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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 FOR THE QUANTITATIVE ANALYSIS OF FUNGAL CELL WALLS by DONALD STEWART CAMERON B.Sc, U n i v e r s i t y of A l b e r t a , 1962 M.Sc, U n i v e r s i t y of 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 the Department of Botany We accept t h i s t h e s i s as conforming t o the requ i red s t a n d a r d . 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 Br i t ish 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 for financial gain shall not be allowed without my written permission. Department of R n + a n y  The University of Bri t ish Columbia Vancouver 8, Canada Date November 2 9 . 1972 i ABSTRACT Cultures of Tremella mesenterica grown as haploid y e a s t - l i k e u n i c e l l s and Saprolegnia diclina were grown on incompletely defined media and the procedures for i so la t ing c e l l wall preparations from each, free of cytoplasmic and capsular mater ia l , are described. A strategy for quant i ta -t i v e analys is of these c e l l wall preparations was devised and tested , se lect ing from the numerous procedures ava i l ab le : extract ion and gravimetry for l i p i d s ; hydrolysis followed by g a s - l i q u i d chromatography for neutral sugars (as trimethyI s i IyI der ivat ives) and automatic amino acid analys is for amino acids and amino sugars. Problems of degradation as a resu l t of hydrolysis have been considered. The extent to which degradation occurs is d i f f i c u l t to estimate because each const ituent polymer is 'contaminated' with the others. This may accelerate degradation, p a r t i c u l a r l y of amino ac ids . Analyt ica l procedures were reproducible but only 90$ of the weight of the c e l l wall was recovered. The remaining 10$ is probably the resu l t of degradation losses that have not been accounted f o r , and further studies are required to improve the estimation of t h i s er ror . Even under c a r e f u l l y standardized condi t ions , c e l l wall preparations show var iab le composition. A complete analys is was therefore performed on a s ing le c e l l wall preparation of each of the two species. 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 ra t ios of components were s i m i l a r , although, for example one preparation, of S. diclina contained more than twice as much to ta l protein as. another. S imi lar recoveries of const ituents suggest that the strategy is appropriate for quant i tat ive ana lys i s . However, a l te rna t i ve i i methods f o r l i p i d a n a l y s i s t h a t p rov ide more s p e c i f i c i n fo rmat ion are a v a i l a b l e and should be adapted f o r c e l l wal l a n a l y s i s . Q u a n t i t a t i v e recovery of u r o n i c a c i d s p rov ides s u b s t a n t i a ! d i f f i c u l t i e s , and improved methods are r e q u i r e d . i i i TABLE OF CONTENTS page I n t r o d u c t i o n 1 M a t e r i a l s and Methods 9 C e l l w a l l p r e p a r a t i o n 9 A n a l y t i c a l procedures 11 C a l c u l a t i o n of r e s u l t s 17 R e s u l t s 19 Degradat ion under h y d r o l y z i n g c o n d i t i o n s 19 P r o t e i n a n a l y s i s 22 P o l y s a c c h a r i d e a n a l y s i s ' 22 L i p i d a n a l y s i s 32 Elemental and ash a n a l y s i s 32 Complete c e l l wa l l a n a l y s i s 55 D i s c u s s i o n 37 A n a l y t i c a l procedures 40 H y d r o l y s i s and degradat ion 46 The c e l l wa l l p r e p a r a t i o n 56 Bi bI iography 62 Appendix 72 Symbols used f o r monomers 72 I V LIST OF TABLES t a b l e page I Amino A c i d s in the C e l l Wa.l I of Tremella mesenterica 24 II Amino A c i d s in the C e l l Wall of Saprolegnia diclina 25 III Neutra l Sugars in the C e l l Wall of r . mesenterica 28 IV Neutra l Sugars in the C e l l Wall of s. diclina 29 V Amino Sugars in the C e l l Wall of T. mesenterica and S. diclina 33 VI L i p i d s in the C e l l Wall of r . mesenterica and S. diclina 33 VII Elemental and Ash A n a l y s i s of C e l l Wall P r e p a r a t i o n s of T . mesenterica and S. diclina 34 V I M Complete A n a l y s i s of the C e l l Wall of T. mesenterica and S. diclina 36 LIST OF FIGURES v f i g u r e page 1 R e s o l u t i o n o f ' B a s i c Compounds from the 13 cm Column of the Amino Ac id Ana lyzer 13 2 R e s o l u t i o n of Neut ra l Sugars (as TMS D e r i v a t i v e s ) on the G a s - l i q u i d Chromatograph 16 3 Recovery of Free Amino A c i d s Under Hydro Iyz ing C o n d i t i o n s 20 4 Recovery of Glucosamine under Hydro Iyz ing C o n d i t i o n s 21 5 Recovery of Free Neutra l Sugars under Hydro Iyz ing C o n d i t i o n s 23 6 Recovery of Amino A c i d s from C e l l Wa l l s of Tremella mesenterica 26 7 Recovery of Amino A c i d s from C e l l Wa l l s of Saprolegnia diclina 27 8 Recovery of Neutra l Sugars from C e l l Wa l l s of T. mesenterica 30 9 Revovery of Neutra l Sugars from C e l l Wa l l s of S. diclina 31 10 Recovery of Glucosamine from C e l l Wa l l s of T. mesenterica and S. diclina 34 11 Mechanism of GIucopyranoside H y d r o l y s i s 49 v i , ACKNOWLEDGMENT T h i s research p r o j e c t was supported by g ran ts t o Dr. I. E. P. T a y l o r from the Nat iona l Research Counc i l of Canada and the 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 the fungi were provided by Dr. R. J . Bandoni , who, w i th Moi ra Dobson, S h i r i e y Reid and Marg Shand int roduced 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 techn iques and were always a v a i l a b l e f o r c o n s u l t a t i o n . 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; I gleaned much in fo rmat ion from d i s c u s s i o n s w i th him and members of h i s l a b o r a t o r y . S y l v i a T a y l o r and Adr ianne Ross helped wi th the t y p i n g of the manuscr ip 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 stage of the development of the p r o j e c t and in the p r e p a r a t i o n of the t h e s i s . I am g r a t e f u l t o him f o r p r o v i d i n g the o p p o r t u n i t y 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 rcumstances t h a t would s o r e l y t r y o r d i n a r y human endurance, 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 . H i 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 research 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 tudents in B i o l o g y 101/102 f o r extending my i n t e r e s t in teach ing and 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 labours s y m p a t h e t i c a l l y and provided welcome d i v e r s i o n s in squash and t e n n i s , a t p l ays and c o n c e r t s , and in v a r i o u s abstemious r e c r e a t i o n s . 1 INTRODUCTI ON P l a n t c e l l s are c h a r a c t e r i s t i c a l l y enc losed by a c e l l wa l l t h a t i s s e m i - r i g i d , ye t capable of growing. There has been c o n s i d e r a b l e i n t e r e s t in the mechanism of the c e l l wa l l ex tens ion t h a t accompanies c e l l growth and severa l exper imenta l approaches a p p l i e d t o a wide range of organisms have y i e l d e d use fu l i n f o r m a t i o n . The problem i s of i n t e r e s t from severa l q u i t e d i f f e r e n t v i e w p o i n t s : g e n e t i c c o n t r o l systems of c e l l wa l l assembly (Katz and Rosenberger 1971; Davies 1972), mo lecu la r c o n t r o l of morphogenesis and d i f f e r e n t i a t i o n (Cabib and Farkas 1971; B a r t n i c k i -G a r c i a and Lippman 1972), i n t e g r a t i o n of components (Steward, I s rae l and S a l p e t e r 1967; Sadava and C h r i s p e e l s 1969; Nor thcote 1969), p r o p e r t i e s of s t r u c t u r a l polymers us ing the e l e c t r o n microscope and X - r a y d i f f r a c t i o n (Mahadevan and Tatum 1967; Aronson and F u l l e r 1969), and chemical c o n s t i t u e n t s (Novaes-Ledieu and J imenez -Mar t fnez 1968; Wang and B a r t n i c k i -G a r c i a 1970; Marks , K e l l e r and Guar ino 1971). Only by a s y n t h e s i s of r e s u l t s from a l l of these approaches w i l l the processes of c e l l wa l l e x t e n s i o n be comple te ly understood. I n v e s t i g a t o r s of c e l l wa l l e x t e n s i o n u l t i m a t e l y must determine the chemical c o n s t i t u e n t s of the c e l l w a l l , the chemis t ry and geometry of the l i nkages between them, and a d e s c r i p t i o n of how these change as the c e l l grows. The fundamental requirements f o r the i n i t i a l i n v e s t i g a t i o n of the whole c e l l wa l l are 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 i t s c o n s t i t u e n t s . These s t u d i e s may be fo l lowed by f r a c t i o n a t i o n of c e l l wa l l macromoI ecu Ies, 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 t h e i r s t r u c t u r e and attempts t o reassemble these molecu les i n to a working model of c e l l wa l l s t r u c t u r e . At each step In f r a c t i o n a t i o n of the c e l l w a l l , q u a n t i t a t i v e recovery of components from each f r a c t i o n i s e s s e n t i a l . .2 T h i s aspect of the problem i s e s s e n t i a l l y chemical in nature and a t l e a s t 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 should be s e l e c t e d t o present the minimum of a n a l y t i c a l problems. Al though secondary d e p o s i t i o n products are important c e l l wa l l c o n s t i t u e n t s , they p rov ide 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 rec lude s a t i s f a c t o r y r e s o l u t i o n of pr imary c e l l wa l l components. V a s c u l a r p l a n t s are an example of t i s s u e s t h a t present numerous problems. With many d i f f e r e n t c e l l types p r e s e n t , the r e s u l t i n g c e l l wa l l p r e p a r a t i o n i s heterogeneous. As the c e l l w a l l s may be l i g n i f i e d t o va ry ing e x t e n t s , a n a l y s i s becomes f a r more d i f f i c u l t . T i s s u e c u l t u r e procedures can p rov ide more un i fo rm, l ess l i g n i f i e d c e l l s , but w i th these procedures t h e r e i s the problem of producing 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 . Furthermore, s i n c e the c e l l s from e i t h e r source tend t o grow at tached together in masses, the p r e p a r a t i o n of c e l l w a l l s f r e e from both cytoplasm and the middle lame l la becomes more d i f f i c u l t . C e r t a i n o ther groups of organisms do not present these problems. B a c t e r i a have a l ready been e x t e n s i v e l y and comprehensive ly s t u d i e d ( f o r a general review see Rogers and P e r k i n s 1968). A lgae and fungi do not g e n e r a l l y produce l i g n i n - l i k e compounds and many s p e c i e s grow as s imple or branched f i l a m e n t s or as u n i c e l l s . A number of a l g a l s p e c i e s have been s tud ied (Aaronson 1970; Parker 1970). Fungi are in general e a s i e r t o c u l t u r e than a lgae and they grow r a p i d l y so t h a t la rge amounts are r e l a t i v e l y easy t o produce. Y e a s t s , p r i m a r i l y Saccharomyces, have been . w ide ly s tud ied (Phaff 1971) and the advantages of fungi as source 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 of an i n c r e a s i n g number of o ther fungal t y p e s . The f i r s t problem a s s o c i a t e d wi th any c e l l wa l l a n a l y s i s i s t h a t of o b t a i n i n g the c e l l wa 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 c o n t a m i n a t i o n . In general t h i s i nvo l ves breaking the c e l l wa l l and 3 washing i t f r e e of contaminants . Depending on the s p e c i e s and the component under i n v e s t i g a t i o n fungal c e l l w a l l s have been d i s r u p t e d wi th a French press (Kanetsuna, C a r b o n e l l , Moreno and Rodr iguez 1969) or broken by mechanical a g i t a t i o n w i th g l a s s beads in a Braun homogenizer (S ie tsma, E v e l e i g h and Haskins 1969), a M i c k l e d i s i n t e g r a t o r ( G r i f f i n and MacWiI Iiams 1969), or a SorvaI I omnimixer (Aronson and F u I l e r 1969). The r e s u l t a n t suspensions are o f t e n 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 then v a r i o u s l y washed wi th water , g l y c e r o l and aqueous s o l u t i o n s of NaCI, NaCI/NaHCC>3, EDTA and s u c r o s e . P h y s i c a l wa l l f ragmentat ion f o l l owed by v igo rous washing wi th aqueous s o l u t i o n s near pH 7 causes minimal degradat ion of the c e l l w a l l , whereas t reatment w i th s t rong a c i d s or bases, a l c o h o l , or heat may cause d e n a t u r a t i o n of p r o t e i n components o r o x i d a t i o n of p o l y s a c c h a r i d e s . The r e s u l t of any of these t reatments i s a c e l l wal l p r e p a r a t i o n which "cannot s e n s i b l y be equated wi th the f u n c t i o n a l wa l l of the organism from which i t was i s o l a t e d " (Crook and Johnston 1962). C e l l wa l l polymers a re most f r e q u e n t l y hydro lyzed a t temperatures near 100 C w i th aqueous a c i d s ; t h i s r e l e a s e s the monomers but may a l s o degrade them a f t e r (and perhaps before) t h e i r intermoI ecu Iar bonds are broken. The e x t e n t of the d e g r a d a t i o n . v a r i e s f o r i n d i v i d u a l monomers depending on the type of l i nkage between them and on the c o n d i t i o n s of h y d r o l y s i s . P r o t e i n s and t h e i r component amino a c i d s a re much more s t a b l e 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 c o n s t i t u e n t monosacchar ides. P r o t e i n s a re g e n e r a l l y hydro lyzed wi th 6 N HCI in vacuo f o r 10 t o 70 hr ( T r i s t r a m and Smith 1963); under these c o n d i t i o n s p o l y s a c c h a r i d e s a re s i g n i f i c a n t l y degraded. P o l y s a c c h a r i d e s may be hydro lyzed wi th 0.1 t o 2 N H2S0U, 24 N HCOOH, or 2 N CF 3C00H f o r pe r i ods up t o 8 hr t o r e l e a s e neut ra l sugars . G l y c o s i d i c bonds i n v o l v i n g amino sugars a re much more s t a b l e t o a c i d h y d r o l y s i s , and are g e n e r a l l y :. 