y  Chapter  these  existed  and g l y c o g e n c o n c e n t r a t i o n s The  of  differences  absence  and t h e is  results and  of  any  activities  somewhat  r e l a t i o n s h i p s between of  these other  deceptive.  Tables  from two-way ANOVA's c a r r i e d  high  intertidal  samples,  enzymes XVIII out  Pgm-2 in  and  the XIX  separately  respectively.  The  210  Table XVIII. A c t i v i t i e s (units/g soluble protein) UDPGP, and GS of f o u r Pgm-2 g e n o t y p i c c l a s s e s i n t e r t i d a l zone.  of HK, PGM, i n t h e low  Enzyme Pgm-2 A l l e l i c Class  N  HK  PGM  Activity' UDPGP  GS  Glycogen'  Homozygotes for 100 A l l e l e  22  6.06+.49  116.2+6.2  31.9+2.7  6.75±.31  171.4+16.0  Homozygotes wlthout lOO A l l e l e  14  6.17±.62  124.917.8  35.413.4  7.751.39  195.2120.0  Heterozygotes for 100 A l l e l e  60  6.571.30  139.213.8  35.3+1.6  7.931.19  220.5+9.7  Heterozygotes wlthout 100 A l l e l e  12  6.90+.67  106.2+8.5  29.113.7  7.801.43  154.6+21.8  F(3.92)  =  0.48  5.67**  * P < .05 ** P < .01 un1ts/g s o l u b l e p r o t e i n * ptnoles g l u c o s y l u n l t s / g wet t i s s u e 1  0.86  3.68*  4.03**  212  Table XIX. A c t i v i t i e s ( u n i t s / g s o l u b l e p r o t e i n ) of HK, PGM, UDPGP, and GS of f o u r Pgm-2 g e n o t y p i c c l a s s e s i n t h e h i g h i n t e r t i d a l zone.  213  Pgm-2 A l l e l i c Class  Enzyme N  HK  PGM  Activity' UDPGP  GS  Glycogen'  Homozygotes for 100 A l l e l e  20  4.77±.44  101.6+6.4  27.8+2.2  6.00+.40  122.0±12.0  Homozygotes wlthout 100 A l l e l e  17  6.04+.49  106.7+7.0  34.8+2.4  6.94+.44  148.7±13.1  Heterozygotes for 100 A l l e l e  63  4.931.25  118.5+3.6  25.8+1.3  5.30±.23  183.116.8  Heterozygotes W1thout 100 A l l e l e  21  6.19+.44  104.7+6.3  29.7+2.2  6.24+.39  145.3+11.7  2.92*  3.25*  4.56**  F(3,105)  =  3.35*  * P < .05 ** P < .01 units/g soluble protein ' *,moles g l u c o s y l u n i t s / g wet t i s s u e 1  7.93***  214  analysis  of  similar  to  the  low  that  intertidal  seen  were o b s e r v e d between Pgm-2  allelic  between  the  overdominance and  activities  relative  slightly below  these other  for of  glycogen allelic  with  analyses  significant  to  were  (Table was  found  showed for  and  the  manifested  104  differed 4.03,  P  activities  .01),  the  exhibited from  the  The HK  allele  were  but m a r g i n a l l y  heterozygotes.  <  of  in  alleles.  significantly  The  Pgm-2-92/100,  Pgm-2-100  of  t h e Pgm  homozygotes.  homozygote g r o u p , class  to  Mantle  between in  a  Pqm-2  pattern  PGM, but n o t any o f  the  enzymes.  activities  sample  their  were a g a i n  indistinguishable 96,  other  =  four  the  not  were  the  These h e t e r o z y g o t e s  for  either  (F(3,92)  contrast  differences their  than  by  was  were  heterozygotes  groups  expressed  Pgm-2-92,  concentrations  pathway  In  that  of  heterozygotes  Pgm-2-100/100  enzymes.  measured i n the  consistent other  the  higher  that  the  heterozygotes  UDPGP and GS a c t i v i t i e s  activities  to  of  that  differences  m u l t i p l e range t e s t s  greater a c t i v i t i e s  100/104  homozygotes  that  f o r PGM a c t i v i t y  of  No s i g n i f i c a n t  HK and UDPGP a c t i v i t i e s  t h e i r GS a c t i v i t i e s  allele  96/jlOO  in Table XVII.  groups. S i g n i f i c a n t differences  be c a u s e d by t h e -2-100  sample p r o d u c e d r e s u l t s  the  observed of  all  four  overall between enzymes  X I X ) . However, the  analysis, Pqm-2 in  level  low and m u l t i p l e r a n g e t e s t s differences  UDPGP and GS a c t i v i t i e s .  between  genotypic the  of  significant  high  groups  intertidal  significance  were  able  allelic  groups o n l y  F o r b o t h enzymes,  homozygotes  in  i n most  to  detect  for for  their the  215  Pgm-2-92,  96,  activities  and  than  heterozygotes. frequent  to  the  allele  class other  had  the  also  A l t h o u g h they  still  the  and  that  slightly  levels  96/100  100/104 h e t e r o z y g o t e s that  Pgm-2-100  of  high  in  the  HK  similar  intertidal  region  the  allele  96/96,  two t i d a l  expressed  the  and  heights,  A the  w i t h any o t h e r  number  of  ANOVA r e s u l t s  pathway  significant presented  weight-by-allelic  sample. these  and  GS  Pgm-2-100/100 Pgm-2-92/100,  to  the  and but  alternate  heterozygotes  for  the  PGM a c t i v i t i e s , As s e e n of  in  but  the  low  genotypic  r e l a t i o n s h i p w i t h PGM  activity,  enzymes.  i n t e r a c t i o n terms were p r e s e n t  in Table XVII.  class  water  the  HK a n d UDPGP p r o d u c e d s i g n i f i c a n t body  differed  104/104). Consequently,  overdominant  showed a s t r o n g p o s i t i v e  not  Pgm-2-100  (Pgm-2-100/100),  glycogen concentrations  but  the  the  intertidal groups  either  comparison  UDPGP  and GS a c t i v i t i e s .  the  In  low  i n t e r m e d i a t e H K , UDPGP, sample,  than  less  t h u s d i s p l a y e d HK, UDPGP,  a r e a were more s i m i l a r  (Pqm-2-92/92,  three  that  to  r e s e m b l e d one homozygote  intertidal  when p o o l e d o v e r  the  for  underdominant  In t h e  GS a c t i v i t i e s  for  o v e r d o m i n a n t PGM a c t i v i t i e s ,  homozygotes.  homozygotes  100/104  and  activities  observed  expressed  expressed  GS  and  low  96/100  heterozygotes  activities,  the  greater  Pgm-2-100 a l l e l e .  groups,  UDPGP,  from  in  significantly  e x h i b i t e d l a r g e r HK a c t i v i t i e s  genotypic  HK,  and  had  Pqm-2-92/100,  possessing  substantially  genotypes  alleles  Homozygotes and h e t e r o z y g o t e s  alleles  genotypic  104  The a n a l y s e s  second-order t i d a l  interactions.  for  in  both  height-by-  F o r HK, t h e  two-way  216  ANOVA f o r  the  high i n t e r t i d a l  weight-by-allelic .05).  In t h i s  class  sample,  sample y i e l d e d a s i g n i f i c a n t  i n t e r a c t i o n term (F(9,105)  the  HK a c t i v i t y  Pgm-2-100  allele  decreased  showed t h e  reverse  t r e n d i n the  low  intertidal  and  allelic  but  activities  to  increase  observed  interaction reversal  HK a n a l y s i s  class  the  other  observed  i n the in  two t i d a l  positions.  UDPGP  differed  in  interaction  terms  UDPGP  evident  i n the  intertidal  activity  displayed a positive weight,  however  genotypic and  groups  body  that  The  body  weight  P >  .80), tended were  caused and  term  by a enzyme  between  observed  for  At b o t h  weight-by-allelic  class  In t h e  a  groups.  for  the  HK.  showed  heterozygotes  the  second-order  weight  seen  high  positive  and body w e i g h t .  association  = 0.59,  but  In  body  Pqm-2-100 a l l e l e  for  intertidal association  No r e l a t i o n s h i p s  In o y s t e r s  between  no r e l a t i o n s h i p s were  groups.  