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A strategy for the quantitative analysis of fungal cell walls Cameron, Donald Stewart 1972

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\510\ A  STRATEGY  ANALYSIS  FOR  OF  THE  FUNGAL  QUANTITATIVE  CELL  WALLS  by DONALD STEWART CAMERON B.Sc, U n i v e r s i t y o f A l b e r t a , M.Sc,  1962  U n i v e r s i t y o f A l b e r t a , 1965  A THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY in t h e  Department of Botany  We a c c e p t t h i s t h e s i s as c o n f o r m i n g t o t h e required standard.  THE UNIVERSITY OF BRITISH COLUMBIA November,  1972  In presenting this thesis in partial  fulfilment  of the requirements for  an advanced degree at the University of B r i t i s h Columbia, I agree that the Library shall make it freely available for reference and study. I further agree that permission for extensive copying of this thesis for scholarly purposes may be granted by the Head of my Department or by his representatives.  It  is understood that copying or publication  of this thesis f o r financial gain shall not be allowed without my written permission.  Department of  R  n  +  a  n  y  The University of B r i t i s h Columbia Vancouver 8, Canada  Date  November 2 9 . 1972  i ABSTRACT  Cultures of Tremella mesenterica u n i c e l l s and Saprolegnia  diclina  grown as haploid y e a s t - l i k e  were grown on incompletely defined media  and the procedures f o r i s o l a t i n g c e l l wall preparations from each, free of cytoplasmic and capsular m a t e r i a l , are described.  A strategy f o r q u a n t i t a -  t i v e a n a l y s i s of these c e l l wall preparations was devised and t e s t e d , s e l e c t i n g from the numerous procedures a v a i l a b l e :  e x t r a c t i o n and gravimetry  for l i p i d s ; hydrolysis followed by g a s - l i q u i d chromatography f o r neutral sugars (as trimethyI s i IyI d e r i v a t i v e s ) and automatic amino acid a n a l y s i s f o r amino acids and amino sugars. Problems of degradation as a r e s u l t of h y d r o l y s i s have been considered.  The extent t o which degradation occurs i s d i f f i c u l t to  estimate because each c o n s t i t u e n t polymer i s 'contaminated' with the others.  This may a c c e l e r a t e degradation, p a r t i c u l a r l y of amino a c i d s . A n a l y t i c a l procedures were reproducible but only 90$ of the  weight of the c e l l wall was recovered. the r e s u l t of degradation  The remaining 10$ i s probably  losses that have not been accounted f o r , and  f u r t h e r studies are required t o improve the estimation of t h i s e r r o r . Even under c a r e f u l l y standardized c o n d i t i o n s , c e l l wall preparations show v a r i a b l e composition.  A complete a n a l y s i s was  therefore performed on a s i n g l e c e l l wall preparation of each of the two s p e c i e s .  Analyses were performed on other c e l l wall preparations  of the' two species and they showed general s i m i l a r i t i e s ; the r a t i o s of components were s i m i l a r , although, f o r example one preparation, of  S. diclina  contained more than twice as much t o t a l protein as. another. S i m i l a r recoveries of c o n s t i t u e n t s suggest that the strategy  is appropriate f o r q u a n t i t a t i v e a n a l y s i s .  However, a l t e r n a t i v e  i i methods f o r  l i p i d a n a l y s i s t h a t p r o v i d e more s p e c i f i c i n f o r m a t i o n  a v a i l a b l e and should be adapted f o r c e l l  wall a n a l y s i s .  Quantitative  r e c o v e r y of u r o n i c a c i d s p r o v i d e s s u b s t a n t i a ! d i f f i c u l t i e s , and methods a r e  required.  are  improved  i ii TABLE OF CONTENTS page Introduction  1  M a t e r i a l s and Methods  9  Cell wall preparation Analytical  9  procedures  11  C a l c u l a t i o n of r e s u l t s  17  Results  19  D e g r a d a t i o n under h y d r o l y z i n g c o n d i t i o n s  19  Protein analysis  22  Polysaccharide analysis  '  22  Lipid analysis  32  Elemental and ash a n a l y s i s  32  Complete c e l l  55  wall a n a l y s i s  Discussion  37  Analytical  procedures  40  H y d r o l y s i s and d e g r a d a t i o n  46  The c e l l  56  wall preparation  Bi bI iography  62  Appendix  72  Symbols used f o r monomers  72  I V  LIST OF TABLES table I II III IV V  page Amino A c i d s i n t h e C e l l Wa.l I of Tremella mesenterica  24  Amino A c i d s i n t h e C e l l Wall o f Saprolegnia  25  diclina  N e u t r a l Sugars i n t h e C e l l Wall of r . mesenterica  28  N e u t r a l Sugars i n t h e C e l l Wall of s. diclina  29  Amino Sugars i n t h e C e l l Wall o f T. mesenterica and 33  S. diclina VI  L i p i d s i n t h e C e l l Wall of r . mesenterica and 33  S. diclina VII  Elemental and Ash A n a l y s i s o f C e l l Wall of T . mesenterica  VIM  Preparations  and S. diclina  34  Complete A n a l y s i s of t h e C e l l Wall o f T. mesenterica and S. diclina  36  v LIST OF FIGURES figure 1  page R e s o l u t i o n o f ' B a s i c Compounds from t h e 13 cm Column of t h e Amino A c i d A n a l y z e r  2  13  R e s o l u t i o n of N e u t r a l Sugars ( a s TMS D e r i v a t i v e s ) on t h e G a s - l i q u i d Chromatograph  16  3  Recovery of Free Amino A c i d s Under Hydro I y z i n g C o n d i t i o n s  20  4  Recovery o f Glucosamine under Hydro I y z i n g C o n d i t i o n s  21  5  Recovery o f Free N e u t r a l Sugars under Hydro I y z i n g Conditions  6  23  Recovery of Amino A c i d s from C e l l W a l l s o f Tremella 26  mesenterica 7  Recovery o f Amino A c i d s from C e l l W a l l s of Saprolegnia 27  diclina 8  Recovery o f N e u t r a l Sugars from C e l l W a l l s of T. mesenterica 30  9  Revovery of N e u t r a l Sugars from C e l l W a l l s o f S. diclina  10  11  Recovery of Glucosamine from C e l l W a l l s o f T.  31  mesenterica  and S. diclina  34  Mechanism o f G I u c o p y r a n o s i d e H y d r o l y s i s  49  vi ,  ACKNOWLEDGMENT T h i s r e s e a r c h p r o j e c t was supported  by g r a n t s t o Dr.  I.  E.  P.  T a y l o r from t h e N a t i o n a l Research C o u n c i l of Canada and t h e U n i v e r s i t y of B r i t i s h Columbia.  C u l t u r e s of t h e f u n g i  B a n d o n i , who, w i t h M o i r a Dobson,  were p r o v i d e d  by Dr.  S h i r i e y Reid and Marg Shand  R. J . introduced  me t o s t e r i l e m i c r o b i o l o g i c a l t e c h n i q u e s and were always a v a i l a b l e consultation.  for  Dr. G. G. S. Dutton shared h i s e x p e r t i s e in monosaccharide  e s t i m a t i o n by g a s - l i q u i d chromatography; d i s c u s s i o n s w i t h him and members of h i s  I gleaned much i n f o r m a t i o n laboratory.  from  S y l v i a T a y l o r and  A d r i a n n e Ross helped w i t h t h e t y p i n g of t h e m a n u s c r i p t .  The f i n a l  copy  was m e t h o d i c a l l y checked by Diane West-Bourke and Arne McRadu. l a i n T a y l o r has a s s i s t e d and encouraged every s t a g e of  the  development of t h e p r o j e c t and in t h e p r e p a r a t i o n of t h e t h e s i s . grateful  t o him f o r p r o v i d i n g  the opportunity  I am  f o r me t o work w i t h him,  f o r h i s g r e a t p a t i e n c e under c i r c u m s t a n c e s t h a t would s o r e l y t r y  ordinary  human endurance,  His  and f o r h i s a p p r e c i a t i o n of my academic g o a l s .  c o n t r i b u t i o n t o my c a r e e r has been s u b s t a n t i a l , both as a r e s e a r c h a d v i s o r and as a f r i e n d . I a l s o want t o thank my c o l l e a g u e s and s t u d e n t s f o r e x t e n d i n g my i n t e r e s t in t e a c h i n g and  in B i o l o g y  101/102  l e a r n i n g , and t o a l l my f r i e n d s  who bore my l a b o u r s s y m p a t h e t i c a l l y and p r o v i d e d welcome d i v e r s i o n s  in  squash and t e n n i s , a t p l a y s and c o n c e r t s , and i n v a r i o u s abstemious  recreations.  1 INTRODUCTI ON  P l a n t c e l l s a r e c h a r a c t e r i s t i c a l l y e n c l o s e d by a c e l l i s s e m i - r i g i d , y e t c a p a b l e of g r o w i n g . interest cell  in t h e mechanism of t h e c e l l  growth and s e v e r a l e x p e r i m e n t a l  of organisms have y i e l d e d u s e f u l from s e v e r a l q u i t e d i f f e r e n t  of morphogenesis  w a l l e x t e n s i o n t h a t accompanies approaches a p p l i e d t o a wide range i s of  interest  systems of  cell  1971; D a v i e s 1972), m o l e c u l a r c o n t r o l (Cabib and F a r k a s 1971;  i n t e g r a t i o n of components  S a l p e t e r 1967; Sadava and C h r i s p e e l s 1969; structural  The problem  viewpoints: genetic control  and d i f f e r e n t i a t i o n  G a r c i a and Lippman 1972),  that  There has been c o n s i d e r a b l e  information.  w a l l assembly ( K a t z and Rosenberger  wall  Bartnicki-  (Steward,  Israel  and  N o r t h c o t e 1969), p r o p e r t i e s  polymers u s i n g t h e e l e c t r o n m i c r o s c o p e and X - r a y  of  diffraction  (Mahadevan and Tatum 1967; Aronson and F u l l e r 1969), and c h e m i c a l c o n s t i t u e n t s (Novaes-Ledieu  and J i m e n e z - M a r t f n e z  G a r c i a 1970; M a r k s , K e l l e r and G u a r i n o  1971).  r e s u l t s from a l l of t h e s e approaches w i l l e x t e n s i o n be c o m p l e t e l y  Only by a s y n t h e s i s of  t h e p r o c e s s e s of c e l l  w a l l e x t e n s i o n u l t i m a t e l y must  t h e c h e m i c a l c o n s t i t u e n t s of t h e c e l l  cell  Bartnicki-  wall  understood.  I n v e s t i g a t o r s of c e l l  the  1968; Wang and  wall,  determine  t h e c h e m i s t r y and geometry  of  l i n k a g e s between them, and a d e s c r i p t i o n of how t h e s e change as t h e grows.  The fundamental  of t h e whole c e l l its constituents.  requirements  for the  initial  investigation  w a l l a r e a q u a l i t a t i v e and a q u a n t i t a t i v e a n a l y s i s of These s t u d i e s may be f o l l o w e d by f r a c t i o n a t i o n of  w a l l macromoI ecu I e s , a n a l y s i s of t h e i r c o m p o s i t i o n , d e t e r m i n a t i o n of  cell their  s t r u c t u r e and a t t e m p t s t o reassemble t h e s e m o l e c u l e s i n t o a w o r k i n g model of c e l l  wall s t r u c t u r e .  At each s t e p  In f r a c t i o n a t i o n of t h e c e l l  q u a n t i t a t i v e r e c o v e r y of components from each f r a c t i o n  wall,  is e s s e n t i a l .  .2 T h i s a s p e c t o f t h e problem at  least in i n i t i a l  s t u d i e s p l a n t m a t e r i a l s h o u l d be s e l e c t e d t o p r e s e n t  t h e minimum of a n a l y t i c a l p r o b l e m s . are important c e l l  i s e s s e n t i a l l y c h e m i c a l i n n a t u r e and  A l t h o u g h secondary d e p o s i t i o n p r o d u c t s  w a l l c o n s t i t u e n t s , they p r o v i d e both q u a l i t a t i v e and  q u a n t i t a t i v e a n a l y t i c a l problems t h a t may p r e c l u d e s a t i s f a c t o r y r e s o l u t i o n of p r i m a r y c e l l  w a l l components.  V a s c u l a r p l a n t s a r e an example o f  t i s s u e s t h a t p r e s e n t numerous p r o b l e m s . present, the r e s u l t i n g c e l l cell  With many d i f f e r e n t c e l l  wall preparation  i s heterogeneous.  types  As t h e  w a l l s may be l i g n i f i e d t o v a r y i n g e x t e n t s , a n a l y s i s becomes f a r more  difficult.  T i s s u e c u l t u r e p r o c e d u r e s can p r o v i d e more u n i f o r m ,  lignified cells,  but w i t h t h e s e p r o c e d u r e s t h e r e  p r o d u c i n g s u f f i c i e n t q u a n t i t i e s of c e l l s .  i s t h e problem of  Furthermore,  from e i t h e r s o u r c e tend t o grow a t t a c h e d t o g e t h e r preparation of c e l l  less  s i n c e the  cells  i n masses, t h e  w a l l s f r e e from both c y t o p l a s m and t h e m i d d l e  lamella  becomes more d i f f i c u l t . C e r t a i n o t h e r groups o f organisms do not p r e s e n t t h e s e p r o b l e m s . B a c t e r i a have a l r e a d y been e x t e n s i v e l y and c o m p r e h e n s i v e l y s t u d i e d ( f o r a general  r e v i e w see Rogers and P e r k i n s 1968).  g e n e r a l l y produce  A l g a e and f u n g i  do not  l i g n i n - l i k e compounds and many s p e c i e s grow as s i m p l e  o r branched f i l a m e n t s o r a s u n i c e l l s . been s t u d i e d (Aaronson  A number of a l g a l s p e c i e s have  1970; P a r k e r 1970).  Fungi a r e i n g e n e r a l  t o c u l t u r e than a l g a e and they grow r a p i d l y so t h a t r e l a t i v e l y easy t o p r o d u c e . widely studied (Phaff  l a r g e amounts a r e  Y e a s t s , p r i m a r i l y Saccharomyces,  have been  .  1971) and t h e advantages o f f u n g i a s s o u r c e m a t e r i a l  have led t o i n v e s t i g a t i o n o f an i n c r e a s i n g number o f o t h e r f u n g a l The f i r s t problem a s s o c i a t e d w i t h any c e l l t h a t of o b t a i n i n g the c e l l contamination.  easier  types.  wall a n a l y s i s i s  w a l l f r e e from c y t o p l a s m i c and i n t r a c e l l u l a r  In g e n e r a l t h i s  involves breaking the c e l l  w a l l and  3 washing  i t f r e e of c o n t a m i n a n t s .  component under a French p r e s s  Depending  i n v e s t i g a t i o n fungal (Kanetsuna,  cell  on t h e s p e c i e s and t h e  w a l l s have been d i s r u p t e d  C a r b o n e l l , Moreno and R o d r i g u e z  by m e c h a n i c a l a g i t a t i o n w i t h g l a s s beads  1969)  (Griffin  (Aronson and F u I l e r  suspensions are often f u r t h e r  t r e a t e d by s o n i c o s c i l l a t i o n and  NaCI/NaHCC>3, EDTA and s u c r o s e .  1969).  broken  (Sietsma,  and MacWiI Iiams  1969), o r a SorvaI I omnimixer  v a r i o u s l y washed w i t h w a t e r , g l y c e r o l  or  in a Braun homogenizer  E v e l e i g h and H a s k i n s 1969), a M i c k l e d i s i n t e g r a t o r  with  The  resultant then  and aqueous s o l u t i o n s of  Physical wall fragmentation  NaCI,  followed  by  v i g o r o u s washing w i t h aqueous s o l u t i o n s near pH 7 c a u s e s minimal degradation  of t h e c e l l  wall,  whereas t r e a t m e n t w i t h s t r o n g a c i d s o r  a l c o h o l , o r heat may cause d e n a t u r a t i o n of  polysaccharides.  preparation  of  p r o t e i n components o r  The r e s u l t of any of t h e s e t r e a t m e n t s  is a cell  of t h e organism from which i t was i s o l a t e d " (Crook and J o h n s t o n w a l l polymers a r e most f r e q u e n t l y  hydrolyzed  near 100 C w i t h aqueous a c i d s ; t h i s r e l e a s e s t h e monomers degrade them a f t e r broken.  (and perhaps  before) t h e i r  The e x t e n t of t h e d e g r a d a t i o n . v a r i e s  depending  on t h e t y p e of  hydrolysis.  1962). temperatures  but may a l s o  for  individual  l i n k a g e between them and on t h e c o n d i t i o n s  10 t o 70 hr  Proteins are generally  hydrolyzed  w i t h 0.1  w i t h 6 N HCI  in vacuo  P o l y s a c c h a r i d e s may be 3  sugars.  