4 hydro lyzed wi th 2 N t o 6 N HCl f o r 8 t o 32 hr (Dutton 1972). Q u a n t i t a t i v e recovery 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 not been accompl ished (Nordstedt and Samuel son 1966; Stacey 1970). A l though the development of paper chromatography prov ided 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 technology 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 . Compounds more s i m i l a r in s t r u c t u r e can now be detected and separated in exceed ing l y small amounts. There are many a n a l y t i c a l procedures in common use t h a t can y i e l d q u a n t i t a t i v e i n fo rmat ion from c e l l w a l l s , prov ided t h a t p r e c a u t i o n s are observed . The procedures developed by Spackman, S t e i n and Moore (1958) have been wide ly 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 data are r e q u i r e d . 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 determine the 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 those t h a t are d i f f i c u l t t o h y d r o l y z e . They emphasized the 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 yp tophan . The n i n h y d r i n r e a c t i o n w i th hydroxypro I ine 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 1970). Separate de te rm ina t ion i s p r e f e r r e d . In the l i t e r a t u r e of c e l l wa 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 requ i rements . Only a few authors (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 t ryptophan (Korn and Northcote 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; Marks, K e l l e r and Guar ino 1971) c r hydroxypro I ine (Crook and Johnston 1962; Dyke 1964; B a r t n i c k i - G a r c i a 1966; Novaes -Led ieu , Jim§nez-Martfnez and V i l l a n u e v a 1967; Aronson and F u l l e r 1969; Reuvers , Tacoronte , G a r c i a Mendoza and Novaes-Ledieu 1969; Pao and Aronson 1970). The recent development of procedures f o r a n a l y z i n g amino a c i d s by g a s - l i q u i d chromatography as t r imethy I s i IyI d e r i v a t i v e s (Gehrke, 5 Nakamoto and ZumwaIt 1969) or as A ' - i r i f I uoroacety I n - b u t y l e s t e r s (Gehrke, Kuo and Zumwalt 1971) may p rov ide a rap id and s e n s i t i v e a l t e r n a t i v e to the n i n h y d r i n 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 degradat ion must s t i I I be a p p I i e d . The procedures f o r monosaccharide e s t i m a t i o n are not as s tandard i zed as those 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 in methodology i s e v i d e n t . Stacey (1970) has d i s c u s s e d the advantages of g a s - ! i q u i d chromatography in q u a n t i t a t i v e a n a l y s i s of sugars , ye t few i n v e s t i g a t i o n s of fungal c e l l w a l l s have made use of t h i s t e c h n i q u e . Sugars have been est imated 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 (A lbershe im, Nev ins , E n g l i s h and Karr 1967; Wang and B a r t n i c k i - G a r c i a 1970), c r t r i m e t h y l s i l y l d e r i v a t i v e s (Namba and Kuroda 1971; S i k i , Mas le r and Bauer 1970). L loyd (1970) and Lloyd and B i toon (1971) used both procedures . Only a few authors (Albersheim e t a l . 1967; App legar th 1967; App legar th and Bozoian 1968) have a p p l i e d c o r r e c t i o n s de r i ved from s e r i a l h y d r o l y s i s t o sugar a n a l y s i s . Amino sugars a re reso l ved on an amino a c i d a n a l y z e r us ing standard procedures and these i n v e s t i g a t o r s who used the a n a l y z e r f o r amino a c i d e s t i m a t i o n s determined amino sugars as w e l l . There are r e p o r t s of the e s t i m a t i o n of amino sugars as t r imethy I s i Iy! d e r i v a t i v e s but the re have been problems 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 or r e s o l u t i o n (Duti-on 1972). Chattaway, Holmes and Barlow (1963) observed t h a t " l o s s e s [of s u g a r s ] dur ing h y d r o l y s i s were found to be c o n s i d e r a b l e and v a r i a b l e " , ye t on ly t h e y , Marks, K e l l e r and Guar inc (1969) and L loyd and B i toon (1971) have exam5ned the degradat ion of sugars under h y d r o ! y z i n g c o n d i t i o n s in c o n j u n c t i o n wi th c e i l wa i l s t u d i a s . U ron ic 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 (Bioko and R icha rds 1970). They are u s u a l i y es t imated as 6 neut ra l sugars a f t e r r e d u c t i o n of the carboxy l group in the i n t a c t p o l y s a c c h a r i d e before h y d r o l y s i s (Dutton and Kab i r 1971) or by r e d u c t i o n of the ca rboxy l group of the f r e e u r o n i c a c i d s or a I d o b i o u r o n i c a c i d s a f t e r h y d r o l y s i s ( B a r t n i c k i - G a r c i a and Reyes 1968; Dutton and Kab i r 1972; Jones and A lbersheim 1972). L i p i d s have been w ide ly determined by the g r a v i m e t r i c method of B a r t n i c k i - G a r c i a and N ickerson (1962) . Dyke (1964), RusseI I, Sturgeon and Ward (1964) and S ie tsma , E v e l e i g h and Haskins (1969) s a p o n i f i e d the l i p i d s and est imated the methyl e s t e r s of the f a t t y a c i d s by g a s - l i q u i d chromatography. Suomalainen and Nurminen (1970) determined c e l l wa l l l i p i d s ( f a t t y a c i d s and p h o s p h o l i p i d s ) in b a k e r ' s yeast and found monosaccharides a s s o c i a t e d wi th some of the p h o s p h o l i p i d components. E r r o r s 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 incomplete c leavage of l i nkages and degradat ion of components under c o n d i t i o n s of h y d r o l y s i s must be taken i n to account in any q u a n t i t a t i v e a n a l y s i s . The r e p r o d u c i b i l i t y of the a n a l y s i s may a l s o depend on the 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 mo lecu la r content i s un i fo rm , and t h i s in t u r n 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 n u t r i t i o n a l c o n d i t i o n s t h a t may d i f f e r from one source of m a t e r i a l t o another . The purpose of the present study was t o s e l e c t and t e s t a p p r o p r i a t e procedures 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 r e p r o d u c i b l e i n fo rmat ion r e l a t i n g t o the s t r u c t u r e of the fungal c e l l w a l l . These procedures in themselves are not intended t o p rov ide any s t r u c t u r a l i n fo rmat ion about c e l l w a l l s . They do, however, form the b a s i s f o r a l l f u t u r e s t r u c t u r a l s t u d i e s which are planned in t h i s l a b o r a t o r y . In o rder t o e l u c i d a t e the 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 u n i t s must be i d e n t i f i e d c h e m i c a l l y and the method of assembly determined. One of the most p r o f i t a b l e approaches to t h i s s o r t of study has been e l e c t r o n 7 m i c r o s c o p i c examinat ion of the c e l l wa l l before and a f t e r s e l e c t i v e removal of 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 s o l u b i l i z a t i o n in v a r i o u s reagents and enzymes (Mahadevan and Tatum 1965, 1967). There i s the p o s s i b i l i t y of e x t e n s i v e degradat ion as a r e s u l t of these p rocedures , e s p e c i a l l y w i th 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 stage 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 in t h i s t h e s i s must be a p p l i e d and extended to ach ieve t h i s end. Only by q u a n t i t a t i n g the e n t i r e a n a l y s i s can a c c u r a t e models of the c e l l wa l l be c o n s t r u c t e d . T h i s i n v e s t i g a t i o n concerns two fungal" s p e c i e s from q u i t e un re la ted groups t h a t have not p r e v i o u s l y been examined - Saprolegnia diclina, a myceIiaI Oomycete, and Tremella mesenterica, a Bas id iomycete t h a t can be grown in a yeast -1 ike form. The a n a l y t i c a l regime i n c l u d e s : 1) s e r i a l h y d r o l y s i s of c e l l wa 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 neut ra l sugars (est imated as t r i m e t h y I s i I y I 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 wa 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 sugars (est imated by n i n h y d r i n r e a c t i o n on the amino a c i d a n a l y z e r ) . 3) e s t i m a t i o n of u r o n i c a c i d s as neut ra l sugars by r e d u c t i o n of the carboxy l groups in c e l l wa 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 f o r neut ra l suga rs . 4) s e r i a l h y d r o l y s i s of c e l l wa 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 (est imated by n i n h y d r i n r e a c t i o n on the amino a c i d a n a l y z e r ) , i n c l u d i n g separate a n a l y s i s f o r h y d r o x y p r o l i n e , t ryptophan and c y s t e i n e / c y s t i n e . 5) - degradat ion s t u d i e s of f r e e neut ra l suga rs , f r e e amino sugars , and f r e e amino a c i d s under the c o n d i t i o n s f o r h y d r o l y s i s of c e l l wa l l polymers, 6) g r a v i m e t r i c d e t e r m i n a t i o n of l i p i d s . 8 7) a n a l y s i s f o r C, H, 0, N , S, P and a s h . The success of t h i s s t r a t e g y can be measured as the percentage of t o t a l c e l l wa l l s t a r t i n g m a t e r i a l which i s recovered by the s e l e c t e d a n a l y t i c a l p rocedures . 9 MATERIALS AND METHODS Tremella mesenterica and Saprolegnia diclina were from the UBC myco log ica l c u l t u r e c o l l e c t i o n . C u l t u r e media were obta ined from D i f c o L a b o r a t o r i e s , D e t r o i t , M i c h i g a n . Reagents were obta ined from the s u p p l i e r s as 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 Instruments, I n c . , Spinco D i v i s i o n , Pa lo A l t o , C a l i f o r n i a ) ; t h i o d i g l y c o l (Bio*Rad L a b o r a t o r i e s , Richmond, C a l i f o r n i a ) ; D - g a l a c t o s e , D - ga lactosamine h y d r o c h l o r i d e , L i B H 4 , 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 , England) ; a l l . a m i n o a c i d s except m e t h y I h i s t i d i n e (Calb iochem, Los Ange les , C a l i f o r n i a ) ; CF3COOH, L-rhamnose monohydrate (Eastman Kodak Company, Rocheste r , New Y o r k ) ; D-mannose, cyc lohexane c e r t i f i e d ACS spect ranaIyzed ( F i s h e r S c i e n t i f i c Company, F a i r Lawn, New J e r s e y ) ; L - 1 - m e t h y I h i s t i d i n e monohydrate, L-3 -methy l -h i s t i d i n e p u r i s s . ( K o c h - L i g h t L a b o r a t o r i e s L t d . , Colnbrook England) ; e r y t h r i t o l , a l l neut ra l monosaccharides except D - g a I a c t o s e , D-mannose, L-rhamnose ( N u t r i t i o n a l B i o c h e m i c a l s C o r p o r a t i o n , C l e v e l a n d , Oh io ) ; methyl c e l l o s o l v e , n i n h y d r i n , hexamethy Id is i Iazane , t r i m e t h y I c h l o r o s i I a n e ( P i e r c e Chemical Company, R o c k f o r d , I I I i n o i s ) ; D -gIucosamine h y d r o c h l o r i d e , myo-i n o s i t o l (Sigma Chemical Company, S t . L o u i s , M i s s o u r i ) . A l l o the r chemica ls were obta ined l o c a l l y . 'Baker Ana lyzed" grade ( J . T. Baker Chemical Company, Ph i I I i p s b u r g , New Je rsey ) 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 of Tremella mesenterica F r i e s (UBC c o l l e c t i o n #2259-6) were mainta ined a t 20 C on n u t r i e n t agar (15.0 g b a c t o - a g a r , 7 .5 g b a c t o -ma 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 bacto - soy tone per l i t r e ) . 