body w e i g h t  the  groups.  largely  interaction  from  two h e t e r o z y g o t e  site,  thus  the  the  No r e l a t i o n s h i p s  groups.  was  for  Pgm-2-100 a l l e l e  were n o n - s i g n i f i c a n t .  b o t h homozygote  between  for  origin  positions,  the  between  second-order  intertidal  sample,  HK  heterozygotes  similar  for  (F(3,92)  P <  weight  i n t e r a c t i o n between  genotypic  for  body  genotypic  body w e i g h t .  relationship  activity  The  increasing  was n o n - s i g n i f i c a n t  a f u n c t i o n of  = 2.15,  in heterozygotes  other  the  in heterozygotes  as  in  with  body  the  from t h e  Pgm-2-100  were low  allele  UDPGP a c t i v i t y and body  observed  The d i f f e r e n t i a l r e s p o n s e s  i n homozygous and h e t e r o z y g o u s  for of  the  other  UDPGP a c t i v i t y  genotypes  between  217  intertidal  positions  interaction  observed  Analysis significant and  tidal  Multiple class  differences  the than  activity tidal  weight  between t h e  positions  height-by-Pqm-2 change  heights  as  g)  class  had  seen  for  in  oysters  48.0  g.  class  interaction class  oysters the  the  GS  high  activity  = 3.98,  in the  P <  smallest  lower  enzyme  significant  weight  groups  animals  GS  reversals over  i n GS  the  significant  t e r m . The s i g n i f i c a n t  level  first  allele  and X I X .  to  last  between t h e  in  activities  two body tidal  i n t e r a c t i o n was a c o n s e q u e n c e  Pgm-2-100  in T a b l e s XVIII  weight,  However,  greater  for  body  No  these  and l a r g e s t  yielded  In  significantly  of  data  (F(3,105  i n d i v i d u a l s . These  responsible  the  that  above  were  allelic  groups  now e x p r e s s e d  smallest  and  class.  i n t e r t i d a l sample.  largest  XVII.  differences  showed  i n r a n k e d GS a c t i v i t y  heterozygotes  allelic  GS a c t i v i t i e s  low  i n the  weight-by-allelic  the  tests  individuals  i n the  measured  and  second-order  synthetase  f o u r body w e i g h t  between t h e  smallest  in Table  between t i d a l h e i g h t  (12.0-23.9  present  significant  enzyme  significant  range  than  the  glycogen  height  sample,  activities  were  the  between t h e  weight  this  interactions  intertidal  .01).  for  of  between  existed  produced  by two  of the  tidal  218  MULTIPLE REGRESSION ANALYSES  S i m u l t a n e o u s measures GS  and  existing  relative  effects  multiple (Table  (F(4,103)  for  of  the  =  variation for  glycogen  overall  enzyme  i n the  a significant  the m u l t i p l e regression  on  significant  (F(4,116)  the  variation  (r  =  the  in  10.8%).  significant equation  obtained  regression  coefficients  sample were  negative.  that 2-100  this  P  low  activities,  yet  groups.  the  <  by  the the  .05).  data  glycogen  squares  The  the  t-ratio  the  sample,  in  the  less  for  to  of  multiple  low w a t e r  also of  sample  PGM  was  regression  the  partial  high i n t e r t i d a l  Table  heterozygotes  expressed  observed t-ratios  explained  In c o n t r a s t water  sample  ( T a b l e X X I ) was  but  the  The d a t a p r e s e n t e d  highest  pathway  o n l y PGM a c c o u n t e d  f o r UDPGP and GS i n t h e  These genotypes  the  significant  16.4% of  found f o r  may have been c a u s e d by t h e  allele.  genotypic  the  highly  that  of  e x p l a i n e d sums of  only  P < .001).  from  showed  the  than  UDPGP,  intertidal  E s t i m a t i o n of  P < .01),  again,  low  was  intertidal  glycogen  = 3.52,  the  2.35,  = 3.49,  Once  (t  =  HK, PGM,  synthetic  explaining  pathway  (t  high  For  levels.  p r o p o r t i o n of  regression  the  regression  P < .001),  of  a l l o w e d an a s s e s s m e n t  on  analysis.  5.06,  activities  levels  i n mantle glycogen  each  2  the  e a c h enzyme  regression XX),  of  4  suggests  for  t h e Pgm-  l o w e s t UDPGP and GS  levels  of  the  four  219  Table XX. R e s u l t s from t h e m u l t i p l e r e g r e s s i o n a n a l y s i s of t h e g l y c o g e n s y n t h e s i s pathway enzyme a c t i v i t i e s on g l y c o g e n l e v e l s i n t h e low i n t e r t i d a l s a m p l e .  220  Regression  Equation:  Glycogen  = 54.4  + 0.527HK  + 0.626PGM + 1.27UDPGP + 2.42GS  Regression Coefficient  Standard Error  t-ratio  Hexokinase  0.527  3.61  0.15  Phosphoglucomutase  0.626  0.27  2.35*  UDP-Glucose Pyrophosphorylase  1 .27  0.72  1 .76  Glycogen  2.42  4.73  0.51  Enzyme  * P <  Synthetase  .05  221  Table XXI. R e s u l t s from t h e m u l t i p l e r e g r e s s i o n a n a l y s i s t h e g l y c o g e n s y n t h e s i s pathway enzyme a c t i v i t i e s on glycogen l e v e l s i n the h i g h i n t e r t i d a l sample.  of  222  Regression  Equation:  Glycogen  = 94.1  + 1.76HK + 0.729PGM -  0.673UDPGP -  0.510GS  Regression Coeff i c i e n t  Standard Error  Hexokinase  1.76  2.90  0.61  Phosphoglucomutase  0.729  0.207  3.52***  UDP-Glucose Pyrophosphorylase  -0.673  0.656  -1 .03  Glycogen  -0.510  2.89  -0.18  Enzyme  ***  Synthetase  P < .001  t-ratio  223  DISCUSSION  The  correlations  s y n t h e s i s pathway Pgm-2  gigas.  glycogen  into  (Newsholme  glycogen. of  effects  enzyme  activity  An a s s o c i a t i o n particular  demonstrated  only  and S t a r t on t h e  the  locus  to  on g l y c o g e n  1973),  between GS a c t i v i t y (Stalmans  PGM w i t h b o t h HK and association  of  GS. with  the  Hexokinase PGM  metabolism  in  1986a,  to  units  between  the  seriously  genotype-dependent on s y n t h e t i c  rates.  activity  strong  is  relationship  glycogen  synthesis  the  I  (Gabbott,  form Cook  1986b).  obtained is  activity exhibited  i n the  and  expected  D-glucose  i n M. e d u l i s  in Table XVI, i t involved  a  between  c o r r e l a t i o n matrices shown  favoring  therefore,  the  and  G a b b o t t and W h i t t l e  samples  multiple  are  of  to  and r a t e s  levels  the  synthetase  of  1976),  glycogen  different  correlations  between PGM and g l y c o g e n  1979;  flux  Pgm-2 l o c u s  because  in  non-equilibrium  and t h u s  overall  glycogen  both hexokinase  catalyze  attributable  importance  of  the  evidence  HK o r GS c o u l d ,  the  of  levels  results  positive  v a r i a t i o n at  observed d i f f e r e n c e s  positive  activity  known  previously  In comparing the intertidal  activities  s y n t h e s i s pathway,  Significant  and m a n t l e  and W h i t t l e  Pgm-2  are  effects  i n mammalian l i v e r activity  the  PGM w i t h e i t h e r  weaken  of  and  the  provide consistent  glycogen  significant  activity  of  synthetase  reactions exert  all  effect  In t h e  their  groups,  analyses  significant C.  enzymes,  genotypic  regression  between  low b u t  for  notable  the  two  that  the  relationships a not  of  significant i n the  high  224  intertidal  sample,  and GS r e v e r s e d these four  and t h e  in sign  differences,  intertidal  locations.  