stable  conditions  t o 2 N H 2 S0U, 24 N HCOOH, o r 2 N CF C00H f o r  up t o 8 hr t o r e l e a s e n e u t r a l  of  constituent  ( T r i s t r a m and Smith 1963); under t h e s e  p o l y s a c c h a r i d e s are s i g n i f i c a n t l y degraded.  are  monomers  P r o t e i n s and t h e i r component amino a c i d s a r e much more  monosaccharides.  hydrolyzed  at  wall  wall  intermoI ecu Iar bonds  t o a c i d h y d r o l y s i s than p o l y s a c c h a r i d e s and t h e i r  for  oxidation  which " c a n n o t s e n s i b l y be equated w i t h t h e f u n c t i o n a l  Cell  bases,  G l y c o s i d i c bonds  amino s u g a r s a r e much more s t a b l e t o a c i d h y d r o l y s i s ,  periods  involving  and a r e  generally  :.  4  h y d r o l y z e d w i t h 2 N t o 6 N HCl f o r 8 t o 32 hr (Dutton 1972).  Quantitative  r e c o v e r y of u r o n i c a c i d s under c o n d i t i o n s of a c i d h y d r o l y s i s has n o t been a c c o m p l i s h e d ( N o r d s t e d t and Samuel son 1966; S t a c e y 1970).  Although the  development of paper chromatography p r o v i d e d a v a l u a b l e t o o l f o r q u a l i t a t i v e a n a l y s i s modern t e c h n o l o g y has surpassed i t s c a p a c i t i e s f o r r e s o l u t i o n and s e n s i t i v i t y . be d e t e c t e d and s e p a r a t e d analytical  procedures  i n f o r m a t i o n from c e l l  Compounds more s i m i l a r i n s t r u c t u r e can now  i n e x c e e d i n g l y s m a l l amounts.  There a r e many  i n common use t h a t can y i e l d q u a n t i t a t i v e w a l l s , provided that precautions are observed.  The p r o c e d u r e s developed by Spackman, S t e i n and Moore (1958) have been w i d e l y used where p r e c i s e q u a n t i t a t i v e amino a c i d d a t a a r e required.  T r i s t r a m and Smith (1963) recommended s e r i a l h y d r o l y s i s t o  d e t e r m i n e t h e c o r r e c t i o n s which must be a p p l i e d f o r amino a c i d s t h a t are  l a b i l e under h y d r o l y t i c c o n d i t i o n s and t h o s e t h a t a r e d i f f i c u l t t o  hydrolyze.  They emphasized t h e need f o r independent a n a l y s i s of  of c y s t e i n e / c y s t i n e and t r y p t o p h a n . hydroxyproIine  The n i n h y d r i n  reaction with  i s not s a t i s f a c t o r y f o r d e t e r m i n a t i o n of microgram amounts  ( M i t c h e l l and T a y l o r l i t e r a t u r e of c e l l  1970).  Separate determination  is preferred.  In t h e  w a l l a n a l y s i s t h e r e have been no r e p o r t s t h a t s a t i s f y  these requirements.  Only a few a u t h o r s  (Wang and B a r t n i c k i - G a r c i a 1970;  Buck and Obaidah 1971) have used s e r i a l h y d r o l y s i s o r attempted t o determine tryptophan  (Korn and N o r t h c o t e 1960; R u s s e l l , Sturgeon and Ward  1964; B a l l e s t a and V i l l a n u e v a 1971; M a r k s , K e l l e r and G u a r i n o 1971) c r hydroxyproIine  (Crook and J o h n s t o n 1962; Dyke 1964; B a r t n i c k i - G a r c i a 1966;  N o v a e s - L e d i e u , Jim§nez-Martfnez and V i l l a n u e v a 1967; Aronson and F u l l e r 1969; R e u v e r s , T a c o r o n t e , G a r c i a Mendoza and N o v a e s - L e d i e u 1969; Pao and Aronson 1970).  The r e c e n t development of p r o c e d u r e s f o r a n a l y z i n g  a c i d s by g a s - l i q u i d chromatography as t r i m e t h y I s i IyI d e r i v a t i v e s  amino  (Gehrke,  5 Nakamoto and ZumwaIt 1969)  o r as A ' - i r i f I u o r o a c e t y I n - b u t y l  esters  (Gehrke,  Kuo and Zumwalt 1971) may p r o v i d e a r a p i d and s e n s i t i v e a l t e r n a t i v e the ninhydrin  r e a c t i o n but c o r r e c t i o n s f o r h y d r o l y t i c d e g r a d a t i o n  to  must  s t i I I be a p p I i e d . The p r o c e d u r e s f o r monosaccharide e s t i m a t i o n a r e not as s t a n d a r d i z e d as t h o s e f o r amino a c i d s and c o n s i d e r a b l e v a r i a t i o n methodology  is evident.  S t a c e y (1970) has d i s c u s s e d t h e advantages  g a s - ! i q u i d chromatography i n v e s t i g a t i o n s of f u n g a l  in of  in q u a n t i t a t i v e a n a l y s i s of s u g a r s , y e t few cell  w a l l s have made use of t h i s  technique.  Sugars have been e s t i m a t e d as a l d i t o l a c e t a t e d e r i v a t i v e s  (Albersheim,  N e v i n s , E n g l i s h and K a r r 1967; Wang and B a r t n i c k i - G a r c i a 1970), c r trimethylsilyl  derivatives  (Namba and Kuroda 1971; S i k i , M a s l e r and Bauer  L l o y d (1970) and Lloyd and B i t o o n (1971) used both p r o c e d u r e s . authors  ( A l b e r s h e i m e t a l . 1967; A p p l e g a r t h  Only a few  1967; A p p l e g a r t h and B o z o i a n  have a p p l i e d c o r r e c t i o n s d e r i v e d from s e r i a l  1968)  h y d r o l y s i s t o sugar a n a l y s i s .  Amino s u g a r s a r e r e s o l v e d on an amino a c i d a n a l y z e r  using  s t a n d a r d p r o c e d u r e s and t h e s e i n v e s t i g a t o r s who used t h e a n a l y z e r amino a c i d e s t i m a t i o n s determined amino s u g a r s as w e l l . of t h e e s t i m a t i o n of amino s u g a r s as t r i m e t h y I s i Iy! have been problems  1970).  for  There a r e  reports  d e r i v a t i v e s but  in o b t a i n i n g q u a n t i t a t i v e r e c o v e r i e s o r  there  resolution  (Duti-on 1972). Chattaway, Holmes and Barlow (1963) observed t h a t " l o s s e s sugars]  during  [of  h y d r o l y s i s were found t o be c o n s i d e r a b l e and v a r i a b l e " ,  y e t o n l y t h e y , M a r k s , K e l l e r and G u a r i n c (1969) and L l o y d and B i t o o n  (1971)  have exam5ned the d e g r a d a t i o n of s u g a r s under h y d r o ! y z i n g  in  conjunction with c e i l  wail  conditions  studias.  U r o n i c a c i cis have proved d i f f i c u l t t o e s t i m a t e by g a s - l i q u i d chromatography  ( B i o k o and R i c h a r d s 1970).  They a r e u s u a l i y e s t i m a t e d as  6 neutral  s u g a r s a f t e r r e d u c t i o n of t h e c a r b o x y l group in t h e  polysaccharide before h y d r o l y s i s  (Dutton and K a b i r 1971)  intact  o r by r e d u c t i o n  of t h e c a r b o x y l group of t h e f r e e u r o n i c a c i d s o r a I d o b i o u r o n i c after hydrolysis  acids  ( B a r t n i c k i - G a r c i a and Reyes 1968; Dutton and K a b i r  Jones and A l b e r s h e i m  1972).  L i p i d s have been w i d e l y determined B a r t n i c k i - G a r c i a and N i c k e r s o n ( 1 9 6 2 ) .  by t h e g r a v i m e t r i c method  Dyke (1964),  RusseI I,  l i p i d s and e s t i m a t e d t h e methyl  lipids  (fatty  Suomalainen and Nurminen  (1970) determined c e l l  wall  in b a k e r ' s y e a s t and found  monosaccharides a s s o c i a t e d w i t h some of t h e p h o s p h o l i p i d  of  the  e s t e r s of t h e f a t t y a c i d s by g a s - l i q u i d  a c i d s and p h o s p h o l i p i d s )  Errors  of  Sturgeon  and Ward (1964) and S i e t s m a , E v e l e i g h and H a s k i n s (1969) s a p o n i f i e d  chromatography.  1972;  in q u a n t i t a t i v e e s t i m a t i o n due t o  components.  incomplete cleavage  l i n k a g e s and d e g r a d a t i o n of components under c o n d i t i o n s of  must be t a k e n i n t o a c c o u n t in any q u a n t i t a t i v e a n a l y s i s .  hydrolysis  The  r e p r o d u c i b i l i t y of t h e a n a l y s i s may a l s o depend on t h e a v a i l a b i l i t y of c e l l u l a r m a t e r i a l whose m o l e c u l a r c o n t e n t  is uniform,  and t h i s  depends on f a c t o r s such as morphology, s t a t e of m a t u r i t y and c o n d i t i o n s t h a t may d i f f e r  from one s o u r c e of m a t e r i a l t o  in t u r n  nutritional  another.  The purpose of t h e p r e s e n t study was t o s e l e c t and t e s t appropriate  p r o c e d u r e s f o r o b t a i n i n g q u a n t i t a t i v e and  information  r e l a t i n g t o t h e s t r u c t u r e of t h e f u n g a l  procedures  in t h e m s e l v e s a r e not  i n f o r m a t i o n about c e l l  walls.  reproducible  cell  wall.  These  intended t o p r o v i d e any s t r u c t u r a l  They do,  however,  form t h e b a s i s f o r  f u t u r e s t r u c t u r a l s t u d i e s which a r e planned  in t h i s  laboratory.  t o e l u c i d a t e t h e complete s t r u c t u r e of c e l l  w a l l s , the s t r u c t u r a l  all  In o r d e r units  must be i d e n t i f i e d c h e m i c a l l y and t h e method of assembly d e t e r m i n e d .  One  of t h e most p r o f i t a b l e approaches t o t h i s s o r t of study has been e l e c t r o n  7 microscopic examination of t h e c e l l  w a l l b e f o r e and a f t e r s e l e c t i v e  removal o f s t r u c t u r a l l y d i s t i n c t u n i t s by d i f f e r e n t i a l  solubilization  in v a r i o u s r e a g e n t s and enzymes (Mahadevan and Tatum 1965,  1967).  is t h e p o s s i b i l i t y of e x t e n s i v e degradation as a r e s u l t of these  There procedures,  e s p e c i a l l y w i t h NaOH e x t r a c t i o n ; q u a n t i t a t i v e r e c o v e r i e s a t each s t a g e must be a c h i e v e d .  The a n a l y t i c a l methods d e s c r i b e d  be a p p l i e d and extended t o a c h i e v e t h i s end.  Only by q u a n t i t a t i n g t h e  e n t i r e a n a l y s i s can a c c u r a t e models o f t h e c e l l This  i n t h i s t h e s i s must  w a l l be c o n s t r u c t e d .  i n v e s t i g a t i o n c o n c e r n s two fungal" s p e c i e s from q u i t e  u n r e l a t e d groups t h a t have n o t p r e v i o u s l y been examined -  diclina,  a m y c e I i a I Oomycete, and Tremella mesenterica,  B a s i d i o m y c e t e t h a t can be grown The a n a l y t i c a l regime 1)  Saprolegnia  a  in a yeast-1 ike form. includes:  s e r i a l h y d r o l y s i s of c e l l  sugars (estimated as t r i m e t h y I s i I y I  wall polysaccharides t o release  neutral  d e r i v a t i v e s by g a s - l i q u i d  chromatography). 2)  s e r i a l h y d r o l y s i s of c e l l  s u g a r s ( e s t i m a t e d by n i n h y d r i n 3)  for neutral  (estimated  r e a c t i o n on t h e amino a c i d  e s t i m a t i o n of u r o n i c a c i d s a s n e u t r a l  t h e c a r b o x y l groups  4)  w a l l p o l y s a c c h a r i d e s t o r e l e a s e amino  in c e l l  s u g a r s by r e d u c t i o n o f  w a l l p o l y s a c c h a r i d e s f o l l o w e d by a n a l y s i s as  sugars.  s e r i a l h y d r o l y s i s of c e l l by n i n h y d r i n  w a l l p r o t e i n s t o r e l e a s e amino a c i d s  r e a c t i o n on t h e amino a c i d a n a l y z e r ) ,  separate a n a l y s i s f o r hydroxyproline, 5) -  analyzer).  degradation  tryptophan  studies of free neutral  including  and c y s t e i n e / c y s t i n e .  s u g a r s , f r e e amino  and f r e e amino a c i d s under t h e c o n d i t i o n s f o r h y d r o l y s i s o f c e l l polymers, 6)  g r a v i m e t r i c determination of l i p i d s .  sugars, wall  8 7)  a n a l y s i s f o r C,  H, 0,  N , S,  P and a s h .  The s u c c e s s of t h i s s t r a t e g y can be measured as t h e of t o t a l c e l l analytical  percentage  w a l l s t a r t i n g m a t e r i a l which i s r e c o v e r e d by t h e s e l e c t e d  procedures.  9 MATERIALS AND METHODS  Tremella mesenterica and Saprolegnia diclina UBC m y c o l o g i c a l c u l t u r e c o l l e c t i o n . Difco Laboratories, Detroit,  were from t h e  C u l t u r e media were o b t a i n e d  from  Michigan.  Reagents were o b t a i n e d from t h e s u p p l i e r s a s i n d i c a t e d : Beckman Amino A c i d C a l i b r a t i o n M i x t u r e Type 1 (Beckman Spinco D i v i s i o n , Palo A l t o , C a l i f o r n i a ) ; Richmond, C a l i f o r n i a ) ;  thiodiglycol  Instruments, I n c . , (Bio*Rad  Laboratories,  D-galactose, D-galactosamine hydrochloride,  p y r i d i n e AnalaR ACS ( B r i t i s h Drug Houses L t d . , P o o l e , E n g l a n d ) ; a c i d s except m e t h y I h i s t i d i n e  (Calbiochem,  CF3COOH, L-rhamnose monohydrate York);  D-mannose,  Company,  erythritol,  L-1-methyIhistidine  ninhydrin,  Chemical Company,  hexamethyIdisiIazane,  Rockford,  locally.  Company, P h i I I i p s b u r g ,  L-3-methyl-  England); D-mannose,  Biochemicals Corporation, Cleveland, Ohio);  IIIinois);  (Sigma Chemical Company,  were o b t a i n e d  (Fisher S c i e n t i f i c  monohydrate,  a l l neutral monosaccharides except D - g a I a c t o s e ,  cellosolve,  all.amino  R o c h e s t e r , New  (Koch-Light L a b o r a t o r i e s L t d . , Colnbrook  (Nutritional  4  California);  c y c l o h e x a n e c e r t i f i e d ACS s p e c t r a n a I y z e d  L-rhamnose  inositol  (Eastman Kodak Company,  F a i r Lawn, New J e r s e y ) ;  histidine puriss.  Los A n g e l e s ,  LiBH ,  trimethyIchlorosiIane  D-gIucosamine  (Pierce  hydrochloride,  S t . Louis, Missouri).  ' B a k e r A n a l y z e d " grade  methyl  myo-  A l l other chemicals  ( J . T . Baker Chemical  New J e r s e y ) was used when a v a i l a b l e .  C e l I WaI I P r e p a r a t i o n C u l t u r e s o f Tremella mesenterica F r i e s (UBC c o l l e c t i o n #2259-6) were m a i n t a i n e d a t 20 C on n u t r i e n t agar  (15.0 g bacto-agar, 7.5 g bacto-  m a l t e x t r a c t , 0 . 5 g b a c t o - y e a s t e x t r a c t and 1.0 g b a c t o - s o y t o n e p e r l i t r e ) .  1Q Inoculum c u l t u r e s were prepared above w i t h o u t b a c t o - a g a r )  i n 50 ml o f l i q u i d medium ( t h a t d e s c r i b e d  and shaken a t 20 C f o r 48 h r .  A l i q u o t s of t h e  inoculum (5 m l ) were t r a n s f e r r e d t o a 2800 ml Fernbach f l a s k c o n t a i n i n g 1  l i t r e o f t h e l i q u i d medium and shaken a t 20 C f o r 48 h r .  c o n d i t i o n s t h e fungus grew a s h a p l o f d ,  Under t h e s e  yeast-like unicells.  C e l l s were h a r v e s t e d by c e n t r i f u g a t i o n a t 9000 * g f o r 10 m i n , washed once w i t h w a t e r , and c o o l e d by s t i r r i n g f o r 30 min i n an i c e b a t h . C e l l s were broken  i n 50 ml p o r t i o n s u s i n g a B l a c k s t o n e U l t r a s o n i c Probe  a t 200 w a t t s f o r 2 m i n , c o o l e d f o r 30 min and fragmented 2 min.  The c e l l s were kept c h i l l e d  was immersed  f o r a further  i n an i c e bath t h r o u g h o u t and t h e probe  i n i c e between t r e a t m e n t s .  The r e s u l t i n g s u s p e n s i o n o f whole  and broken e e l Is was c e n t r i f u g e d a t 750 x gr f o r 5 min a t 2 C. The supernatant  s u s p e n s i o n c o n s i s t e d of broken c e l l s .  l a r g e l y o f whole c e l l s , centrifuged;  The p e l l e t , c o n s i s t i n g  was t r e a t e d a g a i n w i t h t h e u l t r a s o n i c probe and  the supernatant  s u s p e n s i o n s were combined.  As a r e s u l t o f  t h i s t r e a t m e n t a p p r o x i m a t e l y 95% o f t h e c e l l s were b r o k e n .  