1Q Inoculum c u l t u r e s were prepared in 50 ml of l i q u i d medium ( tha t desc r i bed above w i thout bacto -agar ) and shaken a t 20 C f o r 48 h r . A l i q u o t s of the inoculum (5 ml) 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 of the l i q u i d medium and shaken a t 20 C f o r 48 h r . Under these c o n d i t i o n s the fungus grew as h a p l o f d , y e a s t - l i k e u n i c e l l s . C e l l s were harvested by c e n t r i f u g a t i o n a t 9000 * g f o r 10 min , washed once w i th water , and coo led by s t i r r i n g f o r 30 min in an ice bath . C e l l s were broken in 50 ml p o r t i o n s us ing 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 watts f o r 2 min , coo led f o r 30 min and fragmented f o r a f u r t h e r 2 min . The c e l l s were kept c h i l l e d in an i c e bath throughout and the probe was immersed in ice between t r e a t m e n t s . The r e s u l t i n g suspension of whole and broken eel 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 suspension c o n s i s t e d of broken c e l l s . The p e l l e t , c o n s i s t i n g l a r g e l y of whole c e l l s , was t r e a t e d again w i th the u l t r a s o n i c probe and c e n t r i f u g e d ; the supernatant suspensions were combined. As a r e s u l t of t h i s t reatment approx imate ly 95% of the c e l l s were broken. Any remaining whole c e l l s were removed by c e n t r i f u g a t i o n dur ing the course of the washing procedure . C u l t u r e s of Saprolegnia diclina Humphrey (UBC c o l l e c t i o n #145) were mainta ined a t 4 C on s l a n t s of the same medium used f o r Tremella. Inoculum c u l t u r e s prepared on t h i s medium in p e t r i p l a t e s were grown a t 20 C f o r 96 h r . The medium and mycelium were homogenized in a s t e r i l e blendor c o n t a i n i n g 100 ml d i s t i l l e d water ; 20 ml of the 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 of l i q u i d medium (10.0 g d e x t r o s e , 5 .0 g bacto -peptone , 0 .5 g b a c t o - y e a s t e x t r a c t per l i t r e ) . C u l t u r e s were grown a t 20 C f o r 60 hr wi thout s h a k i n g . Examinat ion of samples from each of the c u l t u r e f l a s k s w i th the l i g h t 11 microscope showed no spores t o be p resent . The mycelium was harvested by f i l t r a t i o n on a BUchner funnel and washed once wi th water . The pad c f mycelium was ground t o a f i n e powder in l i q u i d N 2 w i th a mortar and p e s t l e . When the temperature rose above 0 C 50 mI of 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 th the u l t r a s o n i c probe ( tw ice f o r 2 min) as desc r i bed above. As a r e s u l t of t h i s t reatment v i r t u a l l y a l l of the c e l l s were broken. The washing procedure i s based on t h a t of M i t c h e l l and T a y l o r (1969). The suspension of 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 f o r 10 min wi th Tremella, 3000 * g f o r 5 min w i th Saprolegnia) t o recover c e l l w a l l f ragments . The supernatant s o l u t i o n was d i s c a r d e d . T h i s procedure was repeated 4 t i m e s . The c e l l wa l l fragments were then washed 5 t imes wi th i c e - c o l d 1.0 M NaCI s o l u t i o n ( c e n t r i f u g a t i o n a t 12000 x g f o r 10 min w i th Tremella, 3000 x g f o r 5 min w i th Saprolegnia), 5 t imes wi th c o l d water and t w i c e w i th 8 .0 M urea s o l u t i o n ( c e n t r i f u g a t i o n a t 27000 x g f o r 10 min w i th Tremella, 3000 * g f o r 5 min w i th Saprolegnia). The fragments were suspended f o r 12 hr in 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 wi th 8 M urea s o l u t i o n , 5 t imes w i th water , 5 t imes wi th 1.0 N NK^OH s o l u t i o n ( c e n t r i f u g a t i o n a t 12000 x g f o r 10 min w i th Tremella, 3000 x g f o r 5 min wi th Saprolegnia) and 5 t imes w i th water . The c e l l w a l l s recovered from t h i s t reatment were f r e e z e - d r i e d and s to red a t - 20 C. A n a l y t i c a l Procedures L i p i d s were est imated by the procedures of B a r t n i c k i - G a r c i a and N ickerson (1962). Elemental a n a l y s i s f o r C, H, N, 0 , P, S, and ash 12 were performed by Organic M i c r o a n a l y s i s , M o n t r e a l , Quebec. C e l l waI Is»(approximateIy 5 mg) were hydro lyzed in vacuo w i th 0 .5 ml of 6 N HCI c o n t a i n i n g 5% ( v / v ) CH2SH-COOH (Matsubara and Sasaki 1969) a t 110 C f o r 8, 24, 48, 72.and 144 hr ( T r i s t r a m and Smith 1963). Hyd ro l ysa tes were d r i e d in vacuo over concent ra ted H2SO4 and KOH p e l l e t s , then r e d i s s o l v e d in 1.0 ml water . 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 (pH 2.2). and 200 yI of the i n t e r n a l standard s o l u t i o n ( a - a m i n o - 3 - g u a n i d o -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 neut ra l and a c i d i c amino a c i d s ) in pH 2.2 b u f f e r were a p p l i e d t o the a p p r o p r i a t e column of a Beckman Amino Ac id Ana lyzer Model 120C. The a n a l y s i s i s based on the method of Spackman, S t e i n and Moore (1958) . B a s i c amino a c i d s were separated on a 13 cm column; t h i s reso lved g lucosamine from gaIactosamine , 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 ( these were not reso l ved from each o ther ) from h i s t i d i n e (F igu re 1 ) . Tryptophan and gaIactosamine were not reso lved by t h i s system. S ince the a l k a l i n e h y d r o l y s i s procedure t o r e l e a s e t ryptophan degrades ga Iactosamine , and the a c i d i c h y d r o l y s i s procedure t o r e l e a s e gaIactosamine g e n e r a l l y degrades t r y p t o p h a n , t h i s presented no problem. A c i d i c and neut ra l amino a c i d s were reso l ved on a 58 cm column, g lucosamine was separated from pheny la lan ine and thus d id not i n t e r f e r e w i th the pheny la lan ine d e t e r m i n a t i o n . The length of the column i s c r i t i c a l t o t h i s r e s o l u t i o n . Hydroxypro I ine was determined by the spect rophotomet r ic method of Bergman and Loxley (1970) us ing p -d imethy laminobenzaldehyde. Tryptophan was determined us ing the amino 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 w a l l s w i th 4 .2 N NaOH (Hugl i and Moore 1972). Ana lyses were performed in t r i p l i c a t e on a s i n g l e c e l l w a l l 14 p r e p a r a t i o n of Tremella, and once on each of 2 c e l l wa l l p r e p a r a t i o n s of Saprolegnia. A s y n t h e t i c m ix tu re of amino a c i d s present in the c e l l w a l l s (except h y d r o x y p r o l i n e and t ryptophan) and glucosamine h y d r o c h l o r i d e ' was prepared f o r degradat ion s t u d i e s ; i t was t r e a t e d w i t h 6 N HCl by the same procedure as the c e l l wa l l p r e p a r a t i o n s . Degradat ion of both hydroxypro I ine and t ryptophan was determined independent ly . C e l l w a l l s (approx imate ly 2 mg) were hydro lyzed in sea led tubes a t 110 C w i th 0 .5 ml 2 N CF 3C00H f o r 15, 30, 60, 120, 240 and 480 min (Albersheim e t a I 1967) t o r e l e a s e neut ra l sugars . Hyd ro l ysa tes were d r i e d in vacuo over KOH p e l l e t s . U ron ic a c i d s were est imated as neut ra l sugars by reduc ing the carboxy l groups of the p o l y s a c c h a r i d e s w i th LiBHi+ (Dutton and Kab i r 1971). The procedure was adapted f o r micro a n a l y s i s by reducing the p r o p o r t i o n s of reagents . The c e l l wa l l p r e p a r a t i o n (20 to 35 mg) was d i s s o l v e d in 8.0 ml formamide and t r e a t e d wi th 6 .0 ml p y r i d i n e and 4 .0 ml p r o p i o n i c anhydr ide . A f t e r s to rage f o r 2 days the s o l u t i o n was p r e c i p i t a t e d by adding i t t o 150 ml 2% H C l . The t o t a l recovered p r e c i p i t a t e , f i l t e r e d and d r i e d in vacuo, was r e t r e a t e d w i th 10.0 ml p y r i d i n e and 1.5 ml p r o p i o n i c anhydr ide . The t o t a l recovered propionated a c i d was d i s s o l v e d in 15.0 ml t e t r a h y d r o f u r a n , and 10.0 ml d i e t h y l e ther c o n t a i n i n g d i a z o -methane (cooled t o -73 C) were added; the mix tu re was a l lowed t o stand a t - 7 3 C f o r 6 h r . Diazomethane was prepared frcm W - m e t h y I - N - n i t r o s o -p-to. l uenesu I f onam ide accord ing t o Vogel (1956). Subsequent r e a c t i o n s up t o the recovery of carboxy I - reduced 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 th those desc r ibed 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 hydro lyzed wi th 2 N CF 3C00H f o r 2 hr t o r e l e a s e neut ra l sugars . Neut ra l sugars were analyzed by g a s - l i q u i d chromatography as t r i m e t h y I s i l y I (TMS) d e r i v a t i v e s . Hyd ro l ysa tes were d i s s o l v e d 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 th 0.1 ml hexamethyId is i Iazane and 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). The mix tu re was shaken f o r a few seconds, and a f t e r 30 min was f r e e z e - d r i e d t o remove the p y r i d i n e . 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 in 100 y l cyc lohexane and a l i q u o t s of t h i s m ix tu re or s u i t a b l e d i l u t i o n s of i t were i n j e c t e d i n t o a V a r i a n Aerograph dual column gas chromatograph Model 1740, equipped wi th f lame i o n i z a t i o n d e t e c t o r s . The f low r a t e s of N 2 and H 2 were 25 ml / min and a i r 250 ml / min . Columns (4 .9 m * 3 mm) of 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 60 /80 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 S p e c i a l i t i e s , B r o c k v i l l e , Onta r io ) were temperature programmed l i n e a r l y from 130 C (at i n j e c t i o n ) t o 230 C a t 2 degrees / min . F igu re 2 shows the r e s o l u t i o n obta ined by t h i s system. A s y n t h e t i c m ix tu re of f r e e neut ra l sugars was prepared f o r degradat ion 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 th 2 N CF3COOH by the same procedures as the c e l l wa l l p r e p a r a t i o n s . It a l s o served as a c a l i b r a t i o n m i x t u r e . C e l l w a l l s (approx imate ly 2 mg) were hydro lyzed in vacuo w i th 0 .5 ml 2 N HCl (Oates and Schrager 1967) f o r 8, 16, 32 and 72 hr t o r e l e a s e amino sugars . Hyd ro l ysa tes were d r i e d in vacuo over concent rated H2S04 and KOH p e l l e t s , d i s s o l v e d in pH 2 .2 b u f f e r , and est imated on the amino a c i d a n a l y z e r us ing the 13 cm column. The m ix tu re of g lucosamine h y d r o c h l o r i d e and amino a c i d s used f o r degradat ion s t u d i e s was t r e a t e d w i t h 2 N HCl by the same procedures FIGURE 2 RESOLUTION OF NEUTRAL SUGARS (AS TMS DERIVATIVES) ON THE GAS-LIQUID CHROMATOGRAPH 17 as the c e l l wa l l p r e p a r a t i o n s t o determine the degradat ion of g lucosamine under these c o n d i t i o n s . C a l c u l a t i o n of R e s u l t s Peak areas were est imated by the product of peak he ight * peak width a t the h a l f - h e i g h t which B a l l , H a r r i s and Habgood (1967) concluded i s the most r e l i a b l e manual method. The e s t i m a t e s , p a r t i c u l a r l y of the width a t the h a l f - h e i g h t are more a c c u r a t e on amino a c i d chromatograms where the number of dots p r i n t e d per t ime (<* width) can be counted; the measurement of the width on g a s - l i q u i d chromatograms i s l e s s a c c u r a t e because of the width of the ink t r a c e . In terna l s tandards are necessary in 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 i n t e r n a ! s t a n -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 . 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 the c e l l w a l l s examined meant t h a t 1 t o 2 yI of the i n i t i a l s i l y l a t e d p r e p a r a t i o n ( c a . 2 mg d i s s o l v e d in 100 yI cyc lohexane) had t o be i n j e c t e d onto the g a s - l i q u i d chromatograph in o rder t o d e t e c t minor c o n s t i t u e n t s . Cyclohexane i s v o l a t i l e a t room temperature , and even though the p r e p a r a t i o n s were immediately stoppered and p laced a t -20 C, evapora t ion losses were p o s s i b l e . Furthermore, the 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 in cyc lohexane . With as l i t t l e as 1 t o 2 yI in the s y r i n g 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 are d i f f i c u l t t o e s t i m a t e . To de tec t the major c o n s t i t u e n t s 20 y i of the concent ra ted s o l u t i o n were d i l u t e d w i t h 500 y l cyc lohexane ; a s i m i l a r e r r o r from NH^CI cou ld r e s u l t . T h i s i s the 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 the use of . i n t e r n a ! s tandards . Both e r y t h r i t o l and aiyo-inos ? t o I were used; the 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 approx imate ly \% t h a t of i n o s i t o l in o rder t o o b t a i n measurable peaks on s c a l e a t the d i f f e r e n t c o n c e n t r a t i o n s . A l l t r a c e s of CFgCOOH were removed after h y d r o l y s i s and before 50 uI of each i n t e r n a l standard s o l u t i o n were added. . The r a t i o of area per weight was determined f o r each i n t e r n a l standard (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 , the area of the i n t e r n a l standard c a l c u l a t e d from the d i l u t i o n of the sample, was compared wi th the a c t u a l area measured on the chromatogram. The weight of anhydro sugar in the samples was c a l c u l a t e d by comparison of i t s peak area w i t h the area of a known weight of anhydro e q u i v a l e n t in the c a l i b r a t i o n m i x t u r e . Where t h e r e were severa l peaks f o r a sugar , the best r eso l ved one from the sample was compared w i t h the e q u i v a l e n t peak of the 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 the i n t e r n a l standard was used t o compensate f o r the d e t e r i o r a t i o n of the n i n h y d r i n reagent . For every run on the amino a c i d a n a l y z e r 20 nmoles of i n t e r n a l standard were added t o the a p p r o p r i a t e column w i t h the sample or c a l i b r a t i o n m ix tu re 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 the b a s i c amino a c i d column and n o r l e u c i n e on the neut ra l and a c i d i c amino a c i d column) . The area of each amino peak was compared w i t h the area of the i n t e r n a l standard f o r t h a t a n a l y s i s . The weight of the anhydro amino a c i d was c a l c u l a t e d by comparison wi th the 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 the amino a c i d s in the Beckman Amino Ac id C a l i b r a t i o n M i x t u r e Type 1. RESULTS 19 The c e l l w a l l s a f t e r