locus  glycogen  on  appeared to either  48  mantle  second  the  this  correlations  chromosome  of  correlations  reactions,  p a r t i c u l a r l y at  The s i g n i f i c a n t activities trend.  of  (0.58  in  lines).  D. were  lower  with  the  observed  Pacific  activity  discrepancy  of  al.  mantle  significant on  a  changes  seasonal  of  Pgm-2 has  activity  not with  substitution  in t h i s  lines  of  study  activities  hexokinase was  this  by t h e  the  general  significant  of  HK and  PGM  in t h i r d  chromosome  activity  exhibited  strongly  associated  pyrophosphorylase.  edulis,  marked  hexokinase  w i t h the  This  differences  organisms.  activity,and kinetic  coincides  between  highly  and 0.55  UDP-glucose  branch p o i n t .  with  however,  Mytilus  enzymes  enzymic  consistent  lines,  23  between s e q u e n t i a l  w i t h PGM, y e t  that  both  pattern  between t h e  in s p e c i f i c  basis  the  observed a  c o u l d p e r h a p s be e x p l a i n e d  tissue  the  between  i n c a r b o h y d r a t e m e t a b o l i s m between t h e s e g r o u p s of the  at  sample  its  (1982)  observed  oyster,  correlations  of  glucose-6-phosphate  melanogaster  s e c o n d chromosome  In the  much  et  PGM and UDPGP a r e  In  correlations  the  relationships  of  isogenic  Wilton  positive  fall  relationships  Drosophila melanogaster, significant  of  steps.  activity  and t h i r d  in  concentrations  influence  PGM  Despite  identical patterns  any  metabolism  between  tidal positions.  glycogen  Therefore,  t h e HK o r GS r e a c t i o n  of  two  groups e x h i b i t e d  occur through  In a s t u d y in  between t h e  the  Pgm-2 g e n o t y p i c  correlation coefficients  In  exhibits properties  utilization  of  225  endogenous v e r s u s times  of  activity with  the  year  may have  activities seasonal  in  glycogen could  Livingstone  is  adjacent  not  possibility this  enzyme  pathway  of  or  be  Both  relationship to  the  active  supplies),  their  uncoupled v i a  the  catabolic  pathways  locus  glycogen  on  genetic  variation  presumed  subsample  of  and  UDP-glucose no  allozymic  individuals  activity.  Glycogen  yielding synthetase  electrophoretically,  effects  due t o  at  genetic  so  the  variants  of  discounted.  i n Tables XVIII  and  XIX  v a r i a t i o n at  the  Pgm-2 l o c u s  observed  the  other  classes  for  differences  in their  sample,  not  of  monomorphic,  examined  Significant  heights.  Pqm-2  by p r e s e n c e  in the  be  that  were  the  were  presented  intertidal  differences  anabolic  of  confounding  enzymes.  intertidal  effectively  hexokinase  activity  Pgm-2 g e n o t y p i c high  distinct  loci  cannot  independently  be  (due  different  A l t h o u g h PGM  strong  glucose  steps.  not  of  enzyme  a  external  times  of  The r e s u l t s that  show  from  observed  could  to  at  1983).  sample  complicated  resolution  activity  and C l a r k e  seasonal  impact  pyrophosphorylase was  glucose  this  enzymic  variation  of  1981).  apparent  synthesis  expected  at  i n d u c t i o n of  The  good  (Livingstone  been  hexokinase  s y n t h e s i s of  (cf.  exogenous s u p p l i e s  important  caused  d i d not  glycogen  were d e t e c t e d  by t h e  GS a c t i v i t i e s  finding  was  that  unusual behavior  occur  synthesis  HK and UDPGP a c t i v i t i e s  and between t h e i r  An  demonstrate  of  between in  the  at  both these  Pgm-2-  226  92/100,  96/100,  samples,  heterozygotes  overdominance levels  and 100/104 h e t e r o z y g o t e s .  of  for  the  Pqm-2-100  for t h e i r a c t i v i t i e s  HK,  concentrations  UDPGP  or  displayed  indirect  consequence  synthetic  pathway.  GS. by  of  the  of  the  these  Pgm-2-100/100 PGM  high  homozygotes  class  of  area  that  2-100  were  UDPGP,  similar the  lower  overdominant  heterozygotes.  of  had  pathway  (with exception  for  the  lower  enzymes Pgm-2-100 mantle  different  low  glycogen  activity levels  yet  levels. of  the  an  entire  capable  of  of  to  and  resemble  i n the  the  glycogen  possessed  These r e s u l t s  HK, UDPGP,  the  Pgm-  homozygotes these  zone,  activities  PGM) t h a n t h e  the  Despite  than  high i n t e r t i d a l larger  in  intertidal  possessing  and 104/104  their  alternate  zone. low  than  100/104  homozygotes  intertidal  still  Pgm-2-92,  Excluding  96/100,  mantle  considerably  allele,  not  and GS a c t i v i t i e s  heights.  96/96,  In t h e  same h o m o z y g o t e s  glycogen  was of  their  the  the h e t e r o z y g o t e s  quantities  higher  appear  Pgm-2-100/100  Pgm-2-92/92,  in  Homozygotes f o r  a n d GS a c t i v i t i e s to  not  concentrations  both t i d a l  the  expressed  mantle glycogen  but s h i f t e d  in  but the  not.  Pgm-2-92/100,  region,  HK,  allele,  displayed  the  homozygotes  exhibiting  at  approximated the  intertidal  do  intertidal  allele  heterozygotes  had g r e a t e r HK, UDPGP,  activities,  heterozygotes  in  Pgm-2 g e n o t y p e s .  96 and 104 a l l e l e s  both  coordinated elevation  differences  o b s e r v e d between  PGM,  Therefore,  These non-random a s s o c i a t i o n s explaining  In  these  of  all  heterozygotes significantly  i n d i c a t e that  and GS e x p r e s s e d  the  between  227  the  Pqm-2  differing  In  genotypic  groups  mantle glycogen  Chapter  3  "regulatory"  locus,  structural  locus,  it  was  could  r e g u l a t o r y element  w h i c h was a s s o c i a t e d by  activity by t h e but  of  the  two homozygote  these  further  responsible possessing  for the  an u n d e t e c t e d pathway activities  the  Modifier enzymes  have  Potier  1980)  Belote  and  allele. allele  It  w o u l d have  two homozygous  been  observed  1980).  these  than  in  results  properties.  alleles,  The  the  different  any  shown  explanation, pleiotropic observations  null of  other  allele  being  heterozygotes  improbable impact  depressing  that  on  the  the  PGM  groups.  coordinate  If  two  These  seems h i g h l y  other  suggested  exert  Pgm-2  the  was  support t h i s  a  Pgm-2  pathway enzymes  enzymes.  against  having  unusual  104 a l l e l e s .  synthesis  the  allele,  r e g u l a t o r y element  loci  for  for  Pgm-2-100  and  and D . m e l a n o g a s t e r  responsible  extremely  w i t h the  structure  Lucchesi  their  producing It  o v e r d o m i n a n t PGM a c t i v i t i e s  Pqm-2 n u l l  of  with  for  segregating  pathway  evidence  activity  was  96,  this  Pgm-2-100  of  a tightly-linked  Pgm-2 g e n o t y p e s .  