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Respiratory properties of mitochondria from heart and mosaic muscle of rainbow trout (Salmo gairdneri)… Donaldson, Judith Margaret 1985

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RESPIRATORY MUSCLE  P R O P E R T I E S OF MITOCHONDRIA OF RAINBOW  UTILIZATION  TROUT  AND  FROM HEART  (Salmo q a i r d n e r i ) s  MOSAIC  SUBSTRATE  R E S P O N S E TO T E M P E R A T U R E  EXTRAMITOCHONDRIAL  AND  AND  pH  By JUDITH B.Sc.,  A  Mount  MARGARET  Allison  THESIS SUBMITTED  REQUIREMENTS  FOR  DONALDSON  University,  IN P A R T I A L  N.B.,  FULFILLMENT  T H E D E G R E E OF MASTER  OF  19B1  OF  THE  SCIENCE  in THE  F A C U L T Y OF GRADUATE ZOOLOGY  We  accept  this  thesis  THE  DEPARTMENT  as conforming  UNIVERSITY  to the required  OF B R I T I S H  October ©Judith  STUDIES  Margaret  standard  COLUMBIA  1985  Donaldson,  1985  90  In  presenting  degree  this  at the  thesis  in  partial  University of  British  fulfilment  of  department publication  this or of  thesis for by  his  or  scholarly her  Zoology  The University of British Columbia 1956 Main Mall Vancouver, Canada V6T 1Y3 Date  DE-6(3/81)  I further  October 12, 1985  requirements  purposes  may be  representatives.  for  an advanced  Library  shall make it  agree that permission for  It  this thesis for financial gain shall not  permission.  Department of  the  Columbia, I agree that the  freely available for reference and study. copying  of  is  granted  by  understood  the that  extensive  head of  my  copying  or  be allowed without my written  i i  ABSTRACT Mitochondria muscle o-f  were i s o l a t e d  rainbow t r o u t  respiratory pyruvate,  rates  were  The  determined  -final  generate pH p r o f i l e s . all  (Salmo q a i r d n e r i at 5  and  R. ) .  mosaic State  and  15°C  cases,  three  substrates  were  Pyruvate was o x i d i z e d  indicating  carbohydrate metabolism.  good  At 15°C, malate  used  at high  potential  3  using  m a l a t e , l a c t a t e , glutamate or a c e t y l — c a r n i t i n e  substrate.  in  -from heart  as to  rates  for  aerobic  was an  equally  good s u b s t r a t e f o r heart m i t o c h o n d r i a , while a l l  substrates  were o x i d i z e d  in  muscle  of  heart  at  mitochondria.  similar  rates  Maximal  mitochondria were g r e a t e r  to  oxidation  pyruvate rates  than or equal t o those of muscle.  State 3 D i o for oxidation  of most s u b s t r a t e s i n heart  approximately  2 , except f o r  Mitochondrial  oxidation  malate which had  tended  t o be  more  a Q i o of  3.  sensitive  to  decreased temperature i n muscle than i n h e a r t , with r e s p e c t  to a c e t y l — c a r n i t i n e  which i n muscle had Q ± o  particularly  and glutamate  v a l u e s of 4 and 7 ,  was  oxidation  respectively.  Based on RCR v a l u e s at 5 and 15*=^, t h e r e was no  indication  t h a t membrane p e r m e a b i l i t y t o H"*" i o n s was a l t e r e d by a 10C change i n temperature i n  mitochondria from e i t h e r  At pH above 7 . 6 r e s p i r a t o r y pH.  State  3  0  tissue.  r a t e s decreased with i n c r e a s i n g  respiratory  rate  increased  in  mitochondria as pH decreased, below 7 . 6 w h i l e i n muscle  heart  mitochondria,  no  such  values  were  Muscle  mitochondria  with  above  respect  oxidative  rates  sensitive greater  to  heart,  the  that  changes  both  those  less  of  muscle  that  tissue  which  sensitive  and  populations  needs  pH  each  in  high  had  with  of  tissue  for  the  than  and  the  mosaic  face  larger do  those  It  p a t t e r n s and  the  less  to  vitro.  different  pH  higher  was  response  e x t r a m i t o c h o n d r i a l pH was  pH.  were  relative  in vivo  RCR  extreme  and  typically  to  environment of  at  to  i n keeping  substrate utilization  intracellular  except  mitochondria  temperature,  i n temperature  different  supply.  Heart  mitochondria  mitochondrial the  RCR.  observed.  more s e n s i t i v e  i n e x t r a m i t o c h o n d r i a l pH  were  concluded  two  were t h e  decreased  was  a l l experiments  than  Muscle  fluctuations  to  in  dependence  o x i d a t i v e demands of  muscle.  of  to  4  pH  in  the  reflected  mitochondria aerobic  and energy  i v  TABLE QF CONTENTS List  of F i g u r e s  .  List  of T a b l e s .  .  Acknowledgements Introduction  . .  .  .  I s o l a t i o n of  .  .  .  . .  M a t e r i a l s and Methods Chemicals .  .  .  .  .  .  .  .  Spetrophotometric .  Temperature  .  Effect  .  of pH .  .  .  Effect  .  Literature  Cited  .  1  .  .  .  7  .  7  .  .  .  .  .  10  .  16  .  .  .  . .  .  .  . .  .  39 51  .  .  20  .  .  .  .  . .  .  .  .  .  .  .  .  .  .  .  8  15  .  .  vii  Check of LDH Reaction .  .  Temperature  Conclusions  .  .  11  Substrate U t i l i z a t i o n  of pH  .  .  Substrate U t i l i z a t i o n  Discussion  vi  Intact Mitochondria  Consumption Rates  .  .  .  0  .  .  .  .  Results  v  .  .  P r o t e i n Determination 3  .  .  . .  .  . .  .  64 .  . .  65 75 Bl  .  .  .  .  .  .  90  .  .  .  .  .  .  93  V  LIST OF FIGURES 1.  0  3  consumption —  exogenous NADH  .  2.  0  3  consumption —  succinate  .  3.  Mitochondrial  4.  O  5.  State 3 0  3  consumption and RCR at 15 C  6.  State 4 0  2  consumption at lS^C  7.  State 3  s  .  i n h i b i t o r s and uncouplers .  .  State3 0  malate "spark"  consumption at  concentration 9.  D  2  .  consumption —  .  .  .  .  .  .  .  .  .  .  .  .  .  .  30  .  . 3 2  .  .  consumption at 5 and 15 C  .  11.  State 4 D  3  consumption and RCR at 5°C  .  12.  State 3 0  3  consumption and RCR at  0  5 and 15°C: Heart v s .  .  35  .  .  37  .  .  40  .  mosaic muscle  .  42  .  . 4 5  .  .  49  consumption pH p r o f i l e s :  A c e t y l — c a r n i t i n e and glutamate 14.  . 2 7  concentrations  2  3  25  .  .  State 3 D  State 3 Q  23  .  .  10.  13.  .  pyruvate  limiting  o-f pyruvate  . 2 1  15°Cs  mosaic muscle  consumption v s .  2  . 0  Heart v s . 8.  .  .  consumption —  .  .  .  .  .  52  S t a t e 3 0=. consumption pH p r o f i l e s : Lactate  .  .  .  .  .  .  15.  L a c t a t e o x i d a t i o n at v a r i o u s pH v a l u e s  16.  State 4 0  17.  Malate o x i d a t i o n as a f u n c t i o n of pH  Z  . .  consumption and RCR pH p r o f i l e s .  . .  54 .  57  -  .  59  .  .  62  vi  LIST OF TABLES 1.  State 3 O  consumption, RCR and ADP/O  z  at 15 C . D  2.  .  .  Lactate state 3 oxidation 2 and lOmM NAD*  3.  .  .  .  .  .  17  r a t e s with .  .  .  .  .  IS  Conversion of l a c t a t e t o pyruvate as a f u n c t i o n of  CNAD~3  4.  S t a t e 3 and s t a t e 4 Q ,  5.  Some s t a t e 3 o x i d a t i o n various species  6.  .  . values  0  .  .  .  .  19 .  . 4 8  r a t e s from .  .  Comparison of s t a t e 3 o x i d a t i o n  .  .  .  .  72  r a t e s from  b l o w f l y and l o c u s t f a t bodies  .  .  .  73  ACKNOWLEDGEMENTS I wish f i r s t t o thank my s u p e r v i s o r , for h i s advice work.  I  would a l s o  committee, D r s . helpful  and support throughout like  John  to  G o s l i n e and John  s u g g e s t i o n s on the manuscript.  in  The L a b ,  who  technical assistance.  of the study and f o r the manuscript.  the  final  throughout  manuscript  for  for  Tom Mommsen  for  aspects  discussing  for h i s patience in  his  the course of my s t u d i e s .  many  and  I would e s p e c i a l l y l i k e t o  and  my  encouragement  with the t e c h n i c a l  thousand l i t t l e chores  of  J e f f Dunn who i s no  h i s unlimited patience in  Finally,  this  Thanks are due a l s o  S p e c i a l thanks t o  my husband, Michael C a s t e l l i n i , me with the  members  Phillips,  provided both  h i s i n v a l u a b l e a s s i s t a n c e both  Hochachka,  the course of  thank the  t o the members of The L a b , p a r t i c u l a r l y longer  Peter  involved  in  tremendous  thank helping  producing support  1  INTRODUCTION Modulation  and a d a p t a t i o n of metabolic pathways i s  well e s t a b l i s h e d  means  specific functions. cellular  for In  environment  a f f e c t i n g poikilotherms have  order  i n which  taken i n t o c o n s i d e r a t i o n .  -  allowing to  tissues  to  accomplish  on enzyme  Hazel,1984),  kinetics  transport  Simpson,19BO) and, c o n s e q u e n t l y ,  either  energy r e q u i r e d f o r  a e r o b i c a l l y or  study i s source  to  compare  for  two  pH  (Somero, (Hazel  and  (Halestrap,1978;  metabolic r a t e . tissue function i s  glycolytically oxidative  tissue  be  factors  temperature and i n t r a c e l l u l a r  1981p White and Somero,1982), membrane s t r u c t u r e  The  the  must  Two major environmental  far-reaching effects  Prosser,1974;  fulfill this  metabolism o c c u r s  a  and the  metabolism  types  with  derived  aim of as  an  different  this energy  oxidative  requirements and determine how t h i s f u n c t i o n i s a f f e c t e d by environmental to  f a c t o r s such as temperature and pH.  accomplish  respiration  this,  will  several  be i n v e s t i g a t e d .  mitochondria from one t i s s u e of r e s p i r a t i o n  aspects  of  The  first  T h i r d l y , what are  temperature and pH on o x i d a t i v e  physical  from the  chemistry  direct  and enzyme  whether  (ie.  tissue  rates  capacity. fastest)  the e f f e c t s  phosphorylation?  are the aforementioned p r o p e r t i e s Apart  is  e x h i b i t higher maximal  S e c o n d l y , which s u b s t r a t e s are burned best  order  mitochondrial  and how t h i s r e l a t e s t o o x i d a t i v e  i s o l a t e d mitochondria?  In  in of  Finally,  specific?  e f f e c t of  temperature  Michaelis—Menten  on  constants  < K > , temperature induced a l t e r a t i o n  i n membrane  m  may d i s r u p t  the a c t i v i t i e s  and t r a n s p o r t  This  is  membrane-bound  in  documented f o r  some  in  since  plots  mitochondrial  and  many  Modulation  often  Arrhenius  poikilotherms  al.,1977).  transporters  is  enzymes  Hochachka  T h i l o et  are membrane-bound.  enzymes  discontinuities  and P r o s s e r , 1 9 7 4 ;  et a l . , 1 9 8 1 ;  significant  m i t o c h o n d r i a l enzymes  systems  o-f membrane a s s o c i a t e d  p r o c e s s e s (Hazel  and Somero,1973; Hirano  structure  of  indicated which  by  have  been  membrane-bound  (Wodtke,1976;  enzyme  Irving  and  Watson,1976). The  potential  mitochondrial  respiration  intramitochondrial which tend  e f f e c t s of a l t e r e d i n t r a c e l l u l a r  to  pH  have  DiPrisco,1975;).  are  many.  would a f f e c t high pH  H* i o n s are  (Mela  involved  on  Lowered  mitochondrial  optima  pH  et  enzymes al.,1972;  i n the exchange  of  ADP/ATP a c r o s s the i n n e r m i t o c h o n d r i a l membrane (see H i n k l e and  Yu,1979)  as  well  as  other  ( H a l e s t r a p , 1 9 7 8 ; N i c h o l a s et a l . , 1 9 7 4 ; H* i o n g r a d i e n t p l a y s a transport  to  Moyle,1968).  ATP  further  formation  transport  Simpson,1980).  r o l e by c o u p l i n g  (Mitchel1,1961;  Rate of o x i d a t i v e  processes  r e l a t e d t o the magnitude of the pH g r a d i e n t  electron  Mitchell  phosphorylation  is  The  and  directly  (see H i n k l e and  McCarty,1978). T i s s u e s are known t o o x i d i z e s u b s t r a t e s and m i t o c h o n d r i a i s o l a t e d from d i f f e r e n t different  respiratory  properties  which  differentially  sources o f t e n have reflect  in  vivo  tissue function.  For example s e a l heart not o n l y has fewer  mitochondria per  gram of  t i s s u e than  dog heart  but  the  3 mitochondria themselves have lower a c t i v e r e s p i r a t o r y and lower cytochrome c o n t e n t s  (Sordahl et a l . , 1 9 8 3 ) .  when r e s p i r a t i o n i s expressed  as a -function of  content, mitochondria  isolated  different  s p e c i e s may  produce d i f f e r e n t  has been  observed  in  fat  rates  from the  body  of  same  Even  cytochrome tissue  in  maximal r a t e s  as  blowfly  and  locust  ( B a l l a n t y n e and S t o r e y , 1 9 8 3 ) . S i m i l a r o b s e r v a t i o n s were  made with v a r i o u s  tissues  from a s i n g l e s p e c i e s . Guinea p i g white muscle m i t o c h o n d r i a e x h i b i t e d slower  r a t e s of  substrates  except  relatively  active  r e s p i r a t i o n than  red with  a-glycerophsophate, a-glycerophosphate  muscle ( B l a n c h a e r , 1 9 6 4 ) .  indicating  shuttle  in  from  rabbit  Mitochondria  all a  white red  muscle e x h i b i t e d higher r e s p i r a t o r y  r a t e s than those  from  white  acetyl—carnitine  or  muscle  with  either  palmitoyl-carnitine  as  substrate  Blanchaer,1971), i n d i c a t i n g larger  role  in  red  that  muscle.  (Pande  f a t metabolism It  has  been  demonstrated i n f a t body of b l o w f l y and l o c u s t and  Storey,1983)  Phillips,1983) preference  and  locust  that  are  tissue  reflected  rectum function  in  and plays  a  similarly (Ballantyne  (Chamberlin  and  and  substrate  mitochondrial  substrate  u t i 1 i z a t i on. The t i s s u e s mosaic  muscle  chosen f o r  from  rainbow  O x i d a t i v e metabolism i s heart.  phosphorylation i s  to  trout  were h e a r t  (Salmo  the major energy  Heart t i s s u e demands  t h e r e f o r e the a b i l i t y  t h i s study  mandatory.  qairdneri).  source i n  trout  a constant energy supply  maintain high r a t e s of The bulk  of  and  and  oxidative the  mosaic  4 muscle i s composed o-f with  white - f i b r e s which are  small-diameter  red  -fibres  (Boddeke  interspersed et  al.,1959).  According t o ATPase and s u c c i n i n c dehydrogenase  activities,  these mosaic muscle red - f i b r e s c o n s t i t u t e a d i f f e r e n t type  than  those  al.,1975). teleost  of  In g e n e r a l , the  i s active  At high  superficial  at slow  c r u i s i n g speeds  muscle  fibre  (Johnston  et  s u p e r f i c i a l red muscle of  the  c r u i s i n g speeds  i s also  recruited  (Webb,1970; Hudson,1973) and d e r i v e s energy from  oxidative  sources.  mosaic muscle  (Hudson,1973).  White f i b r e s are a l s o capable of producing  anaerobically for short periods.  Clearly,  depending on the  a c t i v i t y of the f i s h , mosaic muscle may or may not energy from o x i d a t i v e  in  require  phosphorylation.  