washing showed no ev idence of c y t o p l a s m i c contaminat ion by phase c o n t r a s t l i g h t mic roscopy , no ev idence of c a p s u l a r m a t e r i a l w i th India ink under the l i g h t microscope and no v i s i b l e r ibosomes, membranes or c a p s u l a r m a t e r i a l w i th e l e c t r o n mic roscopy . The y i e l d of c e l l w a l l s from 6 I of medium was 100 t o 150 mg from Tremella mesenterica and 200 mg from Saprolegnia diclina. These p r e p a r a t i o n s were s to red a t - 20 C and d r i e d in vacuo f o r 12 hr before we igh ing . Degradat ion under Hyd ro l y z inq C o n d i t i o n s An a l i q u o t of the s y n t h e t i c m ix tu re of amino a c i d s and glucosamine h y d r o c h l o r i d e was sub jected t o the h y d r o l y z i n g c o n d i t i o n s used f o r c e l l wa l l p r o t e i n s (6 N HCl in vacuo) f o r each of the t ime pe r iods (8 t o 145 hr) as the c e l l wa l l p r e p a r a t i o n s . A s o l u t i o n of hydroxypro I ine was s i m i l a r l y t r e a t e d . A l i q u o t s of a s o l u t i o n of t ryptophan were t r e a t e d (wi th 4 .2 N NaOH in vacuo) l i k e the c e l l wa l l p r e p a r a t i o n s f o r 8 t o 96 hr . The r e c o v e r i e s of the amino a c i d s are shown in F igu re 3 . A l i q u o t s of the mix tu re of amino a c i d s and glucosamine h y d r o c h l o r i d e were a l s o sub jected t o the 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 amino sugars from c e l l wa l l p o l y s a c c h a r i d e s (2 N HCl in vacuo) f o r 8 t o 96 hr . The r e c o v e r i e s of g lucosamine from the two t reatments are compared in F igu re 4. Glucosamine i s degraded t o a f a r g r e a t e r ex ten t in 6 N H C i . A l i q u o t s of the c a l i b r a t i o n mix tu re of f r e e neu t ra l sugars were sub jected t o 2 N CF 3C00H f o r 15 min to 8 hr ( the same t reatment used t o r e l e a s e neut ra l sugars from c e l i wa l l p o l y s a c c h a r i d e s ) . The r e c o v e r i e s FIGURE 3 RECOVERY OF FREE AMINO ACIDS UNDER HYDR0LY2ING CONDITIONS (6 N HCI in vacuo) 21 FIGURE 4 RECOVERY OF GLUCOSAMINE UNDER HYDROLYZING CONDITIONS A Comparison of 2 N and 6 N HCl Time (hr) are shown in F i g u r e 5 . P r o t e i n Ana i ys i s -A l l of the usual p r o t e i n amino a c i d s except c y s t e i n e / c y s t i n e were, found in the c e l l w a l l s of both s p e c i e s . A p p l i c a t i o n of l a r g e r samples t o the 58 cm column f a i l e d t o show any t r a c e of 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 ryptophan were detected as p r o t e i n c o n s t i t u e n t s in both s p e c i e s . It was not p o s s i b l e t o determine amide in the c e l l wa l l p r e p a r a t i o n s because of r e s i d u a l NH 3 from the washing procedure and degradat ion of amino compounds. The r e s u l t s are presented in Tab les I and I I , and in F i g u r e s 6 and 7 . The amount of each monomer recovered a f t e r h y d r o l y s i s i s determined p r i m a r i l y by two r e a c t i o n s : the r a t e a t which the monomer i s r e leased from the polymer and the r a t e a t which the f r e e monomer i s degraded under the h y d r o l y z i n g c o n d i t i o n s . Curves such as those in F i g u r e s 6 and 7 represent the amount of monomer re leased dur ing hydro lys minus the amount of f r e e monomer degraded. P o l y s a c c h a r i d e A n a l y s i s 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 w a l l s of both organisms. Rhamnose was detected in T. mesenterica but not in S. dieUna; t r a c e s of r i b o s e were detected in S. diclina but not in T . mesenterica. The r e s u l t s are presented in Tab les III and IV, and in F igu res 8 and 9. Amino Sugars Glucosamine was found in the c e l l waI Is of both organisms; 24 TABLE I AMINO ACIDS IN THE CELL WALL OF Tremella mesenterica (yg anhydro amino a c i d recovered / mg c e l l wal l p repa ra t i on ) 8 hr Durat ion 24 hr of Hydro lys 48 hr is w i th 6 72 hr N HCI 145 hr best e s t i mate T r p 2 3 0.1 <0.1 0.1 4 3 0.1 Lys 2 .2 3 .3 2 .9 2 .3 2 .0 3 .3 H i s 0.8 1 .2 1.1 0 .9 0 .8 1 .2 Arg 2 .2 3 .5 3 .2 2 .5 2 .2 3 .5 Asx 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 A l a 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 Hyp 5 3 0.4 0 .3 0 .2 0.1 0 .4 T o t a l Recovery 48.9 1 maximum va lue 2 c e l l wa l l p r e p a r a t i o n hydro lyzed in 4 .2 N NaOH (Hug I I and Moore 1972) 3 not determined hydro lyzed f o r 96 hr 5 determined spect rophotometr ica I Iy (Bergman and Loxley 1970) 25 TABLE II AMINO ACIDS IN THE CELL WALL OF Saprolegnia d i c l i n a (ug anhydro amino a c i d recovered / mg c e l l w a l l p r e p a r a t i o n ) Durat ion of H y d r o l y s i s w i t h 6 N HCl best 8 hr 24 hr 48 hr 72 hr 145 hr e s t i m a t e 1 T r p 2 3 0.2 . <0.1 <0Ah 3 0.2 Lys . 0 .8 1 .2 0.8 0.7 . 0 .6 1 .2 H is 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 A l a 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 Val . 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 Hyp 6 0.6 0.4 0.2 0 .2 0.1 0.6 To ta l Recovery 13.3 1 maximum va I ue 2 ce 1 1 wa1 I p r e p a r a t i o n hydro lyzed in 4 .2 N NaOH (Hug 1i an< Moore 1972) not determined hydro lyzed f o r 96 hr not recovered determined spect rophotomet r ica I Iy (Bergman arid Loxley 1970) 27 RECOVERY OF AMINO ACIDS FROM CELL WALLS OF Saprolegnia diclh ( H y d r o l y s i s in vacuo with 6 N HCl) o Time (hr) Time (hr) 28 TABLE M l NEUTRAL SUGARS IN THE CELL WALL OF Tremella mesenterica (ug anhydro sugar / mg c e l l wa l l p r e p a r a t i o n ) Durat ion of H y d r o l y s i s w i t h 2 N CF3COOH best 15 min 30 min 1 hr 2 hr 4 hr 8 hr e s t i m a t e 1 Rha 13.0 15.0 17.5 " 1 6 . 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 2 8 .5 4 .5 2 2 8 .5 Xyl 156.5 161.0 150.0 118.5 98.0 56.5 1 6 1 . 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 G l c 225.0 338.0 4 6 3 . 5 3 379.5 414.0 • 368.0 414.0 To ta l Recovery 703.0 1 maximum va lue 2 not detected 3 spur ious v a l u e 4 not con f i rmed , i d e n t i f i e d by chromatographic p o s i t i o n on l y 29 TABLE IV NEUTRAL SUGARS IN THE CELL WALL OF Saprolegnia diclina (yg anhydro sugar / mg c e l l w a l l p r e p a r a t i o n ) Durat ion of H y d r o l y s i s w i th 2 N CF 3C00H best 15 min 30 min 1 hr 2 hr 4 hr 8 hr e s t i m a t e 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 Fuc 2 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 G lc 187.0 401.5 610.5 686.5 607.5 636.5 686.5 To ta l Recovery 725.5 maximum va lue not con f i rmed , i d e n t i f i e d by chromatographic p o s i t i o n on l y FIGURE 8 RECOVERY OF NEUTRAL SUGARS FROM CELL WALLS OF Tremella mesenterica -(Hydrolysis with 2 N CF3C00H) FIGURE 9 RECOVERY OF NEUTRAL SUGARS FROM CELL WALLS OF Saprolegnia diclina (Hydrolysis with 2 N CF3C00H) 32 gaIactosamine was not detected in e i t h e r . The ex ten t of w-acety I a t ion was not determined. The r e c o v e r i e s of g lucosamine a f t e r h y d r o l y s i s of the c e l l wa l l p r e p a r a t i o n s w i t h 2 N HCI are presented in Table V. The r e c o v e r i e s of glucosamine a f t e r h y d r o l y s i s w i th 2 N HCI and 6 N HCI are compared in F igu re 10. The massive degradat ion of g lucosamine w i th 6 N HCI p rec ludes i t s use in any study t h a t attempts 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 of carboxy l groups in the c e l l wa l l p o l y s a c c h a r i d e s of T. mesenterica 35.29 mg of c e l l wa l l p r e p a r a t i o n were reduced by the method of Dutton and Kab i r (1971); 10.55 mg of reduced c e l l wa l l p rep -a r a t i o n were recovered . For S. diclina the amounts were 20.93 mg and 3.87 mg r e s p e c t i v e l y . A f t e r h y d r o l y s i s in 2 N CFgCOOH the mole r a t i o s of the sugars recovered were compared w i th those of the unreduced c e l l wa l l p r e p a r a t i o n hydro I y s a t e s . The d i f f e r e n c e s were we l l w i t h i n the exper imenta l e r r o r so t h a t u r o n i c a c i d s are present in very small amounts, i f a t a l l , in the c e l l waI Is of the two s p e c i e s . L i p i d Ana Iys i s I nd i v i dua l l i p i d components were not determined; l i p i d s were separated as 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 ickerson 1962); those e x t r a c t a b i e in e t h a n o I : d i e t h y I e ther and ch lo ro fo rm ( r e a d i l y e x t r a c t a b l e l i p i d s ) and those re leased a f t e r t reatment w i th \% HCI in e t h a n o l : e t h e r (bound l i p i d s ) . The r e s u l t s a re present 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 c e l l wa l l p r e p a r a t i o n s of each s p e c i e s were ana lyzed ; the r e s u l t s a re presented in Table V I I . 33 TABLE V AMINO SUGARS IN THE CELL WALL OF Tremella mesenterica AND Saprolegnia diclina (yg anhydro GlcNx / mg c e l l w a l l p r e p a r a t i o n ) Durat ion of H y d r o l y s i s w i th 2 N HCI best 8 hr 16 hr 32 hr 72 hr 96 hr e s t i m a t e 1 T. mesenterica 6.1 13.1 20.4 23.9 2 23.9 S. diclina 4 .7 7 .5 8 .9 8 .8 8 .0 8 .9 1 maximum va lue 2 not determined TABLE VI LIPIDS IN THE CELL WALL OF Tremella mesenterica AND Saprolegnia diclina (yg / mg c e l l w a l l p repa ra t i on ) Read i I y TotaI E x t r a c t a b l e L i p i d s Bound L i p i d s L i p i d s Recovered T. mesenterica 33 .5 43.0 76 .5 S. diclina 82 .0 37.5 119.5 uc) a n h y d r o G l c l l x / m<) c o l l w a l l p r o p f t f h t I on T : I ' — 1 '—I r 34a TABLE VII ELEMENTAL AND ASH ANALYSIS OF CELL WALL PREPARATIONS OF Tremella mesenterica AND Saprolegnia diclina 1 (yg / mg c e l l wa l l p r e p a r a t i o n ) Tremella mesenterica Saprolegnia diclina #4 2 #5 #1 #2 2 C 377 .1 4 0 4 . 4 3 7 6 . 6 3 8 3 . 6 H 66 .1 7 0 . 9 6 3 . 4 6 5 . 5 0 4 3 1 . 0 4 5 1 . 7 4 1 1 . 7 470 .1 N 1 5 . 2 1 8 . 4 63 .1 1 5 . 0 S " 6 9 . 0 2 9 . 7 20 .1 3 0 . 7 P 1 4 . 0 1 0 . 5 1 8 . 8 4.1 Ash 3 2 . 7 1 6 . 6 3 2 4 . 8 Tota l 1005.1 1 0 0 2 , 2 9 5 3 . 7 9 9 3 . 8 a n a l y s i s performed by Organic M i c r o a n a l y s i s , M o n t r e a l , Quebec, on c e l 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 hr c e l l wa 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 recovery of c e l l w a l l components f o r each s p e c i e s i s summarized in Table V I I I . Approx imate ly 90% of the weight of each c e l l wa l l p r e p a r a t i o n was recovered a f t e r the a n a l y s i s in two q u i t e d i s s i m i l a r f u n g i . The remaining 10$ i s not accounted f o r but the s i m i l a r r e c o v e r i e s obta ined from'two such d i f f e r e n t fungal s p e c i e s suggest t h a t the s t r a t e g y i s r e p r o d u c i b l e . R e s u l t s presented are de r i ved from one c e l l wa l l p r e p a r a t i o n f o r each s p e c i e s . Con f i rmat ion was obta ined from two o ther 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 the r workers in the l a b o r a t o r y . The c e l l wa l l p r e p a r a t i o n s d i f f e r e d in 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 compos i t ions but mole r a t i o s among neut ra l sugars and among amino a c i d s i n d i c a t e d t h a t monomeric compos i t ions f o r each of these components were s i m i l a r . A n a l y t i c a l accuracy was e s t a b I i s h e d from repeat 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 . R e p r o d u c i b i l i t y f o r amino a c i d a n a l y s i s was ±3% ( lower l i m i t of d e t e c t i o n c a . 0 .5 nmole) and f o r sugar a n a l y s i s ±5% ( lower l i m i t of d e t e c t i o n ca. 50 ng) . 36 TABLE VII I COMPLETE ANALYSIS OF THE CELL WALL OF Tremella mesenterica AND Saprolegnia diclina (yg / mg c e l l w a l l p repa ra t i on ) Tremella mesenterica Saprolegnia diclina P o l y s a c c h a r i d e anhydro neut ra l sugars 703.0 725.5 anhydro amino sugars 23 .9 8 .9 anhydro u r o n i c a c i d s <0.5 <0.5 P r o t e i n anhydro amino a c i d s 48.9 13.3 L i p i d 76.5 119.5 Ash • 32.7 24 .8 Tota l Recovery 885.0 892.0 37 DISCUSSI ON Although the c e l l waI Is of n e i t h e r s p e c i e s have been s t u d i e d p r e v i o u s l y , Saprolegnia ferax has been i n v e s t i g a t e d (Crook and Johnston 1962; P a r k e r , P reston and Fogg 1963; Novaes -Led ieu , J imSnez -Mar t fnez and V i l l a n u e v a 1967; S ie tsma, E v e l e i g h and Haskins 1969). None of these 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 but they d id show t h a t the c e l l wa l l conta ined g lucose as the predominant 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 w i th r i b o s e , mannose and glucosamine present as minor c o n s t i t u e n t s . Novaes-Led ieu e t a I. (1967) repor ted the presence of rhamnose in a d d i t i o n t o the o ther sugars . Rhamnose i s not a c o n s t i t u e n t of the c e l l wa I Is of S. diclina; the TMS e t h e r s of rhamnose and r i b o s e are complete ly r e s o l v e d by the g a s - l i q u i d chromatography system employed (see F igu re 2 ) . Only Novaes-Ledieu e t a I. (1967) repor ted the presence of g a l a c t o s e , which in S. diclina i s the second most abundant sugar . A rab inose , fucose and .xyjose were detected in t r a c e amounts in S. diclina but not in S. ferax. Parker e t a l . (1963) s tud ied on ly sugar c o n s t i t u e n t s of the c e l l w a l l s ; they examined two a d d i t i o n a l s p e c i e s of Saprolegnia. The compos i t ion of S. monoica was s i m i l a r t o t h a t of S. ferax; t h a t of S. litoralis had the 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 detected as w e l l . Crook and Johnston (1962) and Novaes-Ledieu e t a l . (1967) est imated q u a n t i t a t i v e amino a c i d compos i t ion from r e l a t i v e i n t e n s i t i e s of n i n h y d r i n - p o s i t i v e spots on paper chromatograms. The p r o t e i n amino a c i d s , i n c l u d i n g h y d r o x y p r o l i n e were p resen t , a l though n e i t h e r t e s t e d f o r t r yp tophan ; Crook and Johnston (1962) repor ted the absence of meth ion ine . The q u a n t i t a t i v e r e s u l t s of the c e l l wa l l ana lyses of S. ferax and S. diclina are compared below. 