glycogen  other  the  responsible  groups appear to  r e q u i r e that  on  provide  is  Pgm-2-92,  levels  also  effects  the  of  that  disequilibrium  be  that  shared  predictors  hypothesized  in complete  enzyme a c t i v i t i e s  one of  poor  concentrations.  different this  are  effects  Mus m u s c u l u s (Bentley  this  and  putative  in C. gigas, First,  on  different  (Womack, Yan and Williamson  regulatory it  displays  in heterozygous  1979; element three  condition  228  it  gives  rise  t o o v e r d o m i n a n t PGM a c t i v i t y  allele  segregating  alleles  produces greater  homozygous  than  Third,  appears  it  relationships  with  the  of  the  three  HK,  UDPGP,  variant to  that  resemble  i n the h i g h i n t e r t i d a l , homozygotes  in Chapter  consistently at  s e c o n d and t h i r d  in  different  regulatory  element.  Pgm-2-100  allele  in  intertidal  the  two  the  intertidal  Pgm-2-100/100  Pgm-2-92/92,  intertidal. the  three  first  96/96,  The  results  property  different  by t h e  the  between since  homozygotes  equivalence the  three  of  an  in  this  overdominant  of  homozygotes  less  frequent  very  heterozygotes similar  UDPGP a c t i v i t i e s  predicted patterns  The  repeatability  b o t h w o u l d be homozygous  possessed  for  data presented  the h y p o t h e s i s  Homozygotes and  96/104 h e t e r o z y g o t e s  is  tissues  seasons.  to determine t h e i r  r e g i o n , and GS a c t i v i t i e s  However, the  the  low  the  dominance  tissues.  is  alleles,  regulatory  and  study  p r e d i c t i o n of  formed  in  suggested  further  locus  heterozygotes  area.  the  the  shown t h a t  positions  seasons and  A specific  high  3 have  effects  require  structural  in  but  the  when  allele.  observed i n the mantle and a d d u c t o r muscle  both i n t e r t i d a l  section  heterozygotes  Pgm-2-100  in  the  structural  activities  the  between  homozygote  presented  GS  r e g u l a t o r y genotypes  such  104/104  and to  Second,  frequent  reversals  positions  and  less  linked  cause  levels.  of  these a l l e l e s .  same  lacking  the  low  Further  were o b s e r v e d between  in  than  92/104, measured  discrepancies their  the  intertidal  Pgm-2-92/96,  were c o n s i d e r a b l y l o w e r  Pgm-2  the  HK a c t i v i t i e s  in the  the  for  and  from  low water HK  229  activities their  a n d h i g h w a t e r GS a c t i v i t i e s .  PGM a c t i v i t i e s ,  less  frequent  similarities  Pgm-2  homozygotes alleles  expected  by  t h e model s u p p o r t e d by t h e s e of  the  two  expressed  at  sample,  intertidal  both t i d a l  An  mantle  between  cells.  of  the  lacking  the  of  and d i s s i m i l a r  of  An  the  however, pathway,  inverse  overdominant  these strong  four  of  disparity  allelic in  classes the  enzymes  low  in  the  concentrations  the  different  c o u l d be t h a t  capacities  zone,  Pgm-2-100  glycogen  C o n v e r s e l y , the allele  homozygotes  glycogen  low  of  that  Virtually  was the  seen  lower three  50%  no e v i d e n c e  in  Pgm-2-100  two g e n o t y p i c  throughout were  simply  depressed  contradicted  activities  predicted  levels  for  and  these  in  mantle v e s i c u l a r  possessed  strongly  levels  heterozygotes.  levels  arose  present  is  relationship  where h e t e r o z y g o t e s  highest  GS a c t i v i t i e s .  of  enzyme  they  glycogen  relationship  supporting this  by e x p r e s s i n g but  all  amount o f  c o n c e n t r a t i o n s and h i g h e r a c t i v i t i e s Pgm-2-100/100  two  glycogen  for  classes  l i m i t e d storage  e x h i b i t e d the and  was t h e  c o n c e n t r a t i o n a n d enzyme a c t i v i t y  Some e v i d e n c e  UDPGP,  the  PGM a c t i v i t i e s  explanation  tissues.  high i n t e r t i d a l  allele  These  levels  Pgm-2 g e n o t y p i c  glycogen  because  however,  groups.  an i n d i r e c t c o n s e q u e n c e  their  the  of  exhibit  for  heights.  alternative  activities as  results  altered  sample,  not  from  r e g u l a t o r y m o d e l . One f e a t u r e  substantially different  intertidal high  heterozygous  apart  and h e t e r o z y g o t e s  did  the  Therefore,  HK,  groups  glycogen  enzymes.  The  this  pattern  the  entire  lower  than  favoring  the this  230  explanation Therefore,  was even  observed  if  glycogen  in  the  low  intertidal  c o n c e n t r a t i o n s had  t h e pathway enzyme a c t i v i t i e s ,  some o t h e r  some  factor(s)  sample.  effect  on  must a l s o  be  involved.  The m u l t i p l e r e g r e s s i o n a n a l y s e s already  evident  enzymes,  o n l y PGM e x p l a i n e d a s i g n i f i c a n t  variation observed more  in  complete  of  and the  also the  gigas  operating  of  below  its  haemolymph g l u c o s e 1981;  by  proportion  the of  in  a  The  concentrations  2)  its  synthetase  g l y c o g e n was  et  of  this  al.  (1986)  hexokinase  that  this  in  reaction  glucose-6-phosphate  of  effects  1)  levels the  because  control  in  the  of  in C.  reaction of  of  reaction,  synthesized  glycogen  metabolism  i n marine b i v a l v e s  1983),  The  identified  on m a n t l e g l y c o g e n  potential  PGM was  The c a t a l y t i c  non-significant  consequence  were  area. or  Torres  suggesting  observed  of  carbohydrate  in supplying  catalytic  glycogen  have  coefficient  be 0 . 7 7 ,  L i v i n g s t o n e and C l a r k e  overridden large  be  hexokinase  Recently,  variation  perhaps  t-ratio  intertidal  hexokinase  site  glycogen.  activity  could  of  a dominant r o l e  synthesis  but t h e  the  was  pathway  results  rather surprising.  glycolytic control  exert  hexokinase  is  four  amount o f Similar  high  either  1973).  l i v e r p r e p a r a t i o n s to  may  the  regulatory Start  levels.  by  properties  a major  (Newsholme estimated  in  effects  and X I X . Of t h e  samples,  in these analyses  regulatory  enzyme as  for  glycogen  significant  absence  synthetase  rat  mantle  for both i n t e r t i d a l  highly  and  from T a b l e s X V I I I  s e r v e d t o c o n f i r m what  the  low  (e.g.  Zaba  flux  being  o r 3) fall  that  a  from  231  gluconeogenic  precursors.  The  of  activity  been  found to  and  Hers  this  enzyme  mantle  directly  1974,  of  PGM a c t i v i t y  corresponding at  first  an  appear  to  distinction of  rates  rate  of  concentrations  their  greater  activities.  be  area  influence  effect  the  96/100,  for  of  synthesis  single  C h a p t e r 4,  these  flux  population  would be o b s c u r e d  i n the  sample, by e f f e c t s  the of  for  PGM. G l y c o g e n  higher  to  flux of  GS,  a  glycogen  the  result  influence  of  the  locus.  