Nutritionally, important r o l e  energy  carbohydrates seem  fish  including trout, thrive  than  in  to play  mammals.  on h i g h - p r o t e i n  a  less  Many  fish,  d i e t s and  derive  energy from o x i d a t i o n of l i p i d s or g l u c o s e d e r i v e d  through  gluconeogenesis from  Teleost  hearts  contain  carbohydrates, Newsholme,1979; Sidel1,1983).  amino  the  fatty  enzymes acids  Driedzic In  (Love,1980). necessary  to  and a c e t o a c e t a t e and  addition  millimolar concentrations acids  acids  of  Stewart,19B2; teleost  (Zammit  and  Hansen  and  plasma  glucose, lactate  (Zammit and Newsholme,1979; Larsson and  utilize  contains and  Fange,1977).  S i g n i f i c a n t c o n t r a c t i l e f a i l u r e i n f u e l - d e p r i v e d sea h e a r t s c o u l d be prevented by l.OmM p a l m i t a t e Hart,1984).  in  the  i n c l u s i o n of  perfusion  Lactate oxidation  fatty  raven  lOmM g l u c o s e  medium  (Driedzic  or and  i n f i s h h e a r t s appears t o be  controlled  by  lactate  oxidase  activity  (Driedzic  et  al. ,  1984). The white  mosaic  muscle,  Perfusion  of  muscle  i s  capable  the  hind  exogenous g l u c o s e , acid  main (Moen  of  and  C0  of  the  only  f o r CQ  i s  7:3  mosaic  muscle  of  limited  and  preference  may  substrate  utilization  i n mosaic  heart  temperature  muscle  fluctuations,  since  the  requirements. temperature  and  Blood in  change  of  temperature  muscle  than  since than  does  perfused and  i n heart larger  i t relies  other  heart. as  more  heart,  fish  end—products.  to  mosaic of  light  on  oxidative  the  same d e g r e e  different  the  pH  of  tends  to  be  vary  pH  white  restricting  a  white  muscle  also  exercise  metabolism  i s not  clearance  et  with  in  during  anaerobic  muscle  pH  higher  changes  with  Rahn  of  White  may  oxidative  may  change  more h e a v i l y on  thereby  red  Mitochondrial  Heisler,1984).  addition  production  Z  (Reeves,1972;  intracellular  much In  (see  have  metabolic- modulation  have  (ApH/AT)  may  red.  intracellular  In  endogenous  muscle.  poikilotherms  Reeves,1977).  the  vascularization  to  however,  and  CQ  in  amino  although  was  total  experience  tissues  al.,1975;  experiences  shed  that  three  muscle  since  relative  metabolism. showed  than  acid),  mitochondria  largely  3-hydroxy-butyric  production  this  substrate  Both  and  quickly  a  to  (Nag,1972) i s  trout  Mosaic  minimally  proportion  muscle  vary  acid  alanine, glutamic  carbon  though  oxidative  rainbow  more  Z  trout,  some  Klungsoyr,1981).  contributed since  of  part  to  (isoleucine, source  rainbow  acetoacetic  were c o n v e r t e d  acids  of  of  as  well  protons  6  Thus c o n d i t i o n s -for differ  substantially  previous  work  m i t o c h o n d r i a l -function i n  i n mosaic  one  might  muscle  and h e a r t .  anticipate  that  Additionally  mitochondria t o be  one  might  l e s s temperature  with a constant demand  intracellular  sensitivities Accordingly,  pH,  one  between  might  the two  characterized according substrate u t i l i z a t i o n and e x t r a m i t o c h o n d r i a l r e l a t e the q u a l i t i e s  to  pH.  expect  keeping  maximal  rates  heart.  fluctuations  different  pH  populations.  respiration  patterns,  heart  metabolism i n  mitochondrial  mitochondrial  substrate  sensitive in  for oxidative  two  expect  S i n c e the two t i s s u e s are s u b j e c t t o d i f f e r e n t in  From  the  m i t o c h o n d r i a l p o p u l a t i o n s would d i s p l a y d i f f e r e n t preferences.  trout  will of  be  oxidation,  and response t o temperature  An attempt  of m i t o c h o n d r i a l  s p e c i f i c f u n c t i o n of the t i s s u e i n  vivo.  w i l l be  made  r e s p i r a t i o n to  to the  7  MATERIALS AND METHODS M i t o c h o n d r i a were i s o l a t e d muscle of rainbow t r o u t  from heart and  (Salmo q a i r d n e r i  R. ) .  mosaic  Trout of  both  sexes weighing 250-500g were obtained from a commercial f i s h farm.  They were kept i n  aerated running water at 5 -  and f e d d a i l y with 1/4 i n c h C l a r k f i s h p e l l e t s C o . , S a l t Lake C i t y ,  Utah).  In a l l  designed t o generate pH p r o f i l e s , c o l l e c t e d during  the  mid-September. however,  The  summer data  0  (Moore-Clark  experiments except  those  the bulk of the data  were  months -  from  15 C  each  from  late  tissue  do  a few measurements made i n e a r l y s p r i n g .  May  to  include, The  data  f o r heart pH p r o f i l e s were c o l l e c t e d i n May and June of 1984 while the analogous measurements f o r mosaic muscle were made l a t e the  following  winter.  Because  d i f f e r e n c e s i n the pH s t u d y , respiration  for  merely t h e i r  each  of p a s s i b l e  a b s o l u t e r a t e s of  t i s s u e are never  seasonal  mitochondrial  compared d i r e c t l y  —  p a t t e r n of change with changing pH.  CHEMICALS Acetyl-DL-carnitine oligomycin, 5'-diphosphate (NAD"*") ,  sodium  (ATP),  B—nicotinamide  (NADH), p y r u v a t e ,  protease  adenine  l a c t a t e dehydrogenase  fatty  (ribonuclease—free)  salt  of  (DNP), adenosine  adenine  dinucleotide  dinucleotide  L—malate, L—glutamate,  (P5255),  essentially  dinitrophenol  rotenone,  B—nicotinamide  (free a c i d ) ,  HC1,  succinate,  (reduced) lactate  (porcine heart)  bovine  serum  albumin  acid  free)  (BSA)  (LDH),  (Fraction and  V  sucrose  were purchased from Sigma Chemical  Co.,  8  St.  Louis  various  MO.  A l l other  commercial  ISOLATION  OF  were  easily  f o r c e p s and  bulbus  arteriosus eight  weight  of  —  with  more  from  the  2  3  2/3  length  the  dissection, buffer  i n an  mosaic  muscle  muscle  pigment The  muscle  then of  was  pigment,  and with  the  muscle  buffer.  tissue  wet  both  heart  Tris-HCl,  to  was  grade.  keep  of  from 50  isolation muscle  the  from minced  two  buffer.  and a  and  and  lateral  line,  extending  along  as  from  the  isolation  of  A  Any  fish, traces  trimmed  away  once  to  of  muscle.  i n the  rinsed  pooled  layer  the  buffer.  were c a r e f u l l y  The  superficial  line.  still  then  the  possible.  surface  was  rinsed  Throughout  cold  isolation  wet  dissected  ice—cold  while  fish  tissue  was  the  in  minced  discarded.  minced  lateral  the  blood  isolation  trunk.  separated  beyond removed  muscle  above  heads  were  obtain  finely  i t as  along  or  to  bathed  their  Hearts  operculum  thouroughly  Muscle  and  -from  steps  away  Mosaic  ice-cold  muscle  weight  The  was  partially  red  was  trout  removed  to  4«=*C.  trimmed  the  the  extends  transferred  -  trout,  carefully  also  analytical  in ice-cold  tissue  behind  attempt was  purchased  isolation  were p o o l e d  muscle  which  was  was  the  of  the  0  buffer.  of  directly  at  washed  The  isolation  beginning  were  d e c a p i t a t i o n and All  out  fish  g.  trunk  were of  by  pithed.  with  from  red  killed  were c a r r i e d  Hearts  and  used  MITOCHONDRIA  were  immediately  dissection  The  sources  INTACT  Trout  chemicals  more  obtain  a  g. b u f f e r was  mitochondria.  210mM m a n n i t o l ,  essentially  the  same  It c o n s i s t e d of  70mM s u c r o s e ,  lOmM  EDTA  for lOOmM  and,  in  9 the case of muscle, 5mM M g C l final  mitochondrial  0.1% BSA  but  and  2  suspension  t h i s was  added  0.IX  BSA at pH 7 . 3 .  from heart as  also  the very  coupled r e s p i r a t i o n .  The  i s o l a t i o n may f u r t h e r product  (personal  a d d i t i o n of BSA  but  step  of  and d u r a t i o n at the s t a r t  i n c r e a s e the s t a b i l i t y  observation)  contained  last  i s o l a t i o n i n order t o i n c r e a s e the s t a b i l i t y  The  t h i s was  of the not  of of  final  realized  when the experiments were begun. The next step i n m i t o c h o n d r i a l homogenization.  The t i s s u e was f i r s t  protease  P5255) and  (Sigma  case of h e a r t ,  2 -  i s o l a t i o n buffer  stirred  i s o l a t i o n was  tissue  incubated i n a general occasionally.  In  3 g of t i s s u e were incubated i n 30 mis of  and 2 2 . 4 mg  protease f o r  t i s s u e was then homogenized very g e n t l y  10 minutes.  teflon pestle.  of muscle, 50 -  were d i v i d e d i n t o two  55 g of t i s s u e  In the case  and each l o t was incubated i n 60 mis of i s o l a t i o n b u f f e r  decanted  protease and  for  14  replaced  minutes.  Homogenization proceeded u s i n g t i s s u e i n 30 mis of  a Sorvall  buffer  was  protease—free  8 -  then  lOg  i s o l a t e d by  differential  Crude t i s s u e homogenates were f i r s t spun i n  RCB-2 at 600g f o r  10  and  buffer.  10 minutes at 4 ° C .  The  cellular  d e b r i s was d i s c a r d e d and the supernatant was c e n t r i f u g e d 9000g f o r  lots  buffer.  t i s s u e p o r t i o n s of  M i t o c h o n d r i a were then centrifugation.  The  with  The  in a Patter-Elvehjem  homogenizer with a l o o s e - f i t t i n g  4 4 . 8 mg  the  minutes at 4 ° C .  p e l l e t was resuspended i n b u f f e r Pasteur p i p e t t e and  The r e s u l t i n g  mitochondrial  by g e n t l e a s p i r a t i o n with a  recentrifuged  was repeated f i v e t i m e s .  at  at 9000g.  The f i n a l  p e l l e t was  This  process  resuspended  10 i n 1.0 ml i s o l a t i o n b u f f e r .  Two 60 ul a l i q u o t s were removed  and  later  frozen  at  A l i q u o t s of the  -80=0  for  white muscle  protein  determination.  i s o l a t i o n buffer  (containing  BSA) were a l s o f r o z e n f o r p r o t e i n d e t e r m i n a t i o n . i s o l a t i o n b u f f e r was  added t o  suspension.  case of  In the  second a d d i t i o n of b r i n g the  final  the remaining the heart  of  preparation,  BSA  mitochondrial protein concentrations were g e n e r a l l y between 7 -  to  this  0.2% BSA O . 1'/..  of these  20 mg per m l .  of  mitochondrial  i s o l a t i o n buffer contained concentration  1.0 ml  to  Final  preparations  Preparations  were  s t a b l e f o r at l e a s t two hours when kept on i c e . PROTEIN DETERMINATION M i t o c h o n d r i a l p r o t e i n c o n c e n t r a t i o n was  determined  a c c o r d i n g t o a m o d i f i c a t i o n of the Lowry technique i n the p r o t e i n i s  p r e c i p i t a t e d by  Na—deoxycholate  (Bensadoun  6% TCA i n  and  technique e l i m i n a t e d i n t e r f e r e n c e EDTA.  The s e n s i t i v i t y  5 — 50 ug allow  for  protein.  of  100 u l  membranes. to  3  mis  The technique of  (30 — 50 u l )  was  Weinstein,  1976).  added  was f u r t h e r  and  modified  to  100  solubilize  distilled  water  Na-deoxycholate c o n c e n t r a t i o n of mixed v i g o r o u s l y  and  allowed  P r o t e i n was then p r e c i p i t a t e d by  to  u l of  10%  mitochondrial  give  167 ug/ml. to  stand  to  volume  A 10 u l a l i q u o t of t h i s s o l u t i o n was d i l u t e d with  of  membranes.  were d i l u t e d t o a f i n a l buffer.  of This  by s u c r o s e , Tris—HC1  mitochondrial  with B S A - f r e e i s o l a t i o n  Na-deoxycholate  the presence  of t h i s technique i s i n the range  solubilization  Protein aliquots  which  for  a  final  Samples 15  a d d i t i o n of 6% TCA  up  were  minutes. (final  11 concentration) minutes i n a  -Followed by  centri-fLigation at  swinging bucket  completely removed u s i n g a resultant reagent.  protein pellet  rotor.  3300g f o r  30  The supernatant  was  Pasteur p i p e t t e a s p i r a t o r .  was d i s s o l v e d  A f t e r mixing v i g o r o u s l y  in 3  The  mis of  Lowry  f o r 30 seconds, 1.5 mis  F o l i n - C i o c a l t e u reagent, d i l u t e d  1:1  with water,  was  of  added  and the c o l o u r allowed t o develop i n the dark f o r 45 minutes (Bensadoun and W e i n s t e i n ,  1976).  660 nm  spectrophotometer.  in a  Pye-Unicam  Absorbance was measured at Bovine  serum  in a  Gilson  albumin was used as the s t a n d a r d . Q  CONSUMPTION RATES  2  Assays were conducted p o l a r o g r a p h i c a l l y  oxygraph with a C l a r k - t y p e 0=. e l e c t r o d e and a w a t e r - j a c k e t e d chamber.  Temperature  was maintained  at e i t h e r  5 or  15 C D  with a constant water bath and c i r c u l a t o r .  Calibration  was  achieved by a l l o w i n g  with a i r at  the  water t o e q u i l i b r a t e  experimental temperature.  Values  for 0  2  content i n  100%  a i r — s a t u r a t e d water at v a r i o u s temperatures are a v a i l a b l e  in  Davis,  O  1975. Sodium s u l f i t e was  added t o determine zero  z  content. Final u l of  assay volume was 2 . 0 mis i n c l u d i n g 200 —  mitochondrial  mitochondrial p r o t e i n . Tris-HCl,  c o n t a i n i n g 1.5  -  3.0  The assay b u f f e r c o n s i s t e d of  210mM m a n n i t o l , 70mM  KH P0^ at pH 7 . 3 . 3  suspension  sucrose,  lOmM EGTA and  The pH of the assay mixture was  400 mg  lOOmM lOmM  measured  with a radiometer pH—meter a f t e r a d d i t i o n of mitochondria then the chamber was s e a l e d .  Assay pH was allowed t o  —  drift  with temperature so at 15°C the pH was 7.4 and at 5°C the pH  12  was  7.6—7.7.  stirrer  Cell  and  dissolved  contents  teflon-coated  i n  assay  rotenone  Substrates,  uncouplers  through  and  mixed  bar.  buffer  •ligomycin,  chamber  were  adjusted  were  magnetic  removable  to  dissolved  and i n h i b i t o r s  a small,  a  A l l substrates  and  DNP  with  were  port  were  pH  in  7.3.  ethanol.  injected into the  using  lOul  Hamilton  syringes. Mitochondrial ADP  pulses  administered  consumption, Estabrook and by  RCR  and  (1967).  Williams a number  measuring  respiration  0  Z  methods.  Membrane  consumption  substrate,  RCR  was  uncouplers  were  administered,  i n order Once  from  each  used  the integrity had  regimes  throughout.  The  rates  15°C.  The s u b s t r a t e s  except the  of D  lactate,  4mM  malate  TCA  were  cycle. 2  was t e s t e d  2  first  regime  included  accompanied  units of  inhibitors  of  LDH,  a n d 5mM  observed  i n the mitochondrial  since  no  suspension.  and  different method  was  measurement  of  substrates  at  pyruvate,  measurements  as  preparations  three  involved  b y 0 . 0 5 mM  ADP.  oligomycin  The ADP—pulse  5mM  by  respiration.  f o rspecific  acetyl-carnitine  addition  and  or succinate  established,  consumption  Lactate  o f NADH  of the mitochondrial  imposed.  