Any r e m a i n i n g  whole c e l l s were removed by c e n t r i f u g a t i o n d u r i n g t h e c o u r s e o f t h e washing  procedure. C u l t u r e s o f Saprolegnia  diclina  Humphrey (UBC c o l l e c t i o n #145)  were m a i n t a i n e d a t 4 C on s l a n t s of t h e same medium used f o r Tremella. Inoculum c u l t u r e s prepared on t h i s medium 20 C f o r 96 h r .  in petri  p l a t e s were grown a t  The medium and mycelium were homogenized  blendor c o n t a i n i n g  in a s t e r i l e  100 ml d i s t i l l e d w a t e r ; 20 ml o f t h e inoculum were  t r a n s f e r r e d t o a 2800 ml Fernbach f l a s k c o n t a i n i n g 1 l i t r e o f l i q u i d medium ( 1 0 . 0 g d e x t r o s e , 5 . 0 g b a c t o - p e p t o n e , per  litre).  0.5 g bacto-yeast extract  C u l t u r e s were grown a t 20 C f o r 60 hr w i t h o u t  shaking.  E x a m i n a t i o n o f samples from each o f t h e c u l t u r e f l a s k s w i t h t h e l i g h t  11  m i c r o s c o p e showed no s p o r e s t o be p r e s e n t . The mycelium was h a r v e s t e d by f i l t r a t i o n on a BUchner and washed once w i t h w a t e r . powder  funnel  The pad c f mycelium was ground t o a f i n e  i n l i q u i d N w i t h a m o r t a r and p e s t l e . 2  When t h e t e m p e r a t u r e  r o s e above 0 C 50 mI o f water were added t o make a t h i c k s l u r r y which was t r e a t e d w i t h t h e u l t r a s o n i c probe ( t w i c e f o r 2 min) above.  as described  As a r e s u l t o f t h i s t r e a t m e n t v i r t u a l l y a l l o f t h e c e l l s  were  broken. The washing procedure (1969). for cell  i s based on t h a t o f M i t c h e l l and T a y l o r  The s u s p e n s i o n o f broken c e l l s was c e n t r i f u g e d a t 2 C (27000 x g  10 min w i t h Tremella, wall fragments.  3000 * g f o r 5 min w i t h Saprolegnia)  The s u p e r n a t a n t  procedure was repeated 4 t i m e s .  s o l u t i o n was d i s c a r d e d .  The c e l l  (centrifugation at  3000 x g f o r 5 min w i t h  5 t i m e s w i t h c o l d water and t w i c e w i t h 8 . 0 M urea s o l u t i o n a t 27000 x g f o r 10 min w i t h Tremella,  This  w a l l fragments were then  washed 5 t i m e s w i t h i c e - c o l d 1.0 M NaCI s o l u t i o n 12000 x g f o r 10 min w i t h Tremella,  t o recover  Saprolegnia), (centrifugation  3000 * g f o r 5 min w i t h  Saprolegnia).  The fragments were suspended f o r 12 hr i n 8 M urea s o l u t i o n a t 4 C, then c e n t r i f u g e d and washed t w i c e more w i t h 8 M urea s o l u t i o n , 5 t i m e s w i t h w a t e r , 5 t i m e s w i t h 1.0 N NK^OH s o l u t i o n  (centrifugation  a t 12000  10 min w i t h Tremella,  3000 x g  with water.  w a l l s r e c o v e r e d from t h i s t r e a t m e n t were  The c e l l  f o r 5 min w i t h Saprolegnia)  x  g for  and 5 t i m e s freeze-dried  and s t o r e d a t - 2 0 C.  Analytical  Procedures L i p i d s were e s t i m a t e d by t h e p r o c e d u r e s o f B a r t n i c k i - G a r c i a  and N i c k e r s o n ( 1 9 6 2 ) .  Elemental a n a l y s i s f o r C, H, N, 0 , P, S , and a s h  12 were performed  Cell  by O r g a n i c M i c r o a n a l y s i s , M o n t r e a l ,  Quebec.  waI Is»(approximateIy 5 mg) were h y d r o l y z e d  i n vacuo w i t h 0 . 5  ml of 6 N HCI c o n t a i n i n g 5% ( v / v ) CH2SH-COOH (Matsubara and S a s a k i 1969) a t 110 C f o r 8 , 2 4 , 4 8 , 72.and H y d r o l y s a t e s were d r i e d then r e d i s s o l v e d  144 hr ( T r i s t r a m and Smith  1963).  i n vacuo over c o n c e n t r a t e d H2SO4 and KOH p e l l e t s ,  i n 1.0 ml w a t e r .  (pH 2.2). and 200 yI of t h e i n t e r n a l  A l i q u o t s of t h i s s o l u t i o n , 50 u l HCI standard s o l u t i o n  (a-amino-3-guanido-  p r o p i o n i c a c i d f o r b a s i c amino a c i d s and n o r l e u c i n e f o r n e u t r a l amino a c i d s )  i n pH 2 . 2 b u f f e r  and a c i d i c  were a p p l i e d t o t h e a p p r o p r i a t e column of  a Beckman Amino A c i d A n a l y z e r Model  120C. The a n a l y s i s i s based on t h e  method of Spackman, S t e i n and Moore ( 1 9 5 8 ) .  B a s i c amino a c i d s were  s e p a r a t e d on a 13 cm column; t h i s r e s o l v e d g l u c o s a m i n e from g a I a c t o s a m i n e , and 1 - m e t h y I h i s t i d i n e  and 3 - m e t h y I h i s t i d i n e  each o t h e r ) from h i s t i d i n e ( F i g u r e 1 ) . not r e s o l v e d by t h i s s y s t e m . release tryptophan  ( t h e s e were not r e s o l v e d from  Tryptophan  and g a I a c t o s a m i n e were  S i n c e t h e a l k a l i n e h y d r o l y s i s procedure t o  degrades g a I a c t o s a m i n e ,  and t h e a c i d i c  hydrolysis  p r o c e d u r e t o r e l e a s e g a I a c t o s a m i n e g e n e r a l l y degrades t r y p t o p h a n , p r e s e n t e d no problem. 58 cm column, not  A c i d i c and n e u t r a l  amino a c i d s were r e s o l v e d on a  g l u c o s a m i n e was s e p a r a t e d from p h e n y l a l a n i n e and t h u s d i d  interfere with the phenylalanine determination.  column i s c r i t i c a l  to this resolution.  The l e n g t h of t h e  HydroxyproIine  was determined  by t h e s p e c t r o p h o t o m e t r i c method of Bergman and L o x l e y (1970) p-dimethylaminobenzaldehyde.  Tryptophan  a c i d a n a l y z e r a f t e r h y d r o l y s i s of c e l l Moore  this  using  was determined u s i n g t h e amino  w a l l s w i t h 4 . 2 N NaOH ( H u g l i and  1972). A n a l y s e s were performed  i n t r i p l i c a t e on a s i n g l e c e l l  wall  14 p r e p a r a t i o n of Tremella, of  and once on each of 2 c e l l  wall  preparations  Saprolegnia.  A s y n t h e t i c m i x t u r e of amino a c i d s p r e s e n t  in the c e l l  ( e x c e p t h y d r o x y p r o l i n e and t r y p t o p h a n ) and g l u c o s a m i n e was prepared f o r d e g r a d a t i o n  studies;  t h e same p r o c e d u r e as t h e c e l l h y d r o x y p r o I i n e and t r y p t o p h a n  Cell  hydrochloride'  i t was t r e a t e d w i t h 6 N HCl by  wall preparations. was determined  w a l l s (approximately  walls  D e g r a d a t i o n of  both  independently.  2 mg) were h y d r o l y z e d  in sealed tubes  a t 110 C w i t h 0 . 5 ml 2 N CF C00H f o r 15, 3 0 , 60, 120, 240 and 480 min 3  ( A l b e r s h e i m e t a I 1967) t o r e l e a s e n e u t r a l dried  sugars.  H y d r o l y s a t e s were  i n vacuo o v e r KOH p e l l e t s . U r o n i c a c i d s were e s t i m a t e d as n e u t r a l  s u g a r s by r e d u c i n g t h e  c a r b o x y l groups of t h e p o l y s a c c h a r i d e s w i t h LiBHi+ (Dutton and K a b i r 1971). The p r o c e d u r e was adapted f o r m i c r o a n a l y s i s by r e d u c i n g t h e p r o p o r t i o n s of  reagents.  The c e l l  wall preparation  (20 t o 35 mg) was d i s s o l v e d i n  8 . 0 ml formamide and t r e a t e d w i t h 6 . 0 ml p y r i d i n e and 4 . 0 ml p r o p i o n i c anhydride. adding  A f t e r s t o r a g e f o r 2 days t h e s o l u t i o n was p r e c i p i t a t e d by  i t t o 150 ml 2% H C l .  and d r i e d  recovered p r e c i p i t a t e ,  filtered  i n v a c u o , was r e t r e a t e d w i t h 10.0 ml p y r i d i n e and 1.5 ml  propionic anhydride.  The t o t a l  i n 1 5 . 0 ml t e t r a h y d r o f u r a n , methane  The t o t a l  r e c o v e r e d p r o p i o n a t e d a c i d was d i s s o l v e d  and 1 0 . 0 ml d i e t h y l  ( c o o l e d t o - 7 3 C) were added;  -73 C for 6 hr.  diazo-  t h e m i x t u r e was a l l o w e d t o stand a t  Diazomethane was prepared  p - t o . l uenesu I f onam ide a c c o r d i n g t o Vogel up t o t h e r e c o v e r y of c a r b o x y I - r e d u c e d  ether containing  frcm  (1956).  W-methyI-N-nitrosoSubsequent  reactions  p o l y s a c c h a r i d e s were i d e n t i c a l  w i t h t h o s e d e s c r i b e d by Dutton and K a b i r .  The reduced p o l y s a c c h a r i d e s  15 were then h y d r o l y z e d Neutral trimethyIsilyI  w i t h 2 N CF C00H f o r 2 hr t o r e l e a s e n e u t r a l 3  s u g a r s were a n a l y z e d by g a s - l i q u i d chromatography  (TMS) d e r i v a t i v e s .  H y d r o l y s a t e s were d i s s o l v e d  0.05 ml t r i m e t h y I c h I o r o s i I a n e  (Sweeley e t a l . 1963).  to  in 100 y l  and a l i q u o t s of t h i s m i x t u r e o r s u i t a b l e d i l u t i o n s of  i t were  i n t o a V a r i a n Aerograph dual column gas chromatograph Model w i t h flame  ionization detectors.  ml / min and a i r 250 ml / m i n .  and  The m i x t u r e was  shaken f o r a few s e c o n d s , and a f t e r 30 min was f r e e z e - d r i e d The TMS d e r i v a t i v e s were r e d i s s o l v e d  as  in 1 ml  p y r i d i n e and t r e a t e d s u c c e s s i v e l y w i t h 0.1 ml h e x a m e t h y I d i s i I a z a n e  the p y r i d i n e .  sugars.  The f l o w r a t e s of N  2  cyclohexane injected  1740,  and H  Columns ( 4 . 9 m * 3 mm) of  remove  2  equipped  were 25  105? s i l i c o n e  f l u i d SF 96 on a c i d washed DMCS t r e a t e d 6 0 / 8 0 mesh f l u x - c a l c i n e d d i a t o m i t e (Chromosorb W, Chromatographic temperature  S p e c i a l i t i e s , B r o c k v i l l e , Ontario)  programmed l i n e a r l y from 130 C ( a t  2 degrees / m i n .  t o 230 C a t  F i g u r e 2 shows t h e r e s o l u t i o n o b t a i n e d by t h i s system.  A s y n t h e t i c m i x t u r e of f r e e n e u t r a l degradation  injection)  were  sugars was prepared  for  s t u d i e s ; a l i q u o t s were' t r e a t e d w i t h 2 N CF3COOH by t h e same  p r o c e d u r e s as t h e c e l l  wall preparations.  It a l s o served as a c a l i b r a t i o n  mixture.  Cell  w a l l s (approximately  2 mg) were h y d r o l y z e d  0 . 5 ml 2 N HCl  (Oates and S c h r a g e r 1967)  amino s u g a r s .  H y d r o l y s a t e s were d r i e d  and KOH p e l l e t s , d i s s o l v e d  f o r 8,  in vacuo w i t h  16, 32 and 72 hr t o r e l e a s e  in vacuo o v e r c o n c e n t r a t e d H2S04  in pH 2 . 2 b u f f e r , and e s t i m a t e d on t h e amino  a c i d a n a l y z e r u s i n g t h e 13 cm column. The m i x t u r e of g l u c o s a m i n e h y d r o c h l o r i d e and amino a c i d s used for degradation  s t u d i e s was t r e a t e d w i t h 2 N HCl by t h e same p r o c e d u r e s  FIGURE 2  RESOLUTION  OF  NEUTRAL  SUGARS (AS  TMS  DERIVATIVES)  ON  THE  GAS-LIQUID  CHROMATOGRAPH  17 as t h e c e l l  w a l l p r e p a r a t i o n s t o d e t e r m i n e t h e d e g r a d a t i o n of  glucosamine  under t h e s e c o n d i t i o n s .  C a l c u l a t i o n of  Results  Peak a r e a s were e s t i m a t e d by t h e p r o d u c t of peak h e i g h t * peak width a t the h a l f - h e i g h t  which B a l l ,  i s t h e most r e l i a b l e manual method. width at the h a l f - h e i g h t  H a r r i s and Habgood  (1967) c o n c l u d e d  The e s t i m a t e s , p a r t i c u l a r l y of  a r e more a c c u r a t e on amino a c i d  where t h e number of d o t s p r i n t e d per t i m e (<* w i d t h ) measurement of t h e w i d t h on g a s - l i q u i d chromatograms  the  chromatograms  can be c o u n t e d ;  the  is less accurate  because of t h e w i d t h of t h e i n k t r a c e . Internal  s t a n d a r d s a r e n e c e s s a r y i n both monosaccharide and  amino a c i d e s t i m a t i o n s .  For g a s - l i q u i d chromatography  the  interna!  dards were used t o check d e t e c t o r response and d i l u t i o n e r r o r s .  stan-  The  g r e a t d i s p a r i t y of sugar c o n c e n t r a t i o n s in t h e c e l l  w a l l s examined meant  t h a t 1 t o 2 yI  ( c a . 2 mg d i s s o l v e d  in 100 yI  of t h e  initial  s i l y l a t e d preparation  c y c l o h e x a n e ) had t o be i n j e c t e d onto t h e g a s - l i q u i d  in o r d e r t o d e t e c t minor c o n s t i t u e n t s . temperature,  chromatograph  C y c l o h e x a n e i s v o l a t i l e a t room  and even though t h e p r e p a r a t i o n s were immediately  and p l a c e d a t - 2 0 C, e v a p o r a t i o n  l o s s e s were p o s s i b l e .  s i l y l a t i o n procedure produces NH^CI, which i s i n s o l u b l e With as l i t t l e as 1 t o 2 yI  in t h e s y r i n g e ,  stoppered  Furthermore,  the  in c y c l o h e x a n e .  p a r t i c l e s of NHuCI may cause  s i g n i f i c a n t volume e r r o r s t h a t a r e d i f f i c u l t t o e s t i m a t e .  To d e t e c t t h e  major c o n s t i t u e n t s 20 y i of t h e c o n c e n t r a t e d s o l u t i o n were d i l u t e d  with  500 y l c y c l o h e x a n e ; a s i m i l a r e r r o r from NH^CI c o u l d r e s u l t .  is  t h e d i l u t i o n e r r o r t h a t was c o r r e c t e d by t h e use o f . i n t e r n a ! Both e r y t h r i t o l  and aiyo-inos  This  standards.  ? t o I were used; t h e c o n c e n t r a t i o n of e r y t h r i t o i  18 was a p p r o x i m a t e l y \% t h a t of  inositol  in o r d e r t o o b t a i n measurable peaks  on s c a l e a t t h e d i f f e r e n t c o n c e n t r a t i o n s . removed after  h y d r o l y s i s and before  A l l t r a c e s of CFgCOOH were  50 uI of each i n t e r n a l s t a n d a r d  solution  were added. . The r a t i o of a r e a per w e i g h t was determined f o r each i n t e r n a l s t a n d a r d (on c a l i b r a t i o n r u n s ) .  For each sample a n a l y z e d , t h e a r e a of  t h e i n t e r n a l s t a n d a r d c a l c u l a t e d from t h e d i l u t i o n of t h e sample, was compared w i t h t h e a c t u a l a r e a measured on t h e chromatogram.  The w e i g h t  of anhydro sugar in t h e samples was c a l c u l a t e d by comparison of a r e a w i t h t h e a r e a of a known w e i g h t of anhydro e q u i v a l e n t c a l i b r a t i o n mixture.  i t s peak  in t h e  Where t h e r e were s e v e r a l peaks f o r a s u g a r ,  the  b e s t r e s o l v e d one from t h e sample was compared w i t h t h e e q u i v a l e n t peak of t h e c a l i b r a t i o n a n a l y s i s .  For amino a c i d a n a l y s i s t h e i n t e r n a l  s t a n d a r d was used t o compensate  f o r t h e d e t e r i o r a t i o n of t h e n i n h y d r i n r e a g e n t . amino a c i d a n a l y z e r 20 nmoles of  internal  For every run on t h e  s t a n d a r d were added t o t h e  a p p r o p r i a t e column w i t h t h e sample o r c a l i b r a t i o n m i x t u r e  Ca-amino-S-  g u a n i d i n o - p r o p i o n i c a c i d on t h e b a s i c amino a c i d column and n o r l e u c i n e on t h e n e u t r a l  and a c i d i c amino a c i d c o l u m n ) .  was compared w i t h t h e a r e a of t h e i n t e r n a l  The a r e a of each amino peak  standard f o r t h a t a n a l y s i s .  The w e i g h t of t h e anhydro amino a c i d was c a l c u l a t e d by comparison w i t h t h e s i m i l a r r a t i o in a c a l i b r a t i o n a n a l y s i s c o n t a i n i n g 20 nmoles of amino a c i d s in t h e Beckman Amino A c i d C a l i b r a t i o n M i x t u r e Type 1.  