the  not  of  PGM,  variation  is  partitioning (cf.  rates  genotypic  Pgm-2  the  a combination  branch point  other  of  controlling  through  should  regulatory  modulation  higher  to  However,  low compared t o  due  PGM a c t i v i t i e s  than observed  and  PGM a c t i v i t y  responding  100/104 h e t e r o z y g o t e s  studies.  The  rates  glucose-6-phosphate In  may  enzymes l i k e  and  significant  w i t h GS a c t i v i t y ,  flux,  likely  that  the  from t h e  growth"  in  overdominant  and  most  (Hue  molluscan  between a  responsible  in oysters  W a l s h and K o s h l a n d 1 9 8 4 ) . by G S , t h e  pathway  The  has  and  made  synthesis.  is  shown  expected to at  be  in  glycogen,  these previous  largely  "scope As  mantle  rates  accumulating  1983).  relationship  overall  measured  intertidal  on  is  control  in Gabbott  must  glycogen  high  of  by n o n - r e g u l a t o r y  s y n t h e t a s e may w e l l net  any  contradict  enzyme's d i c t a t i o n flux  13  synthesis  Evidence  degree  variation  absence of  important  these  Figure  i n mammalian l i v e r  glycogen  1976).  exerts a similar (see  synthetase  parallel  Stalmans  tissue  effect  glycogen  LaPorte, determined  Pgm-2-92/100, in higher classes.  glycogen  rates In  a  synthetase  However  if  Pgm-  232  2 genotype over  a  is  period  correlation reported Gabbott  The be  of  active  between  in  and W h i t t l e  impact of  the  between  Pgm-2 l o c u s  were  the  lacking  were  in turn  synthetase  the  the  were  Although  observed  correlated  Compared  than the to  the  heterozygotes glycogen a major flux  factor  rates  levels,  as  (1979),  and  11%  Pgm-2-100  in  other  in  similar  low  and  on  the  average  homozygotes. genotypic  the  The  classes  Pqm-2-100/100  levels  of  glycogen  in glycogen  classes  that  levels  were d i r e c t l y  The o v e r d o m i n a n t c l a s s  96/96,  and  heterozygote  group,  demonstrate  in determining glycogen  and  104/104  31% more PGM a c t i v i t y  appear s t r o n g l y a f f e c t e d  were  the  groups  PGM a c t i v i t i e s  These p a t t e r n s  In  possessed  measured  by Pgm-2  13%  of more  homozygotes.  the overdominant and  that  levels,  of  intertidal  activities  classes.  these three  these a l l e l i c  Pgm-2-92/92,  both  Pgm-2-100/100  of  would  their activities  these  allele  than the  higher  synthesis  heterozygous  PGM a c t i v i t i e s .  expressed  levels.  in  exhibiting  between  had  However  two  than  if  substantial differences  with their  heterozygotes glycogen  larger  activity,  only  genotypic  concentrations  21%  homozygotes.  equal.  four  17% h i g h e r GS a c t i v i t i e s glycogen  taken  would d e m o n s t r a t e a s t r o n g  on g l y c o g e n  differences  sample,"  homozygotes  measurements  Cook and W h i t t l e  f o r ' d i f f e r e n t genotypes  intertidal  mean  synthesis  by G a b b o t t ,  significant  detected  similar  (1986a).  synthetase  samples,  of  GS a c t i v i t y and m a n t l e g l y c o g e n  M. e d u l i s  similar  glycogen  ignored, a series  but  43%  higher  GS a c t i v i t y the  is  realized  genotype-dependent  233  enzyme a c t i v i t y  variation.  An i n t e r a c t i o n between mantle  glycogen  was a l s o  PGM and  evident  b u t was c o m p l i c a t e d by t h e  highest  c o n c e n t r a t i o n s of four  important of  allelic  assumption that  over  the  been  of  the  inhibiting  glycogen  proportion  of  pathway  the  enzymes  phosphatase  and  levels  in heterozygotes the  depressed  synthetase  1980).  sample  the  inhibitory  of  of  this  total  possible allele  their  genotypic  the  the'GS  were  larger  subsequently  D)  classes  p r e d i c t i o n , but these  by  1965;  that  higher  plus  (I  has  lowering  (Danforth  have been of  activity  own s y n t h e s i s  form  is  but  effect  the  Pgm-2  it  conclusions  Glycogen  and t h u s  (I)  an  remained unchanged  its  Pgm-2-100  collected  E s t i m a t e s of  activity  provided a test  Therefore,  for  was  t h r o u g h the  concentrations.  i n the a c t i v e  light  the  sampling date.  synthetase  the  activities  the  that  have  r e g u l a t o r of  enzyme  Malthus  namely,  a negative  Watts  before  analyses,  sample,  exhibited  This discrepancy brings to  time p e r i o d p r e c e d i n g the  i m p l i c a t e d as  affecting  Pgm-2-92/100,  l o w e s t GS  significantly affects  the m u l t i p l e r e g r e s s i o n  relationships  the  These genotypes  g l y c o g e n and the  classes.  in  high i n t e r t i d a l  u n u s u a l b e h a v i o r of  a n d 100/104 h e t e r o z y g o t e s .  the  activity  in the  96/100,  of  GS  glycogen glycogen  could  assays  have  were  not  by  the  performed.  The  expression  heterozygotes  f o r the  area p r i o r  the  to  of  overdominant  Pgm-2-100  sampling date  allele  GS in  activities  the  high  intertidal  c a n n o t be d i s c o u n t e d . However i n  234  the  high  activity its  this  tidal  levels,  height.  explanation  magnitude  Another  is  (Chapter  inhibition  the  heterozygotes  100/100  fact  t h e GS a c t i v i t i e s  of  great  that  levels the  than  i n the  i n the  the  Pgm-2-100  t h e s e g r o u p s may have  92/100,  96/100,  and 100/104 h e t e r o z y g o t e s  sample  could  indicate  2-100  suited  i n the  with  those present  i m p o r t a n c e of  measured of  in  this  fall  is  this  potential  expected  that to  previously  c o n c e n t r a t i o n s of high  synthesis  of  and h e t e r o z y g o t e s  of  that  been the  to  Pgm-2-  suggest  t h e i r pathway a c t i v i t y the  homozygotes  and the  sample  i n the  at  concentrations  allele  high i n t e r t i d a l  The h i g h e r g l y c o g e n  expressed  be r e c t i f i e d  The c o m p a r a b l e HK and UDPGP a c t i v i t i e s  levels.  optimally  of GS  extent  similar  more  to  inhibition  mantle glycogen  on GS a c t i v i t y  possessing  homozygotes  the  f a c t o r d i s c o u n t i n g the  Therefore,  by g l y c o g e n  of  t h e s e were below  higher  4).  have been n e g l i g i b l e .  f a r too  since  reach considerably  season  the  the  by g l y c o g e n a p p e a r s  observed  other  can  intertidal,  at  Pqm-2-  intertidal levels  glycogen lacking  are than  t h e Pgm-  allele.  In summary, t h e a c t i v i t y synthesis effect  pathway enzymes p r o v i d e s  of  evidence  the  Pgm-2 l o c u s  suggesting  regulatory  element  heterozygotes these other glycogen  r e l a t i o n s h i p s between  additional  the  enzymes,  levels  on g l y c o g e n  producing  for  of  the  but none  evidence  favoring  of  the  overdominant a c t i v i t y  that  allele  is  an some  putative levels  of  on t h e a c t i v i t i e s  of  can account  genotypic  glycogen  metabolism. There  effects  Pgm-2-100  these  further  the  groups.  