to  confirmed  including rotenone,  been  were  maximal  lOmM  integrity  several  2  Chance  was  t o c h a r a c t e r i z e coupled  tissue  experimental  and  by  viability  either pyruvate  monitored  0  according  are defined  i n the presence  measured using  DNP,  states  by 400 nmole  syringe.  calculated  Mitochondrial  ADP/O w a s  or  Hamilton  ADP/O w e r e  Respiratory  (1956). of  by  was i n i t i a t e d  5mM  malate,  glutamate.  malate  to  A l l "spark"  also  required  the  LDH  activity  was  Porcine  heart  LDH  13 suspended i n g l y c e r o l o-f 2mM NAD* i n oxidation  was used.  It  the assay b u f f e r  i n heart  appeared t h a t  inclusion  produced maximal r a t e s  p r e p a r a t i o n s but subsequent  experiments  have i n d i c a t e d t h a t these assays were NAD*-1imited. assays were c a r r i e d out i n the  presence of  second regime was a repeat of 5°C r a t h e r than  15°C.  Muscle  lOmM NAD*.  The  the f i r s t but c a r r i e d out  In the case  of  of l a c t a t e  at  oxidation,  a d d i t i o n of LDH was i n c r e a s e d t o 4 u n i t s / 2 mis assay volume. Sample  size  was  extremely  variable  in  these  experiments because some p r e p a r a t i o n s were more s t a b l e others.  Results  are expressed  r a t e s were measured f o r 3 ADP were v a r i a b l e from one p u l s e they d i d  not vary  Each mean includes  2  or  3  t o any  per  As an a s i d e t o these f i r s t of 0=>  The  predictable  rates  consumption  but  pattern.  a l l observations  preparation  p r e p a r a t i o n s f o r each experimental  the r a t e  Oxidation  t o another i n each a s s a y ,  average of  assays  SEM.  p u l s e s per a s s a y .  according  r e p r e s e n t s the  as mean +  than  and  3  and -  condition. two s e t s of  at v a r i o u s  experiments,  concentrations  pyruvate was measured at l S ^ C .  The malate "spark" and  p u l s e were a d m i n i s t e r e d b e f o r e  the a d d i t i o n of pyruvate  order t o be  sure of the  the onset of  initial  state 3 respiration.  6  pyruvate c o n c e n t r a t i o n  of ADP in at  Pyruvate  concentration  The t h i r d experimental regime monitored  mitochondrial  ranged from 0.01 — lOmM.  o x i d a t i o n as a  f u n c t i o n of pH.  was assayed at lS^C and a v a r i e t y the  assay  suspension  s u b s t r a t e s or ADP.  was  Mitochondrial  respiration  of pH v a l u e s .  measured  before  The pH  addition  The pH was measured again at the end  of of of  14 each assay t o ensure t h a t i t the course of  the experiment.  v a r i e d by more assay  the  pH u n i t  were  discarded.  data  glutamate and  v a l u e s v a r i e d from 5 . 2 buffered mitochondrial  8.3.  cases where  pH  course of  an  d u r i n g the  Substrates  lactate.  included  Assay b u f f e r  A d d i t i o n of 200 -  suspension  was a s i g n i f i c a n t  The f i n a l  In the few  than 0 . 1  acetyl-carnitine,  since i t  had not changed markedly d u r i n g  400uls  of  assay  pH  altered final  f r a c t i o n of t o t a l  assay  volume.  assay pH v a l u e s c o u l d not be d u p l i c a t e d a c c u r a t e l y  from one m i t o c h o n d r i a l p r e p a r a t i o n t o another so each on the  pH  profile plots  s i n g l e assay.  Values  usually  Where pH v a l u e s from than 0.02 pH u n i t s ,  3 - 4  of  as not  Means  ADP p u l s e s per  assay.  not vary by  more to  noted  pH v a l u e s were staggered  among  artificial  having an  r a t e than  a  These o c c u r r e n c e s are  to create  any p r e p a r a t i o n  lower r e s p i r a t o r y  from  v a l u e s from the assays were combined  Experimental  p r e p a r a t i o n s so  point  + SEM.  s e p a r a t e assays d i d  r e p r e s e n t a s i n g l e data p o i n t . on the p l o t s .  represents data  are expressed as mean  r e p r e s e n t average v a l u e s from  result  pH  trends  inherently  others.  A total  of  as  a  higher  or  18 and  16  p r e p a r a t i o n s were used t o generate the heart and muscle data respectively.  using a  Malate o x i d a t i o n as  a f u n c t i o n of pH was  single  of heart  preparation  r e s u l t s have been w i l l be  discussed.  r e p r e s e n t s the  included for  mean from  except where n o t e d .  The  4 ADP  mitochondria.  illustrative  "Each p o i n t  on  the  pulses in  order i n  measured  reasons  pH-profile a single  which the  were made was pH 6 . 8 , 7 . 3 , 7 . 8 , 8 . 2 and 6 . 4 .  The which plot assay  measurements  15 Mitochondrial r a t e s and  diminished  mitochondrial  aging  aging r e s u l t s respiratory  were apparent  i n slowed  control. (if  RCR  respiratory If  signs  and ADP/O  were  suddenly low and the p r e p a r a t i o n was more than one hour the data were d i s c a r d e d . ANOVAs f o l l o w e d  by  the  Data were analysed u s i n g Student-Newman-Keuls  old)  one—way  (SNK)  test.  Transformations were performed as r e q u i r e d i n order t o the a p p r o p r i a t e s t a t i s t i c a l  of  meet  assumptions.  SPECTROPHOTOMETRIC CHECK OF LDH REACTION The LDH r e a c t i o n was measured s p e c t r o p h o t o m e t r i c a l l y room temperature u s i n g m i t o c h o n d r i a l assay b u f f e r was i d e n t i c a l measurements and A d d i t i o n of  to  included  2 units  of LDH  assay c o n d i t i o n s  t h a t used f o r  lOmM l a c t a t e initiated  and the  0  .  at The  consumption  3  2-10mM  NAD"*".  reaction.  The  c o n v e r s i o n of NAD"*" t o NADH was f o l l o w e d t o completion at 340 nm i n  a Pye—Unicam  spectrophotometer.  a l t e r e d by i n c r e a s i n g LDH or l a c t a t e  Results  concentration.  were  not  RESULTS Mitochondria  i s o l a t e d from heart and mosaic muscle  rainbow t r o u t e x h i b i t e d  respiratory  RCR v a l u e s and ADP/O r a t i o s l e a s t two hours. consumption of  Table 1  control,  shows the s t a t e 3 r a t e of  mitochondria at  15°C, o x i d i z i n g  rate  pyruvate of  or malate were  heart  mitochondrial  presence of 2 u n i t s that  of  investigation  that  increased l a c t a t e pyruvate  (Table  It  oxidation 2).  using  (specifically  pH)  which  mitochondrial  oxidation  The  lactate  in  the  mM NAD* was lower  than  r a t e s of  natoms O m i n  - 1  mg  found  to  LDH  reaction  was  b u f f e r s and identical  increasing  CNAD*]  to  It  2-10mM,  lOmM  those  of  checked conditions  those  of  the  The r e a c t i o n  was  ranged from  i n mosaic muscle  (Table 1). from  assay  to  and was NAD*—1imited.  state 3 oxidation - 1  subsequent  relative  being observed with  l a c t a t e as s u b s t r a t e  upon  assays (Table 3 ) .  protein  with h i g h e s t r a t e s  oxidation  rates  were  in  - 1  s u p p l i e d as s u b s t r a t e .  found t o be slow i n t h i s assay b u f f e r Mean  mg p r o t e i n  - 1  NAD* c o n c e n t r a t i o n  The  spectrophotometrically  saturating  when  was  increasing  oxygen  r a t e s being observed  of LDH and 1-2  pyruvate.  at  Mean r a t e s of s t a t e 3  ranged from 70-124 natoms 0 m i n  heart m i t o c h o n d r i a , with h i g h e s t either  as evidenced by  (Table 1) and were s t a b l e f o r  c o n c e n t r a t i o n s of v a r i o u s s u b s t r a t e s . oxidation  of  67-120  mitochondria,  either  pyruvate  or  was demonstrated that  by  lactate  in  conversion  m i t o c h o n d r i a l assay b u f f e r was i n c r e a s e d from 0.02 umoles/ml t o 0 . 0 5 umoles/ml  (Table 3 ) .  Reported v a l u e s f o r  lactate  17  TABLE 1:  S t a t e 3 0 consumption, RCR, ADP/O f o r mitochondria i s o l a t e d from heart and mosaic muscle of rainbow t r o u t at 15°C. Sample s i z e as d e s c r i b e d i n M a t e r i a l s and Metods. Mean ± SEM 2  STATE 3 (natoms O/min/mg p r o t e i n )  RCR  ADP/O  HEART Pyruvate(5mM)  124.49±7.17  7.68±0.54  2.54±0.06  Malate(5mM)  117.79±6.25  5.85±0.53  2.26±0.04  Lactate(lOmM)  82.24±7.06  7.13±0.68  2.56±0.05  Acetyl-carnitine(4mM)  73.69±5.72  7.60±0.42  2.36±0.13  Glutamate(5mM)  70.33±4.54  6.05±0.37  2.29±0.10  Pyruvate(5mM)  95.33±6.71  6.00±0.44  2.82±0.09  Malate(5mM)  6 7 . 0 8 ± 6 . 13  4.84±0.28  2.79±0.14  Lactate(5mM)  120.34±12.71  4.39±0.13  2.58±0.04  Acetyl-carnitine(4mM)  78.53±7.18  5.64±0.27  2.45±0.07  Glutamate(5mM)  87.46±6.88  4.6S+0.19  2.80±0.07  MUSCLE  18  TABLE 2:  S t a t e 3 o x i d a t i o n r a t e s with l a c t a t e as s u b s t r a t e , i n the presence o-f 2mM or lOmM NAD*, compared t o s t a t e 3 o x i d a t i o n r a t e s with s a t u r a t i n g c o n c e n t r a t i o n s of p y r u v a t e . Mean ± SEM. Sample s i z e i n p a r e n t h e s e s . Assays i n c l u d e d lOmM l a c t a t e , 2 u n i t s LDH and 0.05mM malate.  S t a t e 3 0 Consumption Rate (natoms D m i n mg p r o t e i n ) 2  - 1  NAD* <2mM)  - 1  88.06 ± 2.98  (4)  NAD* (lOmM)  115.98 ± 0.92  (6)  Pyruvate  H O . 88 ± 3 . 0 9  (6)  (lOmM)  19  TABLE 3:  Conversion of l a c t a t e t o pyruvate as a f u n c t i o n of NAD*. Assay c o n d i t i o n s d e s c r i b e d i n M a t e r i a l s and Methods. Initial l a c t a t e c o n c e n t r a t i o n was lOumoles/ml i n a r e a c t i o n volume of 1ml.  CNAD-3 (mM)  L a c t a t e -> Pyruvate (umoles)  2  0.021  4  0.033  9  0.042  10  0.054  20 oxidation  i n muscle were assayed i n the presence o-f 2  units  LDH and lOmM NAD*. Mitochondrial standard t e s t s .  i n t e g r i t y was  In the  exogenous  a l s o suggest  NAD"*"-1 inked  confirming  (Fig.l).  t h a t the  were i n t a c t .  of  ADP/0 r a t i o s were substrates.  respiring  2.3 -  2.9  this inhibition  (Fig.  O x i d a t i o n of a l l malate  "spark"  intermediates. capable  of  to  1)  with  inhibit  r a t i o s for  the  1.9 ( F i g .  2).  1.4 -  i n h i b i t e d ADP-stimulated  and an uncoupler of o x i d a t i v e  (Table  (Table 1>  2) and ADP/0  as  mitochondria  appeared  FAD*-1inked s u b s t r a t e , s u c c i n a t e , were Oligomycin completely  neither  mitochondrial  RCR v a l u e s  Rotenone (Fig.  that  High  majority  NAD*-1inked r e s p i r a t i o n  of  mitochondria c o u l d o x i d i z e NADH  substrate,  membranes were i n t a c t  a number  absence of s h u t t l e systems  heart nor mosaic muscle an  assessed by  phosphorylation,  respiration DNP, r e l i e v e d  3). s u b s t r a t e s r e q u i r e d the presence of  (0.05mM)  to  augment  depleted  a TCA  Although both m i t o c h o n d r i a l p o p u l a t i o n s were  oxidizing  malate  as  a  sole  substrate,  the  c o n c e n t r a t i o n of malate used f o r  " s p a r k i n g " was too low  to  contribute  direct  0  significantly  consumption ( F i g .  as  a  substrate  for  3  4).  SUBSTRATE UTILIZATION F i g u r e 5 compares  s u b s t r a t e p r e f e r e n c e between  and muscle mitochondria at oxidation rates  i n heart  exogenous s u b s t r a t e s .  lS^C.  Figure  5a shows s t a t e  mitochondria u s i n g  The r a t e s were  pyruvate or malate as s u b s t r a t e .  heart  a variety  h i g h e s t with  The r a t e of  lactate  3 of  either  21  FIGURE Is Representative mitochondrial 0 consumption t r a c e s . C o n c e n t r a t i o n of each a d d i t i o n i n a f i n a l volume of 2 . 0 miss NADH - 2mM, ADP - 200uM, malate - 0.05mM, glutamate - 5mM, Pyruvate - 5mli 2  A. B.  Heart; 1.75 mg p r o t e i n ; lS^C Muscle; 2.33 mg p r o t e i n ; 15°C  NADH  I  ADP NADH  N3  23  FIGURE 2: Representative mitochondrial 0 consumption t r a c e s . C o n c e n t r a t i o n s f o r each a d d i t i o n i n a f i n a l volume of 2 . 0 miss ADP - 200uM; rotenone lmg/ml; s u c c i n a t e — 5mM 2  A. B.  Heart; 1.05 mg p r o t e i n ; 15°C Muscle; 4.00 mg p r o t e i n ; 5°C  ADP/D=1.9 ADP/0=1.4  5mM PYRUVATE SmM MALATE  I  ADP ROTENONE  25  FIGURE 3 : Representative mitochondrial 0 consumption t r a c e s . C o n c e n t r a t i o n s f o r each a d d i t i o n i n a f i n a l volume of 2 . 0 mis: ADP — 200uM; o l i g o m y c i n - 5 ug/ml; DNP - 0.04uM 2  A. B.  Heart; 1 . 2 9 mg p r o t e i n ; 5°C Muscle; 3 . 6 3 mg p r o t e i n ; 5°C  27  FIGURE 4s R e p r e s e n t a t i v e m i t o c h o n d r i a l consumption t r a c e s .  A. B.  D  2  Hearts 1.38 mg p r o t e i n s 15*=*C; c o n c e n t r a t i o n of each a d d i t i o n i n a f i n a l volume of 2 . 0 miss malate - O.OSmM, ADP - 200uM, pyruvate - 0.05mM Muscle; 2.40 mg p r o t e i n ; 15°C; c o n c e n t r a t i o n f o r each a d d i t i o n i n a f i n a l volume of 2 . 0 miss malate - O.OSmM, ADP - 200uM, pyruvate - 0.02mM  28  ADP |  PYRUVATE  29 oxidation  was  significantly  lower  than  pyruvate or m a l a t e ,  although l a c t a t e  not maximized under  these c o n d i t i o n s of  acetyl-carnitine which was malate.  of  either  oxidation rates  were  low CNAD*3.  Both  and glutamate were burned at the same r a t e ,  significantly  lower  than t h a t  There were no s i g n i f i c a n t  the RCR v a l u e s  that  with t h i s  pyruvate  or  d i f f e r e n c e s among any  of  treatment -  of  where  state 3  were h i g h , s t a t e 4 r a t e s were s i m i l a r l y e l e v a t e d The p a t t e r n of  <Fig.  s u b s t r a t e p r e f e r e n c e i n mosaic  mitochondria was q u i t e d i f f e r e n t same temperature  rates  (Fig.  5b).  from  6). muscle  t h a t of heart at  L i k e h e a r t , muscle  the  exhibited  high r a t e s of o x i d a t i o n with pyruvate as s u b s t r a t e , malate produced the lowest mean r a t e s i n muscle.  however  In  muscle  a s s a y s , where l a c t a t e o x i d a t i o n was maximized, s t a t e 3 r a t e s of l a c t a t e pyruvate.  o x i d a t i o n were In f a c t ,  at  l e a s t as  the mean r a t e  although the d i f f e r e n c e was not While f o r  5a), for  three  lactate,  acetyl—carnitine t o pyruvate  s u b s t r a t e s were  based on maximal r a t e s of s t a t e 3 o x i d a t i o n .  t h e r e was  no s i g n i f i c a n t  difference  and (Fig.  equally  good,  Other than the  d i f f e r e n c e between malate and l a c t a t e o x i d a t i o n r a t e s 5),  of  significant.  substrates r e l a t i v e  muscle a l l  those  was higher f o r  heart mitochondria  glutamate were poor  high as  (Fig.  i n muscle s t a t e  3  r a t e s among any of the s u b s t r a t e s examined. There appeared t o be s i g n i f i c a n t d i f f e r e n c e s among RCR's f o r (Fig.  5b).  