38 Saprolegnia ferax S. diclina Novaes-Ledieu Sietsma e t a I. e t a I. neut ra l sugars 93 % 84 % 12.5% amino sugars 1.7 2.7 0 .9 u r o n i c a c i d s not detected not determined not detected p r o t e i n 1.2 3 .0 1.4 l i p i d 1.0 5 .0 12.0 ash not determined 3 .2 2 .5 There i s genera l agreement about the compos i t ion of the c e l l w a l l s of the two s p e c i e s ; the d i f f e r e n c e s observed between s p e c i e s and between two ana lyses of the same s p e c i e s may be the r e s u l t of severa l f a c t o r s . Both Novaes-Ledieu e t a I. (1967) and Sietsma e t a I. (1969) est imated t o t a l carbohydrates w i th the anthrone reagent . Aminoff (1970) has s t a t e d t h a t " q u a n t i t a t i v e de te rm ina t ion i s p o s s i b l e o n l y when the i d e n t i t y of sugar components t o be assayed i s known. Severa l o ther subs tances , i n c l u d i n g t r y p t o p h a n . . . . , i n t e r f e r e in the [anthrone] r e a c t i o n The f a c t t h a t the c e l l w a l l s c o n t a i n g lucose as the predominant sugar reduces the magnitude c f the f i r s t problem but the substances t h a t cause i n t e r f e r e n c e (and t ryptophan was determined in n e i t h e r of the s t u d i e s ) may r e s u l t in 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 re h igher than the t r u e carbohydrate c o n t e n t . 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 id not r e p o r t the age of t h e i r c u l t u r e o r the temperature a t which they were grown; such f a c t o r s may a f f e c t the compos i t ion of eel I W a l l s . The c u l t u r e medium used by Sietsma e t a I. (1969) conta ined c h o l e s t e r o l ; t h i s may have a f f e c t e d the l i p i d content of the c e i l w a l l s or the l i p i d d e t e r m i n a t i o n . There have been no r e p o r t s of c e i l w a l l compos i t ion of Tremella, and Ustilago maydis growing in y e a s t - l i k e form appears t o be the on ly othe / . 3 9 Heterobas id iomycete i n v e s t i g a t e d (Crook and Johnston 1962). They found g lucose and glucosamine t o be the major monosaccharides of the c e l l wa l l w i th less g a l a c t o s e and t r a c e s of mannose. They detected a l l the p r o t e i n amino a c i d s except hydroxypro I ine and methion ine (they d id not t e s t f o r t r y p t o p h a n ) . O ' B r i e n and Ralph (1966) examined the sugar components of c e l l w a l l s of severa l Bas id iomycetes and found g lucose and glucosamine t o be the major c o n s t i t u e n t s , w i th l e s s mannose and x y l o s e , and t r a c e s of fucose p r e s e n t . G a l a c t o s e was absent in a l l s p e c i e s examined except Cnniophora cerebella. Tremella mesenterica ( i n t h e . h a p l o i d y e a s t - l i k e form) shows two s t r i k i n g d i f f e r e n c e s from these Bas id iomycetes ; the glucosamine content was found t o be very low (Table V ) , ' a n d hydroxypro I ine was detected (Table 1) . In a d d i t i o n t o g a l a c t o s e , both rhamnose and a rab inose 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 tud ied in a large number of f u n g i ; Gor in and Spencer (1968) d i scussed the types and l i nkages of monosaccharide c o n s t i t u e n t s in the fung i and how they might be use fu l in taxonomic s t u d i e s . B a r t n i c k i - G a r c i a (1968) cons idered the p o l y s a c c h a r i d e s of fungal c e l l w a l l s and dev ised a scheme based on the 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 chemical compost i ion of the c e l l wa l l and major taxonomic groupings e labo ra ted on morpho log ica l c r i t e r i a . The data t o extend t h i s scheme w i l l come from c a r e f u l q u a l i t a t i v e and quant - • i t a t i v e s t u d i e s not on ly of the sugar components present but of the types of l i nkages between them in many more fungal s p e c i e s than have been examined t o t h i s t ime . The p r o t e i n c o n s t i t u e n t s in fungal c e l l w a l l s have not been as w ide l y s tud ied as the p o l y s a c c h a r i d e s . At present too few s p e c i e s have been examined t o make more than very genera l s ta tements . A i l the p r o t e i n amino a c i d s are present except c y s t e i n e / c y s t i n e which appears to be absent in many fungi (Roy and Landau 1972). E i t h e r a s p a r t a t e and g lu tamate , o r 40 th reon ine and s e r i n e are f r e q u e n t l y the most abundant amino a c i d s . Hydroxy-p r o l i n e and tryptophan have not been w ide ly reported but they have not been w ide l y sought. HydroxyproI ine has been found in s p e c i e s of the Oomycetes ( i t c o n s t i t u t e s 20.4$ of the t o t a l amino a c i d component of Atkinsiella dubia, Aronson and F u l l e r 1969) and in Candida albicans (Chattaway e t a l . 1968). It had not been p r e v i o u s l y reported in fungi w i th 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 of eel Iu Ios i c ceII waI Is of f u n g i , a l g a e , o r h igher p l a n t s ( B a r t n i c k i - G a r c i a 1968). The d i s c o v e r y of hydroxypro I ine i s Tremella mesenterica appears t o be the f i r s t r epo r t in a Bas id iomycete . Fu r the r s t u d i e s w i l l revea l whether the c e l l wa l l of T. mes-enterica lacks c h i t i n , whether some of the g lucan i s c e l l u l o s i c , o r whether hydroxypro I ine 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 wa l l p r o t e i n s . A n a l y t i c a l Procedures The e s t i m a t i o n of most amino a c i d s i s based on the procedures of Moore, Spackman and S t e i n (1958) and Spackman, S t e i n and Moore (1958). A c i d 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 th 6 N H C l ; s e r i a l h y d r o l y s i s t o es t imate incomplete c leavage and degradat ion i s recommended ( T r i s t r a m and Smith 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 yp tophan , w i th the r a t e a c c e l e r a t e d in the presence of carbohydrate (Spencer 1963). Matsubara and Sasaki (1969) attempted t o min imize the d e s t r u c t i o n of t ryptophan by a d d i t i o n of CH2SH-COOH t o the p r o t e i n before h y d r o l y s i s . However t h i s t reatment improves the recovery of t ryptophan on ly in the absence of carbohydrate (Hug I i and Moore 1972; James 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 of a c h i e v i n g q u a n t i t a t i v e recovery of t r yp tophan . Hug I i and Moore (1972) have d i scussed the l i m i t a t i o n s of methods t h a t have been employed and 41 proposed a s imp le q u a n t i t a t i v e procedure us ing the amino a c i d a n a l y z e r : NaOH i s used f o r h y d r o l y s i s of the p r o t e i n r a t h e r than Ba(0H)2 t o avo id a d s o r p t i o n of t ryptophan on BaSOi* o r BaC03, s t a r c h i s added as an a n t i -o x i d a n t ( f o r samples r i c h in carbohydrate such as c e l l w a l l s , s t a r c h i s not r e q u i r e d ) , and the h y d r o l y s a t e i s d i s s o l v e d in pH 4.25 b u f f e r t o avo id degradat ion of t ryptophan in a h i g h l y a c i d i c medium (amino a c i d s are r o u t i n e l y d i s s o l v e d in pH 2 .2 b u f f e r f o r e s t i m a t i o n on the amino a c i d a n a l y z e r ) . HydroxyproI ine has been est imated us ing the amino a c i d a n a l y z e r but under the standard 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 w i t h a s p a r t a t e . I t can be detected by peak enhancement of the absorbance a t 440 nm o r est imated by a l t e r n a t i v e programming of the a n a l y z e r So t h a t the two amino a c i d s a re r e s o l v e d . However the procedure i s not s a t i s f a c t o r y because of the low s e n s i t i v i t y of the n i n h y d r i n . M i t c h e l l and T a y l o r (1970) examined a number of a l t e r n a t i v e procedures us ing p -d imethylaminobenzaldehyde as the spect rophotomet r ic agent and found t h a t of Bergman and Loxley (1970) t o be the most s u i t a b l e and the most s e n s i t i v e f o r de te rm ina t ion of hydroxypro I ine in c e l l w a l l s . There are some problems in q u a n t i t a t i v e recovery of c y s t e i n e / c y s t i n e a f t e r a c i d h y d r o l y s i s ( 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 large excess of h y d r o l y s a t e s of c e l l w a l l s of T. mesenterica and S. diclina were a p p l i e d t o the 58 cm column of the amino a c i d a n a l y z e r , 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 observed . Under these c o n d i t i o n s c y s t e i n e , i f present a t a l l , was below the l i m i t s of d e t e c t i o n and thus i s cons idered t o be absent . James (1972) has repor ted 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 recove red . 42 In the present s t u d i e s , c e l l w a l l s of T. mesenterica were hydro lyzed w i th and wi thout CH 2SH-C00H; the re was no s i g n i f i c a n t d i f f e r e n c e in the recovery of p r o l i n e and no c y s t e i n e was detected wi th e i t h e r t rea tment . Ross (personal communication) has examined the c e l l w a l l s of Cryptococcus laurentii and C. neoformans, which may be r e l a t e d t o Tremella ( S l o d k i , Wickerham and Bandoni 1966); h y d r o l y s i s w i thout CH 2SH-C00H showed no c y s t e i n e t o be present in e i t h e r s p e c i e s . C e l l wa l l p o l y s a c c h a r i d e s have been hydro lyzed wi th v a r i o u s a c i d s . h^SO^ has been w ide ly used but n e u t r a l i z a t i o n w i th BaC03 produces a p r e c i p i t a t e of BaSO^ t h a t may adsorb monosaccharide c o n s t i t u e n t s , e s p e c i a l l y u r o n i c a c i d s . HCl i s v o l a t i l e , but i t i s g e n e r a l l y agreed t h a t i t causes more degradat ion than H 2 S 0 4 (Dutton 1972). A lbersheim et a l . (1967) used CF3COOH as the h y d r o l y z i n g a c i d because of i t s v o l a t i l i t y and thus n e u t r a l i z a t i o n of the excess h y d r o l y z i n g a c i d was not r e q u i r e d . The sugars re leased by h y d r o l y s i s have been est imated by g a s - l i q u i d chromatography as 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 l d i t o l a c e t a t e s have been more w ide ly used in c e l l wa l l i n v e s t i g a t i o n s but TMS d e r i v a t i v e s may be s u p e r i o r in two r e s p e c t s : ( i ) they can be prepared d i r e c t l y w i thout the i n t e r v e n t i o n of a r e d u c t i o n s t e p , and ( i i ) they produce m u l t i p l e peaks ( u s u a l l y two t o four ) w i t h the a and B pyranos ides as the major components. The number and p r o p o r t i o n of the peaks f o r each sugar depend on the s o l v e n t in which the sugar comes t o e q u i l i b r i u m , the s o l v e n t in which the d e r i v a t i v e i s i n j e c t e d onto the column, and the s t a t i o n a r y phase of the column. S ince the number of peaks and t h e i r r e l a t i v e p r o p o r t i o n s are found t o be constant f o r each sugar under g iven 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 , 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 on expected peak p r o p o r t i o n s t o c a l c u l a t e the t o t a l amounts of sugar from 43 any complete ly reso l ved peak ( H o l l i g a n 1971). S i n c e in b i o l o g i c a l m a t e r i a l t h e r e 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 the 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 presence 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 th known r e t e n t i o n and peak area p r o p o r t i o n s a l l o w s i d e n t i f i c a t i o n of most monosaccharides w i t h con f i dence ( B h a t t i , Chambers and Clamp 1970). Amino sugars a re c u s t o m a r i l y r e leased 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 f o r the recovery of l i b e r a t e d amino sugars . F i g u r e 10 shows the massive degradat ion of g lucosamine dur ing h y d r o l y s i s of c e l l w a l l s w i t h 6 N HCI compared w i th 2 N HCI. T h i s matter apparen t l y i s not w ide l y a p p r e c i a t e d as many recent 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 ra t zne r 1972) r e p o r t g lucosamine compos i t ion data obta ined a f t e r h y d r o l y s i s w i th 6 N HCI. 