for  the  differing  These  results  235  demonstrate  a surprisingly  at  locus  the  Pgm-2  expected  from  branch p o i n t  on  strong  metabolic  a partitioning  discussed  effect  of  flux,  consistent  effect  in Chapter  4.  at  the  allozymic  variation with  that  glucose-6-phosphate  236  CHAPTER 6  GENERAL DISCUSSION  Heterozygotes been  at  shown by F u j i o  than  homozygotes  five  (1982) in  20  gigas.  Although s i m i l a r  in  present  the  that  the  study,  Columbia  of  (e.g.  and  1985).  Fujio  these  example allele  the  i n the  identical  studied  of  gigas if  a  exist  i n the  body  of  been  reasons in  for  believing  British  Columbia  of  genetic  the  frequency  i n the  diverged  of  heterozygosity  studied.  Pacific.  For  the  most  common  was  almost  populations similarity  coasts  responsible it  at any  The c l o s e  relationships,  Ozaki  to  (0.595)  between b o t h P a c i f i c is  nearly  1979a;  23 J a p a n e s e  (1985).  are  distributions  and e a s t e r n  population  mechanism(s)  B . C . population  not  effects  variability:  regions  frequency  have  western  and F u j i o  British  a c c o m p a n i e d by f o u n d e r  loci  locus,  weights  Crassostrea  colonization  of  have  demonstrated  H e r s h b e r g e r and Chew 1975,  mean f r e q u e n c y  genetic  not  from b o t h g e o g r a p h i c  populations  multiple-locus  The  levels  Bay s t u d y  by O z a k i  have  Pgm-2)  adult  populations  present  been  between t h e Pgm-2  its  (0.596) C.  not  enzyme  Nanoose  to  greater  Furthermore, a l l e l i c  extent  at  be  species.  Buroker,  polymorphic  appreciable  that  this  (including  are h i s t o r i c a l  would  estimates  identical  exhibit  Japanese  reducing  heterozygosity  loci  relationships  by C . g i g a s has  significantly  to  there  same p a t t e r n s  populations  enzyme  suggests  for should  these also  237  Fujio  (1982)  explanation loci  proposed  for  the  that  greater  because a s i g n i f i c a n t  between  the  magnitude showed  their the  20 p o p u l a t i o n s  with  inbreeding coefficients  fact per  only  the  activity  is  was  others all  homozygotes, If  the  s h o u l d have  been  high F  values.  shows t h a t for  into  nearly  than  the data  the  The  inbreeding  larger  body  three  that  overdominance  most  common h e t e r o z y g o t e s  gigas.  identical  this  these  in three  both  the  show  (confined same  These  only to  size results  of are  at this  mantle  and  at  both  seasons.  Since,  limited  inter-  three  less  the  genotypes  enzyme  of  observed  different  locus  increased  study.  in  for  The m a g n i t u d e  and was c o n s i s t e n t l y  sampled  the  and  In  pattern  samples  Crassostrea  at  his  inbreeding.  exhibiting  and  partitioned  unity.  responsible  by t h e  tissues,  in F u j i o ' s  this  observed  However,  apparent  in a l l  differentiation  for  to  five  oysters.  nearly  alleles),  responsible  the  correct,  been  in  frequencies  frequent  of  demonstrated  locations  populational  reported  has  muscle  intertidal allele  have  locus  overdominance  samples  y i e l d i n g high F values,  populations  expressed  Pqm-2  adductor  is  heterozygous  study  population  c o u l d be e q u a l l y  likely all  was  the  close  was p r e s e n t  c o u l d not  My  extent  i n the  it  weights of  the  of  most  relationship  i n d i v i d u a l s were l a r g e r  hypothesis  that se  ones  heterozygous  inbreeding observed  groups;  the  h e t e r o z y g o t e s at  inbreeding c o e f f i c i e n t s .  distinct  irrespective  of  negative  two  samples,  sizes  mean body w e i g h t s of  of  that  i n b r e e d i n g was  must  Pgm-2 unique  have  been  heterozygotes in providing  238  evidence  favoring  involved  in  a  allele,  and  with  and t h e  unidentified recessives,  association  growth  by d i f f e r e n t  incompatible  o v e r d o m i n a n c e e x p l a n a t i o n f o r one  positive  heterozygosity expressed  the  rate.  the  associative  may t h e r e f o r e  Although  entirely  "regulatory" appealing activity  levels  genetic  ten  the  the  Pgm-2  in Chapter  genotypes,  populations,  protein levels of  pathway.  These  vicinity  of  the  adjacent  deleterious  i s unknown. polymorphic  is  intuitively  explain  the  allelic  frequency  by  distilling  that  that  may  of the  polymorphism e x h i b i t i n g  with  enzymes  enzyme  and the maintenance  the  overdominance  of  the  and  the  glycogen  heterozygosity  scored in m u l t i p l e - l o c u s studies  f o r the  invoking  were p l e i o t r o p i c e f f e c t s  suggest  number o f m e t a b o l i c e f f e c t s explanation(s)  3  measured i n b o t h t i s s u e s  results  loci  associated locus  null  o v e r d o m i n a n t PGM  overdominant  i n t o a two a l l e l e  However,  for  Pqm-2-100 a l l e l e  simultaneously Pgm-2  natural  system  at  activities  can  the  v a r i a t i o n in a balanced s t a t e ,  overdominance.  soluble  of  in  multi-allelic  observed  it  were  unnecessary.  the  l o c u s model d i s c u s s e d  distributions this  for  levels  an u n d e t e c t e d  hypothesis,  producing  speculative,  because  of  segregating  be deemed  heterozygotes  activity  and h e t e r o z y g o t e s  overdominance loci  multiple-locus  enzyme  predicted effects  tightly-linked  of  The  Pgm-2 h o m o z y g o t e s  The u n d e r l y i n g mechanism activities  between  locus  act  c o r r e l a t i o n s observed.  to  on  the  mantle  synthesis in  the  could exert  complicate  a  the  239  Genotype-dependent locus  was  shown  differing  in Chapter  glycogen  adductor muscle interactions glycogen  heterozygotes two to  tissue.  to  be  relative  reversed  in  positions  of  the  genotypes allows kinetic  versus  specific  activity  behaved  as  four  enzyme a c t i v i t y  a  data,  homozygotes  acted  as  -2-92  allozyme  have  advantage of  this  over  the  variant  concentrations  shown of  manner c o n s i s t e n t genotypes tendency  having to  others in  the  low o b s e r v e d  frequency  themselves that  give  the  of  locus.  2.  enjoy  However,  enzyme  activity  allele  showed  as  expected  These  Pgm-2-92  For  Pgm-2-100  specific  a  allele,  the  the  activity t h e Pgm  catalytic properties  the  glycogen  differed  in a  differences:  from  no  their  combined w i t h  argue  could  be  n e u t r a l markers of  rise  to  the  differences  of  allele  absolutely  results,  a  Pgm-2  importance  and h e t e r o z y g o t e s  properties.  In a d d i t i o n  p o p u l a t i o n samples;  to  the  polymorphism,  this  the  and  and o v e r  superior kinetic  Chapter  others  of  this  relative  for  mantle  homozygotes  genotypes p o s s e s s i n g  the  Pgm-2-92  outperform  in  and w i n t e r ,  expected  only with t h e i r  kinetic  differences  due t o  homozygotes  advantageous  allozymes  been  the  the  homogeneous  these groups,  not  of  group i n a l l  W i t h i n b o t h of  but  analysis  v a r i a t i o n at  group.  