the  19 RESULTS  The c e l l  w a l l s a f t e r washing showed no e v i d e n c e of c y t o p l a s m i c  c o n t a m i n a t i o n by phase c o n t r a s t m a t e r i a l w i t h India  l i g h t m i c r o s c o p y , no e v i d e n c e of c a p s u l a r  i n k under t h e l i g h t m i c r o s c o p e and no v i s i b l e  r i b o s o m e s , membranes o r c a p s u l a r m a t e r i a l w i t h e l e c t r o n m i c r o s c o p y . The y i e l d of c e l l from Tremella  w a l l s from 6 I o f medium was 100 t o 150 mg  and 200 mg from Saprolegnia  mesenterica  p r e p a r a t i o n s were s t o r e d a t - 2 0 C and d r i e d  These  diclina.  i n vacuo f o r 12 hr b e f o r e  weighing.  Degradation  under H y d r o l y z i n q  Conditions  An a l i q u o t of t h e s y n t h e t i c m i x t u r e o f amino a c i d s and g l u c o s a m i n e hydrochloride  was s u b j e c t e d t o t h e h y d r o l y z i n g  wall  proteins  (6 N HCl i n vacuo)  hr)  as t h e c e l l  (8 t o 145  A s o l u t i o n of h y d r o x y p r o I i n e was  A l i q u o t s of a s o l u t i o n of t r y p t o p h a n  ( w i t h 4 . 2 N NaOH i n vacuo) hr.  f o r each of t h e t i m e p e r i o d s  wall preparations.  similarly treated.  c o n d i t i o n s used f o r c e l l  like the c e l l  were t r e a t e d  wall preparations  f o r 8 t o 96  The r e c o v e r i e s of t h e amino a c i d s a r e shown i n F i g u r e 3 . A l i q u o t s of t h e m i x t u r e of amino a c i d s and g l u c o s a m i n e  were a l s o s u b j e c t e d t o t h e h y d r o l y s i s c o n d i t i o n s f o r r e l e a s i n g s u g a r s from c e l l  w a l l p o l y s a c c h a r i d e s (2 N HCl i n vacuo)  hydrochloride amino  f o r 8 t o 96 h r .  The r e c o v e r i e s of g l u c o s a m i n e from t h e two t r e a t m e n t s a r e compared i n Figure 4.  Glucosamine  i s degraded t o a f a r g r e a t e r e x t e n t i n 6 N H C i .  A l i q u o t s of t h e c a l i b r a t i o n m i x t u r e of f r e e n e u t r a l  sugars  were s u b j e c t e d t o 2 N CF C00H f o r 15 min t o 8 hr ( t h e same t r e a t m e n t 3  to release neutral  s u g a r s from c e l i  wall polysaccharides).  used  The r e c o v e r i e s  FIGURE 3  RECOVERY OF FREE  AMINO ACIDS  (6 N HCI i n vacuo)  UNDER HYDR0LY2ING CONDITIONS  21 FIGURE 4  RECOVERY OF GLUCOSAMINE UNDER HYDROLYZING CONDITIONS A Comparison of 2 N  Time  and 6 N HCl  (hr)  a r e shown i n F i g u r e 5 .  P r o t e i n Ana i ys i s All were, found  o f t h e usual  in the c e l l  p r o t e i n amino a c i d s e x c e p t c y s t e i n e / c y s t i n e  w a l l s o f both s p e c i e s .  A p p l i c a t i o n of larger  samples t o t h e 58 cm column f a i l e d t o show any t r a c e o f c y s t e i n e / c y s t i n e or c y s t e i c a c i d .  HydroxyproI ine and t r y p t o p h a n  p r o t e i n c o n s t i t u e n t s i n both s p e c i e s . amide i n t h e c e l l  wall preparations  were d e t e c t e d as  I t was n o t p o s s i b l e t o d e t e r m i n e  because of r e s i d u a l  washing procedure and d e g r a d a t i o n o f amino compounds. presented  in Tables  NH  3  from t h e  The r e s u l t s a r e  I and I I , and i n F i g u r e s 6 and 7 .  The amount o f each monomer r e c o v e r e d a f t e r h y d r o l y s i s i s determined  primarily  by two r e a c t i o n s :  t h e r a t e a t which t h e monomer  i s r e l e a s e d from t h e polymer and t h e r a t e a t which t h e f r e e monomer i s degraded under t h e h y d r o l y z i n g  conditions.  Curves such a s t h o s e i n  F i g u r e s 6 and 7 r e p r e s e n t t h e amount o f monomer r e l e a s e d d u r i n g  minus t h e amount of f r e e monomer  hydrolys  degraded.  Polysaccharide Analysis  Neutral  Sugars A r a b i n o s e , x y l o s e , f u c o s e , mannose, g a l a c t o s e and g l u c o s e  were i d e n t i f i e d  in the c e l l  d e t e c t e d i n T. mesenterica d e t e c t e d i n S. diclina presented  in Tables  w a l l s o f both o r g a n i s m s . b u t n o t i n S. dieUna;  but n o t i n T . mesenterica.  I I I and IV, and  Rhamnose was  t r a c e s o f r i b o s e were The r e s u l t s a r e  i n F i g u r e s 8 and 9 .  Amino Sugars Glucosamine was found  in the c e l l  waI Is o f both  organisms;  24 TABLE I  AMINO ACIDS IN THE CELL WALL OF Tremella  (yg anhydro amino a c i d r e c o v e r e d / mg c e l l  wall  mesenterica preparation)  D u r a t i o n of H y d r o l y s i s w i t h 6 N HCI 8 hr Trp  24 h r  48 h r  72 h r  3  0.1  <0.1  0.1  Lys  2.2  3.3  2.9  His  0.8  1 .2  Arg  2.2  Asx  145 h r  best e s t i mate  3  0.1  2.3  2.0  3.3  1.1  0.9  0.8  1 .2  3.5  3.2  2.5  2.2  3.5  2.6  5.0  4.7  3.3  2.7  5.0  Thr  1.3  2.3  2.3  1 .7  1 .4  2.5  Ser  1 .1  1 .5  1 .8  1 .4  1 .2  1 .8  Glx  4.3  7.5  7.2  5.2  3.9  7.8  Pro  1 .8  2.6  2.4  2.0  1 .6  2.6  Gly  1 .6  2.1  2.3  1 .6  1.4  2.3  Ala  2.1  3.0  3.3  2.1  1 .7  3.3  Cys  0.0  0.0  0.0  0.0  0.0  0.0  Val  1 .2  2.3  . 2.8  2.1  1 .8  2.8  Met  1.1  1 .2  1 .2  0.7  0.5  1 .3  1 le  0.9  2.1  2.5  1 .9  1 .7  2.5  Leu  2.5  3.6  4.3  3.7  2.6  4.3  Tyr  1 .4  2.0  1 .9  1 .4  1 .1  2.0  Phe  1.1  1 .6  2.2  1 .7  1.5  2.2  0.4  0.3  0.2  0.1  0.4  Hyp  2  3  5  T o t a l Recovery  48.9  1  maximum v a l u e  2  cell Moore  3  .  4  w a l l p r e p a r a t i o n h y d r o l y z e d i n 4 . 2 N NaOH (Hug I I and 1972)  n o t determined h y d r o l y z e d f o r 96 hr  5  determined s p e c t r o p h o t o m e t r i c a I Iy (Bergman and L o x l e y 1970)  25 TABLE II  AMINO ACIDS  IN THE CELL WALL OF Saprolegnia  (ug anhydro amino a c i d r e c o v e r e d / mg c e l l  wall  diclina  preparation)  D u r a t i o n of H y d r o l y s i s w i t h 6 N HCl 8 hr Trp  24 hr 0.2 .  3  2  48 hr  72 hr  <0.1  <0A  h  145 hr  best estimate  3  0.2  Lys .  0.8  1 .2  0.8  0.7 .  0.6  1 .2  His  0.3  0.5  0.2  0.2  0.2  0.5  Arg  0.6  0.8  0.6  0.4  0.4  0.8  Asx  1 .0  1-1  1 .2  1 .2  5  1 .2  Thr  0.9  1 .0  1.1  1.1  1 .0  1 .1  Ser  0.4  0.4  0.5  0.4  0.4  0.5  Glx  1 .0  1 .2  1 .3  1 .4  1 .3  1 .4  Pro  0.6  0.8  0.8  0.8  0.-7  0.8  Gly  0.5  0.5  0.6  0.6  0.6  0.6  Ala  0.7  0.9  0.9  0.7 .  0.7  1 .0  Cys  0.0  0.0  0.0  0.0  0.0  0.0  0.4  0.6  0.7  0.7  0.7  0.7  Met  0.3  0.3  0.3  0.2  0.2  0.3  1 le  0.3  0.4  0.5  0.5  0.5  0.5  Leu  0.6  0.8  0.9  0.9  0.8  0.9  Tyr  0.5  0.5  0.5  0.5  0.4  0.5  Phe  0.4  0.5  0.5  0.5  0.4  0.5  0.6  0.4  0.2  0.2  0.1  0.6  Val  Hyp  .  6  T o t a l Recovery 1  2  1  13.3  maximum va I ue ce 1 1 wa1I Moore not  in  4 . 2 N NaOH (Hug 1i an<  1972)  determined  hydrolyzed not  preparation hydrolyzed  f o r 96 hr  recovered  determined s p e c t r o p h o t o m e t r i c a I Iy (Bergman arid L o x l e y  1970)  27  RECOVERY  OF  AMINO  ACIDS  FROM  CELL  WALLS  OF  Saprolegnia  ( H y d r o l y s i s i n vacuo w i t h 6 N H C l )  o  Time ( h r )  Time ( h r )  diclh  28  TABLE M l  NEUTRAL SUGARS IN THE CELL WALL OF Tremella (ug anhydro s u g a r / mg c e l l  wall  mesenterica  preparation)  D u r a t i o n o f H y d r o l y s i s w i t h 2 N CF3COOH  best estimate  15 min  30 min  1 hr  2 hr  4 hr  8 hr  Rha  13.0  15.0  17.5  "16.0  13.5  7.5  17.5  Ara  1.0  1.0  1.0  2.5  1.0  1.0  2.5  Fuc*  1.0  8.5  4.5  2  2  8.5  2  Xyl  156.5  161.0  150.0  118.5  98.0  56.5  161.0-  Man  21.5  40.5  88.5  85.0  96.5  78.0  96.5  Gal  1.5  1.5  3.0  3.0  2.5  2.0  3.0  Glc  225.0  338.0  463.5  379.5  414.0  • 368.0  414.0  T o t a l Recovery  1  maximum v a l u e  2  not d e t e c t e d  3  spurious value  4  not c o n f i r m e d ,  3  703.0  i d e n t i f i e d by c h r o m a t o g r a p h i c p o s i t i o n o n l y  1  29  TABLE IV  NEUTRAL SUGARS IN THE CELL WALL OF Saprolegnia (yg anhydro sugar / mg c e l l  wall  diclina  preparation)  D u r a t i o n o f H y d r o l y s i s w i t h 2 N CF C00H 3  best estimate  15 min  30 min  1 hr  2 hr  4 hr  8 hr  Ara  0.5  0.5  2.0  1.5  1.5  1.5  2.0  Rib  <0.5  <0.5  0.5  <0.5  <0.5  <0.5  0.-5  1.0  1.0  3.0  1.5  1.5  0.5  3.0  Xyl  0.5  0.5  1.0  0.5  0.5  <0.5  1.0  Man  2.0  1.5  2.0  4.0  3.0  3.0  4.0  Gal  22.5  21.0  21.5  26.5  28.5  19.0  28.5  Glc  187.0  401.5  610.5  Fuc  2  Total  686.5  607.5  636.5  Recovery  686.5 725.5  maximum v a l u e not c o n f i r m e d ,  i d e n t i f i e d by c h r o m a t o g r a p h i c p o s i t i o n  only  FIGURE 8  CELL WALLS OF Tremella mesenterica -  RECOVERY OF NEUTRAL SUGARS FROM  (Hydrolysis with 2 N CF C00H) 3  FIGURE  9  RECOVERY OF  NEUTRAL  SUGARS  FROM  CELL  (Hydrolysis with 2 N CF C00H) 3  WALLS  OF  Saprolegnia  diclina  32 g a I a c t o s a m i n e was not d e t e c t e d i n e i t h e r . was n o t d e t e r m i n e d . the c e l l  The e x t e n t o f w - a c e t y I a t ion  The r e c o v e r i e s o f g l u c o s a m i n e a f t e r h y d r o l y s i s o f  w a l l p r e p a r a t i o n s w i t h 2 N HCI a r e p r e s e n t e d  i n T a b l e V . The  r e c o v e r i e s o f g l u c o s a m i n e a f t e r h y d r o l y s i s w i t h 2 N HCI and 6 N HCI a r e compared  i n F i g u r e 10. The m a s s i v e d e g r a d a t i o n o f g l u c o s a m i n e w i t h 6 N  HCI p r e c l u d e s i t s use i n any study t h a t a t t e m p t s t o be q u a n t i t a t i v e .  Uronic  Acids For r e d u c t i o n o f c a r b o x y l groups  of T. mesenterica  3 5 . 2 9 mg o f c e l l  method o f Dutton and K a b i r (1971); a r a t i o n were r e c o v e r e d . 3.87 mg r e s p e c t i v e l y .  in t h e c e l l  wall polysaccharides  w a l l p r e p a r a t i o n were reduced by t h e 10.55 mg o f reduced c e l l  F o r S. diclina  wall  prep-  t h e amounts were 20.93 mg and  After hydrolysis  i n 2 N CFgCOOH t h e mole r a t i o s o f  t h e s u g a r s r e c o v e r e d were compared w i t h t h o s e o f t h e unreduced c e l l preparation error  hydro I y s a t e s .  The d i f f e r e n c e s were w e l l w i t h i n t h e e x p e r i m e n t a l  so t h a t u r o n i c a c i d s a r e p r e s e n t  in t h e c e l l  wall  i n v e r y small amounts,  if at a l l ,  waI Is o f t h e two s p e c i e s .  L i p i d Ana Iys i s Individual  l i p i d components were n o t d e t e r m i n e d ;  l i p i d s were  s e p a r a t e d a s two f r a c t i o n s ( B a r t n i c k i - G a r c i a and N i c k e r s o n 1962); extractabie  i n e t h a n o I : d i e t h y I e t h e r and c h l o r o f o r m  (readily  l i p i d s ) and t h o s e r e l e a s e d a f t e r t r e a t m e n t w i t h \% HCI (bound  lipids).  The r e s u l t s a r e p r e s e n t  those  extractable  in ethanol:ether  in Table V I .  Elemental and Ash A n a l y s i s Two d i f f e r e n t  cell  the r e s u l t s a r e presented  w a l l p r e p a r a t i o n s o f each s p e c i e s were a n a l y z e d ; in Table V I I .  33 TABLE V  AMINO SUGARS IN THE CELL WALL OF  Tremella mesenterica  AND Saprolegnia  (yg anhydro GlcNx / mg c e l l  wall  diclina  preparation)  D u r a t i o n o f H y d r o l y s i s w i t h 2 N HCI 8 hr  16 h r  32 h r  72 h r  T. mesenterica  6.1  13.1  20.4  23.9  S. diclina  4.7  7.5  8.9  8.8  1  maximum v a l u e  2  n o t determined  TABLE VI  96 hr  best estimate  23.9  2  8.0  8.9  LIPIDS IN THE CELL WALL OF  Tremella mesenterica (yg / mg c e l l Read i I y Extractable Lipids  AND Saprolegnia wall  diclina  preparation)  Bound L i p i d s  TotaI L i p i d s Recovered  T. mesenterica  33.5  43.0  76.5  S. diclina  82.0  37.5  119.5  1  uc)  anhydro Glcllx  T  :  /  I  m<) c o l l  wall  '—  p r o p f t f h t Ion  1  '—I  r  34a  TABLE VII  ELEMENTAL AND ASH ANALYSIS OF CELL WALL PREPARATIONS OF AND Saprolegnia  Tremella mesenterica (yg / mg c e l l  Tremella #4  wall  mesenterica  diclina  1  preparation)  Saprolegnia  diclina  #5  #1  #2  404.4  376.6  383.6  2  2  C  377.1  H  66.1  70.9  63.4  65.5  0  431.0  451.7  411.7  470.1  N  15.2  18.4  63.1  15.0  69.0  29.7  20.1  30.7  P  14.0  10.5  18.8  4.1  Ash  32.7  16.6  S  Total  "  1005.1  1002,2  a n a l y s i s performed Quebec, on c e l l  3  953.7  24.8  993.8  by O r g a n i c M i c r o a n a l y s i s , M o n t r e a l ,  w a l l p r e p a r a t i o n s t h a t were d i a l y z e d f o r  72 h r  cell  w a l l p r e p a r a t i o n used f o r complete a n a l y s i s  not determi ned  35 Complete C e l l Wall A n a l y s i s The t o t a l r e c o v e r y of c e l l summarized  in T a b l e V I I I .  w a l l components f o r each s p e c i e s i s  A p p r o x i m a t e l y 90%  of t h e w e i g h t of each c e l l  w a l l p r e p a r a t i o n was r e c o v e r e d a f t e r t h e a n a l y s i s i n two q u i t e d i s s i m i l a r fungi.  The r e m a i n i n g  10$ i s not accounted f o r but t h e s i m i l a r r e c o v e r i e s  o b t a i n e d f r o m ' t w o such d i f f e r e n t strategy  fungal  s p e c i e s suggest t h a t t h e  is reproducible. R e s u l t s p r e s e n t e d a r e d e r i v e d from one c e l l  each s p e c i e s .  wall preparation  C o n f i r m a t i o n was o b t a i n e d from two o t h e r p r e p a r a t i o n s of  each s p e c i e s , i n c l u d i n g some prepared by o t h e r workers in t h e The c e l l  for  wall preparations d i f f e r e d  laboratory.  i n t o t a l e l e m e n t a l , p r o t e i n and  p o l y s a c c h a r i d e c o m p o s i t i o n s but mole r a t i o s among n e u t r a l  s u g a r s and among  amino a c i d s i n d i c a t e d t h a t monomeric c o m p o s i t i o n s f o r each of  these  components were s i m i l a r . Analytical  a c c u r a c y was e s t a b I i s h e d from r e p e a t a n a l y s i s of  amino a c i d and sugar c a l i b r a t i o n m i x t u r e s . a c i d a n a l y s i s was ±3% ( l o w e r sugar a n a l y s i s ±5% (lower  Reproducibility for  amino  l i m i t of d e t e c t i o n c a . 0 . 5 nmole) and f o r  l i m i t of d e t e c t i o n ca.  50 n g ) .  36  TABLE V I I I  COMPLETE ANALYSIS OF THE CELL WALL OF AND Saprolegnia  Tremella mesenterica (yg / mg c e l l  wall  Tremella  diclina  preparation)  mesenterica  Saprolegnia  Polysaccharide anhydro n e u t r a l s u g a r s  703.0  725.5  anhydro amino s u g a r s  23.9  8.9  anhydro u r o n i c a c i d s  <0.5  <0.5  48.9  13.3  76.5  119.5  • 32.7  24.8  885.0  892.0  Protei n anhydro amino a c i d s Lipid Ash T o t a l Recovery  diclina  37 DISCUSSI ON  Although t h e c e l l p r e v i o u s l y , Saprolegnia  waI Is o f n e i t h e r s p e c i e s have been  studied  ferax has been i n v e s t i g a t e d (Crook and J o h n s t o n  1962; P a r k e r , P r e s t o n and Fogg 1963; N o v a e s - L e d i e u , J i m S n e z - M a r t f n e z and V i l l a n u e v a 1967; S i e t s m a , E v e l e i g h and H a s k i n s 1969).  None o f t h e s e  s t u d i e s were s p e c i f i c a l l y q u a n t i t a t i v e b u t t h e y d i d show t h a t t h e c e l l w a l l c o n t a i n e d g l u c o s e as t h e predominant  polysaccharide constituent with  r i b o s e , mannose and g l u c o s a m i n e p r e s e n t a s minor c o n s t i t u e n t s . Led ieu e t a I. (1967) r e p o r t e d t h e presence o f rhamnose the other sugars.  Rhamnose  Novaes-  in addition t o  i s n o t a c o n s t i t u e n t of t h e c e l l  wa I Is o f  t h e TMS e t h e r s of rhamnose and r i b o s e a r e c o m p l e t e l y  S. diclina;  by t h e g a s - l i q u i d chromatography  system employed  resolved  (see Figure 2 ) .  