different  substrates  The usual  in  mosaic muscle  transformations did  data homogeneous, however,  not render  and the c o n c l u s i o n s  t e s t must be viewed c a u t i o u s l y .  at  of the  S t a t e 4 r a t e s were  the lS^C the SNK  clearer.  30  FIGURE 5: S t a t e 3 0 consumption at 15°C. Sample s i z e as d e s c r i b e d i n M a t e r i a l s and Methods (mean ± SEM). S i g n i f i c a n t d i f f e r e n c e among means (P<0.05) i n d i c a t e d by * ( 0 consumption) or + (RCR) 2  3  A. B.  Heart Muscle  State 3 0  RCR  2  consumption  31  -  A  f f  ir  12  -h  -  BO  B  f  f  12 3D  ACETYLCARNITINE  GLUTAMATE  MALATE  PYRUVATE  LACTATE  32  FIGURE 6s S t a t e 4 0 consumption at 15°C. Sample s i z e as d e s c r i b e d i n M a t e r i a l s and Methods (mean ± SEM). S i g n i f i c a n t d i f f e r e n c e between means (P < 0.05) i n d i c a t e d by * . 2  A. B.  Heart Muscle  STATE 4 O , CONSUMPTION (natonta O/mln/mg protein)  4 O , CONSUMPTION  (natoma O/mln/ms prolalnj  T  >o  STATE  T  OD  T  State 4  rates  were  l a c t a t e which  the same  had a  for  all  significantly  substrates  higher r a t e  except  (Fig.  6).  L a c t a t e o x i d a t i o n produced the lowest mean RCR. At 15°C, maximal s t a t e 3 o x i d a t i o n r a t e s were always at l e a s t as high i n h e a r t m i t o c h o n d r i a as i n muscle and i n case  of  both  significantly heart  malate  pyruvate,  higher i n h e a r t  mitochondria  phosphorylation difference  and  than  in  RCR,  accompanied by higher s t a t e  had  substrate.  a  significantly  state 4  values  higher  In the cases  where  of no  state rate  oxidative significant  3  (Fig.  s i g n i f i c a n t with  in  than  heart in  a c e t y l - c a r n i t i n e or glutamate ( F i g . h e a r t had the same  were  was  elevated  was o n l y  rates  rate  there  an  an e l e v a t e d  RCR  7).  higher  muscle so  4 rate  (Fig.  the  maximal r a t e of  rate  6).  significant,  the  Although mean RCR  muscle m i t o c h o n d r i a f o r a l l Pyruvate  was s u p p l i e d  malate  muscle 7).  for  was  s u b s t r a t e s was s u p p l i e d . for  each  tissue  c o n c e n t r a t i o n was state 3 oxidation  at  Although muscle and  higher  was not  f o r heart  (Fig.  8).  in  always  than  at a c o n c e n t r a t i o n  for  of 5mM  S i n c e malate or  of d i r e c t l y when  either  in  order  t o maintain  to  oxidized oxidizable of  these varied  discover  maximal  to  lactate  Pyruvate c o n c e n t r a t i o n was  required  the  s u b s t r a t e s at l o ^ C .  unknown  15°C  were  state 3 oxidation,  was higher  the c o n c e n t r a t i o n  (pyruvate)  as  either  must f i r s t be converted t o pyruvate i n order t o be  substrate  This  mitochondria  the d i f f e r e n c e  ensure maximal r a t e s of o x i d a t i o n .  by m i t o c h o n d r i a ,  was  6).  s t a t e 4 r a t e f o r both s u b s t r a t e s was s i g n i f i c a n t l y muscle ( F i g .  the  rates  what of  Each p o i n t r e p r e s e n t s o n l y one  35  FIGURE 7: S t a t e 3 0 consumption at 15°C. Sample s i z e as d e s c r i b e d i n M a t e r i a l s and Methods (mean ± SEM). S i g n i f i c a n t d i f f e r e n c e between p a i r s (P < 0.05) i n d i c a t e d by * ( 0 consumption) or + (RCR) 2  a  Heart s t a t e 3 0  Muscle s t a t e 3 0  RCR  consumption  3  3  consumption  37  FIGURE Ss S t a t e 3 0 consumption as a f u n c t i o n of pyruvate c o n c e n t r a t i o n . Values were obtained from a t o t a l of 4 m i t o c h o n d r i a l p r e p a r a t i o n s f o r each t i s s u e . Number of assays are i n d i c a t e d i n parentheses and v a l u e s are expressed as mean ± SEM. 2  A. B.  Heart Muscle  38  [PYRUVATE] (mM)  39 s t a t e 3 measurement made d u r i n g the f i r s t p u l s e of ADP, when the i n i t i a l  pyruvate  apparent t h a t at  concentration  were  only  known.  pyruvate c o n c e n t r a t i o n s as  maximal r a t e s of s t a t e 3 0 both p o p u l a t i o n s  was  (Fig.  3  low as  was  O.OSmM  consumption c o u l d be a t t a i n e d by  8) .  obviously  It  State 3  limited  when  0  3  consumption  the  initial  rates  pyruvate  c o n c e n t r a t i o n was O.OlmM. A pyruvate c o n c e n t r a t i o n of O.OlmM i n 2 mis of i s equivalent sufficient  to  20 nmoles  substrate  400 nmoles of ADP 9).  of  to allow  pyruvate.  This  solution was  not  complete p h o s p h o r y l a t i o n  the amount c o n t a i n e d i n one p u l s e  of (Fig.  A second a d d i t i o n of 20 nmoles of pyruvate r e s u l t e d  an e q u i v a l e n t r a t e  of •=> consumption  the f i r s t p u l s e of pyruvate. 400 nmoles of ADP and D . 2  If  the o x i d a t i o n of  were  0.05  Clearly,  and  consumption,  umoles  O.OlmM pyruvate  was 0.08  given umoles of  consumption i s e q u i v a l e n t  to  of p y r u v a t e .  a 2.0  ml  mM pyruvate are e q u i v a l e n t  to  0.032 umoles  0.02  2  with  consumed f o r each molecule  then t h i s 0  volume, 0 . 0 5 , 0.025 and 0.01 0.1,  0  saturating pyruvate,  5 oxygen atoms  pyruvate o x i d i z e d ,  Typical  as was obtained  in  2  of  In  pyruvate,  respectively.  i n t h i s volume would not s u s t a i n 0  3  consumption t o phosphorylate the e n t i r e ADP p u l s e TEMPERATURE For mitochondria  isolated  from e i t h e r  p a t t e r n of s u b s t r a t e p r e f e r e n c e was d i f f e r e n t 15 C a  (Fig.  s u b s t r a t e of  10a,b).  For  p r e f e r e n c e at  heart, 5°C.  pyruvate  the  at 5°C than at remained  M a l a t e , which  e q u a l l y good at 1 5 C , produced s i g n i f i c a n t l y Q  tissue  had  the been  lower s t a t e 3  40  FIGURE 9s R e p r e s e n t a t i v e 0 consumption t r a c e from h e a r t ; 1 . 3 8 mg p r o t e i n ; 1 5 ° C . C o n c e n t r a t i o n of each a d d i t i o n i n a f i n a l volume of 2 . 0 miss malate - 0.05mM ADP - 200uM; pyruvate - O.Olmli 3  MALATE ADP PYRUVATE ADP PYRUVATE ADP PYRUVATE  42  FIGURE 10: S t a t e 3 0 consumption at 5 and 15°C. Sample s i z e as d e s c r i b e d i n M a t e r i a l s and Methods (mean ± SEM). S i g n i f i c a n t d i f f e r e n c e s among means (P < 0.05) i n d i c a t e d by * (15=C), + ( 5 ° C ) . 2  A. B.  Heart Muscle  15°C  STATE 3 O,  CONSUMPTION  (natoms O/min/mg protein)  44 r a t e s than malate  pyruvate at  were  5°C  (Fig.  to  those  equivalent  lOa).  The  produced  a c e t y l - c a r n i t i n e or glutamate as s u b s t r a t e . h e a r t at 5°C were q u i t e v a r i a b l e . o x i d a t i o n of  pyruvate.  (Fig.  11) a n d ,  with  with either  RCR v a l u e s  for  RCR was h i g h e s t f o r  the  L a c t a t e was  s i g n i f i c a n t l y higher s t a t e 4  rates  characterized  r a t e than any other  consequently, a r e l a t i v e l y  by  a  substrate  low RCR.  There  was no s i g n i f i c a n t d i f f e r e n c e i n RCR between 5 and 15°C  for  any given s u b s t r a t e . The p a t t e r n of  mosaic muscle m i t o c h o n d r i a l  p r e f e r e n c e a l s o changed as 10b). At  5°C  pyruvate  a f u n c t i o n of temperature  and l a c t a t e  continued  to  s u b s t r a t e s which produced the h i g h e s t s t a t e 3 Q rates.  Malate was  pyruvate or  oxidized s i g n i f i c a n t l y  l a c t a t e , while  substrates.  p a t t e r n observed at  2  This i s  5b)  be  more s l o w l y  from  t h e r e was  r e a l d i f f e r e n c e i n s t a t e 3 r a t e s among s u b s t r a t e s . not vary s i g n i f i c a n t l y  among  than  lower than those  i n which  the  glutamate  quite different  15°C ( F i g .  (Fig.  consumption  a c e t y l - c a r n i t i n e and  produced r a t e s which were s i g n i f i c a n t l y the other  substrate  of the no  RCR  did  s u b s t r a t e s at 5 ° C , except  for  a c e t y l - c a r n i t i n e which e x h i b i t e d a remarkably high degree of respiratory  control.  3 r a t e s at 5°C ( F i g . and l a c t a t e  10b,  proceeded  w h i l e glutamate state 4 rates.  and With  was s i g n i f i c a n t l y t h e r e was no  S t a t e 4 o x i d a t i o n r a t e s mimicked s t a t e 11).  most r a p i d l y ,  O x i d a t i o n of followed  a c e t y l — c a r n i t i n e produced  pyruvate  by  malate,  the  lowest  a c e t y l - c a r n i t i n e as s u b s t r a t e the  higher  at 5°C  than at  significant difference  15 C f o r any s u b s t r a t e . 0  Fig.  lS^C.  i n RCR  RCR  Otherwise  between 5  and  45  FIGURE 11: S t a t e 4 0 consumption and RCR at 5 ° C . Sample s i z e as d e s c r i b e d i n M a t e r i a l s and Methods (mean ± SEM). S i g n i f i c a n t d i f f e r e n c e among means (P < 0.05) i n d i c a t e d by + ( 0 consumption) or + (RCR) 2  2  A. B.  Heart Muscle  State 4 0  RCR  2  consumption  STATE  4 O,  CONSUMPTION  STATE 4 O,  (natoms O/mln/mg protein)  o  I  <•> I  a 1  1  ro 1  CONSUMPTION  (natoms O/mln/mg protein)  1  o r  '  •> i  1  « i  1  n 1  -r  •P-  boa  uou  47 Q i o v a l u e s -for s t a t e 3 and s t a t e 4 Q a r e shown i n Table  4.  Q  d i f f e r e n c e between mean O  i 0  values  2  consumption r a t e s  are simply based on  the  consumption r a t e s at 5 and  lS^C.  They should t h e r e f o r e be c o n s i d e r e d as approximations  only.  z  S t a t e 3 Qjo v a l u e s f o r heart mitochondria were approximately 2 for a l l State 3  s u b s t r a t e s except malate which was higher Q  values  t o  higher than f o r  f o r muscle  Qio's  were  glutamate o x i d a t i o n . acetyl-carnitine  Except  high  with malate  In f a c t ,  r a t e s were always at the corresponding  muscle  either  t o  and  Muscle s t a t e  acetyl—carnitine  3 or on  reflected  values.  or l a c t a t e ,  heart  of o x i d a t i v e  phosphorylation  at  muscle  mitochondria at  both 5 and 1 5 ° C , heart s t a t e  rate,  and o f t e n higher  except  for  3  than  oxidation  of  than those  of  1).  5 ° C , heart RCR with  for  l e a s t as h i g h ,  l a c t a t e at 15°C (Table At  muscle.  and glutamate o x i d a t i o n was a l s o  high s t a t e 4 Q  12).  be  The dramatic e f f e c t of temperature  e x h i b i t e d the higher r a t e s 5°C ( F i g .  to  v a l u e s f o r malate  x o  2 in  particularly  in strikingly  mitochondria tended  h e a r t , although Q  pyruvate were approximately  (3.08).  v a l u e s were higher  malate  or  pyruvate  as  substrate.  Otherwise no s i g n i f i c a n t  d i f f e r e n c e occurred between RCRs of  different  12).  tissues  t h e r e were muscle s t a t e  no  (Fig.  significant 4  r a t e s with  In c o n j u n c t i o n differences either  between  malate or  s u b s t r a t e , even though heart had s i g n i f i c a n t l y 3 r a t e s under these  conditions.  with  this,  heart  or  pyruvate  as  higher  state  48  TABLE 4:  S t a t e 3 and s t a t e 4 D consumption Q v a l u e s based on mean D consumption r a t e s at 5 and 15 C 3  1 0  z  0  STATE 3 Q Heart Muscle 1 0  STATE 4 Q Heart Muscle 1 0  Acetyl-carnitine  2.25  4.09  2.01  7.19  Glutamate  1.99  6.89  1.69  7.51  Malate  3.08  2.03  3.38  1.98  Pyruvate  1.75  2.30  2.34  1.76  Lactate  1.87  2.75  1.34  2.88  49  FIGURE 12: S t a t e 3 0 consumption and RCR of heart and muscle mitochondria at 5 ° C . Sample s i z e as d e s c r i b e d i n M a t e r i a l s and Methods (mean ± SEM). S i g n i f i c a n t d i f f e r e n c e between means (P < 0.05) i n d i c a t e d by * ( 0 consumption) or + (RCR). 2  3  Heart s t a t e 3 0  Muscle  RCR  3  state 3 0  consumption  3  consumption  51  EFFECT OF pH Three lactate -  substrates  acetyl-carnitine,  and b  acetyl-carnitine  show the and  decreased, state 3 decreased t o  0  2  pH p r o f i l e  respectively.  consumption  rate increased.  3 rate  measurements  conclusive.  pH (6.8)  appeared t o  are  required  State 3 r e s p i r a t i o n  7.6  state  r e s p i r a t i o n was heart.  3  rate  measured  burning As  level  off,  pH  13c,d).  decreased.  at lower  pH  although  6.8  to  At pH  i n muscle  from  those  change i n s t a t e 3 r a t e  as  more p h y s i o l o g i c a l  acetyl-carnitine  c e r t a i n s i m i l a r i t i e s were e v i d e n t  (Fig.  (Fig.  14a),  while  pH-dependence between pH 7 . 0 (7.8),  muscle s t a t e 3 r a t e  observed with  the  pH v a l u e s  first  7.6  (Fig.  decreased.  State 3  showed 14b).  no  the  D  =  heart clear  At high  U n l i k e the  two s u b s t r a t e s ,  (Fig.  glutamate,  7.0 — 8.0 in  muscle  of  substrate  or  14a,b).  consumption was pH—dependent between pH mitochondria  the  w i t h i n the range  p r o f i l e s u s i n g l a c t a t e as of  above  in  3 r a t e was  differed  no  than  physiological  Although the pH  be  Mitochondrial  Even at pH 5 . 8 , well beyond  r a t e s measured at h i g h e r ,  pH this  i n mosaic muscle showed  There was no s i g n i f i c a n t  range, s t a t e  pH  As  pH dropped as low as 6 . 3 .  13c).  and  and beyond,  below  c l e a r pH-dependence below pH 7.4 ( F i g . or  f o r heart  glutamate,  low p h y s i o l o g i c a l  i n c r e a s e of s t a t e  7.4  glutamate  were used t o generate pH p r o f i l e s f o r each t i s s u e .  F i g u r e s 13a  more  -  pH  patterns  lactate  pH  p r o f i l e f o r each t i s s u e showed a d r a s t i c decrease i n s t a t e 3 respiratory  r a t e below pH 7 . 0  In some muscle  mitochondrial  a s s a y s , pyruvate was added and s t a t e 3 r a t e s measured a f t e r  52  FIGURE 13s S t a t e 3 D consumption pH p r o f i l e f o r a c e t y l - c a r n i t i n e and glutamate o x i d a t i o n at 1 5 C . Sample s i z e as d e s c r i b e d i n M a t e r i a l s and Methods (mean ± SEM). 3  0  A. B. C. D.  Heart; acetyl—carnitine Heart; glutamate Muscle; a c e t y l — c a r n i t i n e Muscle; glutamate  STATE 3 O, CONSUMPTION  ( n . I o m . O/mln/mg prot.ln)  « o  • I-  O  ES  54  FIGURE 14: S t a t e 3 0 consumption pH p r o f i l e f o r l a c t a t e o x i d a t i o n at 1 5 C . Sample s i z e as d e s c r i b e d i n M a t e r i a l s and Methods (mean ± SEM). 3  a  A. B.  Heart Muscle  STATE 3 0 CONSUMPTION (natoms O/min/mg protein) 2  _ at o  r •  * o  r  o> o  r  r •  ro o  r - -•'T  r  -  > 1  o  1  b _  1—o—|  1Q 1 r  '  b M  CD  b I  o  b M  '•*>  •oH  >  b  j  i-  L  I  1  i  l a c t a t e o x i d a t i o n r a t e s had r a t e s of  lactate  pyruvate  (Fig.  been measured.  o x i d a t i o n were  15).  the same  At pH 7.S  the  as o x i d a t i o n  of  At pH 7 . 