may  mantle,  environment  the  another  with  concentrations  heterozygotes  homogeneous  associated  by  i m p a c t of  of  Pqm-2  summer s a m p l e .  glycogen  an a s s e s s m e n t  the  genotype  fall  w i t h i n the  mantle  the  measured  between t h e  demonstrating a p h y s i o l o g i c a l  comparison  in  the  levels  v a r i a t i o n at  directly  Complex Pgm-2  observed  the  intertidal  4  activity  concentrations  were  data:  enzyme  that the  in  the  genetic specific  240  activity.  The  adaptive  realized  only  if  it,  Pgm-2 g e n o t y p e s . this  of  fuel  for  effect  on  viability  glycogen  stores  Since  glycogen  for  Pqm-2-100  advantage.  more of  for  The  the  allele  as  (de  strongly  marine  in older at  the  components;  on  virginica size  1983).  al.  in Mytilus  This  of  their  1983),  major  heterozygotes  between  somatic  g r o w t h and  Rodhouse  (1978)  is  a  the  fecundity  has  viability  as  i n immature  increase thus  to  expected  been d e s c r i b e d  to  2 and 3 y e a r s  1983), edulis  shift  in age.  individuals. Interestingly,  (Zouros et  class  important  ( H o l l a n d and Hannant  enjoy  bivalves  Pgm l o c u s  adverse  net  energy  shown by  reserves  under  i m p l i c a t e d as  also  important  proportion  (Gabbott  levels,  glycogen  Zwaan  w i n t e r months  of  differential  pronounced  p o l y m o r p h i s m on f e c u n d i t y  significant  largest  of  two  carbohydrate is  higher  could  fitnesses  The  synthesize  more  much  been  influence  particularly  gametogenesis  latter  this  Crassostrea  1986).  has  allocation  heterozygosity  the  to  be  the  p r o d u c t i o n has been  fitness  of  a  during  source  i m p a c t of  to  fecundity.  when t h i s  should  relative  on m a n t l e g l y c o g e n  potential  viability,  utilize  energy  towards  effects  o v e r d o m i n a n c e w o u l d be  the  supporting basal metabolism  which  gamete  this  affected  genotypes  conditions  oysters,  the  the  affect  environmental  its  of  v i a b i l i t y and  Pqm-2  could d i r e c t l y  1976).  has  components:  abilities  in t u r n ,  Through  polymorphism  fitness  a  -significance  and on t h e (Rodhouse  to  an  be  effect  for of  The  both age  in  fecundity et  al.  241  In been  Crassostrea  significantly  Since  these  differing  qigas,  correlated  results  age  were  In o t h e r  physiological  parameters  This  been  advantage  relationships consumption  al.  lower  heterozygosity  Understanding  the  provide a foundation  weight and t h e  are  Garton,  al.  loss  for  or  the  of  number  heterozygous  than  homozygotes.  i n the  rates  of  Koehn  and  oxygen  that  Bayne stress  in  some  correlated  with  environmental  and Beaumont responsible  enzyme h e t e r o z y g o t e s of  Scott  (Hawkins,  stressful  explanation  negative  under n u t r i t i v e  Gentili  of  that  significantly  1983;  multiple-locus a  observation  more  efficiency  and o t h e r  Recently,  and  b i o c h e m i c a l mechanism(s)  metabolic  in b i v a l v e s  of  under  et  growth r a t e s  and p r o t e i n t u r n o v e r  rates  (Green  if  presumably r e f l e c t e d  1984),  only  determined  with  suggest  weights. of  marine b i v a l v e s ,  heterozygosity  rates  growth  increased  of  has  20 p o p u l a t i o n s  with d i f f e r e n t i a l  which  1985)  (Rodhouse and G a f f n e y  body  be  correlated  is  between  et  and Day 1 9 8 6 ) ,  conditions  cannot  (Koehn and Shumway 1982;  Diehl  instances  adult  a r e m e t a b o l i c a l l y more e f f i c i e n t  energetic  1984;  it  species  has  with  heterozygosity  o b t a i n e d by p o o l i n g  was a s s o c i a t e d  heterozygosity  individuals  only  structures,  heterozygosity viabilities.  multiple-locus  these  1988).  for  the  may  thus  relationships  species.  Koehn,  relative  c o n t r i b u t i o n of  between  multiple-locus  Diehl 15  and loci  Scott  (1988)  to  positive  heterozygosity  a  and  examined  growth  the  correlation rate  in a  242  single was  cohort  unique  of  the  in  enzymes t h a t  functioned  not  for  or  the  generation  enzyme  those  and t o  of  cost  and  degradation  of  of  reactions  metabolic  more  advantage  first  allelic  developed  by  balance  for  these  effects  (p.  (i.e.,  of  128):  biosynthesis  enzymes)  is  inversely  variation  among  pathway."  expenditures  of  physiological  diversity  by E a s t  the  more h e t e r o z y g o u s  efficient  of  redox  that  catalytic  in a  that  for  proposed  constituent  study  catabolism,  To a c c o u n t  pathway m a i n t e n a n c e  predicts  a  protein  explanation  magnitude  sequence of  in  and  This  contribution  in maintaining c e l l u l a r  a r e m a i n t a i n e d by l o w e r  resulting  ideas  of  the  explanation  significant  these authors  the  to  lateralis.  reducing equivalents.  "...  pathways  a  provide a general  related  This  Mulinia  in glycolysis  involved  heterozygosity,  the  clam,  demonstrating  but  results  coot  (1936),  is  an  biochemical  energy,  thus  phenotype.  This  extension  Lerner  (1954)  may be  interpreted  of  and B e r g e r  (1976).  The r e s u l t s manner  consistent  expression acting  from t h e  of  on t h e  with  this  the  well-founded  that  the  "averaging  out"  of  two homozygotes differentiation shown  study  above  overdominance.  suggested  allozymes  present  basis  "advantage" the  (i.e.  premises,  Koehn, of  of  Diehl  exhibited  heterozygosis  in Chapter  the  2 appears  four  to p r e c l u d e  the  intermediacy, arises  from  the  of  the  The minor  most  in  a  (1988),  performances  overdominance). by  and S c o t t  heterozygote  differing catalytic marginal  but d i f f e r  in  common this  kinetic Pgm-2  mechanism  243  from  operating  activities  at  of  heterozygotes  this  the  locus.  Instead,  Pgm-2-92/100,  the  greater  96/100,  has p r o v i d e d d i r e c t e v i d e n c e  for  and the  unconditional  b i o c h e m i c a l overdominance. Because  a monomer, t h e  l a r g e r enzyme a c t i v i t i e s  a r e most PGM  easily  enzyme.  e n v i s a g e d as  Steady-state  t h r o u g h a b a l a n c e between degradation enzyme  (Goldberg  activities  heterozygotes experience However, the  for  higher, if  overdominant  St. from  1976).  allele  lower,  a r e the  activity  levels  metabolic  of  decreased  rates  the  their  increased  arguments suggest  Pgm-2  locus  altered  feature  higher  heterozygotes through  of  1)  rates  may  not  that be  the  the  heterozygotes, of  and  the overdominant  synthetic  rates,  w o u l d be e x p e c t e d  genotypes, lower  would r e s u l t reserves.  