Only  N o v a e s - L e d i e u e t a I. (1967) r e p o r t e d t h e presence of g a l a c t o s e , which i n i s t h e second most abundant s u g a r .  S. diclina  were d e t e c t e d  i n t r a c e amounts  i n S. diclina  Arabinose,  b u t n o t i n S. ferax.  e t a l . (1963) s t u d i e d o n l y sugar c o n s t i t u e n t s o f t h e c e l l examined two a d d i t i o n a l  s p e c i e s o f Saprolegnia.  S. monoica was s i m i l a r t o t h a t o f S. ferax;  f u c o s e and .xyjose  walls;  Parker they  The c o m p o s i t i o n of  t h a t of S. litoralis  had t h e  same c o n s t i t u e n t s and u r o n i c a c i d s were d e t e c t e d a s w e l l . Crook and J o h n s t o n (1962) and N o v a e s - L e d i e u e t a l . (1967) e s t i m a t e d q u a n t i t a t i v e amino a c i d c o m p o s i t i o n from r e l a t i v e of n i n h y d r i n - p o s i t i v e acids,  s p o t s on paper chromatograms.  including hydroxyproline  for tryptophan; methionine.  were p r e s e n t ,  intensities  The p r o t e i n  although  neither  amino  tested  Crook and J o h n s t o n (1962) r e p o r t e d t h e absence o f  The q u a n t i t a t i v e r e s u l t s of t h e c e l l  S. ferax and S. diclina  are compared  below.  wall analyses of  38  Saprolegnia Novaes-Ledieu e t a I. neutral  sugars  S.  diclina  S i e t s m a e t a I.  93 %  84 %  12.5%  1.7  2.7  0.9  amino sugars uronic acids  ferax  not detected  n o t determined  not detected  protein  1.2  3.0  1.4  lipid  1.0  5.0  12.0  3.2  2.5  ash  n o t determined There  i s general  agreement about t h e c o m p o s i t i o n o f t h e c e l l  w a l l s of t h e two s p e c i e s ; t h e d i f f e r e n c e s observed between s p e c i e s and between two a n a l y s e s o f t h e same s p e c i e s may be t h e r e s u l t o f s e v e r a l factors.  Both N o v a e s - L e d i e u e t a I. (1967) and Sietsma e t a I.  estimated t o t a l carbohydrates with t h e anthrone has s t a t e d t h a t " q u a n t i t a t i v e d e t e r m i n a t i o n  reagent.  including t r y p t o p h a n . . . . ,  The f a c t t h a t t h e c e l l  Aminoff  (1970)  i s p o s s i b l e o n l y when t h e  i d e n t i t y o f sugar components t o be assayed i s known. substances,  (1969)  interfere  Several  other  i n t h e [anthrone]  w a l l s c o n t a i n g l u c o s e a s t h e predominant  reaction  sugar  r e d u c e s t h e magnitude c f t h e f i r s t problem b u t t h e s u b s t a n c e s t h a t cause interference may r e s u l t  (and t r y p t o p h a n  was determined  in n e i t h e r of t h e s t u d i e s )  i n a n a l y t i c a l d e t e r m i n a t i o n s t h a t a r e h i g h e r than t h e t r u e  carbohydrate content.  The c e l l s were grown  in d i f f e r e n t media.  Sietsma  e t a I. (1969) d i d n o t r e p o r t t h e age of t h e i r c u l t u r e o r t h e t e m p e r a t u r e a t which t h e y were grown; Walls.  such f a c t o r s may a f f e c t t h e c o m p o s i t i o n of e e l I  The c u l t u r e medium used by S i e t s m a e t a I. (1969)  contained  cholesterol;  t h i s may have a f f e c t e d t h e l i p i d c o n t e n t o f t h e c e i l  or t h e l i p i d  determination.  There have been no r e p o r t s o f c e i l and Ustilago  maydis growing  wall composition of  walls  Tremella,  i n y e a s t - l i k e form appears t o be t h e o n l y  othe  /  .  3  Heterobasidiomycete  investigated  (Crook and J o h n s t o n 1962).  They  found  g l u c o s e and g l u c o s a m i n e t o be t h e major monosaccharides of t h e c e l l with  l e s s g a l a c t o s e and t r a c e s of mannose.  amino a c i d s e x c e p t h y d r o x y p r o I i n e tryptophan). cell  9  wall  They d e t e c t e d a l l t h e p r o t e i n  and m e t h i o n i n e  ( t h e y d i d not t e s t f o r  O ' B r i e n and Ralph (1966) examined t h e sugar components of  w a l l s of s e v e r a l B a s i d i o m y c e t e s and found g l u c o s e and g l u c o s a m i n e  t o be t h e major c o n s t i t u e n t s , w i t h l e s s mannose and x y l o s e , and t r a c e s of fucose present. Cnniophora  G a l a c t o s e was a b s e n t  cerebella.  Tremella  i n a l l s p e c i e s examined e x c e p t ( i n t h e . h a p l o i d y e a s t - l i k e form)  mesenterica  shows two s t r i k i n g d i f f e r e n c e s from t h e s e B a s i d i o m y c e t e s ; c o n t e n t was found t o be v e r y (Table  1).  low ( T a b l e V ) , ' a n d  t h e glucosamine  hydroxyproIine  was d e t e c t e d  In a d d i t i o n t o g a l a c t o s e , both rhamnose and a r a b i n o s e were d e t e c t e a . P o l y s a c c h a r i d e s have been s t u d i e d  i n a l a r g e number of f u n g i ;  G o r i n and Spencer (1968) d i s c u s s e d t h e t y p e s and l i n k a g e s o f monosaccharide constituents studies. fungal  in the fungi  and how t h e y might be u s e f u l  i n taxonomic  B a r t n i c k i - G a r c i a (1968) c o n s i d e r e d t h e p o l y s a c c h a r i d e s of  cell  w a l l s and d e v i s e d a scheme based on t h e c l o s e c o r r e l a t i o n  t h a t can be e s t a b l i s h e d between c h e m i c a l c o m p o s t i i o n of t h e c e l l major taxonomic g r o u p i n g s  e l a b o r a t e d on m o r p h o l o g i c a l  data t o extend t h i s scheme w i l l  criteria.  w a l l and The  come from c a r e f u l q u a l i t a t i v e and q u a n t - •  i t a t i v e s t u d i e s not o n l y of t h e sugar components p r e s e n t but of t h e t y p e s of  l i n k a g e s between them i n many more f u n g a l  s p e c i e s t h a n have been  examined t o t h i s t i m e . The p r o t e i n c o n s t i t u e n t s i n f u n g a l w i d e l y s t u d i e d as t h e p o l y s a c c h a r i d e s .  cell  w a l l s have not been as  A t p r e s e n t t o o few s p e c i e s have  been examined t o make more than v e r y g e n e r a l  statements.  A i l the protein  amino a c i d s a r e p r e s e n t e x c e p t c y s t e i n e / c y s t i n e which appears t o be absent in many f u n g i  (Roy and Landau 1972).  E i t h e r a s p a r t a t e and g l u t a m a t e , o r  40 t h r e o n i n e and s e r i n e a r e f r e q u e n t l y t h e most abundant amino a c i d s . p r o l i n e and t r y p t o p h a n widely sought.  have n o t been w i d e l y r e p o r t e d b u t t h e y have n o t been  HydroxyproIine  has been found  i n s p e c i e s o f t h e Oomycetes  ( i t c o n s t i t u t e s 2 0 . 4 $ of t h e t o t a l amino a c i d component o f dubia,  1968).  Hydroxy-  Aronson and F u l l e r 1969) and i n Candida I t had n o t been p r e v i o u s l y r e p o r t e d  albicans  Atkinsiella  (Chattaway e t a l .  in fungi with c h i t i n o u s c e l l  waI Is and has been sa i d t o be c h a r a c t e r i s t i c o f e e l I u I o s i c c e I I waI Is of f u n g i , a l g a e , o r h i g h e r p l a n t s ( B a r t n i c k i - G a r c i a 1968). hydroxyproIine Basidiomycete. enterica  mesenterica  Further studies w i l l  lacks c h i t i n ,  hydroxyproIine  Analytical  i s Tremella  The d i s c o v e r y o f  appears t o be t h e f i r s t r e p o r t r e v e a l whether t h e c e l l  w a l l o f T. mes-  whether some o f t h e g l u c a n i s c e l l u l o s i c ,  i s not r e s t r i c t e d in i t s d i s t r i b u t i o n in c e l l  in a  o r whether  wall  proteins.  Procedures The e s t i m a t i o n o f most amino a c i d s i s based on t h e p r o c e d u r e s  of Moore, Spackman and S t e i n (1958) and Spackman, S t e i n and Moore Acid h y d r o l y s i s of p r o t e i n s  i s u s u a l l y performed w i t h 6 N H C l ; s e r i a l  h y d r o l y s i s t o e s t i m a t e i n c o m p l e t e c l e a v a g e and d e g r a d a t i o n ( T r i s t r a m and Smith  (1958).  i s recommended  1963).  H y d r o l y s i s under such c o n d i t i o n s g e n e r a l l y b r i n g s about complete d e s t r u c t i o n of t r y p t o p h a n ,  with t h e r a t e a c c e l e r a t e d i n t h e presence of  c a r b o h y d r a t e (Spencer 1963).  Matsubara and S a s a k i (1969) attempted t o  minimize t h e d e s t r u c t i o n of tryptophan p r o t e i n before h y d r o l y s i s . of t r y p t o p h a n o n l y James 1972).  by a d d i t i o n o f CH2SH-COOH t o t h e  However t h i s t r e a t m e n t  i n t h e absence o f c a r b o h y d r a t e  improves t h e r e c o v e r y (Hug I i and Moore 1972;  A l k a l i n e h y d r o l y s i s has proved a s u c c e s s f u l means o f  achieving q u a n t i t a t i v e recovery of tryptophan.  Hug I i and Moore (1972)  have d i s c u s s e d t h e l i m i t a t i o n s o f methods t h a t have been employed and  41 proposed a s i m p l e q u a n t i t a t i v e p r o c e d u r e u s i n g t h e amino a c i d  analyzer:  NaOH i s used f o r h y d r o l y s i s o f t h e p r o t e i n r a t h e r t h a n Ba(0H)2 t o a v o i d adsorption of tryptophan oxidant  (for  on BaSOi* o r BaC03, s t a r c h i s added as an a n t i -  samples r i c h i n c a r b o h y d r a t e such as c e l l  not r e q u i r e d ) ,  and t h e h y d r o l y s a t e  avoid degradation of tryptophan are r o u t i n e l y d i s s o l v e d acid  i s dissolved  w a l l s , starch i s  i n pH 4 . 2 5 b u f f e r t o  i n a h i g h l y a c i d i c medium (amino a c i d s  i n pH 2 . 2 b u f f e r  f o r e s t i m a t i o n on t h e amino  analyzer). HydroxyproIine  has been e s t i m a t e d u s i n g t h e amino a c i d a n a l y z e r  but under t h e s t a n d a r d c o n d i t i o n s used f o r p r o t e i n h y d r o l y s a t e s , i t co-chromatographs with a s p a r t a t e .  I t can be d e t e c t e d by peak enhancement  of t h e absorbance a t 440 nm o r e s t i m a t e d by a l t e r n a t i v e programming o f t h e a n a l y z e r S o t h a t t h e two amino a c i d s a r e r e s o l v e d . procedure ninhydrin.  i s not s a t i s f a c t o r y because o f t h e M i t c h e l l and T a y l o r  However t h e  low s e n s i t i v i t y o f t h e  (1970) examined a number o f a l t e r n a t i v e  procedures using p-dimethylaminobenzaldehyde  as t h e  spectrophotometric  agent and found t h a t o f Bergman and L o x l e y (1970) t o be t h e most s u i t a b l e and t h e most s e n s i t i v e f o r d e t e r m i n a t i o n o f h y d r o x y p r o I i n e There a r e some problems cystine after acid hydrolysis  walls.  i n q u a n t i t a t i v e recovery of c y s t e i n e /  (e.g.  o x i d a t i o n t o c y s t e i c a c i d may o c c u r ) .  Even when a l a r g e e x c e s s o f h y d r o l y s a t e s o f c e l l and S. diclina  in c e l l  w a l l s o f T. mesenterica  were a p p l i e d t o t h e 58 cm column o f t h e amino a c i d  analyzer,  no d i s c e r n a b l e peaks f o r e i t h e r c y s t e i n e o r c y s t e i c a c i d were o b s e r v e d . Under t h e s e c o n d i t i o n s c y s t e i n e , of d e t e c t i o n and t h u s  i f p r e s e n t a t a l l , was below t h e  limits  i s c o n s i d e r e d t o be a b s e n t .  James (1972) has r e p o r t e d t h a t when CH2SH-COOH i s added t o p r o t e i n s before h y d r o l y s i s ,  recovery of p r o l i n e  i s g r e a t e r than from  h y d r o l y s a t e s t h a t do not c o n t a i n mercaptans and no c y s t e i n e i s r e c o v e r e d .  42 In t h e p r e s e n t s t u d i e s , c e l l  w a l l s o f T. mesenterica  were  hydrolyzed  w i t h and w i t h o u t CH SH-C00H; t h e r e was no s i g n i f i c a n t d i f f e r e n c e 2  r e c o v e r y o f p r o l i n e and no c y s t e i n e was d e t e c t e d w i t h e i t h e r Ross ( p e r s o n a l  laurentii  communication)  treatment.  w a l l s o f Cryptococcus  and C. neoformans, which may be r e l a t e d t o Tremella  Wickerham and Bandoni  1966);  c y s t e i n e t o be p r e s e n t  Cell acids.  has examined t h e c e l l  in the  (Slodki,  h y d r o l y s i s w i t h o u t CH SH-C00H showed no 2  in either species.  w a l l p o l y s a c c h a r i d e s have been h y d r o l y z e d  with various  h^SO^ has been w i d e l y used but n e u t r a l i z a t i o n w i t h BaC03 produces  a p r e c i p i t a t e o f BaSO^ t h a t may adsorb monosaccharide c o n s t i t u e n t s , especially uronic acids. that al.  HCl i s v o l a t i l e , b u t i t i s g e n e r a l l y  i t c a u s e s more d e g r a d a t i o n than H S 0 2  4  (1967) used CF3COOH a s t h e h y d r o l y z i n g  (Dutton 1972).  agreed  Albersheim e t  a c i d because o f i t s v o l a t i l i t y  and t h u s n e u t r a l i z a t i o n o f t h e e x c e s s h y d r o l y z i n g  a c i d was n o t r e q u i r e d .  The s u g a r s r e l e a s e d by h y d r o l y s i s have been e s t i m a t e d by g a s - l i q u i d chromatography  a s TMS d e r i v a t i v e s o r as a l d i t o l a c e t a t e s .  a c e t a t e s have been more w i d e l y used d e r i v a t i v e s may be s u p e r i o r d i r e c t l y without the  in c e l l  i n two r e s p e c t s :  wall  Alditol  i n v e s t i g a t i o n s but TMS  ( i ) t h e y can be prepared  i n t e r v e n t i o n o f a r e d u c t i o n s t e p , and ( i i ) t h e y  produce m u l t i p l e peaks ( u s u a l l y two t o f o u r ) w i t h t h e a and B p y r a n o s i d e s as t h e major components.  The number and p r o p o r t i o n o f t h e peaks f o r each  sugar depend on t h e s o l v e n t the solvent  i n which t h e sugar comes t o e q u i l i b r i u m ,  i n which t h e d e r i v a t i v e  s t a t i o n a r y phase o f t h e column. r e l a t i v e proportions  i s i n j e c t e d o n t o t h e column, and t h e  S i n c e t h e number o f peaks and t h e i r  a r e found t o be c o n s t a n t f o r each sugar under  c o n d i t i o n s of d e r i v a t i v e p r e p a r a t i o n , on e x p e c t e d peak p r o p o r t i o n s  given  i t i s p o s s i b l e from a p p r o p r i a t e  data  t o c a l c u l a t e t h e t o t a l amounts o f sugar from  43 any c o m p l e t e l y r e s o l v e d peak ( H o l l i g a n material there  1971).  Since in b i o l o g i c a l  i s u s u a l l y some degree of background c o n t a m i n a t i o n ,  the  p o s i t i v e i d e n t i f i c a t i o n of a monosaccharide from t h e appearance of a s i n g l e peak may o f t e n be i m p o s s i b l e .  The p r e s e n c e of a c h a r a c t e r i s t i c  m u l t i p l e peak p a t t e r n w i t h known r e t e n t i o n and peak a r e a  proportions  a l l o w s i d e n t i f i c a t i o n of most m o n o s a c c h a r i d e s w i t h c o n f i d e n c e  (Bhatti,  Chambers and Clamp 1970). Amino s u g a r s a r e c u s t o m a r i l y r e l e a s e d from p o l y s a c c h a r i d e s w i t h HCI.  