3 s t a t e 3 l a c t a t e o x i d a t i o n was  somewhat reduced r e l a t i v e  t o pyruvate  and at  pH 6 . 5  this  r e d u c t i o n was f u r t h e r a c c e n t u a t e d . RCR and s t a t e F i g u r e 16.  4 v a l u e s at d i f f e r e n t p H ' s are shown  In most cases  the RCR's and s t a t e 4 r a t e s  almost m i r r o r - i m a g e s of each o t h e r . 4 was h i g h .  The  were  When RCR was low,  RCR p a t t e r n s were not normally  by the s t a t e 3 p a t t e r n s  (Fig.  13,14).  was heart mitochondria  burning l a c t a t e .  in  state  duplicated  The e x c e p t i o n t o  this  In t h i s case  RCR  v a l u e s r e f l e c t e d s t a t e 3 p a t t e r n s more c l o s e l y than s t a t e (Fig.  13).  State 4 rates  h e a r t than i n muscle.  fell  i n t o a more narrow range  Heart s t a t e 4  - 1  min  - 1  mg p r o t e i n  more narrow  - 1  ,  10 natoms 0 m i n  mg  - 1  r a t e s r a r e l y reached 20 natoms  although,  i n heart  in  The lowest s t a t e 4 r a t e s measured f o r  both p o p u l a t i o n s of m i t o c h o n d r i a were protein .  4  a d m i t t e d l y , the pH range  than muscle.  In  several  muscle s t a t e 4 r a t e s approached 30 natoms m i n  - 1  0  was  instances  mg p r o t e i n  - 1  and were o f t e n well above 2 0 , although with a c e t y l - c a r n i t i n e as s u b s t r a t e s t a t e 4 r a t e remained r e l a t i v e l y 6.B — 7 . 4  With l a c t a t e as s u b s t r a t e , muscle  e x h i b i t e d the  highest  state  measurements except one natoms 0  low between pH  min  - 1  mg  4  (pH 6.7)  protein . - 1  rates  of  mitochondria  all,  f a l l i n g between RCR v a l u e s  with  all  24 -  rarely  52  dropped  below 4 i n e i t h e r t i s s u e except at very high or very low pH. Based on d a t a from muscle  mitochondria, state 4 rates  e l e v a t e d at high pH (>7.5) and RCRs were c o n s i s t e n t l y 4.  O x i d a t i o n with heart mitochondria was not  were below  consistently  57  FIBURE 15: R e p r e s e n t a t i v e heart m i t o c h o n d r i a l 0 consumption t r a c e s of l a c t a t e o x i d a t i o n at v a r i o u s values. Assay b u f f e r i n c l u d e d lOmM NADH and 2 u n i t LDH. C o n c e n t r a t i o n s of each a d d i t i o n i n a f i n a l volume of 2 . 0 mis: ADP — 200uM; pyruvate — 5mM. 2  A. B. C.  pH 6.54 pH 7.32 pH 7.84  10mM LACTATE O.OSmM MALATE  oo  59  FIGURE 16: S t a t e 4 O consumption and RCR pH p r o - f i l e s at 15°C. Sample s i z e as d e s c r i b e d i n M a t e r i a l s and Methods (mean ± SEM). a  A. B. C. D. E. F.  Heart; a c e t y l - c a r n i t i n e H e a r t ; glutamate Heart; l a c t a t e Muscle; a c e t y l - c a r n i t i n e Muscle; glutamate Muscle; l a c t a t e  State 4 0 o  o RCR  2  consumption  STATE 4 O CONSUMPTION a (natoma O/min/mg protaln)  aoa 09  monitared at high enough  pH t o make  a v a l i d comparison  to  muscle. The p o i n t t h a t s t a t e 3 r e s p i r a t o r y d e c r e a s i n g pH i n s e t of (Fig.  h e a r t was f u r t h e r  assays i n 17).  It  which 5mli  i s important  done i n the f o l l o w i n g order  rate increased  emphasized by a  malate was t o note t h a t  used as  single  substrate  the assays  were  : pH 6 . 8 , 7 . 3 , 7 . 8 , 8 . 2 and 6 . 5 .  The f i n a l a s s a y , at the lowest pH, y i e l d e d the h i g h e s t 3 r a t e s and RCR's.  with  state  62  FIGURE 17s Malate o x i d a t i o n <5mM> by heart mitochondria at lS^C as a -function o-f pH. Each p o i n t r e p r e s e n t s 4 o b s e r v a t i o n s -from a s i n g l e assay except where noted (*). Values expressed as mean ± SEM. All p o i n t s were d e r i v e d from a s i n g l e m i t o c h o n d r i a l preparation.  )  8  STATE 4 CONSUMPTION  STATE 3 O,  (natoma O/mln/mg protein)  CONSUMPTION  (natoma O/mln/mg protein)  ro  o  o  T"  M  J  L  T  ro o  T"  9 O  T"  64 DISCUSSION M i t o c h o n d r i a from exhibited  a high degree  both  heart  and  of r e s p i r a t o r y  mosaic  control,  muscle  indicating  t h a t p r e p a r a t i o n s from each t i s s u e were e q u a l l y v i a b l e . protocol  for  p r e p a r a t i o n of heart mitochondria d i d not  significantly  from  t h a t of  While the technique f o r  Chappell and  it  was  the f i r s t  such i s o l a t i o n  i s o l a t i o n of  mitochondria  somewhat  mitochondria-rich rarely  been  difficult  mosaic  undamaged  than  heart.  presumably  of  from  The problem  because  the  role  mitochondria i n energy p r o d u c t i o n of white or mosaic i s minor r e l a t i v e  to  t h a t of other  s t u d i e s of t i s s u e a d a p t a t i o n is interesting  t o see  the r e s p i r a t o r y  for  requirement,  oxidative  metabolism i s  Mitochondrial  the  inner  efficient  mitochondrial oxidative  oxidation  of  shuttle  system  a~glycerophosphate  such  for it  properties  of  has a  low  high. judged on  RCR and ADP/O. membrane  phosphorylation  exogenous  of  from those of heart whose need  v i a b i l i t y was  exogenous NADH o x i d a t i o n ,  had  level,  mosaic muscle, which  differ  a  muscle  However,  at the s u b — c e l l u l a r  how  mitochondria i s o l a t e d from oxidative  tissues.  for  tissue.  white or  reasonable y i e l d s  such as  approached,  from t h i s  in teleost  more  tissue  mosaic  an a d a p t a t i o n of t h a t used  The low d e n s i t y of mitochondria muscle makes  vary  (1972).  i s o l a t i n g mitochondria from  muscle of rainbow t r o u t was heart,  Hansford  The  NADH should as  shuttle  the  is  a  the b a s i s  of  Impermeability  of  requirement  (Mitchel1,1961) require  a  et  and  functional  malate-aspartate  (Williamson  of  or  al.,1973;  65 Buecher,1964s Hochachka et presented  here,  al.,1979).  exogenous  measurable r a t e s ,  NADH  In the  was  not  experiments oxidized  i n d i c a t i n g membrane i n t e g r i t y .  Both p o p u l a t i o n s of mitochondria had a c c e p t a b l e ratios,  ranging from 2 . 3 -  both 5  and  substrate, fell  15°C.  below  NAD--1inked and Hunter,1951; Davis et  The  ADP/O r a t i o  the  2 for  transport  and  although  ions are  1.9.  according  v a l u e s are  required  functions.  for  Perfectly  FAD"*"-1 inked s u b s t r a t e s , results  mitochondrial  from  values  Lardy,1952;  study  of  3  for  (Ochoa,1943;  Estabrook,1967; and  probably not various  respectively  this  values  to Hinkle  coupled  at  FAD*-1inked  Both s e t s of  have r e a l ADP/O r a t i o s of 2 . 0 and 1.33  ADP/O  the  FAD--1inked substrates  (1979) these t r a d i t i o n a l because H*  -  for  theoretical  Coopenhaver  al.,1974),  ADP/O  2 . 8 f o r NAD*-1inked s u b s t r a t e s  s u c c i n a t e , was 1.4  somewhat  at  inner  Yu  realized membrane  mitochondria  may  f o r NAD~-1inked  and  (Hinkle and  indicate  that  Yu,1979). in  both  p o p u l a t i o n s 0= consumption was t i g h t l y coupled  t o ATP p r o d u c t i o n .  T h i s was confirmed  by high RCR  values  (>4) . SUBSTRATE UTILIZATION At 15=*C a l l r a t e s of  oxidative  populations. r a t e s of indicating  substrates  phosphorylation  Pyruvate  oxidation, a  good  (except  in  potential  metabolism i n both t i s s u e s .  in  particular  not only  at for  NADH) produced both  mitochondrial  had c o n s i s t e n t l y  lS^C but aerobic  high  also  at  high 5°C,  carbohydrate  66  Lactate and, i n  i s a potential  fact,  oxidatively  oxidative  teleost hearts  (Lanctin  et  are  substrate in  known t o  al.,1980;  heart  burn  Driedzic  et  lactate  al.,1984).  L a c t a t e produced as an endproduct of anaerobic metabolism i n mosaic muscle i s (Jones  and  Randal1,1978;  ultimate fate eventually  is a  Turner  et  oxidative  and  al,  fuel  A  periods and  p a r t of  its it  the l i v e r ,  glycogen  Wardle,1979;  1983).  for  long  al.,1983)  blood t o  converted t o  (Batty  Turner  et  for  debate.  v i a the  be  situ  Brooks,1983; potential  may  in  the muscle  matter of  transported  some f r a c t i o n oxidized  retained in  Thus  in  while  situ  or  Donovan lactate  mosaic muscle  is  and is  as well as  a for  heart. The r a t e of l a c t a t e o x i d a t i o n surprisingly pyruvate  low,  assuming t h a t  extramitochondrially.  by heart mitochondria was l a c t a t e was  Doubling the  converted  to  concentration  of s u b s t r a t e or LDH and i n c r e a s i n g NAD"*" from 1-2 mM made d i f f e r e n c e to  state  3  oxidation  rates.  State  3  rates  obtained under these c o n d i t i o n s were assumed at the time be maximal. state  3  However,  oxidation  presence of  1—2  in later rates  mM NAD"*".  achieved with lOmM  assays,  beyond  those  The r a t e s  NAD"*" were equal  assay c o n d i t i o n s ,  significantly  more l a c t a t e  lOmM NAD"*" than  t o pyruvate  i n the presence  probable t h a t the  h e a r t mitochondria were NAD"*"—1 i mi t e d .  the  oxidation  In a d d i t i o n ,  in  with given  converted  the presence  of 2mM NAD*.  reported rates for  in  t o those obtained  the LDH r e a c t i o n  It  to  stimulated  produced  of l a c t a t e  s a t u r a t i n g c o n c e n t r a t i o n s of p y r u v a t e . mitochondrial  lOmM NAD"*"  no  is  of  quite  lactate oxidation  in  67 In mosaic muscle,  no s i g n i f i c a n t  between s t a t e 3 r a t e s of This i s  what  one  occurred  o x i d a t i o n of l a c t a t e or  would  converted t o pyruvate  difference  predict  if  lactate  q u i c k l y enough  pyruvate. were  t o supply  being  saturating  c o n c e n t r a t i o n s of pyruvate t o r e s p i r i n g m i t o c h o n d r i a . both p o p u l a t i o n s were r a t e s , the a b i l i t y depend  on  the  capable of burning  t o use  ability  extramitochondrially  et  to  convert  high  to  on l a c t a t e oxidase  to  pyruvate lactate activity  al.,1984).  assumed  that  of  l a c t a t e oxidation to t h i s  lactate  extramitochondrially  and  pyruvate.  The  should not  be overlooked  oxidative  lactate  which would be c o n s i s t e n t with  The d i s c u s s i o n has  pyruvate at  l a c t a t e as a s u b s t r a t e seemed  o x i d a t i o n r a t e being dependent (Driedzic  Since  was  was  possibility  converted  to  t r a n s p o r t e d and of  an  point  pyruvate  oxidized  intramitochondrial In  such a  LDH  case,  the  r a t e may be a f f e c t e d not o n l y by L D H k i n e t i c s  but  by t r a n s p o r t  however.  as  of l a c t a t e i n t o the m i t o c h o n d r i a l m a t r i x .  only known i n t r a m i t o c h o n d r i a l  is  LDH  LDHx,  whose  The  synthesis  i s c o n t r o l l e d by a unique g e n e t i c l o c u s and which occurs  in  s p e c i a l i z e d sperm  de  mitochondria  Domenech et a l . , 1 9 7 2 ;  Blanco et  (Clausen,1969; al.,1975).  Machado  Van Dop et  (197B) have provided evidence t h a t the pyruvate of  bovine  sperm  mitochondria  l a c t a t e and p y r u v a t e . of  rat  liver  has  In c o n t r a s t ,  mitochondria  does  dual  translocase  specificity  the pyruvate not  al.  for  translocase  transport  lactate  (Halestrap and Denton,1974). While i n t r a m i t o c h o n d r i a l i n sperm  cells,  Skilleter  and  LDH has only been Kun  (1972)  observed  reported  the  68 presence of LDH a s s o c i a t e d rat  liver  mitochondria.  demonstrated  that  mitochondrial  fraction  outer s u r f a c e .  LDH  in  Hultin,1970)  and  observed  associated was l o c a t e d  trout  skeletal  specifically in  brain  muscle <Lluis,1984)  of  uncertain,  efficiency noted  with  the  on the  rat  liver  outer  LDH  than s o l u b i l i z e d  muscle  While  such  membrane  with  (Lluis,1984).  skeletal  aid It  in  muscle form  no evidence  of  Lactate  oxidation  r e q u i r e d the a d d i t i o n of exogenous LDH.  While pyruvate of 5mM, a  was normally s u p p l i e d at a c o n c e n t r a t i o n  c o n c e n t r a t i o n as low  r a t e s of s t a t e 3 o x i d a t i o n .  as 0.05mM produced  Low r a t e s of l a c t a t e  maximal oxidation  would i n d i c a t e minimal l a c t a t e c o n v e r s i o n t o pyruvate itself  oxidation.  was  somehow  interfering  In a study i n which o x i d a t i v e  with  unless pyruvate  phosphorylation  malate/glutamate i n r a t b r a i n mitochondria was i n h i b i t e d the presence of l a c t a t e , the cause of per se  be  t r o u t heart or mosaic  muscle mitochondria i n the present study.  lactate  the  should  a c t i v i t y i n the bound  m i t o c h o n d r i a l — a s s o c i a t e d LDH i n e i t h e r  eel  physiological  skeletal  There was  has  muscle of  may  rabbit  and  LDH  and  substrate supply.  associated  been  (Melnick  any p o s s i b l e  an o r i e n t a t i o n  in  <1973)  mitochondrial-bound  mitochondria had somewhat lower  clearly  Krebs  (Vdovichenko,1978)  of m i t o c h o n d r i a l  that  and  mammals and s k e l e t a l  (Mattison et a l . , 1 9 7 2 ) . role is  Brdiczka  LDH bound t o s u b c e l l u l a r p a r t i c l e s has  demonstrated  been  with the intermembrane space  inhibition  ( H i l l e r e d et  it  was concluded  rather  al,1984).  that  low pH  than the presence of In the  of in was  lactate  present study  all  s u b s t r a t e s were n e u t r a l i z e d p r i o r t o i n c l u s i o n i n the a s s a y ,  69 so t h e r e  was  pyruvate.  no  difference  State 3 i n h i b i t i o n  (19S4) was  accompanied  Respiratory control the s t a t e 3 addition  of  5mM  that  of  and  was high i n the present s t u d y ,  even when  l a c t a t e o x i d a t i o n was  pyruvate  to  lactate  r a t e s of  lactate  assays  did  low.  in  Finally,  which  It  not,  inhibit  pyruvate  mitochondria.  rates,  pyruvate o x i d a t i o n . in  any  in t h i s  way,  One  substrate,  i n order t o be  would not  was  study.  s u p p l i e d as the s o l e exogenous  must a l s o be converted t o  muscle  at sub-maximal  p h o s p h o r y l a t i o n of pyruvate  by i s o l a t e d  lactate  i n the study of H i l l e r e d et a l loss  oxidizing  M a l a t e , when  between  control.  r e s u l t e d i n maximal concluded  by  pH  respiratory  r a t e of  m i t o c h o n d r i a were  oxidative  in  oxidized  expect  maximal  r a t e s of malate s t a t e 3 o x i d a t i o n t o be higher than those of pyruvate maximal  unless rates  the of  o x i d a t i o n were  pyruvate  oxidation.  