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 in doubt f o r they may rep resent as l i t t l e as 5 t o 10$ of the r e a l g lucosamine c o n t e n t . There are c o n f l i c t i n g r e p o r t s on the r e s o l u t i o n and the q u a n t i t a t i v e recovery of TMS d e r i v a t i v e s of amino sugars (Dutton 1972). Moore and S t e i n (1948) showed t h a t g lucosamine r e a c t s q u a n t i t a t i v e l y w i t h n i n h y d r i n under c o n d i t i o n s of assay f o r amino a c i d s . Glucosamine and gaIactosamine can be reso l ved on the 13 cm column of the amino a c i d a n a l y z e r (F igu re 1 ) . At present t h i s seems t o be the p r e f e r a b l e method of a n a l y s i s f o r amino sugars . G I y c o s i d u r o n i c a c i d l i nkages are r e s i s t a n t t o a c i d h y d r o l y s i s and under c o n d i t i o n s t h a t r e l e a s e neut ra l sugars on i y p a r t i a l c leavage of such bonds may occur (Dutton 1972). The r e s u l t i n g a I d o b i o u r o n i c a c i d s can be hydro iyzed by prolonged a c i d t reatment t h a t degrades neut ra l sugars (Adams 1965). Free u r o n i c a c i d s are l a b i l e in a c i d media and 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 products of unknown compos i t ion (Aminoff 1970). ' Jones and A lbersheim (1972) hydro lyzed c e l l wa l l p o l y s a c c h a r i d e s wi th d i l u t e a c i d (0 .2 N CF3COOH), then t r e a t e d the p a r t i a l l y depolymer ized p r e p a r a t i o n w i th a mix tu re of ext race I IuIar po Iysacchar ide -deg rad ing enzymes from Sclerotium rolfsii. The l i b e r a t e d sugars and u r o n i c a c i d s were reduced wi th 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 separated us ing an ion exchange r e s i n . The a l d o n i c a c i d s were reduced wi th NaBH4 t o a l d i t o l s . The a l d i t o l s were est imated by g a s - l i q u i d chromatography as a c e t a t e d e r i v a t i v e s . Dutton and K a b i r (1972) methylated p o l y s a c c h a r i d e s from corn leaves and s t a l k s , then hydro lyzed them t o r e l e a s e methylated neut ra l sugars and methylated a I d o b i o u r o n i c a c i d s , which were separated us ing ion exchange r e s i n s . The methylated a I d o b i o u r o n i c a c i d s were hydro lyzed and the neut ra l sugars analyzed t o revea l the l i n k a g e s . Dutton and Kab i r (1971) a l s o pub l i shed a procedure f o r reducing the carboxy l groups of the u r o n i c a c i d s in p o l y s a c c h a r i d e s wi th LTBHt+ before h y d r o l y s i s . Th is was the method chosen t o es t imate u r o n i c a c i d s in the present s t u d i e s . However the method was found to be u n s u i t a b l e f o r the in fo rmat ion requ i red in the present s t u d i e s . A f t e r r e d u c t i o n of the carboxy l groups the p o l y s a c c h a r i d e s were hydro lyzed t o r e l e a s e neut ra l sugars . The amount of u r o n i c a c i d s cou ld be est imated on l y by d i f f e r e n c e from the h y d r o l y s i s products of the unreduced p o l y s a c c h a r i d e . A l d o b i o u r o n i c a c i d s unhydrolyzed in the unreduced p o l y s a c c h a r i d e should rep resent an increase in the neut ra l sugar components when the reduced p o l y s a c c h a r i d e i s h y d r o l y z e d . If the a l d o b i o u r o n i c a c i d s (from the unreduced p o l y s a c c h a r i d e ) a re hydro lyzed under the c o n d i t i o n s chosen, the u r o n i c a c i d es t imate w i l l be low. P a r t i c u l a r l y if the u r o n i c a c i d 45 content of the 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 in t h i s es t imate as the 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 on l y about 5%. Furthermore the a n a l y s i s does not revea l upon which component the ca rboxy l group was l o c a t e d . The method i s u s e f u l , however, in check ing the u r o n i c a c i d compos i t ion 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 i s a I ready ava i I a b I e . Whi le each of these th ree methods i s use fu l in q u a l i t a t i v e a n a l y s i s , they present problems of q u a n t i t a t i v e recovery v i z . incomplete h y d r o l y s i s , losses on ion exchange r e s i n s , incomplete d e r i v a t i z a t i o n r e a c t i o n s . At p resen t , the problem of q u a n t i t a t i v e recovery of s p e c i f i c u r o n i c a c i d s has not been complete ly so lved (Norstedt and Samuel son 1966; B lake and R ichards 1970). 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 d e c a r b o x y l a t i o n i s the method of c h o i c e (Aminoff 1970). The present s t u d i e s have been p a r t i c u l a r l y concerned wi th 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 the c e l l w a l l . A study of c e l l w a l l l i p i d s of b a k e r ' s yeast (Suomalainen and Nurminen 1970) 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 the c e l l wa l l and the whole c e l l . Procedures e x i s t f o r the r e s o l u t i o n of l i p i d c o n s t i t u e n t s . The e x t r a c t i o n method of F o l c h , Lees and SIoane-StanIey (1957) has been recommended. Dyke (1964) and S ie tsma , E v e l e i g h and Haskins (1969) q u a n t i t a t i v e l y est imated l i p i d s and methyl e s t e r s of f a t t y a c i d s by g a s - l i q u i d chromatography. Suomalainen and Nurminen (1970) extended the i n v e s t i g a t i o n t o i nc lude p h o s p h o l i p i d s . Kuks is (1966) has pub l i shed an e x c e l l e n t review of q u a n t i t a t i v e procedures designed p r i m a r i l y 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 wa l l i n v e s t t g a t r o n s . 46 The r e s u l t s of the elemental a n a l y s i s (Table V I I ) show reasonable agreement between the d i f f e r e n t c e l l wa 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 , However they do show t h a t c e l l w a l l s from d i f f e r e n t p r e p a r a t i o n s are not I d e n t i c a l . The amount of N i s r a t h e r h igh in both s p e c i e s examined. In T. mesenterica the amounts of N de r i ved from amino groups and NH3 account f o r p r a c t i c a l l y a l l of the N found. The high 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 the washing procedure i s not comp le te l y removed even a f t e r the 72 hr d i a l y s i s p e r i o d . In S. diclina the sum of N from amino groups and NH3 accounts f o r on l y 33% of the t o t a l N. Even in p rep -a r a t i o n #1, which conta ined s i g n i f i c a n t l y more p r o t e i n , the recovery of N from amino groups and NH3 was of the same o r d e r . E v i d e n t l y the re are o ther N - con ta in ing substances present in S. diclina. There i s no b a s i s f o r s p e c u l a t i o n as t o t h e i r i d e n t i t y . The presence of la rge amounts of S cannot be e x p l a i n e d in 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 absent and meth ion ine accounts f o r on l y a small p r o p o r t i o n of the t o t a l S found) . Phosphodtester l i nkages have been repor ted in fungal mannans (Cawley and L e t t e r s 1968), and Lloyd (1970b) has examined a p e p t i d o -phosphogaIactomannan in Cladosporium werneckii which c o n t a i n s 3.2% phosphate. T h i s might a l s o account f o r the P l e v e l s in T. mesenterica and S. diclina. H y d r o l y s i s and Degradat ion The C-N pept ide bond e x h i b i t s c o n s i d e r a b l e double bond c h a r a c t e r and thus i s s t a b i l i z e d by resonance (Spencer 1963). A l though a l l of the pept ide bonds in a p r o t e i n a re 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 , the 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 the approach of . E l e c t r o -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 i n f l u e n c e the s t a b i l i t y of each bond 47 t o h y d r o l y s i s ; the most important f a c t o r s i n f l u e n c i n g r a t e a re the e f f e c t i v e s i z e of the amino a c i d s i d e cha ins on e i t h e r s i d e of the pept ide 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 the bond. Pep t ide bonds i n v o l v i n g v a l i n e , w i th a bulky i sopropy l group, are most s t a b l e ; those w i th the group f a r t h e r removed, as in i s o l e u c i n e are l e s s s t a b l e ; those w i th s t e r i c f a c t o r s a t a minimum, l i k e g l y c i n e and a l a n i n e , are s t i l l more l a b i l e . When the s i d e cha in i s pa r t of the amino a c i d c o n t r i b u t i n g the ca rboxy l t o the pept ide l i n k i t has a g r e a t e r e f f e c t of the r a t e of h y d r o l y s i s than when i t i s pa r t of the amino a c i d t h a t c o n t r i b u t e s the amino group. In a c i d s o l u t i o n s ca rboxy l groups are uncharged and b a s i c groups tend t o repe l H + . When the b a s i c group i s in the s i d e cha in i t has l ess e f f e c t on h y d r o l y s i s than a f r e e a-amino group ad jacent t o the pept ide bond. T h i s i s i n d i c a t e d by an accumulat ion of d i p e p t i d e s in p a r t i a l h y d r o l y s i s of p r o t e i n s ( H a r r i s , Co le and Pon 1956; Spencer 1963). Pep t ide bonds i n v o l v i n g the amino groups of s e r i n e and t h r e o n i n e a re among the most l a b i l e in a c i d s o l u t i o n . Spencer (1963) p resents hypotheses t h a t have been advanced t o e x p l a i n t h i s l a b i l i t y ; they i n v o l v e p a r t i c i p a t i o n of the 8-0 in an o x a z o l i n e r i n g . In a d d i t i o n he d i s c u s s e s hypotheses t o e x p l a i n the p r e f e r e n t i a l r e l e a s e of a s p a r t a t e from p r o t e i n w i t h d i I u t e a c i d . Except under s p e c i a l c o n d i t i o n s of h y d r o l y s i s f o r t r y p t o p h a n , 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 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 of l i b e r a t e d amino a c i d s and p roduct ion of a r t i f a c t s (Spencer 1963). The g e n e r a l l y accepted mechanism of h y d r o l y s i s of g I y c o p y r a n o s i d i c bonds 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 the g l y c o s i d i c oxygen (a l though the p r o t o n a t i o n of the r i n g oxygen cannot 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 ev idence f a v o r s the g l y c o s i d i c oxygen) t o g i v e the con jugate a c i d . The con jugate a c i d decomposes t o a g l y c o s y l carbon ium-oxonium i o n , which then adds water (De Bruyne and Wouters-Leysen 1971). The carbonium-oxoniurn ion most probably e x i s t s in the h a l f - c h a i r c o n f i g u r a t i o n ( B e M i l l e r 1967). The mechanism i s . iI I us t ra ted w i th a 1 ,4 -8-D -gIucopyranose polymer in F i g u r e 11. De Bruyne and Wouters -Leysen (1971) showed t h a t in HCI the r e a c t i o n proceeds v i a carbonium-oxoniurn ions generated unimolecu Iar Iy from the con jugate a c i d . In H 2 S O 4 some of the c r i t e r i a were not in accordance w i th the un imo lecu la r mechanism but the authors a t t r i b u t e d t h i s not t o a change in the mechanism but t o the 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 general m e c h a n i s t i c c r i t e r i a . Few exper iments of t h i s type have been performed w i th f u r a n o s i d e s and the 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 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 ycopy ranos id ic l i nkages are u s u a l l y more r e a d i l y hydro lyzed than 3-D l i n k a g e s ; f u r a n o s i d i c l i n k a g e s are hydro lyzed under very m i l d c o n d i t i o n s . When the g l y c o s i d i c l i nkage i n v o l v e s the reduc ing group of a 2- -amino-2-deoxy a ldose the N H 3 + formed 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 the ne ighbor ing g l y c o s i d i c c o n s t i t u e n t s from a t t a c k by H + ; such bonds are much more s t a b l e t o a c i d h y d r o l y s i s (Jones and Per ry 1963). The g l y c o s i d i c l i nkage i n v o l v i n g the reducing group of the g l y u r o n i c a c i d shows st rong 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 the s t a b i l i t y of t h i s l i n k a g e . 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 are e a s i l y o x i d i z e d by atmospher ic oxygen, and, even when the r e a c t i o n s are c a r r i e d out under o x y g e n - f r e e n i t r o g e n , some degree of d e g r a d a t i o n , probably occas ioned by t r a c e s of oxygen, i s d i f f i c u l t t o avo id (Bouveng and L indberg 1960). 49 FIGURE II MECHANISM OF GLUCOPYRANOSIDE HYDROLYSIS alter BeMiller (1967) H.OH + H + 50 H y d r o l y s i s i s the predominant r e a c t i o n under c o n d i t i o n s where both the a c i d and polymer a re in d i l u t e s o l u t i o n s a t temperatures near 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 r e a c t i o n s and dehydrat ion r e a c t i o n s t h a t r e s u l t in the fo rmat ion of anhydro sugars and f u r f u r a l d e r i v a t i v e s ( B e M i l l e r 1967). Condensat ion products 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 , e s p e c i a l l y from the degradat ion of t ryptophan (James 1972). The comp iex i t y and compos i t ion of the 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 ime of heat ing (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 condensat ion products t h a t a re 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 in automat ic amino a c i d a n a l y s i s p rocedures . The use of CH 2SH-C00H in p r o t e i n h y d r o l y s a t e s (Matsubara and Sasaki 1969) t o prevent t ryptophan d e s t r u c t i o n a l s o serves t o reduce humin p r o d u c t i o n . In the presence of p o l y s a c c h a r i d e s t ryptophan i s degraded even w i th CH 2SH-C00H and humin p roduct ion i s i n c r e a s e d . The e f f e c t s of CH 2SH-C00H on amino a c i d 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 recommend the a d d i t i o n of CH 2SH-C00H o r CH 3-CHSH-C00H t o p r o t e i n h y d r o l y s a t e s c o n t a i n i n g t r y p t o p h a n . 