The c o n c e n t r a t i o n of HCI used i s c r i t i c a l  l i b e r a t e d amino s u g a r s . glucosamine during 2 N HCI.  f o r t h e r e c o v e r y of  F i g u r e 10 shows t h e m a s s i v e d e g r a d a t i o n  h y d r o l y s i s of c e l l  This matter apparently  of  w a l l s w i t h 6 N HCI compared w i t h  i s not w i d e l y a p p r e c i a t e d as many  r e c e n t papers ( i n c l u d i n g Pao and Aronson 1970; Wang and B a r t n i c k i - G a r c i a 1970; G r a t z n e r  1972)  r e p o r t glucosamine composition data obtained  h y d r o l y s i s w i t h 6 N HCI.  after  The q u a n t i t a t i v e s i g n i f i c a n c e of such r e p o r t s  may be i n doubt f o r t h e y may r e p r e s e n t as l i t t l e as 5 t o 10$ of  the  real glucosamine content.  resolution  There a r e c o n f l i c t i n g r e p o r t s on t h e  and t h e q u a n t i t a t i v e r e c o v e r y of TMS d e r i v a t i v e s of amino s u g a r s 1972).  (Dutton  Moore and S t e i n (1948) showed t h a t g l u c o s a m i n e r e a c t s q u a n t i t a t i v e l y  with ninhydrin  under c o n d i t i o n s of assay f o r amino a c i d s .  Glucosamine  and g a I a c t o s a m i n e can be r e s o l v e d on t h e 13 cm column of t h e amino a c i d analyzer  (Figure 1).  A t p r e s e n t t h i s seems t o be t h e p r e f e r a b l e  method  of a n a l y s i s f o r amino s u g a r s . GIycosiduronic a c i d linkages are r e s i s t a n t to a c i d and under c o n d i t i o n s t h a t r e l e a s e n e u t r a l of such bonds may o c c u r (Dutton 1972). can be h y d r o i y z e d  sugars oniy p a r t i a l  cleavage  The r e s u l t i n g a I d o b i o u r o n i c  by prolonged a c i d t r e a t m e n t t h a t degrades  s u g a r s (Adams 1965).  hydrolysis  Free u r o n i c a c i d s a r e  neutral  l a b i l e in a c i d media and  acids  44  r e a d i l y undergo d e c a r b o x y l a t i o n t o g i v e p r o d u c t s o f unknown (Aminoff 1970).  composition  '  Jones and A l b e r s h e i m (1972) h y d r o l y z e d  cell  wall polysaccharides  w i t h d i l u t e a c i d ( 0 . 2 N CF3COOH), then t r e a t e d t h e p a r t i a l l y preparation with a mixture of extraceI IuIar enzymes from Sclerotium  rolfsii.  depolymerized  poIysaccharide-degrading  The l i b e r a t e d s u g a r s and u r o n i c a c i d s  were reduced w i t h NaBHi* t o a l d i t o l s and a l d o n i c a c i d s r e s p e c t i v e l y , which were s e p a r a t e d u s i n g a n i o n exchange r e s i n . reduced w i t h NaBH4 t o a l d i t o l s . chromatography  The a l d o n i c a c i d s were  The a l d i t o l s were e s t i m a t e d by g a s - l i q u i d  as acetate d e r i v a t i v e s .  Dutton and K a b i r (1972) m e t h y l a t e d p o l y s a c c h a r i d e s from c o r n leaves and s t a l k s , then h y d r o l y z e d  them t o r e l e a s e m e t h y l a t e d  s u g a r s and m e t h y l a t e d a I d o b i o u r o n i c ion exchange r e s i n s . and t h e n e u t r a l  neutral  a c i d s , which were s e p a r a t e d  The m e t h y l a t e d a I d o b i o u r o n i c a c i d s were  using hydrolyzed  sugars analyzed t o reveal t h e l i n k a g e s .  Dutton and K a b i r (1971) a l s o p u b l i s h e d a procedure f o r r e d u c i n g t h e c a r b o x y l groups o f t h e u r o n i c a c i d s i n p o l y s a c c h a r i d e s w i t h LTBHt+ before h y d r o l y s i s .  T h i s was t h e method chosen t o e s t i m a t e u r o n i c a c i d s  in t h e p r e s e n t s t u d i e s . for the information  However t h e method was found t o be u n s u i t a b l e  required  in t h e present s t u d i e s .  After  of t h e c a r b o x y l groups t h e p o l y s a c c h a r i d e s were h y d r o l y z e d neutral  sugars.  reduction t o release  The amount of u r o n i c a c i d s c o u l d be e s t i m a t e d o n l y by  d i f f e r e n c e from t h e h y d r o l y s i s p r o d u c t s o f t h e unreduced A l d o b i o u r o n i c a c i d s unhydrolyzed  i n t h e unreduced p o l y s a c c h a r i d e should  r e p r e s e n t an i n c r e a s e i n t h e n e u t r a l polysaccharide i s hydrolyzed.  sugar components when t h e reduced  If t h e a l d o b i o u r o n i c a c i d s (from t h e  unreduced p o l y s a c c h a r i d e ) a r e h y d r o l y z e d the uronic a c i d estimate w i l l  polysaccharide.  be low.  under t h e c o n d i t i o n s c h o s e n ,  P a r t i c u l a r l y if t h e u r o n i c a c i d  45 c o n t e n t of t h e p o l y s a c c h a r i d e i s low, t h e r e may be c o n s i d e r a b l e e r r o r t h i s e s t i m a t e as t h e r e p r o d u c i b i l i t y of a n a l y s i s i s o n l y about Furthermore  in  5%.  t h e a n a l y s i s does not r e v e a l upon which component t h e c a r b o x y l  group was l o c a t e d .  The method  is useful,  however,  in c h e c k i n g t h e  u r o n i c a c i d c o m p o s i t i o n of a p o l y s a c c h a r i d e f o r which an e s t i m a t e  is  a I ready ava i I a b I e . W h i l e each of t h e s e t h r e e methods  is useful  in q u a l i t a t i v e  a n a l y s i s , they p r e s e n t problems of q u a n t i t a t i v e r e c o v e r y v i z . hydrolysis,  l o s s e s on ion exchange r e s i n s ,  reactions.  At p r e s e n t ,  incomplete  incomplete  derivatization  the problem of q u a n t i t a t i v e r e c o v e r y of s p e c i f i c  u r o n i c a c i d s has not been c o m p l e t e l y s o l v e d B l a k e and R i c h a r d s 1970).  ( N o r s t e d t and Samuel son  On a m i l l i g r a m s c a l e q u a n t i t a t i v e  i s t h e method of c h o i c e (Aminoff  1966;  decarboxylation  1970).  The p r e s e n t s t u d i e s have been p a r t i c u l a r l y concerned w i t h q u a n t i t a t i v e a n a l y s i s of p r o t e i n and p o l y s a c c h a r i d e c o n s t i t u e n t s of cell  wall.  A study of c e l l  and Nurminen  1970)  and t h e whole c e l l . constituents.  wall  l i p i d s of b a k e r ' s y e a s t  the  (Suomalainen  showed q u a n t i t a t i v e d i f f e r e n c e s between t h e c e l l P r o c e d u r e s e x i s t f o r t h e r e s o l u t i o n of  The e x t r a c t i o n method of F o l c h , Lees and  (1957) has been recommended.  wall  lipid  SIoane-StanIey  Dyke (1964) and S i e t s m a , E v e l e i g h and  H a s k i n s (1969) q u a n t i t a t i v e l y e s t i m a t e d a c i d s by g a s - l i q u i d chromatography.  l i p i d s and methyl  e s t e r s of  Suomalainen and Nurminen  extended t h e i n v e s t i g a t i o n t o i n c l u d e p h o s p h o l i p i d s .  (1970)  K u k s i s (1966) has  p u b l i s h e d an e x c e l l e n t r e v i e w of q u a n t i t a t i v e p r o c e d u r e s d e s i g n e d f o r animal t i s s u e s t h a t might be adapted f o r c e l l  wall  fatty  primarily  investtgatrons.  46 The r e s u l t s o f t h e e l e m e n t a l a n a l y s i s ( T a b l e V I I ) show r e a s o n a b l e agreement between t h e d i f f e r e n t c e l l However t h e y do show t h a t c e l l Identical.  w a l l p r e p a r a t i o n s t h a t were a n a l y z e d ,  w a l l s from d i f f e r e n t  preparations a r e not  The amount of N i s r a t h e r h i g h i n both s p e c i e s examined.  In  t h e amounts of N d e r i v e d from amino groups and NH3 a c c o u n t  T. mesenterica  f o r p r a c t i c a l l y a l l of t h e N f o u n d .  The h i g h NH3 l e v e l s a p p a r e n t l y  i n d i c a t e t h a t NHi+OH from t h e washing p r o c e d u r e even a f t e r t h e 72 hr d i a l y s i s p e r i o d .  In S.  i s n o t c o m p l e t e l y removed  diclina  t h e sum o f N from  amino groups and NH3 a c c o u n t s f o r o n l y 33% o f t h e t o t a l N.  Even i n p r e p -  a r a t i o n #1, which c o n t a i n e d s i g n i f i c a n t l y more p r o t e i n , t h e r e c o v e r y of N from amino groups and NH3 was of t h e same o r d e r . other N-containing  substances present  f o r s p e c u l a t i o n as t o t h e i r  i n S.  diclina.  Evidently there are There i s no b a s i s  identity.  The p r e s e n c e of l a r g e amounts of S cannot be e x p l a i n e d i n terms of S - c o n t a i n i n g amino a c i d s ( c y s t e i n e i s a b s e n t and m e t h i o n i n e a c c o u n t s f o r only a small p r o p o r t i o n of t h e t o t a l S found). Phosphodtester  l i n k a g e s have been r e p o r t e d  (Cawley and L e t t e r s 1968), phosphogaIactomannan phosphate. and S.  in fungal  mannans  and L l o y d (1970b) has examined a p e p t i d o -  i n Cladosporium  werneckii  which c o n t a i n s  T h i s might a l s o a c c o u n t f o r t h e P l e v e l s i n T.  3.2% mesenterica  diclina.  H y d r o l y s i s and D e g r a d a t i o n The C-N p e p t i d e bond e x h i b i t s c o n s i d e r a b l e d o u b l e bond c h a r a c t e r and t h u s i s s t a b i l i z e d by resonance (Spencer 1963).  A l t h o u g h a l l of t h e  p e p t i d e bonds i n a p r o t e i n a r e s u s c e p t i b l e t o h y d r o l y s i s by a c i d , t h e r a t e of h y d r o l y s i s w i l l  depend on f a c t o r s a f f e c t i n g t h e approach of  s t a t i c and s t e r i c p r o p e r t i e s g r e a t l y  .  Electro-  i n f l u e n c e t h e s t a b i l i t y o f each bond  47 to hydrolysis;  t h e most i m p o r t a n t f a c t o r s i n f l u e n c i n g r a t e a r e t h e  e f f e c t i v e s i z e of t h e amino a c i d s i d e c h a i n s on e i t h e r s i d e of p e p t i d e bond and t h e i r p o s i t i o n s r e l a t i v e t o t h e bond. i n v o l v i n g v a l i n e , with a bulky t h o s e w i t h t h e group f a r t h e r  isopropyl  group,  still  more  labile.  bonds  a r e most s t a b l e ; less s t a b l e ;  l i k e g l y c i n e and a l a n i n e ,  l i n k i t has a g r e a t e r  effect  h y d r o l y s i s than when i t i s p a r t of t h e amino a c i d t h a t  c o n t r i b u t e s t h e amino g r o u p .  In a c i d s o l u t i o n s c a r b o x y l groups  uncharged and b a s i c groups tend t o r e p e l H .  are  When t h e b a s i c group  +  in t h e s i d e c h a i n i t has l e s s e f f e c t on h y d r o l y s i s than a f r e e group a d j a c e n t t o t h e p e p t i d e bond. of d i p e p t i d e s  in p a r t i a l  1956;  1963).  Spencer  P e p t i d e bonds  This  h y d r o l y s i s of p r o t e i n s  (Harris,  C o l e and Pon  Spencer (1963)  hypotheses t h a t have been advanced t o e x p l a i n t h i s in an o x a z o l i n e r i n g .  hypotheses t o e x p l a i n t h e p r e f e r e n t i a l  l a b i l i t y ; they  involve  In a d d i t i o n he d i s c u s s e s protein  acid.  a l k a l i n e h y d r o l y s i s of p r o t e i n s  hydrolysis for  tryptophan,  i s not used because of e x t e n s i v e d e s t r u c t i o n  l i b e r a t e d amino a c i d s and p r o d u c t i o n of a r t i f a c t s (Spencer  The g e n e r a l l y a c c e p t e d mechanism of bonds  threonine  presents  r e l e a s e of a s p a r t a t e from  E x c e p t under s p e c i a l c o n d i t i o n s of  of  a-amino  i n v o l v i n g t h e amino groups of s e r i n e and  p a r t i c i p a t i o n of t h e 8-0  is  i s i n d i c a t e d by an a c c u m u l a t i o n  a r e among t h e most l a b i l e in a c i d s o l u t i o n .  with diIute  are  When t h e s i d e c h a i n i s p a r t of t h e amino a c i d  c o n t r i b u t i n g the carboxyl t o the peptide of t h e r a t e of  Peptide  removed, as i n i s o l e u c i n e a r e  t h o s e w i t h s t e r i c f a c t o r s a t a minimum,  the  h y d r o l y s i s of  1963).  gIycopyranosidic  i n v o l v e s a r a p i d , • e q u i I i b r i u m - c o n t r o l l e d ' p r o t o n a t i o n of t h e g l y c o s i d i c  oxygen ( a l t h o u g h  t h e p r o t o n a t i o n of t h e r i n g oxygen c a n n o t be e n t i r e l y  48  e x c l u d e d , a v a i l a b l e e v i d e n c e f a v o r s t h e g l y c o s i d i c oxygen) conjugate a c i d . oxonium  to give  The c o n j u g a t e a c i d decomposes t o a g l y c o s y l  carbonium-  i o n , which then adds water (De Bruyne and W o u t e r s - L e y s e n  The carbonium-oxoniurn configuration  ion most p r o b a b l y e x i s t s in t h e  ( B e M i l l e r 1967).  8 - D - g I u c o p y r a n o s e polymer (1971) showed t h a t  1971).  half-chair  The mechanism i s . iI I u s t r a t e d w i t h a  i n F i g u r e 11.  De Bruyne and  in HCI t h e r e a c t i o n proceeds v i a  1,4-  Wouters-Leysen  carbonium-oxoniurn  i o n s g e n e r a t e d unimolecu I a r I y from t h e c o n j u g a t e a c i d . of t h e c r i t e r i a were not  the  In H 2 S O 4  some  in a c c o r d a n c e w i t h t h e u n i m o l e c u l a r mechanism  but t h e a u t h o r s a t t r i b u t e d t h i s not t o a change i n t h e mechanism but t o t h e f a i l u r e of a c i d i t y f u n c t i o n s as g e n e r a l m e c h a n i s t i c c r i t e r i a . e x p e r i m e n t s of t h i s t y p e have been performed mechanism of  h y d r o l y s i s of g I y c o f u r a n o s i d i c  w i t h f u r a n o s i d e s and t h e bonds has not been e s t a b l i s h e d .  I t has been observed t h a t a - D - g I y c o p y r a n o s i d i c u s u a l l y more r e a d i l y h y d r o l y z e d are hydrolyzed  than 3-D  linkages are  linkages; furanosidic  under v e r y m i l d c o n d i t i o n s .  aldose the N H  in a c i d s o l u t i o n e l e c t r o s t a t i c a l l y s h i e l d s t h e n e i g h b o r i n g +  hydrolysis  (Jones and P e r r y  linkages  When t h e g l y c o s i d i c l i n k a g e  i n v o l v e s t h e r e d u c i n g group of a 2--amino-2-deoxy  c o n s t i t u e n t s from a t t a c k by H ;  Few  3  +  formed  glycosidic  such bonds a r e much more s t a b l e t o a c i d  1963).  The g l y c o s i d i c l i n k a g e i n v o l v i n g  the  r e d u c i n g group of t h e g l y u r o n i c a c i d shows s t r o n g r e s i s t a n c e t o a c i d h y d r o l y s i s ; B e M i l l e r (1967) d i s c u s s e s t h e o r i e s p o s t u l a t e d t o e x p l a i n t h e s t a b i l i t y of t h i s  linkage.  In a l k a l i n e medium p o l y s a c c h a r i d e s a r e e a s i l y o x i d i z e d by a t m o s p h e r i c o x y g e n , and, even when t h e r e a c t i o n s a r e c a r r i e d out oxygen-free  nitrogen,  t r a c e s of o x y g e n ,  some degree of d e g r a d a t i o n ,  is d i f f i c u l t to avoid  under  p r o b a b l y o c c a s i o n e d by  (Bouveng and L i n d b e r g  1960).  49 FIGURE  II  MECHANISM  OF  GLUCOPYRANOSIDE  alter  H.OH  +  H  +  HYDROLYSIS  BeMiller (1967)  50 Hydrolysis  i s t h e predominant  r e a c t i o n under c o n d i t i o n s where  both t h e a c i d and polymer a r e in d i l u t e s o l u t i o n s a t t e m p e r a t u r e s 100 C.  With p o l y s a c c h a r i d e s a c i d s a l s o c a t a l y z e e p i m e r i z a t i o n  and d e h y d r a t i o n and f u r f u r a l  reactions that result  derivatives (BeMiller  (James 1972).  reactions  in t h e f o r m a t i o n of anhydro  1967).  sugars  C o n d e n s a t i o n p r o d u c t s may a l s o  be formed w i t h a c i d h y d r o l y s i s of p r o t e i n s , of t r y p t o p h a n  near  e s p e c i a l l y from t h e  degradation  The c o m p i e x i t y and c o m p o s i t i o n of t h e r e a c t i o n  m i x t u r e depends on a number of v a r i a b l e s , such as c o n c e n t r a t i o n of reagents, temperature,  and t i m e of h e a t i n g  (Aminoff  1970).  