never higher  transporter In  fact,  rates  than pyruvate  Lower malate r a t e s must have been a r e s u l t pyruvate transport  supply i n t r a m i t o c h o n d r i a l l y of  malate i n t o  were  limiting of  malate  in this  study.  of r e s t r i c t i o n  e i t h e r because of  the mitochondria  or poor  of  slow malate  c o n v e r s i o n v i a m a l i c enzyme. Most mitochondria i s o l a t e d from animal t i s s u e s are able to rapidly Skorkowski et some  oxidize al.,1984).  organisms  by  a  malate as  the only s u b s t r a t e  This l i m i t a t i o n high  malic  i s overcome  enzyme  -  (Henderson,1966; Simpson  B r d i c z k a and  Pette,1971;  al.,1972;  Lin  and  and  Frenkel,1971,1972;  Davis,1974;  Skorkowski  (see in  activity.  NADP*"—! inked m a l i c enzyme i s present i n the mitochondria many t i s s u e s  not  of  Estabrook,1969; Bartholome et  et  al.,1977).  70 Skorkowski et a l  (1984) demonstrated t h a t the a b i l i t y  o-f cod  heart mitochondria t o o x i d i z e malate at s i g n i f i c a n t l y  higher  r a t e s than those of r a b b i t or r a t heart was l i n k e d t o  higher  combined a c t i v i t i e s  enzyme  of NADP"*"-  and NAD*-1 inked m a l i c  i n the cod m i t o c h o n d r i a . In  the present  transported across  study,  malate appeared  the i n n e r  t o be  easily  m i t o c h o n d r i a l membrane  since  the requirement of many s u b s t r a t e s f o r a malate "spark" met by e x t r a m i t o c h o n d r i a l  malate c o n c e n t r a t i o n s of  The "spark" was not of s u f f i c i e n t l y oxidized d i r e c t l y .  0.05mM.  high c o n c e n t r a t i o n t o be  M i t o c h o n d r i a from e i t h e r heart or  muscle mitochondria of rainbow m a l i c enzyme a c t i v i t y  was  mosaic  t r o u t c o n t a i n e d high  t o allow o x i d a t i o n  enough  of 5mM malate  r a t e s equal t o those of pyruvate under some c o n d i t i t o n s . contrast,  t r o u t l i v e r mitochondria were i n c a p a b l e of  malate at high r a t e s  (Suarez  been p o s t u l a t e d  Davis  al.,1972;  by  and Hochachka,1981a). and  his  co-workers  at In  burning It  has  (Davis  et  Davis and Bremer,1973; L i n and D a v i s , 1 9 7 4 ; Lee and  Davis,1979;  H i l t u n e n and Davis,1981)  t h a t m a l i c enzyme  may  be important i n the r e g u l a t i o n  of the c o n c e n t r a t i o n of  TCA  cycle  the  intermediates  and  metabolism i n muscle.  in  regulation  Furthermore, the a b i l i t y  of  energy  to  convert  TCA c y c l e i n t e r m e d i a t e s t o pyruvate may p l a y a s p e c i a l i n the c o n v e r s i o n of the amino  acids  al.,1976;  to  alanine  trout.  in  skeletal  muscle  chain  (Barber  et  G o l d s t e i n and Newsholme,1976; Lee and D a v i s , 1 9 7 9 ) .  L i p i d s and amino f o r both  carbon s k e l e t o n of branched  role  heart and  acids (protein)  mosaic muscle  Acetyl-carnitine,  are p o t e n t i a l  mitochondria of  palmitoyl-carnitine  and  fuels rainbow several  71 amino a c i d s  have proven  good s u b s t r a t e s  i s o l a t e d from a number of sources assess  relative  acetyl-carnitine State 3 were high i n  value,  oxidative  heart  for  (see  Table 5)  i n terms  of  although somewhat  of  source  lower high  s u b s t r a t e s appeared t o respiratory  control.  l i p i d or  exists  mitochondrial substrate  lS^C  RCR v a l u e s remained  using carbohydrate,  fuel  in  p r o t e i n as  heart,  utilization.  Carbohydrate  on would  oxidative  The  may  have  reflected  high a  need  an  based  at l e a s t i n terms of r a t e  phosphorylation.  be The  appear t o be the best s o u r c e ,  utilization  to  phosphorylation  r a t e s of o x i d a t i o n of both s u b s t r a t e s at  equally e f f i c i e n t  oxidative  In order  or glutamate was monitored f o r each t i s s u e .  ADP/O was unchanged and a l l  potential  mitochondria  (Table 5 , 6 ) .  than those of pyruvate or malate. (>6),  for  of  rate  of  pyruvate  to  burn  lactate  oxidatively. According t o m i t o c h o n d r i a l lS^C, carbohydrate, oxidative  lipid  substrates  substrate u t i l i z a t i o n  and p r o t e i n  in  trout  are  mosaic  all  potential  muscle.  Both  a c e t y l — c a r n i t i n e and glutamate produced s t a t e 3 r a t e s were s i m i l a r t o those of pyruvate. variable,  State 4 respiratory  a l l s u b s t r a t e s except l a c t a t e  produced s i g n i f i c a n t l y  higher r a t e s .  the f a c t t h a t l a c t a t e  had the lowest RCR.  high s t a t e 4  r a t e s with  l a c t a t e as  which  T h i s was r e f l e c t e d The reason  substrate i s  ADP/O r a t i o s were s i m i l a r t o one another f o r a l l In terms of r a t e and c o n t r o l  which  Although RCR v a l u e s were  they were a l l g r e a t e r than 4.  r a t e s were s i m i l a r f o r  at  of o x i d a t i v e  in for  unknown.  substrates.  phosphorylation,  72 TABLE 5;  SUBSTRATE  State 5 O  Q  Consumption  2  Pyruvate  s  Consumption of v a r i o u s  species  SUBSTRATE  Q  Consumption  z  Glutamate  5mM  8.8"  5mH  74.2 + 17.0  5oH  330 i 8  5ffiH  335 j 13  b  C  d  Pyruvate and Halate  1(BH  22.2 + 1.2-  5mH  116.8 + 10.0  5mH + 3mH malate  35.4*  5sH + O.loH pyruvate  33.4 + 8.5  + malate  3sH  50.8*  lOaH + 5BH i a l a t e  22"  5mH  0.5aH  115.2  10aH + 5mH malate  55  iii>M  15.6 + 0.8"  Hal ate ifsM  20.2 + 0.4-  3mH  8.7*  5fflH  92.2 + 14,8"  Succinate  1  Proline 30mH  209.0 i 22.4  IfflH  27.6 + 1.2-  5mH + 0.05 fflH pyruvate  71.2 + 15.6*  50uM + 3mM malate  27.1* 94.8"  64.3*  7.5 uH + 0.05aH malate  5aiH  54.2 i 15.6"  30uli  278 + 10  5ffiH  81.0 + 11.2*  30uM  159 ± 7  + rotenone  2188 38  lOuH + rotenone  9B  c  d  Acetyl-carnitine  h  0.63iH  244 t l l  l  0.63mH  132 t 5*  Oxygen consumption i n natoms Q/min/oq p r o t e i n  b  Palmitoyl-carnitine  5IBH  lOcifl + rotenone  f  2349  5DIH  b  b  c  a. Trout l i v e r , 15°, (Suarez and Hochachka, 1981a) b. locust  rectum, 25°C, (Chamberlin and P h i l l i p s , 19835 c. rabbit r e d muscle, unknown T°, (Pande and Blanchaer,1971) d. r a b b i t white muscle, unknown T°,(Pande and Blanchaer, 1971) e. ribbed mussel, 25°, (Burcham et al.,1984! f. squid heart, 15°C, (Hommsen and Hochachka, 1981) g. dog heart, unknown T°,(Hukherjee e t al.,1979) h. dog l i v e r , unknown T , (Fry et a l . , 1980) i . dog kidney, unknown T°, (Fry e t a l . , 1980) D  73  TABLE 6:  S t a t e 3 0=. consumption of l o c u s t and b l o w f l y body mitochondria at 30°C ( B a l l a n t y n e and S t o r e y , 1 9 8 3 ) . Mean ± S E M  SUBSTRATE  fat  D CONSUMPTION (natoms O/min/molecule c y t a) LOCUST BLOWFLY 2  Pyruvate(5.71mM)  N.D.  195.8 ± 5 4 . 3  Pyruvate(5.71mM) malate(0.1ImM) Malate(5.71mM)  N.D.  283.3 ± 8 2 . 6  Malate(5„71mM) pyruvate(0.1ImM) Succinate(5.71mM) rotenone Glutamate(5.71mM) malate(0.llmM) Proline(22.9mM) pyruvate(0.1ImM) Palmitoyl-carnitine(lOuM) malate<0.llmM)  9 3 . 0 ± 30.4  130.3 ± 3 5 . 9  101.0 ± 10.0  211.2 ± 7 0 . 2  6 6 . 9 ± 37. 1  424.7 ± 7 8 . 0  130.6 ± 3 5 . 2  133.8 ± 7 1 . 5  N.D.  165.O ± 4 1 . 5  187.8 ± 2 1 . 8  594.3 ± 2 0 . 8  74 acetyl-carnitine, e q u a l l y well  glutamate  and  were  utilized  by mosaic muscle m i t o c h o n d r i a .  State  3 rates  for  all  substrates in  compared f a v o u r a b l y  to oxidation  workers  particularly  liver  pyruvate  (Table  5),  (Suarez  and  mitochondria  p h o s p h o r y l a t i o n than pyruvate or  r a t e s measured those  for  Hochachka,1981a).  exhibited  higher  did  malate as  both by  other  rainbow  trout  15°C  heart  At  rates  of  oxidative  muscle m i t o c h o n d r i a  substrate.  tissues  In terms  with of  either  oxidative  carbohydrate metabolism, heart not only has the advantage of more mitochondria higher  rate  of  per  given mass  substrate  mitochondrial p r o t e i n . utilization  of  t i s s u e , but  oxidation  as  a  function  The e f f i c i e n c y with which  i s coupled t o ATP  also  a of  substrate  p r o d u c t i o n appears t o be  the  same f o r both t i s s u e s . Acetyl-carnitine  and glutamate were  same r a t e by mitochondria  from both t i s s u e s  values  heart  were  higher  in  indicating  a  more  utilization  t o ATP p r o d u c t i o n .  with  efficient  i n the two m i t o c h o n d r i a l  at  the  at 15°C.  RCR  these  coupling  substrates, of  substrate  Rates of l a c t a t e  utilization  p o p u l a t i o n s c o u l d not be  compared because of sub—maximal in  utilized  directly  r a t e s of l a c t a t e  oxidation  heart. Substrate u t i l i z a t i o n  availability  and  o x i d a t i o n of f a t t y levels,  including  phosphofructokinase  in vivo  competition. acids  (PFK)  Neely and Morgan,1974).  Both  can i n h i b i t  transport  depends on  of  presence  glycolysis  glucose  and pyruvate Oxidation  the  substrate  into  at  and  several  the  cell,  dehydrogenase  of a c y l - c a r n i t i n e s ,  (see in  75 particular  palmitoyl-carnitine,  d e c a r b o x y l a t i o n o-f pyruvate liver  (Batenburg and  Hochachka,19Blb) mitochondria. heart  by  chicken  liver  carnitine  liver  (Jagow  was p a r t l y  and  may  inhibit  (Bremer, 1965) ,  01sen,1976), t r o u t  and  i n c r e a s e d l e v e l s of  been shown t o  i n r a t heart  This inhibiton  free  have  rat  (Suarez et  and  al.,1968)  alleviated in  have  been  a c y l - C o A and decreased  rat  caused  l e v e l s of  by free  CoA (Bremer,1965). In a d d i t i o n t o s u b s t r a t e a v a i l a b i l i t y power output p l a y s a Glycogen can  and  competition,  r o l e in aerobic substrate  s u s t a i n high—power  l i m i t e d p e r i o d i n man, w h i l e  preference.  a e r o b i c metabolism  f a t s p r o v i d e a more  competition for  a  long—term  energy supply at the c o s t of reduced power output. presumably mediated by  for  This  ADP (Hochachka  is and  Somero,1984). TEMPERATURE Temperature may a f f e c t several l e v e l s . however,  all  mitochondrial r e s p i r a t i o n  Once a s u b s t r a t e has entered the TCA  s t e p s t o the f i n a l r e d u c t i o n of oxygen are  same no matter what the o r i g i n a l  substrate.  Any e f f e c t  temperature has on any of these s t e p s would a f f e c t of a l l  substrates equally.  Each  s u b s t r a t e used  study has i t s own d i s c r e e t t r a n s p o r t e r use e x a c t l y  the  TCA c y c l e .  The f a c t t h a t  of the enzymes  required for  membrane—bound  increases  sensitivity  the  cycle the that  oxidation in  this  and no two s u b s t r a t e s  same enzyme complement all  at  for entry into  of the t r a n s p o r t e r s e n t r y i n t o the likelihood  and  TCA c y c l e of  s i n c e temperature change i s known t o  the some are  temperature alter  76 membrane - f l u i d i t y . induced membrane reviewed by  The general restructuring  Hazel  temperatures  (1984).  known  to  phenomenon of in  rainbow t r o u t  Acclimation  a-ffect  the  membranes i n c l u d i n g m i t o c h o n d r i a l activities  and  lipid  (Hazel  and  Bomero,1973p S m i t h ,  has  been  environmental compositon  membranes, can  of enzymes which are f u n c t i o n a l l y  membranes  temperature  Prosser,1974;  o-f  influence  bound t o  these  Hochachka  and  1973,1974,1976).  The e f f e c t of temperature on the t r a n s m i t o c h o n d r i a l gradient  is  not c l e a r .  temperature i s t i s s u e the pH  gradient  is  Change  dependent, but uncertain.  affect intramitochondrial if  the  of i n t r a c e l l u l a r  pH.  Temperature It  transmitochondrial  whether  pH  affects  may  directly  i s important t o determine  pH  of o x i d a t i v e  with  this  gradient  varies  temperature because the magnitude of t h i s g r a d i e n t a f f e c t s the r a t e  pH  phosphorylation  with directly  (see  Hinkle  and McCarty,1978) . Temperature had utilization heart  an e f f e c t on the p a t t e r n of  i n mitochondria i s o l a t e d from both t i s s u e s .  mitochondria  pyruvate  remained  the  p r e f e r e n c e at 5 C , while a c e t y l - c a r n i t i n e at r e l a t i v e l y  reduced r a t e s .  State  malate were a l s o  significantly  5°C.  in substrate u t i l i z a t i o n  Qio  T h i s switch  of s t a t e 3 o x i d a t i o n .  State 3 Q  2 for a l l  s u b s t r a t e s except  mean  of  Q  i 0  3.08  for  respiration.  It  m a l i c enzyme  activity  lower than  1  D  3  of were  3 rates  for  f o r pyruvate  at  was r e f l e c t e d  by  v a l u e s were c l o s e  to  for malate,  state  In  substrate  or glutamate  a  oxidized  substrate  and  i s p o s s i b l e that e i t h e r  which produced 3.38  state  4  malate t r a n s p o r t  or  were s i g n i f i c a n t l y  for  a  reduced  at  low  77 temperature i n t r o u t heart m i t o c h o n d r i a . significantly  as a r e s u l t  as s u b s t r a t e .  H"* ion  -  permeability.  those of Pye change i n  RCR  4  over a  rates  mitochondria  is  of  of temperature  broad  i n d i c a t e d no  lactate  although  with  consistent  temperature range  in  either  The reason f o r  oxidation  at  5 C  in  Q  may  be  at  alter  were c o n s i s t e n t  (Tinea t i n e a ) .  unknown  -  15°C d i d not s i g n i f i c a n t l y  (1976) which  muscle or l i v e r of tench state  a change  These r e s u l t s  et a l .  vary  of temperature except with l a c t a t e  Apparently,  l e a s t i n the range of 5  RCR d i d not  high heart  related  to  sub-maximal o x i d a t i o n c o n d i t i o n s i n the a s s a y . The net e f f e c t of temperature on heart mitochondria was that  even  at  5 C, D  mitochondrial  maintained at reasonable r a t e s  (for  oxidation  could  comparison see Table 5 ) .  The maximal r a t e s of o x i d a t i o n of p y r u v a t e ,  acetyl-carnitine  and glutamate a l l changed by approximately the same As at  15 C, 0  oxidative  carbohydrate  fuel  would  available  at  appear 5°C.  to The  be  amount. the  l a r g e decrease i n o x i d a t i o n r a t e of a l l  major  Recently, heart  decrease  Moffitt  rate  conjunction  and  dropped with  in  Crawshaw in  hour.  carp  metabolic  environmental temperature. were r a p i d .  metabolic  Both  No compensation  However w h i l e  of  rate  (Cyprinus with  results  (Prosser,1973).  demonstrated carpi o small  the response and  L.) drops  r a t e was  that in in  recovery  occurred over a p e r i o d of  the heart  the 0  (1983)  rate  of  s u b s t r a t e s at 5 C .  