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 suggests t h a t samples be hydro lyzed 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 before h y d r o l y s i s , t reatment w i th 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 pe r fo rmic a c i d before h y d r o l y s i s . N e v e r t h e l e s s in samples t h a t c o n t a i n s i g n i f i c a n t amounts of p o l y s a c c h a r i d e s , some humin w i l l be produced w i th a c i d h y d r o l y s i s . Some f u r f u r a l d e r i v a t i v e s ( i n c l u d i n g those de r i ved from hexoses) are converted t o l e v u l i n i c a c i d (Zachar ius and T a l l e y 1962; Anet 1972). Levu I i n i c ' a c i d r e a c t s w i th n i n h y d r i n t o produce a c o l o r e d product which i s e l u t e d from 51 the 58 cm column of the 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 (Zachar ius and T a l l e y 1962; Sentandreu and Nor thcote 1968). T a y l o r (1970) has detected 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 higher absorbance a t 440 nm than a t 570 nm, in h y d r o l y s a t e s of a mix tu re of hydroxypro I ine and s u c r o s e . Zachar ius and P o r t e r (1967) have examined a number of o the r non -n i t rogenous compounds (mainly monosacchar ides , d i s a c c h a r i d e s and r e l a t e d compounds) t h a t produce n i n h y d r i n - p o s i t i v e d e r i v a t i v e s . These peaks are a lmost a l l e l u t e d before a s p a r t a t e on the 58 cm column of the amino a c i d a n a l y z e r and have h igher absorbance a t 440 nm than a t 570' nm. They a l l show much lower c o l o r i n t e n s i t i e s than amino a c i d s . T a y l o r (1970) remarked t h a t the h y d r o l y s a t e s were pa le y e l l o w before 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 wi thout r e a c t i o n w i th n i n h y d r i n . In the present s t u d i e s , t h e r e were numerous very la rge peaks most of which were e l u t e d from the 13 cm column before g lucosamine , and from the 58 cm column before a s p a r t a t e . However the e l u t i o n was not sharp and the peaks o f t e n t a i l e d i n t o the amino a c i d peaks, render ing t h e i r area 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 these e f f e c t s cou ld be s i g n i f i c a n t l y reduced by d e l a y i n g the a d d i t i o n of n i n h y d r i n t o the column e l u a t e s u n t i l j u s t be fore the f i r s t amino a c i d s were e l u t e d . For the 13 cm column n i n h y d r i n was added t o the r e a c t i o n c o i l 10 min a f t e r the e l u t i o n s t a r t e d ; f o r the 58 cm column i t was added a t 33 min . T h i s procedure 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 these a r t i f a c t s of h y d r o l y s i s , t h e r e were severa l n i n h y d r i n - p o s i t i v e products w i th absorbances t h a t resemble those of amino a c i d - n i n h y d r i n peaks (h igher absorbance a t 570 nm than a t 440 nm). F i v e of these peaks were r e g u l a r l y observed in amino a c i d a n a l y s i s of a l l h y d r o l y s a t e s from T. mesenterica and S. diclina. T h e i r p o s i t i o n s do not correspond t o any p r o t e i n amino a c i d s , 1 - or 3 - m e t h y I h i s t i d i n e , 52 glucosamine or ga lac tosamine . The s i z e of these peaks tended t o decrease dur ing the course of h y d r o l y s i s but four were s t i l l de tected a f t e r h y d r o l y s i s f o r 145 h r . A s i m i l a r a r t i f a c t has been reported from h y d r o l y s i s of c e l l w a l l s t h a t c o n t a i n amino sugars (App legar th and Bozoian 1967; Kanetsuna e t a l . 1969). The s y n t h e t i c m ix tu re of amino a c i d s and g l u c o s -amine showed on l y the expected peaks on an amino a c i d a n a l y z e r chromato-gram. When t h i s m i x t u r e was sub jected t o h y d r o l y z i n g c o n d i t i o n s in 6 N H C l , the same f i v e a d d i t i o n a l peaks were observed . S ince the r e c o v e r i e s of the amino a c i d s were g e n e r a l l y high (F igu re 3) and t h a t of g lucosamine very low (F igure 4) these would appear t o be degradat ion products of glucosamine h y d r o l y s i s . T h i s view i s supported by the f a c t t h a t amino a c i d a n a l y z e r chromatograms of c e l l wa l l h y d r o l y s a t e s of Phaseolus vulgaris hypocoty ls (Chang, personal communication) and Avena sativa c o l e o p t i l e s ( O ' S u l i i v a n , personal communicat ion) , which do not c o n t a i n amino sugars , d i d not show any of these peaks. A l l of these a r t i f a c t s of h y d r o l y s i s a re presumably de r i ved from degradat ion or i n t e r a c t i o n of the monomers f o r e s t i m a t e s of which the a n a l y s e s were performed. The aim c f the a n a l y s i s , however, i s to determine the amounts of these monomers. Robe I and Crane (1972) have examined the ques t ion of degradat ion c f amino a c i d s dur ing p r o t e i n hydro -l y s i s . They observe t h a t the t r u e amino a c i d compos i t ion 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 determin ing the amino a c i d s when t h e i r pept ide bonds have been broken and before degradat ion 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 dur ing h y d r o l y s i s are 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 determin ing t r u e o r o r i g i n a l amounts a t zero t ime w i th data i n v o l v i n g s imultaneous y i e l d and decay. 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 polymer. The d e r i v a t i o n of equat ions assumes t h a t the monomers must be in one of t h r e e s t a t e s . 53 State A. The monomers are bound in the polymer and not observab le by a n a l y s i s . State B. The monomers a re hydro lyzed from the polymer ( S t a t e A) and observab le by a n a l y s i s . State C. Hydrolyzed anhydro monomers from S t a t e B are degraded and no longer observab le by a n a l y s i s . As the monomers proceed through the 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 are taken in S t a t e B. The problem r e s t s , t h e r e f o r e , in f i n d i n g the number of monomers i n i t i a l l y in S t a t e A us ing the o b s e r v a t i o n s taken in 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 cons tan t f o r each monomer of a g iven t y p e . The r a t e f o r each type i s then p ropor t iona I t o i t s number remaining in S t a t e A. T h i s assumption i s expressed by the f o l l o w i n g d i f f e r e n t i a l equat ion dA / dt = ~hA where A = the number of mo lecu les remaining in S t a t e A, and h = h y d r o l y s i s cons tan t in u n i t s of f r a c t i o n of S t a t e A per hour (h. i s assumed t o cons tan t throughout the experiment f o r any g i ven monomer). The r a t e of change of the number of hydro lyzed ( S t a t e B) mo lecu les equa ls the r a t e they a re coming out S t a t e A minus the r a t e they a re being l o s t t o S t a t e C. If 1 i s de f ined as the loss c o n s t a n t , and i t i s assumed t h a t the loss r a t e i s p r o p o r t i o n a l t o the number in S t a t e B, then the loss r a t e i s I B , thus dB / dt = (-da / dt ) - IB. The authors develop a method of n o n - l i n e a r l e a s t - s q u a r e s t o e s t i m a t e the o r i g i n a l monomer compost i ion of the polymer. A computer i s r e q u i r e d t o execute the program. A s i m i l a r approach t o the 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 Gheorgh iu , Oette and Baumann (1970). I t p rov ides approx imat ions f o r the 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 5 4 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 data suggest t h a t any r e s t r i c t i o n of f l e x i b i l i t y of a p o l y s a c c h a r i d e cha in reduces the r a t e of 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 the conformat iona l changes necessary f o r forming the h a l f - c h a i r carbonium-oxoniurn ion (F igu re 11) in more h i g h l y ordered systems. Sentandreu and Nor thcote (1968) have found O -g lycosy l l i nkages t o s e r i n e and t h r e o n i n e in yeast c e l l w a l l s . They a l s o suggested the e x i s t e n c e of a w - g l y c o s y l bond between w-acety lg Iucosamine and a s p a r a g i n e . Lamport (1967) has repor ted the presence of O - g lycosy l l i nkages t o hydroxypro I ine in the c e l l waI Is of h igher p l a n t s . Such l i nkages are 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 in fungal c e l l w a l l s such as T. mesenterica and S. diclina. T h i s s i t u a t i o n cou ld cause p o l y s a c c h a r i d e s in c e l l 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 the p r o t e i n s held t h e p o l y s a c c h a r i d e s in c o n f i g u r a t i o n s t h a t were l ess f l e x i b l e than those in pure p o l y s a c c h a r i d e s . 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 monosaccharides would not s i g n i f i c a n t l y a f f e c t pept ide bonds, and i f the g Iycosy I -amino a c i d l i nkages were even moderate ly s t a b l e the s t a b i l i t y of the l i nkages between the monosaccharide u n i t s might we l l be a l t e r e d . Bonds t h a t were l a b i l e i n the f r e e p o l y s a c c h a r i d e might become less so in the c e l l w a l l u n i t . T h i s s o r t of l i nkage i s impl ied by the recovery curves of 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 in p u r i f i e d p r o t e i n s show a negat i ve s lope throughout the course of h y d r o l y s i s ( f o r example, Robel and Crane 1972). The increased s t a b i l i t y of the t h r e o n i n e and s e r i n e pept ide bonds might be e x p l a i n e d t h u s : i f the 3-0 were invo lved in a g l y c o s y l bond, then i t cou id not forrp the o x a z o l i n e r i n g which i s thought t o l a b i l i z e the 55 pept ide bond. T h i s would leave the pept ide bond more r e s i s t a n t t o a c i d h y d r o l y s i s than 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 wa l l chemist ry because the p r o t e i n a n a l y s i s i s c a r r i e d out on a p r o t e i n t h a t i s contaminated w i th approx imate ly 90% of o the r components, most ly p o l y s a c c h a r i d e s . The p o l y s a c c h a r i d e i s on ly 80% ' p u r e ' . 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 degradat ion are d i f f i c u l t t o d e v i s e . It i s r e l a t i v e l y easy t o determine the r a t e of degradat ion of f r e e amino a c i d s and f r e e monosaccharides under the h y d r o l y z i n g c o n d i t i o n s used f o r c e l l wa l l p r e p a r a t i o n s . Neutra l and a c i d i c amino a c i d s are r e l a t i v e l y s t a b l e t o a c i d h y d r o l y s i s ; b a s i c amino a c i d s a re much less so (F igure 3 ) . About 15% of the neut ra l sugars was recovered a f t e r 8 hr in h y d r o l y z i n g c o n d i t i o n s (F igu re 5); mannose i s the most s t a b l e (85% recovery ) and x y l o s e the l e a s t (50%). Glucosamine i s as s t a b l e a f t e r 8 hr in 2 N HCl as mannose in 2 N CF 3 C00H, but on l y 30% i s recovered a f t e r 97 h r . Degradat ion 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 on ly 15$ i s recovered) and degradat ion i s v i r t u a l l y complete a f t e r 97 hr ( F igu re 5). These curves show the e f f e c t of the loss r a t e , IB, de f i ned by Robel and Crane (1972). F i g u r e s 6 t o 10 show curves i n v o l v i n g s imul taneous y i e l d and decay. In the case of neut ra l sugars the r e l a t i v e r a t i o s of degradat ion a f t e r maximal r e l e a s e in F i g u r e s 8 and 9 are of the same order as those in F i g u r e 5. For the amino a c i d s re leased from c e l l w a l l s ( F i g u r e s 6 and 7) the curves genera I l y have negat i ve s lopes t h a t are s teeper than those in F igu re 3 ( f r e e amino a c i d s ) . T h i s suggests t h a t the amino a c i d s in the c e l l wa l l p r e p a r a t i o n s are being degraded t o a g r e a t e r ex ten t than f r e e amino a c i d s under the same c o n d i t i o n s of h y d r o l y s i s . H y d r o l y s i s in 6 N HCl causes d e s t r u c t i o n of monosacchar ides; humin degrades and forms 56 complexes w i t h amino a c i d s . Thus the 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 throughout an experiment may in t roduce a s e r i o u s e r r o r i n t o s t u d i e s of c e l l w a l l h y d r o l y s i s . For t h i s reason the 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 ze ro t ime has not been used. The study of h y d r o l y s i s of c e l l w a l l s in 2 N HCI and 6 N HCI show the g ross inaccuracy of such a p l o t i f degradat ion i s e x t e n s i v e . The degradat ion of most amino a c i d s in the c e l l w a l l s seems e x t e n s i v e and a l though the t o t a l es t imate of amino a c i d s may be low, i t does represent a r e a l f i g u r e , based on a c t u a l recovery of each i n d i v i d u a l amino a c i d . The problem r e q u i r e s f u r t h e r s tudy . The 1 0 $ of the c e l l w a l l s not recovered may be accounted f o r when such problems are s o l v e d . A n a l y s i s of fungal c e l l w a l l s i s f u r t h e r compl icated by the f a c t t h a t r e p r o d u c i b l e c e l l wa l l p r e p a r a t i o n s are o f t e n d i f f i c u l t t o o b t a i n . B i o l o g i c a l d i v e r s i t y c o m p l i c a t e s q u a n t i t a t i v e a n a l y s i s . In s tudy ing the c e l l wa l l components in f u n g i , t h i s must be taken i n t o account , e s p e c i a l l y when making compar isons . The C e l l Wall P r e p a r a t i o n The components t h a t a fungus assembles t o produce the c e l l w a l l a re de r i ved through the a c t i o n of i t s m e t a b o l i c pathways on n u t r i e n t s from the medium on which i t i s g rowing . D i f f e r e n t s t r a i n s of the same fungus growing on the same medium may produce d i f f e r e n t c e l l wa l l components o r the same components in d i f f e r e n t p r o p o r t i o n s . 