The b l a c k p r e c i p i t a t e s and h i g h l y c o l o r e d s o l u b l e  condensation  p r o d u c t s t h a t a r e c o l l e c t i v e l y c a l l e d humin cause i n t e r f e r e n c e a u t o m a t i c amino a c i d a n a l y s i s p r o c e d u r e s . protein hydrolysates  The use of CH SH-C00H t o prevent  d e s t r u c t i o n a l s o s e r v e s t o reduce humin p r o d u c t i o n .  production  is increased.  tryptophan  In t h e presence of  i s degraded even w i t h CH SH-C00H and 2  humin  The e f f e c t s of CH SH-C00H on amino a c i d 2  r e c o v e r i e s have a l r e a d y been d i s c u s s e d .  James (1972) does not  t h e a d d i t i o n of CH SH-C00H o r CH -CHSH-C00H t o p r o t e i n 2  containing tryptophan.  in  2  (Matsubara and S a s a k i 1969)  polysaccharides tryptophan  in  3  recommend  hydrolysates  For a c c u r a t e amino a c i d e s t i m a t i o n he s u g g e s t s  t h a t samples be h y d r o l y z e d  in q u a d r u p l i c a t e :  a d d i t i o n of o x a l i c a c i d and  m e r c a p t o s u c c i n i c a c i d b e f o r e h y d r o l y s i s , t r e a t m e n t w i t h ion exchange r e s i n and N o r i t a f t e r h y d r o l y s i s , and o x i d a t i o n w i t h p e r f o r m i c a c i d  before  hydrolysis. N e v e r t h e l e s s i n samples t h a t c o n t a i n s i g n i f i c a n t amounts polysaccharides, furfural to  some humin w i l l  derivatives  (including  be produced w i t h a c i d h y d r o l y s i s . t h o s e d e r i v e d from hexoses) a r e  l e v u l i n i c a c i d ( Z a c h a r i u s and T a l l e y 1962; Anet 1972).  of Some  converted  LevuIinic' acid  r e a c t s w i t h n i n h y d r i n t o produce a c o l o r e d p r o d u c t which i s e l u t e d  from  51 t h e 58 cm column o f t h e amino a c i d a n a l y z e r between c y s t e i c a c i d and a s p a r t a t e ( Z a c h a r i u s and T a l l e y 1962; Sentandreu and N o r t h c o t e 1968). T a y l o r (1970) has d e t e c t e d a s i m i l a r peak, which i s c h a r a c t e r i z e d by a h i g h e r absorbance a t 440 nm than a t 570 nm, i n h y d r o l y s a t e s o f a m i x t u r e of h y d r o x y p r o I i n e  and s u c r o s e .  Z a c h a r i u s and P o r t e r (1967) have examined  a number o f o t h e r n o n - n i t r o g e n o u s compounds ( m a i n l y m o n o s a c c h a r i d e s , d i s a c c h a r i d e s and r e l a t e d compounds) derivatives.  t h a t produce  ninhydrin-positive  These peaks a r e a l m o s t a l l e l u t e d b e f o r e a s p a r t a t e on t h e  58 cm column o f t h e amino a c i d a n a l y z e r and have h i g h e r absorbance a t 440 nm than a t 570' nm. amino a c i d s .  They a l l show much lower c o l o r i n t e n s i t i e s than  T a y l o r (1970) remarked t h a t t h e h y d r o l y s a t e s were p a l e  y e l l o w b e f o r e a n a l y s i s but t h a t t h e r e was no absorbance a t 440 nm without reaction with ninhydrin. numerous v e r y  In t h e p r e s e n t s t u d i e s , t h e r e were  l a r g e peaks most o f which were e l u t e d from t h e 13 cm  column b e f o r e g l u c o s a m i n e , and from t h e 58 cm column b e f o r e a s p a r t a t e . However t h e e l u t i o n was not sharp and t h e peaks o f t e n t a i l e d i n t o t h e amino a c i d p e a k s , r e n d e r i n g t h e i r a r e a e s t i m a t i o n s d i f f i c u l t .  I t was  found t h a t t h e s e e f f e c t s c o u l d be s i g n i f i c a n t l y reduced by d e l a y i n g t h e a d d i t i o n o f n i n h y d r i n t o t h e column e l u a t e s u n t i l j u s t b e f o r e t h e amino a c i d s were e l u t e d . reaction coil  first  For t h e 13 cm column n i n h y d r i n was added t o t h e  10 min a f t e r t h e e l u t i o n s t a r t e d ; f o r t h e 58 cm column  i t was added a t 33 m i n .  T h i s p r o c e d u r e produced s t a b l e b a s e l i n e s .  In a d d i t i o n t o t h e s e a r t i f a c t s o f h y d r o l y s i s , t h e r e were s e v e r a l n i n h y d r i n - p o s i t i v e p r o d u c t s w i t h a b s o r b a n c e s t h a t resemble t h o s e o f amino a c i d - n i n h y d r i n peaks ( h i g h e r absorbance a t 570 nm than a t 440 nm). F i v e o f t h e s e peaks were r e g u l a r l y observed all  i n amino a c i d a n a l y s i s o f  h y d r o l y s a t e s from T. mesenterica and S. diclina.  do not c o r r e s p o n d t o any p r o t e i n amino a c i d s ,  1 - or  Their positions 3-methyIhistidine,  52 glucosamine o r galactosamine.  The s i z e of t h e s e peaks tended t o d e c r e a s e  d u r i n g t h e c o u r s e o f h y d r o l y s i s but f o u r were s t i l l h y d r o l y s i s f o r 145 of c e l l  hr.  detected a f t e r  A s i m i l a r a r t i f a c t has been r e p o r t e d from h y d r o l y s i s  w a l l s t h a t c o n t a i n amino s u g a r s ( A p p l e g a r t h and B o z o i a n  1967;  Kanetsuna e t a l . 1969). The s y n t h e t i c m i x t u r e of amino a c i d s and g l u c o s amine showed o n l y t h e e x p e c t e d peaks on an amino a c i d a n a l y z e r c h r o m a t o gram.  When t h i s m i x t u r e was s u b j e c t e d t o h y d r o l y z i n g c o n d i t i o n s i n 6 N  H C l , t h e same f i v e a d d i t i o n a l  peaks were o b s e r v e d .  Since the recoveries  of t h e amino a c i d s were g e n e r a l l y h i g h ( F i g u r e 3) and t h a t of g l u c o s a m i n e very  low ( F i g u r e 4) t h e s e would appear t o be d e g r a d a t i o n p r o d u c t s o f  glucosamine h y d r o l y s i s .  T h i s v i e w i s supported by t h e f a c t t h a t amino  a c i d a n a l y z e r chromatograms o f c e l l  vulgaris  h y p o c o t y l s (Chang,  coleoptiles (O'Suliivan,  w a l l h y d r o l y s a t e s of Phaseolus  p e r s o n a l communication) and Avena  sativa  p e r s o n a l c o m m u n i c a t i o n ) , which do n o t c o n t a i n  amino s u g a r s , d i d n o t show any of t h e s e p e a k s . All  of t h e s e a r t i f a c t s of h y d r o l y s i s a r e presumably  derived  from d e g r a d a t i o n o r i n t e r a c t i o n of t h e monomers f o r e s t i m a t e s of which t h e a n a l y s e s were p e r f o r m e d .  The aim c f t h e a n a l y s i s , however,  d e t e r m i n e t h e amounts of t h e s e monomers.  i s to  Robe I and Crane (1972)  have  examined t h e q u e s t i o n of d e g r a d a t i o n c f amino a c i d s d u r i n g p r o t e i n lysis.  hydro-  They o b s e r v e t h a t t h e t r u e amino a c i d c o m p o s i t i o n of a p r o t e i n  i s determined  i d e a l l y by q u a n t i t a t i v e l y d e t e r m i n i n g t h e amino a c i d s when  t h e i r p e p t i d e bonds have been broken and b e f o r e d e g r a d a t i o n o c c u r s . C o r r e c t i o n s f o r amino a c i d d e s t r u c t i o n d u r i n g h y d r o l y s i s a r e d i s c u s s e d and a method of e x t r a p o l a t i o n f o r d e t e r m i n i n g t r u e o r o r i g i n a l  amounts  a t z e r o t i m e w i t h d a t a i n v o l v i n g s i m u l t a n e o u s y i e l d and d e c a y .  These  p r i n c i p l e s can be a p p l i e d t o h y d r o l y s i s of any p o l y m e r .  The d e r i v a t i o n  of e q u a t i o n s assumes t h a t t h e monomers must be i n one of t h r e e s t a t e s .  53 State  The monomers a r e bound  A.  i n t h e polymer and n o t o b s e r v a b l e  by a n a l y s i s . State  B.  The monomers a r e h y d r o l y z e d from t h e polymer ( S t a t e A) and  o b s e r v a b l e by a n a l y s i s . State  C.  H y d r o l y z e d anhydro monomers from S t a t e B a r e degraded and  no longer o b s e r v a b l e by a n a l y s i s . As t h e monomers proceed t h r o u g h t h e d i f f e r e n t s t a t e s , a n a l y t i c a l o b s e r v a t i o n s a r e t a k e n i n S t a t e B. of monomers  The problem r e s t s , t h e r e f o r e ,  in f i n d i n g t h e number  i n i t i a l l y i n S t a t e A u s i n g t h e o b s e r v a t i o n s t a k e n i n S t a t e B.  The r a t e of h y d r o l y s i s from S t a t e A t o S t a t e B i s assumed t o be c o n s t a n t f o r each monomer of a g i v e n t y p e . proportionaI  t o i t s number r e m a i n i n g  The r a t e f o r each t y p e  i n S t a t e A.  e x p r e s s e d by t h e f o l l o w i n g d i f f e r e n t i a l  i s then  T h i s assumption i s  equation  dA / dt = ~hA where A = t h e number o f m o l e c u l e s r e m a i n i n g  in S t a t e A, and h = h y d r o l y s i s  c o n s t a n t i n u n i t s of f r a c t i o n of S t a t e A per hour throughout  (h. i s assumed t o c o n s t a n t  t h e e x p e r i m e n t f o r any g i v e n monomer).  The r a t e of change of t h e number o f h y d r o l y z e d  ( S t a t e B)  m o l e c u l e s e q u a l s t h e r a t e t h e y a r e coming o u t S t a t e A minus t h e r a t e t h e y a r e being  l o s t t o S t a t e C.  If 1 i s d e f i n e d as t h e l o s s c o n s t a n t , and i t  i s assumed t h a t t h e l o s s r a t e i s p r o p o r t i o n a l  t o t h e number  i n S t a t e B,  then the loss r a t e i s I B , thus  dB / dt = (-da  / dt) -  The a u t h o r s d e v e l o p a method of n o n - l i n e a r  least-squares t o estimate the  o r i g i n a l monomer c o m p o s t i i o n of t h e p o l y m e r . e x e c u t e t h e program.  IB.  A computer  i s required t o  A s i m i l a r approach t o t h e problem based on p o l y -  s a c c h a r i d e s t u d i e s i s d i s c u s s e d by G h e o r g h i u , O e t t e and Baumann  (1970).  I t p r o v i d e s a p p r o x i m a t i o n s f o r t h e c o r r e c t i o n f a c t o r s d i s c u s s e d by Robel  54  and Crane (1972) t h a t do not r e q u i r e computer a s s i s t a n c e .  A v a i l a b l e d a t a s u g g e s t t h a t any r e s t r i c t i o n o f f l e x i b i l i t y o f a p o l y s a c c h a r i d e c h a i n reduces t h e r a t e o f h y d r o l y s i s ( B e M i I l e r  1967).  T h i s may be r e l a t e d t o t h e c o n f o r m a t i o n a l changes n e c e s s a r y f o r t h e h a l f - c h a i r carbonium-oxoniurn systems.  ion ( F i g u r e 11)  i n more h i g h l y  forming ordered  Sentandreu and N o r t h c o t e (1968) have found O - g l y c o s y l  t o s e r i n e and t h r e o n i n e  in yeast c e l l  walls.  linkages  They a l s o suggested t h e  e x i s t e n c e o f a w - g l y c o s y l bond between w - a c e t y l g I u c o s a m i n e and a s p a r a g i n e . Lamport (1967) has r e p o r t e d t h e p r e s e n c e o f O - g l y c o s y l hydroxyproIine  i n the c e l l  waI Is o f h i g h e r p l a n t s .  linkages t o  Such l i n k a g e s a r e  l i k e l y t o e x i s t between p r o t e i n and p o l y s a c c h a r i d e i n f u n g a l c e l l such as T. mesenterica and S. diclina. polysaccharides  in c e l l  walls  T h i s s i t u a t i o n c o u l d cause  w a l l s t o be more r e s i s t a n t t o h y d r o l y s i s i f t h e  p r o t e i n s h e l d t h e p o l y s a c c h a r i d e s i n c o n f i g u r a t i o n s t h a t were f l e x i b l e than t h o s e i n pure p o l y s a c c h a r i d e s .  less  The h y d r o l y s i s c o n d i t i o n s  t h a t r e l e a s e m o n o s a c c h a r i d e s would not s i g n i f i c a n t l y a f f e c t  peptide  bonds, and i f t h e g I y c o s y I - a m i n o a c i d l i n k a g e s were even m o d e r a t e l y s t a b l e the s t a b i l i t y o f the be a l t e r e d .  l i n k a g e s between t h e monosaccharide u n i t s might w e l l  Bonds t h a t were l a b i l e i n t h e f r e e p o l y s a c c h a r i d e might  become l e s s s o i n t h e c e l l  wall  unit.  T h i s s o r t o f l i n k a g e i s i m p l i e d by t h e r e c o v e r y c u r v e s o f s e r i n e and t h r e o n i n e  ( F i g u r e s 6 and 7 ) .  C h a r a c t e r i s t i c a l l y the recovery curves  of t h r e o n i n e and s e r i n e i n p u r i f i e d p r o t e i n s show a n e g a t i v e s l o p e throughout the course of h y d r o l y s i s ( f o r  example, Robel and Crane 1972).  The i n c r e a s e d s t a b i l i t y o f t h e t h r e o n i n e and s e r i n e p e p t i d e bonds might be e x p l a i n e d t h u s :  i f t h e 3 - 0 were i n v o l v e d  i n a g l y c o s y l bond,  then  i t c o u i d not forrp t h e o x a z o l i n e r i n g which i s t h o u g h t t o l a b i l i z e t h e  55 p e p t i d e bond.  T h i s would  h y d r o l y s i s than  leave t h e p e p t i d e bond more r e s i s t a n t t o a c i d  in a pure p r o t e i n .  Such c o m p l i c a t i o n s a r i s e in c e l l  w a l l c h e m i s t r y because t h e  p r o t e i n a n a l y s i s i s c a r r i e d o u t on a p r o t e i n t h a t approximately  i s contaminated  of o t h e r components, m o s t l y p o l y s a c c h a r i d e s .  90%  p o l y s a c c h a r i d e i s o n l y 80%  'pure'.  with  The  Thus a p p r o p r i a t e c o r r e c t i o n f a c t o r s  f o r degradation are d i f f i c u l t to d e v i s e . It  i s r e l a t i v e l y easy t o d e t e r m i n e t h e r a t e of d e g r a d a t i o n  f r e e amino a c i d s and f r e e monosaccharides under t h e h y d r o l y z i n g used f o r c e l l  wall preparations.  Neutral  of  conditions  and a c i d i c amino a c i d s a r e  r e l a t i v e l y stable to acid hydrolysis;  b a s i c amino a c i d s a r e much l e s s so  (Figure 3).  s u g a r s was r e c o v e r e d a f t e r 8 hr  About 15%  in h y d r o l y z i n g recovery) hr  of t h e n e u t r a l  c o n d i t i o n s ( F i g u r e 5); mannose  and x y l o s e t h e  in 2 N HCl as mannose  97 h r . only  15$  Degradation  Glucosamine  i s as s t a b l e a f t e r 8  in 2 N C F C 0 0 H , but o n l y 30% 3  and d e g r a d a t i o n  i s recovered  after  i s v i r t u a l l y complete a f t e r 97  These c u r v e s show t h e e f f e c t of t h e  by Robel and Crane  (85%  i s much more e x t e n s i v e in 6 N HCl (even a f t e r 8 hr  i s recovered)  hr ( F i g u r e 5).  l e a s t (50%).  i s t h e most s t a b l e  l o s s r a t e , IB,  defined  (1972).  F i g u r e s 6 t o 10 show c u r v e s i n v o l v i n g s i m u l t a n e o u s y i e l d and decay.  In t h e c a s e of n e u t r a l  s u g a r s t h e r e l a t i v e r a t i o s of  degradation  a f t e r maximal r e l e a s e in F i g u r e s 8 and 9 a r e of t h e same o r d e r as t h o s e in F i g u r e 5.  For t h e amino a c i d s r e l e a s e d from c e l l  walls (Figures  6  and 7) t h e c u r v e s genera I l y have n e g a t i v e s l o p e s t h a t a r e s t e e p e r than t h o s e in F i g u r e 3 ( f r e e amino a c i d s ) . in t h e c e l l  T h i s s u g g e s t s t h a t t h e amino a c i d s  w a l l p r e p a r a t i o n s a r e being degraded t o a g r e a t e r e x t e n t than  f r e e amino a c i d s under t h e same c o n d i t i o n s of 6 N HCl causes d e s t r u c t i o n of m o n o s a c c h a r i d e s ;  hydrolysis.  Hydrolysis  humin degrades and  in  forms  56 complexes w i t h amino a c i d s . Thus t h e assumption t h a t h y d r o l y s i s c o n s t a n t s do not change t h r o u g h o u t an e x p e r i m e n t may i n t r o d u c e a s e r i o u s e r r o r cell  wall hydrolysis.  