Movement of f i s h t o lower temperature g e n e r a l l y a  best  advantage  carbohydrate o x i d a t i o n may be emphasized as a r e s u l t  in  be  diminished,  presumably i t s demand f o r energy was slowed, the heart  one and must  78  have s t i l l  continued f u n c t i o n i n g a e r o b i c a l l y .  cannot maintain Driedzic  a  workload  Hart,1984).  and  If  supply at low temperature substrate  which  can  using  it  heart  energy  (see  anaerobic  heart must may depend  produce  Teleost  high  maintain  energy  more h e a v i l y  rates  of  on  a  oxidative  phosphory1ati on. The  substrate s p e c i f i c i t y  indicates  specific  translocators i n t o the  action  of the temperature  of  temperature  response  on  substrate  or on the b i o c h e m i c a l s t e p s r e q u i r e d f o r  TCA c y c l e .  It  has been  previously  stated  these f u n c t i o n s may be a f f e c t e d by membrane s t r u c t u r e is  altered  by  temperature.  In  contrast  show l i n e a r  Arrhenius  plots for  enzymes with a  over  Raison,1970s (1976)  the  McMurchie  has suggested  entire et  which  mammals,  reported to  activation  that  to  p o i k i 1 o t h e r m s have been mitochondrial  entry  range  constant energy  studied  al.,1973;  (Lyons  Smith,1973).  t h a t the g r e a t e r  of and  Smith  f l e x i b i l i t y of  fish  m i t o c h o n d r i a l enzymes at low temperature might be a p r o p e r t y which, t o g e t h e r  with  membrane l i p i d s ,  would account f o r  and  constant  temperature. general  the  activation However,  view  of  Arrhenius p l o t s  phase  change  the maintenance of a over  transition,  reported f o r  t h r e e s p e c i e s of t r o p i c a l s u c c i n a t e oxidase and  energy  a  a  wide  range  more r e c e n t data has c h a l l a n g e d  phase being  absence of  fish  with  low of this  discontinuous  succinate  oxidase  ( I r v i n g and W a t s o n , 1 9 7 6 )  cytochrome oxidase i n  in  liver  and  in and red  muscle of carp ( W o d t k e , 1 9 7 6 ) . The  pattern  of  substrate  utilization  d r a m a t i c a l l y a f f e c t e d by temperature i n muscle  was  more  mitochondria  79 than i n  heart.  At  15°C  all  substrates  appeared  tc  e q u a l l y good -for a e r o b i c muscle metabolism based on state 3 rates. significantly  As  t h a t supply of pyruvate at low temperatures. was a f f e c t e d heart.  by  burned  pyruvate at 5 C ,  indicating  from malate was somehow  restricted  The  a  degree t o which malate  temperature was  less  oxidation  i n muscle  than  In g e n e r a l , TCA c y c l e enzymes do not occur i n  s p e c i f i c isozymic  forms  M a l i c enzyme i s n o t , but i t  maximal  i n heart mitochondria malate was  more s l o w l y than  (see Hochachka  transporter  and  with the c y c l e .  k i n e t i c s of t h i s  from both t i s s u e s  in  tissue  Somero,1984).  s t r i c t l y s p e a k i n g , a TCA c y c l e  i s closely associated  of the temperature  be  A  enzyme,  comparison  enzyme and the  might e x p l a i n the  malate  difference  i n Q i o v a l u e s observed between the two t i s s u e s . While  a c e t y l — c a r n i t i n e and glutamate were u t i l i z e d  muscle mitochondria at 1 5 ° C , low r a t e s of o x i d a t i v e drop i n o x i d a t i o n  p h o s p h o r y l a t i o n at 5 ° C .  was r e f l e c t e d  state 4 Q i o values. in freeze—tolerant  gall  ( B a l l a n t y n e and  other s u b s t r a t e s  i n very high  fly  (Eurosta  switched o f f  of the  Storey,1984a).  was not  oxidative  between the  mitochondria  explanation  seems  state 3  the e l e c t r o n  and  t h a t the  was  d i d not l i e at  the  transport  system  s i t e of  citrate  synthase. this  of  affected, it  of  in  larvae  cold—acclimated  transfer  likely  and  observed  Since oxidation  so d r a s t i c a l l y  p h o s p h o r y l a t i o n and  would occur into  TCA c y c l e ,  in  very  extreme  solidaqinis)  concluded by the authors t h a t i n h i b i t i o n level  This  A s i m i l a r s i t u a t i o n has been  where l i p i d metabolism was larvae  both s u b s t r a t e s produced  by  or  inhibition  palmitoyl—L—carnitine  study  A given  similar that  80 acetyl-carnitine  and  more d r a s t i c a l l y  lowered than p y r u v a t e ,  According  glutamate  to mitochondrial  carbohydrate would  be  muscle at  5°C.  glutamate  oxidation  the  The  rate  taken  in  the  other  acetyl-carnitine s u b s t r a t e at result  be any  in  terms  of  fuel  source  low  be  poor  these would  RCR  or  temperature.  This  advantage  ADP/O  except  may have  of o v e r a l l  or  with  which had a higher mean RCR than any  either  for  acetyl-carnitine  conjunction  of  much  utilization,  No s u b s t r a t e had an  of temperature i n h i b i t i o n  oxidation.  substrate  i n d i c a t e s that  substrates for t h i s tissue.  were  l a c t a t e or malate.  best a e r o b i c  slow  mitochondrial density  over  oxidation rates  other been  a  acetyl-carnitine  L i k e heart m i t o c h o n d r i a , t h e r e d i d not appear t o  change i n  permeability,  membrane i n t e g r i t y ,  over  t h i s temperature  ADP/O v a l u e s were e s s e n t i a l l y  s p e c i f i c a l l y H"*" range.  In  ion  addition,  unchanged f o r a l l  substrates  at 5 ° C . Pye et a l .  (1976) measured s t a t e 3 r a t e as a  of temperature i n t e n c h .  As with  those of B a l l a n t y n e and Storey  a  wide  temperature had respiration  a  range more  i n muscle  the present r e s u l t s  (1984a) they d i s c o v e r e d  the r e l a t i o n s h i p v a r i e d a c c o r d i n g displaying  function  of  t o s u b s t r a t e and Q»o  drastic  than i n  values.  effect  heart.  on  State  that  tissue,  In  trout,  mitochondrial 3 Q  values  i 0  were higher f o r muscle except with malate as s u b s t r a t e . i m p l i c a t i o n of metabolism  in  significantly d e n s i t y and  this is  t h a t at  mosaic  muscle  curtailed. somewhat  lower  Siven  low temperature of  rainbow the  respiratory  low  and  The  oxidative trout  was  mitochondrial  rates  with  some  81 substrates in t h i s tissue r e l a t i v e t h a t mosaic  muscle - f i b r e s  were not  work d u r i n g acute temperature metabolism i n mosaic total  to heart,  drop.  muscle i s not  muscle energy s u p p l y .  it  is  possible  recruited for  aerobic  Unlike heart,  aerobic  the major component  of  Other energy sources which  may  be a v a i l a b l e at low temperature i n c l u d e anaerobic metabolism i n mosaic muscle and muscle, whose different It  a e r o b i c metabolism i n s u p e r f i c i a l  mitochondrial  respiratory  metabolism i n heart as t o or  g r e a t e r than  i n muscle.  Note t h a t l a c t a t e  s t i m u l a t e d i n heart m i t o c h o n d r i a .  microscopy elaborate  that  muscle  cristae  r a t e s per given  content,  5°C  has been  A  oxidation  muscle has  density  by  shown  mitochondria  (Nag,1972).  s u r f a c e area r e l a t i v e t o lower cytochrome  It  At  was not maximally  Clearly,  mitochondrial  mitochondrial a c t i v i t y .  protein  s u b s t r a t e s except malate were  i s not i n c l u d e d i n t h i s statement s i n c e i t  low  oxidative  a f u n c t i o n of m i t o c h o n d r i a l  i n heart than i n muscle.  compensated f o r  somewhat  lo^C maximal r a t e s of  maximal o x i d a t i o n r a t e s of a l l higher  have  properties.  was noted t h a t at  were always equal  p o p u l a t i o n may  red  tend small  not  increasing by  to  electron have  inner  less  membrane  heart mitochondria may i n d i c a t e resulting  mass of m i t o c h o n d r i a l  in  lower protein  a  respiratory (Sordahl  et  al.,1983). EFFECT OF pH A critical phosphorylation i s thought t o  f a c t o r which  may a f f e c t r a t e of  i s extramitochondrial be d r i v e n  by movement  pH.  oxidative  S y n t h e s i s of  of H"*"  ions into  ATP the  82 mitochondrion i n (Mitchell,1961; gradient If  is a  response Mitchell  result  t o an and  of a  electrochemical  Moyle,1968).  Part  is  driven  of  t r a n s m i t o c h o n d r i a l pH  t h i s g r a d i e n t i s i n c r e a s e d , ATP s y n t h e s i s ,  consumption  gradient  more  quickly  this  gradient.  coupled t o  0  Hinkle  and  (see  3  McCarty,1978). Intracellular Increased  pH  may be  temperature  intracellular  pH i n  often  white  muscle  pH,  at  (see  least  particularly  be l e s s f o r  Heisler,1984).  In  anaerobic metabolism transiently.  This  ways.  decreased Wilson,1970; species  heart than addition,  alter would  for  acidic  intracellular be  a  factor  i n mosaic muscle where anaerobic metabolism  the major energy  source.  expected t o e x p e r i e n c e pH.  in  Data from a number of f i s h  change would  endproducts from  results  several  poiki1otherms (Reeves and  Malan and Reeves,1973). suggest t h a t the  altered in  Clearly,  the l a r g e r  The next q u e s t i o n i s -  mosaic muscle would  is be  shifts in  intracellular  how do changes i n  intracellular  pH a f f e c t the t r a n s m i t o c h o n d r i a l pH g r a d i e n t s ? It  has r e c e n t l y been demonstrated t h a t the presence  of  phosphate or b i c a r b o n a t e i s r e q u i r e d i n order f o r a decrease of e x t r a m i t o c h o n d r i a l g r a d i e n t of  non—respiring rabbit  l i v e r mitochondria buffer  used  According t o gradient  pH t o r e s u l t  in  (Simpson this  i n an i n c r e a s e of the  pH  kidney c o r t e x ,  or  and Hager,1984).  study  contained  M i t c h e l l ' s chemiosmotic  i s produced i n a c t i v e l y  The  lOmM  theory  heart  assay  phosphate.  (1961),  a  r e s p i r i n g mitochondria  c o u p l i n g of ADP p h o p h o r y l a t i o n t o e l e c t r o n t r a n s p o r t .  pH via  It  is  not c l e a r  if  decreased  intracellular  pH  results  in  an  i n c r e a s e d pH g r a d i e n t i n such m i t o c h o n d r i a . With e i t h e r a c e t y l — c a r n i t i n e or glutamate as  substrate  heart mitochondria d i s p l a y e d a p r o g r e s s i v e decrease of  state  3 r a t e as pH  might  expect i f  a  i n c r e a s e d above 7 . 0 .  T h i s i s what one  decreased pH g r a d i e n t  t r a n s p o r t and r a t e  was a f f e c t i n g  of o x i d a t i v e p h o s p h o r y l a t i o n .  sample s i z e and high  variability  points d i f f i c u l t ,  between 6 . 8 and 7 . 1 s t a t e 3  r a t e s began t o l e v e l  h i g h e s t r a t e s of o x i d a t i v e physiological  observed as i n  A  small  between p r e p a r a t i o n s  d e t e r m i n a t i o n of t r a n s i t i o n  t o medium  substrate  but  made  at a off.  The  p h o s p h o r y l a t i o n o c c u r r e d at pH.  t i s s u e s of  No  sharp  pH  other organisms  clam (fiercenaria mercenaria)  optimum  such as  ( B a l l a n t y n e and  Again t h e r e was no c l e a r pH optimum.  H"*"  ions  was  extramitochondrial  not pH  p h y s i o l o g i c a l range. attributed  as  preparations.  at  least  RCR and  easily It  significantly  to  As f a r as  the  state 4 v a r i a b i l i t y among  i s i n t e r e s t i n g t o note t h a t ,  4 one  permeability  affected  within  variability  was  S t o r e y , 1984b) .  can judge on the b a s i s of RCR v a l u e s , membrane to  low  marine  RCR v a l u e s were q u i t e v a r i a b l e and tended t o m i r r o r s t a t e rates.  pH  by normal  could  be  individual r e g a r d l e s s of  the mechanism by which pH a f f e c t s s t a t e 3 r a t e s , between  pH  7 . 4 — 7 . 0 a b s o l u t e s t a t e 3 r a t e s d i d not change d r a s t i c a l l y . They ranged between 60 either  -  80 natoms  - 1  mg p r o t e i n  - 1  for  substrate. The  variability  i n measured  p h o s p h o r y l a t i o n was q u i t e high next.  min  This d i f f i c u l t y  parameters of  oxidative  from one p r e p a r a t i o n t o  i s normally  overcome by  the  repeating  84 s p e c i f i c experimental preparations.  c o n d i t i o n s on  several  In the case of t h i s s t u d y ,  assays c o u l d be  performed from  mitochondrial  only four  separate  each p r e p a r a t i o n .  The  pH  c o u l d only be approximated p r i o r t o assay and confirmed once the assay was i n p r o g r e s s or f i n i s h e d . inability  t o repeat s p e c i f i c  why each p o i n t on the single preparation.  The  preparations  artificial  trends.  of  populations.  pH v a l u e s a c c u r a t e l y which  is  pH v a l u e s were s c a t t e r e d in  The  real  an  pH p r o f i l e u s u a l l y o n l y r e p r e s e n t s  among the  masking  This resulted in  order t o  avoid  consequence of  trends  becauseof  This i s i l l u s t r a t e d  Measurements  were  made  as  randomly  generation  t h i s was  between  by the malate pH  described  in  of  possible  variability  which was generated from a s i n g l e m i t o c h o n d r i a l  a  profile  preparation.  Materials  and  Methods, t a k i n g c a r e t h a t a decrease i n s t a t e 3 r a t e was not caused p r i m a r i l y by the e f f e c t seems c l e a r  from t h i s  profile  o x i d a t i o n of malate i n In a d d i t i o n , RCR d i d where low  RCR  of m i t o c h o n d r i a l a g i n g . t h a t the  r a t e of  not vary very much  and high  state  t i s s u e , where the  was i n c r e a s e d by  state  heart i n c r e a s e d with d e c r e a s i n g  4 rates  occurred.  the need  variability  t o use  3 pH.  except at high  r e s u l t s were s i m i l a r t o those observed with other i n the same  It  pH  These  substrates  of the  response  several preparations  in  order t o encompass a wide range of pH. Mosaic muscle mitochondria changes i n  pH  than  did  those  responded d i f f e r e n t l y of  heart.  With  to  either  a c e t y l — c a r n i t i n e or glutamate as s u b s t r a t e t h e r e was a c l e a r decrease i n s t a t e 3 r a t e above pH 7 . 6 . no c l e a r pH  dependence of  state 3  Otherwise,  except at  t h e r e was  very low  pH  85 where s t a t e 3  r a t e s appeared t o  decrease somewhat.  There  was no i n d i c a t i o n t h a t an i n c r e a s e d pH g r a d i e n t was oxidative  p h o s p h o r y l a t i o n more r a p i d l y  pH was decreased.  