of  to  requirements.  for a l l  genotypes  of  growth  rates,  through the  The  would  but  increased  in  turn  be  and  also  in  oxygen c o n s u m p t i o n .  These  "efficiency"  The  of  these  is  achieved  synthesis  are degraded at  c e l l u l a r energy  in  If  are  energetic  same  r e d u c e d e x p e n d i t u r e of  reflected  enzyme  of  heterozygotes  of  increased  Pqm-2-100  rates  efficiency  this  concentrations  John  enzyme e n s e m b l e s  enzyme  expression  these  opposing rates  r a t h e r than  synthetic  heterozygous  the  and  the  100/104  a r i s i n g through increased l e v e l s enzyme  result  of  specific  but  routine metabolism,  Vmax/Km could  ratios produce  responding  more  of a  more  overdominance observed cause  of  the  at  increased  rather  an  such as  protein turnover.  the  overdominant  efficient  effectively  effect  to  phenotype  the  flux  of  an  Pgm-2 only rates  244  dictated  by  regulatory  simply c a t a l y z i n g efficiently (1986).  as  is  biosynthetic  If  no  the  direct  these  of  this  impact  4.  The  of  this  point.  First,  degradation  fecundity activity  The  not  i n the  a  dual  region  an i n d i r e c t  the  on g l y c o g e n  expression for  the  the  effect  of  reduced  the  Pgm-2  necessarily  predicted  efficiency in higher  mantle  observation  argues flux,  for  as  a  major  expected  might in  of  from  branch  result  from  Crassostrea  from r e d u c e d r a t e s  of  protein  overdominant  enzyme  impact this  of  between PGM a c t i v i t y  Pgm-2 l o c u s  direct  effect  than  glucose-6-phosphate  expression  a  either  consistent  fall  the  Watt  turnover,  result  a  of  protein  advantage  of  i n the  second,  through  at  of  more  from  at  increased  relationship  substrate  i n the  and  heterozygotes  rate  2)  explanation.  heterozygosity  the  was  levels  (resulting  activities),  this  lower  or  G6P  analysis  alternative  p o l y m o r p h i s m on m e t a b o l i c  Therefore,  heterozygosity  much  indirectly  strong  and g l y c o g e n  p a r t i t i o n i n g of  gigas.  could  and  result  polymorphism i s  Although  concentrations,  variation  the  of  metabolism.  Chapter  probably  of  G1P  would  from a d e c r e a s e d  heterozygotes  glycogen  but  of  synthetase,  theoretical  that  advantage  solely  effect  on g l y c o g e n  the  p r e d i c t e d by t h e  metabolic  resulted  by  advantage  unknown, costs  glycogen  interconversion  suggested  The e n e r g e t i c  mechanism  locus  the  enzymes l i k e  on  viability  variation  in  or  enzyme  metabolism.  of  overdominant  Pgm-2-100  allele  is  enzyme  activities  extremely  unusual,  by and  245  thus  unlikely  associations related locus  traits in  Zouros,  provide  Singh  these  PGI  and  growth  Miles  (1979)  allozymes  three  most  However,  in  activity  their  advantageous conditions.  in Given  PGI l o c u s  rate  face  these  genotypic  has  1985),  (Lap)  allele  the  of  dependent  locus.  greatest  allelic effect  variation  on in  in  PGI  positive  Zouros  1978;  (Zouros et  al.  properties  of  between  specific  fluctuating  enzyme  intermediacy as  being  environmental  relationships,  heterozygosity  into  the  general  (1988).  polymorphism in M. e d u l i s ,  implicated  in  1984),  relationships  morphological  ( D i e h l and  et  al.  enzyme  Koehn  1985),  Genotypes  variant  the  interpreted  and S c o t t  viability  at  strict  fit  the  fitness-  differences  their  were  caused  at with  variation 1985)  and  involves  the  possessing  activities  (Koehn and Immerman 1981)  this  physiological  been  (Diehl  aminopeptidase-1  kcat  viability  could  and G a f f n e y  consumption  environments  Diehl  and  and  exhibited  of  well-studied  (Koehn  exhibit  (Singh  in  for  involved  biochemical  that  in C. v i r g i n i c a  ( M i t t o n and Koehn oxygen  the  the  which h e t e r o z y g o s i t y  been  and  genotypes  levels  particularly  growth  1980)  heterozygotes  scheme p r o p o s e d by K o e h n ,  A  has  and f o u n d s i g n i f i c a n t  common  activities.  heterozygosity  rate  examined  explanation  Heterozygosity  virginica  with both  Martin  the  general  in marine b i v a l v e s .  Crassostrea  1983).  at  a  between m u l t i p l e - l o c u s  correlations  the  to  the  in high  salinity  the  greater  (Koehn and S i e b e n a l l e r  1981). A  osmoregulation activity  has  been  of  by  Lap-94  this  documented  genotype(Hilbish,  246  Deaton  and  Koehn  significance 1987). is  of  Hilbish  this  at  d e p e n d i n g on t h e  94/94  the  locus  at  with  the  Foltz  (1986a,  segregating resulting the  at  low  potential  functional  these  and  alleles  Zouros,  results  has  Lap  misclassification  deficiencies  1978;  1986b)  of  three  mechanism involving explanation  to  of  in  not  be  multiple-locus these  studies.  This  is  apparent  a  the L a p -  homozygotes  in  expression although  The p r e v a l e n c e  other  polymorphic  detected  null  heterozygotes explain  of loci  alleles  et  that for  al.  a  1983).  single  the  two  The at  genetic  correlations  Rather, to  for  relationships  these  heterozygosity.  Although delaying  heterozygote  ( S i n g h and Z o u r o s  genotypic  may have  homozygotes  heterozygotes  Foltz  responsible  as  the  studies  suggest  patterns  groups  C r a s s o s t r e a v i r g i n i c a . The  and t h e  may  and  unknown.  1980;  strongly  locus-specific  at  reported in e a r l i e r  loci  biochemical  p o o l i n g of  dominance.  null  other  dominance  Lap-94 a l l e l e  s u p e r i o r performance of  study  that  94 a l l e l e .  the  the  simultaneously  present  for  the  recently  locus  S i n g h and M i l e s  the  is  the  in the  for  strict  adaptive  by Koehn and H i l b i s h  activity  would r e s u l t  relationships  the  of  since  two  in these c o r r e l a t i o n s  the  form two homogeneous  or absence result,  studies,  dominance  both  Lap genotypes  u n d e r l y i n g mechanism i s  involved  explain  have d e m o n s t r a t e d  o v e r d o m i n a n c e by h e t e r o z y g o t e s  similar  has  this  important  multiple-locus of  (1985)  presence  genotype  can  polymorphism (reviewed  levels:  particularly  which  and Koehn  expressed  physiological  1982)  depend on ultimate  a  full  separate goal  of  247  explaining  these  undoubtedly relevance selective systems.  stimulating  increase of  enzyme  process,  and  our  observations,  understanding  polymorphisms, the  evolution  the of  this  of  approach  the  functional  intricacies complex  will  of  the  biochemical  248  LITERATURE CITED  A l e m a n y , M . , and M . 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