i n t o s t u d i e s of  For t h i s reason t h e c o r r e c t i o n of e x t r a p o l a t i o n t o  z e r o t i m e has not been u s e d .  The study of h y d r o l y s i s of c e l l  HCI and 6 N HCI show t h e g r o s s i n a c c u r a c y of such a p l o t is extensive.  if  w a l l s in 2 N degradation  The d e g r a d a t i o n of most amino a c i d s in t h e c e l l  walls  seems e x t e n s i v e and a l t h o u g h t h e t o t a l e s t i m a t e of amino a c i d s may be low,  i t does r e p r e s e n t a r e a l f i g u r e ,  i n d i v i d u a l amino a c i d .  based on a c t u a l r e c o v e r y of each  The problem r e q u i r e s f u r t h e r  The 1 0 $ of t h e c e l l  study.  w a l l s not r e c o v e r e d may be accounted f o r  when such problems a r e s o l v e d .  A n a l y s i s of f u n g a l that reproducible c e l l Biological cell  cell  w a l l s i s f u r t h e r c o m p l i c a t e d by t h e f a c t  wall preparations are often d i f f i c u l t t o o b t a i n .  d i v e r s i t y complicates quantitative a n a l y s i s .  In s t u d y i n g t h e  w a l l components in f u n g i , t h i s must be t a k e n i n t o a c c o u n t , e s p e c i a l l y  when making c o m p a r i s o n s .  The C e l l Wall  Preparation  The components t h a t a fungus assembles t o produce t h e c e l l a r e d e r i v e d t h r o u g h t h e a c t i o n of from t h e medium on which i t  wall  i t s m e t a b o l i c pathways on n u t r i e n t s  is growing.  Different  s t r a i n s of t h e same  fungus growing on t h e same medium may produce d i f f e r e n t c e l l o r t h e same components in d i f f e r e n t p r o p o r t i o n s .  w a l l components  The same s t r a i n grown  on d i f f e r e n t media may a l s o produce t h i s s o r t of d i f f e r e n c e .  The  q u e s t i o n of whether a fungus s y n t h e s i z e d c e l l  w a l l from components t h a t  a r e a l r e a d y a v a i l a b l e i n abundance o r whether  i t r e q u i r e s c e r t a i n components  57 for a specific cell answered.  w a l l assembly p a t t e r n cannot a t p r e s e n t be p r o p e r l y  F u r t h e r m o r e , many f u n g i  be c o m p l e t e l y c h e m i c a l l y d e f i n e d .  will  grow o n l y on media t h a t cannot  I t t h e r e f o r e becomes more d i f f i c u l t t o  r e l a t e s p e c i f i c s t r u c t u r a l elements i n t h e medium w i t h t h o s e u l t i m a t e l y incorporated  into the c e l l  wall.  For example, from t h e p r e s e n t s t u d i e s , S. diclina  was grown i n  a medium c o n t a i n i n g peptone, y e a s t e x t r a c t , and D - g l u c o s e .  If c e l l u l o s e  made up p a r t of t h e p o l y s a c c h a r i d e would 3 - D - x y l o s e , 6-D-mannose o r a-Lf u c o s e r e p l a c e o r even p a r t l y r e p l a c e B - D - g l u c o s e s i n c e t h e y have t h e same c o n f i g u r a t i o n a t C-1 and C - 4 , o r would o t h e r s u g a r s be c o n v e r t e d t o D - g l u c o s e and assembled i n t o c e l l u l o s e ?  If t h e l a t t e r a l t e r n a t i v e o c c u r s ,  would  t h e r e be changes i n t h e morphology o f t h e fungus? There have been few i n v e s t i g a t i o n s o f t h i s s o r t . Sans (1968) c u l t u r e d Cryptococcus d i f f e r i n g only  Bulmer and  neoformans on c o m p l e t e l y d e f i n e d media  i n t h e sugar p r o v i d e d .  T h e i r study was n o t o f c e l l  wall  s t r u c t u r e b u t t h e y d i d r e p o r t d i f f e r e n t r e s p o n s e s i n c a p s u l e development. A n g l u s t e r and T r a v a s s o s (1972) grew Torulopsis  pintolopesii  in defined  media w i t h c h o l i n e o r m e t h i o n i n e and found q u a n t i t a t i v e d i f f e r e n c e s , i n t h e c a r b o h y d r a t e s and amino a c i d s  in t h e c e l l  walls.  and A l b e r s h e i m (1967) c u l t u r e d h i g h e r p l a n t c e l l s  Nevins,  (sycamore)  English  i n media  c o n t a i n i n g d i f f e r e n t s u g a r s and fcund d i f f e r e n c e s i n t h e p r o p o r t i o n s ' o f t h e sugar components o f t h e c e l l  walls.  I d e a l l y a l l s t u d i e s o f a fungus s h o u l d be performed on t h e same strain or culture,  i n t h e same medium ( p r e f e r a b l y  grown f o r t h e same l e n g t h o f t i m e under cells will  c o m p l e t e l y d e f i n e d ) and  identical conditions.  be more u n i f o r m when grown i n synchronous c u l t u r e .  a n a l y s i s of t h e same s p e c i e s a r e compared, t h e s t r a i n , and medium must be t a k e n i n t o a c c o u n t .  Yeast-1 ike When  age o f c u l t u r e  Both T. mesenterica  (  and 5 . diclina  were grown on u n d e f i n e d  m e d i a , and even under c a r e f u l s t a n d a r d i z a t i o n of o t h e r c o n d i t i o n s t h e r e was v a r i a t i o n among c e l l  wall  preparations.  The p r o c e d u r e s f o r b r e a k i n g f u n g a l  c e l l s a r e dependent on t h e  s p e c i e s , t h e c u l t u r e c o n d i t i o n s and t h e form of t h e f u n g u s . methods f o r c e l l  breakage have been d e s c r i b e d .  Various  S p e c i f i c information"  about t h e s i z e of g l a s s beads ( i n t h e o r y t h e y should be of s u c h . a d i a m e t e r t h a t t h e space formed when f o u r beads come t o g e t h e r t e t r a h e d r a I Iy i s . s l i g h t l y s m a l l e r than t h e d i a m e t e r of t h e c e l l s being b r o k e n ) , t h e p r o p o r t i o n of c e l l s and l i q u i d , t h e speed of r o t a t i o n of t h e b r e a k i n g d e v i c e (where a d j u s t a b l e )  and t h e d u r a t i o n o f t h e t r e a t m e n t ( s )  is essential.  In t h e p r e s e n t s t u d i e s , s o n i c o s c i l l a t i o n c o m p l e t e l y broke y e a s t - l i k e c e l l s of T. mesenterica  but had l i t t l e e f f e c t on y e a s t c e l l s  o r f i l a m e n t s of S. diclina.  cerevisiae)  (Saccharomyces  For each s e t of c i r c u m s t a n c e s  t h e o p t i m a l c o n d i t i o n s f o r b r e a k i n g c e l l s must u s u a l l y be determined by t r i a l and e r r o r . such f i n e c e l l  W i t h some t r e a t m e n t s complete c e l l  breakage produces  w a l l fragments t h a t they a r e d i f f i c u l t t o c o l l e c t and wash,  [n such c a s e s i t i s p r e f e r a b l e t o reduce t h e breakage t r e a t m e n t so t h a t cell  wall fragments'are  l a r g e r , and t o s e p a r a t e t h e i n t a c t c e l l s from t h i s  s u s p e n s i o n by c e n t r i f u g a t i o n . Once a s u c c e s s f u l ceI I - b r e a k i n g p r o c e d u r e has been f o u n d , a r e o n l y a few p r e c a u t i o n s t o o b s e r v e . all  times.  there  The c e l l s s h o u l d be kept c o l d a t  Aqueous s o l u t i o n s near pH 7 s h o u l d be u s e d .  The c e l l  walls  s h o u l d be washed f r e e of c y t o p l a s m i c c o n t a m i n a t i o n immediately a f t e r breakage t o m i n i m i z e e n z y m a t i c d e g r a d a t i o n . in b r e a k i n g t h e c e l l s ,  Where g l a s s beads a r e used  c a r e must be t a k e n t o a s s u r e t h e c o r r e c t i o n s f o r  g l a s s fragments a r e a p p l i e d ( B a r t n i c k i - G a r c i a and N i c k e r s o n 1962:  59  i  H o r i k o s h i and  I i d a 1964).  Kanetsuna e t a l . (1969) used a  medium (855?) s u c r o s e ) t o s e p a r a t e c e l l  high-density  w a l l s from whole c e l l s and g l a s s  debris.  The purpose of washing t h e broken c e l l s of c y t o p l a s m and any components w i t h i n t h e c e l l of  it.  i s t o remove a l l  traces  w a l l t h a t a r e not a p a r t  G e n e r a l l y t h e broken c e l l s a r e washed w i t h water and d i l u t e  s o l u t i o n s of NaCI, of c y t o p l a s m . filtration.  sucrose or both.  The c e l l  T h i s procedure removes  large  aqueous  amounts  w a l l fragments a r e r e c o v e r e d by c e n t r i f u g a t i o n  Care must be e x e r c i s e d in f i l t r a t i o n p r o c e d u r e s t o a v o i d  p o l y s a c c h a r i d e c o n t a m i n a t i o n from t h e f i l t e r paper.  Further  washings  a r e r e q u i r e d t o a s s u r e t h a t o t h e r components a r e c o m p l e t e l y removed the c e l l  or  walls.  M i t c h e l l and T a y l o r  s u c c e s s i v e l y with 8.0 M urea,  (1969) washed t h e c e l l  from  walls  1.0 M NHI+OH and 0 . 5 N HC00H.  The urea  s o l u t i o n was chosen because i t d i s s o l v e s many p r o t e i n s and i s u n l i k e l y c l e a v e c o v a l e n t bonds.  NHt+OH and HC00H were chosen t o remove  components c o m p a r t m e n t a l i z e d  to  ionizable  in t h e c y t o p l a s m of t h e i n t a c t c e l l . When  t h e s e compounds a r e r e l e a s e d t h e y may become a s s o c i a t e d w i t h charged (-C00H in u r o n i c a c i d s and d i c a r b o x y l i c amino a c i d s , -NH2 a c i d s and amino s u g a r s ) of t h e c e l l h y d r o l y s i s of v e r y weak bonds.  wall.  groups  in b a s i c amino  Both t r e a t m e n t s c o u l d cause  In t h e p r e s e n t s t u d i e s t h e HCOOH t r e a t m e n t  was o m i t t e d because i t caused f l o c c u l a t i o n of c e l l  wall  fragments.  Other t r e a t m e n t s have been used but t h e y may a f f e c t t h e c e l l  wall.  L i p i d s have been w i d e l y r e p o r t e d as e e l I w a l l c o n s t i t u e n t s and any washings that  involve ethanol, ether  (Moreno,  (Troy and K o f f l e r 1969) o r d e t e r g e n t s  Kanetsuna and CarboneI I 1969), (Troy and K o f f l e r 1969;  and B a r t n i c k i - G a r c i a 1969) may a l s o remove some of t h e  lipid  glycerol  Zevenhuizen components.  P r o t e o l y t i c enzymes have been used t o remove adsorbed p r o t e i n s  (Shah and  60 K n i g h t 1968) Taylor  but they may a l s o d i g e s t t h e c e l l  1969);  in g e n e r a l  wall  itself  (Mitchell  t h e y should not be used in t h e primary  and  preparation  of e e l I waI I s .  Crook and J o h n s t o n (1962) have d i s c u s s e d t h e d i f f e r e n c e s the c e l l  wall preparation  a reproducible c e l l aim. cell  and t h e f u n c t i o n a l  cell  w a l l f r a c t i o n from d i f f e r e n t  wall. cell  Preparation  preparations  of  is the .  Removing contaminant m a t e r i a l may a l s o remove c e r t a i n weakly bound w a l l components.  preparation  is preferable  can be o b t a i n e d . refined  At p r e s e n t t h e r e p r o d u c i b i l i t y of any c e l l  (even though i t may not  constituents)  i n c l u d e a l l of t h e  so t h a t a body of  When t h e fundamental  p r o c e d u r e s can be developed  are associated with the c e l l The ' p u r i t y '  information  i s e s t a b l i s h e d , more  t o i n v e s t i g a t e t h o s e components  w a l l by l a b i l e  of t h e c e l l  basic structural  structure  w i d e l y determined  wall preparation  by phase o r d a r k f i e l d  is usually defined  l i g h t microscopy;  t h e absence of c y t o p l a s m i c (and c a p s u l a r )  for  contamination.  not w i d e l y u s e d .  wall preparations.  Chemical t e s t s f o r  as  This  is  rigorous confirm  Most  i n v e s t i g a t o r s a c c e p t t h e e l e c t r o n m i c r o s c o p e assay as s u f f i c i e n t cell  that  linkages.  q u a n t i t a t i v e a n a l y s i s t h e e l e c t r o n m i c r o s c o p e s h o u l d be used t o  'purified'  wall  functional  t h e absence of c y t o p l a s m (and c a p s u l e i f t h e c e l l s p o s s e s s o n e ) .  for  between  evidence  'purity'  Absence of n u c l e i c a c i d s o r t h e i r p u r i n e and  are  pyrimidine  bases i s u s u a l l y t a k e n t o show l a c k of c y t o p l a s m , y e t B a r t n i c k i - G a r c i a and N i c k e r s o n (1962) and Moreno e t a i . in c e l l  wall preparations  (1969) found t r a c e s of t h e s e  t h a t they a c c e p t e d as  'pure'.  The p r e s e n t s t u d i e s have c o n f i r m e d t h a t q u a n t i t a t i v e of  fungal  cell  walls is possible with c e r t a i n  substances  limitations.  analysis  The  methods  61  chosen f o r e s t i m a t i o n of n e u t r a l procedures t h a t are required  sugars (plus other  in many c a s e s ) , amino s u g a r s and amino  a c i d s a r e s u i t a b l e f o r m i c r o a n a l y s i s of c e l l q u a n t i t a t i v e uronic acid recovery selected  wall preparations.  possible a l t e r n a t i v e s are a v a i l a b l e . a n a l y s i s provided gross  Reliable  i s not a t p r e s e n t p o s s i b l e ; t h e method  i s not s u i t a b l e t o p r o v i d e t h e r e q u i r e d  elemental  complementary  information.  The p r o c e d u r e s used f o r  information;  a r e a l r e a d y a v a i I a b I e must be adapted f o r c e l l  Several l i p i d and  more r e f i n e d t e c h n i q u e s which wall  studies.  The p r o c e d u r e s have been assembled and t e s t e d t o p r o v i d e foundation  f o r a comprehensive  t o b u i l d a model of t h e c e l l growth.  long-range  project  in t h i s  w a l l monomers.  e l u c i d a t i o n of c e l l  w a l l and t o e x p l a i n t h e mechanisms of  This  information  wall s t r u c t u r e at the  l e v e l of fundamental  organisms.  wall structure  in f u n g i  wall a n a l y s i s .  constituents  improvements and o t h e r  In t h i s r e s p e c t t h e study may r e p r e s e n t t h e f i r s t  attempt t o completely q u a n t i t i z e c e l l  information  Their application  intended t o be broad so t h e t e c h n i q u e s and subsequent  can be a p p l i e d t o t h e s t u d y of c e l l  its  i s e s s e n t i a l to complete  and a t each s u c c e e d i n g l e v e l of s t r u c t u r a l a n a l y s i s . is  laboratory  They have been s e l e c t e d t o p r o v i d e b a s i c q u a n t i t a t i v e  about c e l l  the  comprehensive  62 BIBLIOGRAPHY  AARONSON, S .  1970. 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Chemical  w a l l g l u c a n o f Phytophthora  8 : 1496-1502.  72 APPENDIX Symbols Used f o r  Monomers  Ala  a 1 an i ne  lie  i s o l e u c i ne  Ara  arab i nose  Ino  myo-i nos i t o 1  Arg  a r g i n i ne  Leu  l e u c i ne  Asp  aspartate  Lys  lysine  Asx  Man  mannose  Cys  aspartate or asparagine (undef i ned) c y s t e i ne  MeHis  methy1histldine  Ery  erythritol  Met  methionine  Fuc  fucose  Phe  phenylalanine  Gal  ga1actose  Pro  p ro1 i ne  GalN  ga1actosam!ne ( 2 - a m i n o 2-deoxy-ga1actose) g1ucose  Qpa  a-amino-8-guanidino  Rha  rhamnose  glucosamine (2-amlno2-deoxy-gIucose glucosamine or w - a c e t y l glucosamine (undefined) g1utamate  Rib  ribose  Ser  s e r i ne  Thr  threonine  Trp  tryptophan  Gly  glutamate or (undefined) g l y c i ne  Tyr  t y r o s i ne  His  histidine  Val  va 1 i ne  Hyp  4 - h y d r o x y p r o 1 i ne  Xyl  xylose  Glc GlcN GlcNx Glu Glx  glutamine  propionate  

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