driving  as e x t r a m i t o c h o n d r i a l  RCR v a l u e s were h i g h e s t between pH 6 . 9  7 . 4 with a c e t y l - c a r n i t i n e  and pH  7.0 — 7.4  with  -  glutamate  i n d i c a t i n g t h a t membranes tended t o be more leaky at  either  low or high pH.  muscle  than i n  S t a t e 4 r a t e s were higher  heart  with glutamate  as  s u b s t r a t e and,  e x t e n t , with a c e t y l - c a r n i t i n e as w e l l . be lower at both  i n mosaic  low and high pH  to  some  RCR v a l u e s tended t o  i n muscle than i n  heart.  In a d d i t i o n , s u b s t r a t e u t i l i z a t i o n  data presented  previously  has  to  in  indicated  that  RCR  tended  mitochondria than i n h e a r t .  be  lower  muscle  According t o these d a t a , muscle  m i t o c h o n d r i a l membranes may have been more l e a k y than heart c o u n t e r p a r t s extreme pH. directly  Low  to  logically,  and  may  have been  RCR and high s t a t e  increased  more  sensitive  4 r a t e s may  permeability  o-f  H"" 1  membrane.  It  reduced e x t r a m i t o c h o n d r i a l pH g r a d i e n t  because of  T h i s may have  is  possible,  changes i n  been the reason  which, gradient  therefore,  pH d i d not r e s u l t  that  i n an i n c r e a s e d  membrane  that s t a t e 3  to  translate  ions  would be t r a n s l a t e d t o a reduced H~*~ ion  a c r o s s the  their  permeability. rates did  not  appear pH—dependent except at high pH. At  very high pH, o x i d a t i v e  proceeded s l o w l y .  Unfortunately,  phosphorylation  invariably  few data are a v a i l a b l e  on  RCR or ADP/0 r a t i o s because the slow r a t e s of o x i d a t i o n made s t a t e 4 very available it  difficult  to  measure.  From  the sparse  would appear t h a t t h e r e was a severe  in respiratory  control  data  breakdown  and p o s s i b l y membrane damage at  high  86 pH.  In  ultrastructure  s t u d i e s , mitochondrial—swel1ing  was  demonstrated at high pH (Chang and M e r g n e r , 1 9 7 3 ) » The  DMO technique  t r a n s m i t o c h o n d r i a l pH. tool  if  commonly  This  f o r examining f u r t h e r  pH on r e s p i r a t o r y  is  the e f f e c t of  activity.  tissues  gradient,  It  as  to  technique would  decreased e x t r a m i t o c h o n d r i a l  i n c r e a s e d pH  used  measure  be a  extrami tochondri a l  c o u l d be used t o  determine  pH does i n f a c t r e s u l t  has been  observed  (Simpson and H a g e r , 1 9 8 4 ) , and i f  same i n both heart and muscle.  with  3 1  i n an other  this effect is  Another technique  f o r measuring pH g r a d i e n t s i s  useful  the  available  P NMR (Ogawa et  al.,1981>.  RCR v a l u e s and s t a t e 4 r a t e s only a l l o w one t o s p e c u l a t e the nature  of  H"" i o n  permeability  and pH  on  gradients  and  s h o u l d , consequently be used c a u t i o u s l y . Regardless mitochondrial  of the  mechanism  metabolism,  the  by which  result  is  pH  that  affects  state  3  o x i d a t i o n appears t o be l e s s dependent on e x t r a m i t o c h o n d r i a l pH i n  mosaic  muscle than  experience s i g n i f i c a n t result  in  heart.  changes  in i n t r a c e l l u l a r  of temperature or e x e r c i s e ,  experience any  significant  phosphorylation experience  large  r a t e s because muscle.  as  a  result.  intracellular  Otherwise,  in pH  dropped.  In  physiological  when  oxidative  rate  Heart oxidative is  of  would  pH  may as  Z  flux  to  oxidative also  not  than  state 3 rates in  for 0  a  phosphorylation  less variable  terms, one  l a r g e decrease i n i n t r a c e l l u l a r ischemia  in  pH-dependece of  would cause i n c r e a s e d c a p a c i t y  which  would not be expected  change  variation  Muscle,  in  heart  i n heart as  pH  would only expect  a  pH d u r i n g c a r d i a c hypoxia  or  metabolism  is  already  severely  87 Q —1imited.  Increased r a t e s  2  may be u s e f u l d u r i n g r e c o v e r y  of o x i d a t i v e  phosphorylation  from such a s t a t e .  The e f f e c t of a c i d o s i s on m i t o c h o n d r i a l r e s p i r a t i o n has long been respect  of to  al.,1972;  hypoxia  Kahles  al,1984). swelling  interest  ischemic  et a l , 1 9 7 9 ; is  often  It be  particularly  cell  Fry et  irreversible  Chaudry,1985). a c i d o s i s may  researchers,  and  Ischemia and  to  damage  al,1980s  characterized  (Mela  et  Hillered  et  by  mitochondrial  with  cellular  damage  (see  has been p o s t u l a t e d t h a t the accompanying partially  responsible  for  this  damage.  M i t o c h o n d r i a i s o l a t e d from i s c h e m i c h e a r t have been shown t o display  reduced  RCR  Ganote,1976; Schwartz  and  state  3  et a l . , 1 9 7 3 ;  rate  (Jennings  Peng et a l . , 1 9 7 7 ;  and Kahles  et a l . , 1 9 7 9 ) . Numerous mammalian s t u d i e s have i n d i c a t e d t h a t s t a t e respiration  of  many  extramitochondrial al.,1972; Fry  et  pH  tissues (Chance  M i t c h e l s o n and al.,1980;  Hillered  i n c r e a s e d above pH suggested t h a t  t h a t dog heart 6.3 for  7.1.  was  range.  to  Mela  The  (1959) observed  The  a  general  3 very  Mukherjee  decreased sensitive et a l .  et a l . at  (1979)  m i t o c h o n d r i a , incubated with  pH  pH  (1972)  extreme  to  pH,  significant  m i t o c h o n d r i a o n l y as  work of Tobin  et  al.,1983;  s t a t e 3 r a t e with d e c r e a s i n g  state not  Conrad,1959;  al.,19B4).  r a t e of heart  while  m i t o c h o n d r i a l pH physiological  et  and Conrad  decrease i n s t a t e 3  sensitive  H i r d , 1 9 7 3 ; Sharyshev et  response i s a decrease i n although Chance  and  is  3  in  pH, the  demonstrated l a c t a t e at  pH  3 hours showed no change i n s t a t e 3 although RCR and  SB ADP/O  were  depressed  with  glutamate  or  succinate  as  substrate. The mechanism by  which H"*" i o n c o n c e n t r a t i o n  s t a t e 3 r a t e s i n these s t u d i e s i s unknown. was not  depressed except  at f a i r l y  et a l .  (19B0)  have found t h a t  best preserved d u r i n g inhibition while  the  is  not  al.,1972;  Mukherjee  response i s not the r e s p i r a t o r y  et  chain  medium  affected  that  (Tobin  et  that  the  a pH—induced change  pH may  in  The f a c t  that  pH has  been  high optimal  would  cause  acidification.  ischemia  respiration  i n c l u d e ECa *"]  and 35  and  the  affect  respiration  T h i s would imply t h a t a c i d i f i c a t i o n  a l t e r e d by  Clactate3  was  specific,  indicating  caused by  by which  Fry  However,  substrate  least  enzymes have  (Mela e t a l . , 1 9 7 2 ) .  intramitochondrial  be  level.  (Tobin et a l . , 1 9 7 2 ) .  a means  external  to  the  et  and have suggested  al.,1979)  entirely  many m i t o c h o n d r i a l suggested as  appears always  (Tobin  succinate oxidation  a pH change  response  ADP/O  Fry et a l . , 1 9 8 0 ) .  occurred at the NAD*—NADH redox  succinate  the  Typically,  extreme pH  a l . , 1972.; i i i t c h e l s o n and H i r d , 1 9 7 3 ;  affected  which  degree  of  Other f a c t o r s which  are  may  some  affect  mitochondrial  (Mela at a l . , 1 9 7 2 ) ,  dilution  caused  of  by  [phosphate],  cellular  swelling  (Mukherjee et a l . , 1 9 7 9 ) . According t o  r e s u l t s from  decrease of s t a t e 3 r e s p i r a t o r y t r o u t heart physiological irreversible  or  mosaic  muscle.  a c i d o s i s alone  t h i s study  t h e r e was  no  r a t e with d e c r e a s i n g pH  in  It  would be  seems  unlikely  that  capable of  creating  m i t o c h o n d r i a l damage and concommitant  cellular  a b n o r m a l i t i e s i n these t i s s u e s i n the same manner p o s t u l a t e d  89 by some mammalian r e s e a r c h e r s . freezing-tolerant a l s o had  a  l a r v a e of  broad pH  Mitochondria i s o l a t e d  gall  optimum  hepatopancreas of ( B a l l a n t y n e and  a  straightforward, to  mitochondria i s o l a t e d  marine  pH  clam  (Mercenaria Clearly,  on m i t o c h o n d r i a l  varying  the same  range authors  s i m i l a r t o t h a t observed  Storey,1984b).  extramitochondrial  solidaqinis)  physiological  However,  have demonstrated a pH p a t t e r n studies in  (Eurosta  i n the  ( B a l l a n t y n e and S t o r e y , 1 9 8 4 a ) .  many mammalian  fly  from  in  from  the  mercenaria)  the  effect  respiration  of  is  from s p e c i e s to s p e c i e s and  not  tissue  tissue. The e f f e c t of pH  different  at  low  pH  on l a c t a t e o x i d a t i o n was than had  s u b s t r a t e s i n t h i s study. increase in state 3 rate Below pH 7 . 0 , however,  been  observed  strikingly with  other  For heart mitochondria a d i s t i n c t was observed as pH was  decreased.  r e s p i r a t i o n dropped suddenly.  m i t o c h o n d r i a had a broader pH  Muscle  optimum, i n keeping with  the  general t r e n d observed p r e v i o u s l y ,  but below pH 7 . 0 s t a t e  rates rapidly  r a p i d drop at low pH  decreased.  been p r e v i o u s l y pyruvate  No such  observed.  formation  would tend t o s h i f t  from  S i n c e H* i o n s lactate,  are a product  increased  In  used i n  t h i s study,  has  (Lindahl  and Mayeda,1975).  a d d i t i o n , heart a pH  by the after  from l a c t a t e .  a d d i t i o n of  of  lactate  LDH, which  optimum g r e a t e r  than  was 10.0  Rapid decreases i n l a c t a t e - b a s e d  s t a t e 3 r a t e s at low pH probably i n d i c a t e l i m i t e d of pyruvate  had  acidification  the r e a c t i o n toward formation of  r a t h e r than pyruvate.  3  This interpretation  pyruvate t o  the muscle  l a c t a t e metabolism had been measured.  conversion  was confirmed assay  mixture  The higher  the  90 pH, the  closer  lactate  oxidation rates  pyruvate o x i d a t i o n r a t e s , s t a t e 3 o x i d a t i o n was a t o convert  l a c t a t e to  indicating  heart o x i d i z i n g  higher than i n h e a r t ,  4  particularly state  4  lactate.  RCR  previously  seemed  at high pH.  values  pH  reflected  high s t a t e 4  i n muscle  inordinately  oxidation  known but i s  of  and  mitochondria.  high,  The reason f o r  during  m i t o c h o n d r i a i s not  ability  and unchanging over a wide  i n accordance with  rates  values  of a reduced  of  In muscle, s t a t e 4 r a t e s were much  low RCR v a l u e s observed state  maximal  pyruvate.  changes i n s t a t e 3 r a t e .  The  to  t h a t pH i n h i b i t i o n  direct result  S t a t e 4 r a t e s were low range i n  were  however,  low RCR and lactate  by  c o n s i s t e n t with  high  muscle previous  f i n d i n g s at 5 and 15°C.  CONCLUSIONS Mitochondrial substrate u t i l i z a t i o n t h a t carbohydrates have the a e r o b i c energy  supply i n  potential heart.  at 15°C  indicates  for highest rates  Lactate i s  a  potential  s u b s t r a t e depending on c o n v e r s i o n of l a c t a t e t o pyruvate LDH.  L i p i d and p r o t e i n are a l s o p o t e n t i a l  on high o x i d a t i o n All  r a t e s of  substrates, particularly  produce a high muscle, a l l  degree of  substrates  a c e t y l — c a r n i t i n e or  based  glutamate.  respiratory  control. at  In  state 3 oxidation is similar  rates.  to that  mosaic  15°C»  At 5°C carbohydrate appears t o be the p r e f e r r e d  m i t o c h o n d r i a the p a t t e r n  by  a c e t y l - c a r n i t i n e and g l u t a m a t e ,  s u b s t r a t e s have equal p o t e n t i a l  energy source based on  of  In  aerobic heart  at 15°C,  but  91 lower o v e r a l l  rates  at the lower  higher pyruvate o x i d a t i o n may be s e l e c t e d . and glutamate  In  temperature suggest  rates relative  t o other  substrates  mosaic muscle mitochondria both  metabolism  are d r a m a t i c a l l y  mitochondria  are  for state  dependence, p a r t i c u l a r l y  no  2  for  to d i s p l a y for  membrane  temperature range i n v e s t i g a t e d ,  most  of  down,  source. by  heart  substrates. temperature  acetyl-carnitine  each t i s s u e ' s s p e c i f i c  energy g e n e r a t i o n  change i n  rates  a greater  oxidation  T h i s may r e f l e c t  t o maintain a e r o b i c There i s  oxidation  approximately  Muscle mitochondria tend  or glutamate.  3  lipid  switched  l e a v i n g carbohydrate as the p r e f e r r e d a e r o b i c f u e l Q i o values  that  at low  temperature.  i n t e g r i t y over  which i s  need  the  10C  presumably why  decrease i n temperature i s not accompanied by a  o  a  predictable  change i n RCR. Given i t s higher m i t o c h o n d r i a l d e n s i t y heart d i s p l a y s a higher c a p a c i t y f o r p r o v i d i n g mosaic muscle. does  not  appear  anything,  to  be  compensated  State 3 r e s p i r a t i o n  approximately  state 3 pH  6.8.  display a limited a b i l i t y  degree  by  mitochondrial  protein.  i s l e s s pH—dependent i n muscle than r a t e s i n c r e a s e as Mitochondria  pH decreases  from  both  to  tissues  t o produce energy a e r o b i c a l l y  at  RCR and s t a t e 4 data suggest a higher degree of H"*"  i o n leakage i n leakage  any  r a t e s of the mitochondria themselves.  s t a t e 3 r a t e s per mg of m i t o c h o n d r i a l  high pH.  to  heart has the advantage of higher  i n heart where  does  Mosaic muscle metabolism, on the other hand,  increasing respiratory If  energy a e r o b i c a l l y than  may  muscle mitochondria affect  the  at pH  maintenance  of  extremes. a  pH  Such  gradient  92 resulting  in  a  decreased  pH  dependence  in  muscle  mitochondria. As i n a l l high r a t e s of higher r a t e s  s p e c i e s , t r o u t heart has a constant need o x i d a t i v e metabolism. of m i t o c h o n d r i a l  mosaic muscle,  which  is  r e s p i r a t i o n as  less oxidative.  f u r t h e r emphasized by the f a c t t h a t more s e v e r e l y a f f e c t e d  by a  temperature i t  as c r i t i c a l  i s not  This i s reflected  larger  mosaic  fluctuations  respiratory  muscle in  The  point  by to is  muscle mitochondria are  drop i n temperature. for trout  o x i d a t i v e metabolism i n mosaic muscle two t i s s u e s ,  compared  for  to  as i n h e a r t .  is likely  intracellular  to pH.  At  low  maintain Of  the  experience  the  Mitochondrial  r a t e s of t h i s t i s s u e are l e s s dependent on pH i n  the p h y s i o l o g i c a l  range. 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