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 ques t ion of whether a fungus syn thes i zed c e l l wa l l from components t h a t are a l r e a d y a v a i l a b l e in abundance o r whether i t r e q u i r e s c e r t a i n components 57 f o r a s p e c i f i c c e l l wa l l assembly p a t t e r n cannot a t present be p r o p e r l y answered. Furthermore, many fung i w i l l grow on l y on media t h a t cannot be complete ly c h e m i c a l l y d e f i n e d . It 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 in the medium wi th those u l t i m a t e l y incorporated i n to the c e l l w a l l . For example, from the present s t u d i e s , S. diclina was grown in a medium c o n t a i n i n g peptone, yeast e x t r a c t , and D -g lucose. If c e l l u l o s e made up par t of the p o l y s a c c h a r i d e would 3 - D - xy lose , 6-D-mannose or a-L-fucose r e p l a c e or even p a r t l y r e p l a c e B-D -g lucose s i n c e they have the 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 ther sugars be converted t o D -g lucose and assembled i n to c e l l u l o s e ? If the 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 in the morphology of the fungus? There have been few i n v e s t i g a t i o n s of t h i s s o r t . Bulmer and Sans (1968) c u l t u r e d Cryptococcus neoformans on comp le te l y de f ined media d i f f e r i n g on ly in the sugar p r o v i d e d . T h e i r study was not of c e l l wa l l s t r u c t u r e but they d i d r e p o r t d i f f e r e n t responses in c a p s u l e development. A n g l u s t e r and Travassos (1972) grew Torulopsis pintolopesii in de f ined media w i th c h o l i n e or meth ion ine 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 , in the carbohydrates and amino a c i d s in the c e l l w a l l s . Nev ins , E n g l i s h and A lbersheim (1967) c u l t u r e d h igher p l a n t c e l l s (sycamore) in media c o n t a i n i n g d i f f e r e n t sugars and fcund d i f f e r e n c e s in the p r o p o r t i o n s ' o f the sugar components of the c e l l w a l l s . I d e a l l y a l l s t u d i e s of a fungus should be performed on the same s t r a i n o r c u l t u r e , in the same medium ( p r e f e r a b l y comp le te l y de f ined ) and grown f o r the same length of t ime under i d e n t i c a l c o n d i t i o n s . Yeast -1 ike c e l l s w i l l be more uni form when grown in synchronous c u l t u r e . When a n a l y s i s of the same s p e c i e s are compared, the s t r a i n , age of c u l t u r e and medium must be taken i n to account . ( Both T. mesenterica and 5 . diclina were grown on undef ined media , 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 ther c o n d i t i o n s the re was v a r i a t i o n among c e l l wa l l p r e p a r a t i o n s . The procedures f o r breaking fungal c e l l s a re dependent on the s p e c i e s , the c u l t u r e c o n d i t i o n s and the form of the fungus. Va r i ous methods f o r c e l l breakage have been d e s c r i b e d . S p e c i f i c information" about the s i z e of g l a s s beads ( in theory they should be of such .a d iameter t h a t the space formed when four beads come together te t rahedra I Iy i s . s l i g h t l y s m a l l e r than the diameter of the c e l l s being broken) , the p r o p o r t i o n of c e l l s and l i q u i d , the speed of r o t a t i o n of the breaking d e v i c e (where a d j u s t a b l e ) and the d u r a t i o n of the t rea tment (s ) i s e s s e n t i a l . In the present s t u d i e s , s o n i c o s c i l l a t i o n comple te ly 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 yeast c e l l s (Saccharomyces cerevisiae) or f i l a m e n t s of S. diclina. For each se t of c i rcumstances the opt imal c o n d i t i o n s f o r breaking 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 . With some t reatments complete c e l l breakage produces such f i n e c e l l wa l l fragments t h a t they are d i f f i c u l t t o c o l l e c t and wash, [n such cases i t i s p r e f e r a b l e t o reduce the breakage t reatment so t h a t c e l l wa l l f r agments 'a re l a r g e r , and t o separate the i n t a c t c e l l s from t h i s suspension 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 reak ing procedure has been found, t h e r e are on ly a few p recau t ions t o observe . The c e l l s should be kept c o l d a t a l l t i m e s . Aqueous s o l u t i o n s near pH 7 should be used. The c e l l w a l l s should be washed f r e e of c y t o p l a s m i c contaminat ion immediately a f t e r breakage to min imize enzymatic d e g r a d a t i o n . Where g l a s s beads are used in breaking the c e l l s , ca re must be taken t o assure the c o r r e c t i o n s f o r g l a s s fragments are a p p l i e d ( B a r t n i c k i - G a r c i a and N ickerson 1962: 59 i H o r i k o s h i and I ida 1964). Kanetsuna e t a l . (1969) used a h i g h - d e n s i t y medium (855?) sucrose) t o separate c e l l w a l l s from whole c e l l s and g l a s s d e b r i s . The purpose of washing the broken c e l l s i s t o remove a l l t r a c e s of cytoplasm and any components w i t h i n the c e l l wa l l t h a t are not a par t of i t . G e n e r a l l y the broken c e l l s are washed wi th water and d i l u t e aqueous s o l u t i o n s of NaCI, sucrose or both . T h i s procedure removes la rge amounts of cy top lasm. The c e l l wa l l fragments are recovered by c e n t r i f u g a t i o n or f i l t r 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 procedures t o avo id p o l y s a c c h a r i d e contaminat ion from the f i l t e r paper. Fu r the r washings are requ i red t o assure t h a t o ther components are complete ly removed from the c e l l w a l l s . M i t c h e l l and T a y l o r (1969) washed the c e l l w a l l s s u c c e s s i v e l y w i th 8 .0 M u rea , 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 t o c l e a v e c o v a l e n t bonds. NHt+OH and HC00H were chosen t o remove i o n i z a b l e components compartmental ized in the cytoplasm of the i n t a c t c e l l . When these compounds are re leased they may become a s s o c i a t e d w i th charged groups (-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 in b a s i c amino a c i d s and amino sugars) of the c e l l w a l l . Both t reatments cou ld cause h y d r o l y s i s of very weak bonds. In the present s t u d i e s the HCOOH t reatment was omit ted because i t caused f l o c c u l a t i o n of c e l l wa l l f ragments . Other t reatments have been used but they may a f f e c t the c e l l w a l l . L i p i d s have been w ide ly repor ted as eel I w a l l c o n s t i t u e n t s and any washings t h a t i n v o l v e e t h a n o l , e ther (Moreno, Kanetsuna and CarboneI I 1969), g l y c e r o l (Troy and K o f f l e r 1969) or de te rgents (Troy and K o f f l e r 1969; Zevenhuizen 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 the l i p i d 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 Knight 1968) but they may a l s o d i g e s t the c e l l w a l l i t s e l f ( M i t c h e l l and T a y l o r 1969); in general they should not be used in the pr imary p r e p a r a t i o n of ee l I waI I s . Crook and Johnston (1962) have d i s c u s s e d the d i f f e r e n c e s between the c e l l wa l l p r e p a r a t i o n and the f u n c t i o n a l c e l l w a l l . P r e p a r a t i o n of a r e p r o d u c i b l e c e l l wa l l f r a c t i o n from d i f f e r e n t c e l l p r e p a r a t i o n s i s the . a im. 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 c e l l wa l l components. At present the r e p r o d u c i b i l i t y of any c e l l wa l l p r e p a r a t i o n (even though i t may not i nc lude a l l of the f u n c t i o n a l c o n s t i t u e n t s ) i s p r e f e r a b l e so t h a t a body of b a s i c s t r u c t u r a l i n fo rmat ion can be o b t a i n e d . When the fundamental s t r u c t u r e i s e s t a b l i s h e d , more r e f i n e d procedures can be developed t o i n v e s t i g a t e those components t h a t are a s s o c i a t e d w i t h the c e l l wa l l by l a b i l e l i n k a g e s . The ' p u r i t y ' of the c e l l wa l l p r e p a r a t i o n i s u s u a l l y de f i ned as the absence of cytoplasm (and c a p s u l e i f the c e l l s possess one) . T h i s i s w ide l y determined by phase or dark f i e l d l i g h t m ic roscopy ; f o r r i g o r o u s q u a n t i t a t i v e a n a l y s i s the e l e c t r o n microscope should be used t o con f i rm the absence of c y t o p l a s m i c (and c a p s u l a r ) c o n t a m i n a t i o n . Most i n v e s t i g a t o r s accept the e l e c t r o n microscope assay as s u f f i c i e n t ev idence f o r ' p u r i f i e d ' c e l l wa l l p r e p a r a t i o n s . Chemical t e s t s f o r ' p u r i t y ' are not w ide ly used. Absence of n u c l e i c a c i d s o r t h e i r pu r ine and p y r i m i d i n e bases i s u s u a l l y taken t o show lack of cy top lasm, yet B a r t n i c k i - G a r c i a and N ickerson (1962) and Moreno e t a i . (1969) found t r a c e s of these substances in c e l l wa l l p r e p a r a t i o n s t h a t they accepted as ' p u r e ' . The present s t u d i e s have conf i rmed t h a t q u a n t i t a t i v e a n a l y s i s of fungal c e l l w a l l s i s p o s s i b l e w i th c e r t a i n l i m i t a t i o n s . The methods 61 chosen f o r e s t i m a t i o n of neut ra l sugars (p lus o ther complementary procedures t h a t are requ i red in many c a s e s ) , amino sugars and amino a c i d s a re 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 wa l l p r e p a r a t i o n s . R e l i a b l e q u a n t i t a t i v e u r o n i c a c i d recovery i s not a t p resent p o s s i b l e ; the method s e l e c t e d i s not s u i t a b l e t o p rov ide the requ i red i n f o r m a t i o n . Severa l p o s s i b l e a l t e r n a t i v e s a re a v a i l a b l e . The procedures used f o r l i p i d and elemental a n a l y s i s prov ided gross i n f o r m a t i o n ; more r e f i n e d techn iques which are 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 w a l l s t u d i e s . The procedures have been assembled and t e s t e d t o p rov ide the foundat ion f o r a comprehensive long- range p r o j e c t in t h i s l abo ra to ry t o b u i l d a model of the c e l l wal l and to e x p l a i n the mechanisms of i t s growth . They have been s e l e c t e d t o p rov ide b a s i c q u a n t i t a t i v e i n fo rmat ion about c e l l wa l l monomers. T h i s i n fo rmat ion i s e s s e n t i a l to complete e l u c i d a t i o n of c e l l wa l l s t r u c t u r e a t the leve l of fundamental c o n s t i t u e n t s and a t each succeeding leve l of s t r u c t u r a l a n a l y s i s . T h e i r a p p l i c a t i o n i s intended t o be broad so the techn iques and subsequent improvements can be a p p l i e d t o the study of c e l l wa l l s t r u c t u r e in fungi and o the r o rgan isms. In t h i s r espec t the study may rep resent the f i r s t comprehensive attempt t o comple te ly q u a n t i t i z e c e l l wa l l a n a l y s i s . 62 BIBLIOGRAPHY AARONSON, S . 1970. M o l e c u l a r ev idence f o r e v o l u t i o n in the a l g a e : a p o s s i b l e a f f i n i t y between p l a n t c e l l w a l l s and animal s k e l e t o n s . Ann. N. Y. Acad. Sci. 175: 531-540. ADAMS, G. A. 1965. Complete a c i d h y d r o l y s i s , p. 269-276. In Methods in Carbohydrate Chemistry V. ALBERSHEIM, P . , D. J . NEVINS, P. D. ENGLISH and A. KARR. 1967. A method f o r the a n a l y s i s of sugars in p l a n t c e l l - w a l l p o l y s a c c h a r i d e s by g a s -l i q u i d chromatography. Carbohyd. Res. 5: 340-345. AMINOFF, D. 1970. 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Biochemistry 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 arg i n i ne Leu l euc i ne Asp a s p a r t a t e Lys l y s i n e Asx a s p a r t a t e o r asparag ine Man mannose (undef i ned) Cys c y s t e i ne MeHis m e t h y 1 h i s t l d i n e Ery e r y t h r i t o l Met methionine Fuc fucose Phe p h e n y l a l a n i n e Gal ga1actose Pro p ro1 i ne GalN ga1actosam!ne (2 -amino - Qpa a - a m i n o - 8 - g u a n i d i n o p rop ionate 2 -deoxy -ga1actose) Glc g1ucose Rha rhamnose GlcN glucosamine (2 -amlno - Rib r i b o s e 2 -deoxy -g Iucose GlcNx glucosamine o r w - a c e t y l - Ser s e r i ne glucosamine (undef ined) Glu g1utamate Thr t h r e o n i n e Glx glutamate o r g lutamine Trp t ryptophan (undef ined) Gly g l y c i ne Tyr t y r o s i ne His h i s t i d i n e Val va 1 i ne Hyp 4-hydroxypro1 i ne Xyl x y l o s e 

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