15.9. 1972

Specialia

on Gas C h r o m Q. The m a j o r p e a k (95% of t h e m a s s of t h e sample) h a d t h e s a m e r e t e n t i o n t i m e as cholesterol a c e t a t e . A m i n o r p e a k was p r e s e n t w i t h a r e t e n t i o n t i m e of 0.18 'relative to cholesterol a c e t a t e . This c o m p o n e n t b e h a v e d in a m a n n e r similar t o cholestane, leading t o t h e conclusion t h a t it was a h y d r o c a r b o n of s o m e sort. Mass s p e c t r o m e t r y of t h e b r i n e s h r i m p sterol a c e t a t e was p e r f o r m e d b y t h e M o r g a n - S c h a e f e r Corp. (Montreal, Quebec). T h e s p e c t r u m o b t a i n e d , a g r e e d well w i t h a s p e c t r u m of cholesterol a c e t a t e 1l. A second p a r e n t ion was f o u n d a t m/e 430 i n d i c a t i n g t h a t c h o l e s t a n o l (cholestane3-oi) was p r e s e n t . This is n o t s u r p r i z i n g as c h o l e s t a n o l a c e t a t e is p o o r l y s e p a r a t e d f r o m cholesterol a c e t a t e in m o s t gas c h r o m a t o g r a p h i c s y s t e m s t~. On t h e basis of t h e s e studies, we h a v e c o n c l u d e d t h a t t h e sterol f r a c t i o n of A. salina f r o m Mono L a k e is comp o s e d p r i m a r i l y of c h o l e s t e r o l w i t h a s i g n i f i c a n t perc e n t a g e of c h o l e s t a n o l also p r e s e n t . The m i n o r c o m p o n e n t on t h e gas c h r o m a t o g r a p h m a y h a v e b e e n c a n t h a x a n t h i n or a n o t h e r c a r o t e n i o d c o m m o n l y p r e s e n t in b r i n e s h r i m p 13 I t is difficult to c o m p a r e t h e results of t h e p r e s e n t s t u d y w i t h t h o s e of o t h e r w o r k e r s as t h e p o p u l a t i o n of b r i n e s h r i m p used here is physiologically d i s t i n c t ~, 2 from t h o s e used in o t h e r studies. I n a d d i t i o n , TYsoN ~4 has o b s e r g e d a s p i r o c h e t e - l i k e o r g a n i s m in t h e tissues of s o m e s p e c i m e n s of A. salina. As our s h r i m p s were h a r v e s t e d f r o m a n a t u r a l source, we c a n n o t rule out t h e p o s s i b i l i t y that i n f e c t e d individuals were p r e s e n t . TESHIMA a n d ~4~ANAZAWAt5 h a v e d e m o n s t r a t e d t h a t A. salina can c o n v e r t d i e t a r y ergosterol into cholesterol. I n this s t u d y gas c h r o m a t o g r a p h i c d a t a was p r e s e n t e d s h o w i n g cholesterol t o be t h e o n l y sterol in Euglena-fed

1023

b r i n e s h r i m p . H o w e v e r , t h e i r s y s t e m , like ours, w o u l d h a v e separated cholesterol a n d c h o l e s t a n o l w i t h d i f f i c u l t y 1~. A s s u m i n g t h a t c h o l e s t a n o l was also p r e s e n t in t h e i r sterol sample, it n o w a p p e a r s e v i d e n t t h a t t h e e x t r e m e e n v i r o n m e n t of Mono L a k e has n o t r e q u i r e d a c h a n g e in t h e q u a l i t a t i v e sterol c o m p o s i t i o n oI A. salina.

Zusammen/assung. Artemis salina v o m Mono-See, K a lifornien, U . S . A . , e n h ~ l t Cholesterin u n d C h o l e s t a n o l als ihr haupts~tchlichstes Sterin. Diese Z u s a m m e n s e t z u n g ist ~hnlich wie die jenige y o n im L a b o r a t o r i u m e r z e u g t e n S a l z - K r a b b e n u n d z w a r t r o t z d e m das W a s s e r des MonoSees ein p H yon 9.6 u n d einen Salzgehalt y o n 2.23 M h a t ~6. T m PAYNE a n d S. S. KUWAItARA 17

Department of Chemistry, Cali/ornia State College at Long Beach, Long Beach (Cali/ornia 90801, USA), 22 February 7972.

11 B. A. KNIGHTS,J. Gas Chromat. 5,273 (1967). 12 G. W. PATTERSON,Analyt. Chem. 43, 1165 (1971). t~ B. H. DAvl~s, W.-]. Hsu and C. O. CHICHESTER,Comp. Biochem. Physiol. 33, 601 (1970). 14 G. E. TYSON, J. Invert. Path. 75, 145, (1970). 15 S. TESHIJ~IAand A. KANAZAWA,Coinp. Bioehem. Physiol. 38B, 603 (1971). 16 Acknowledgment. This work was supported, in part, by a grant from the Long Beach (California) Heart Association. 17 Present address: Department of Developraental and Celi Biology, University of California, Irvine (CaliIornia 92664, USA).

A Site of A c t i o n of Light on 14C-Acetate I n c o r p o r a t i o n into H u m a n S k i n S t e r o l s I t was r e c e n t l y s h o w n t h a t b r o a d s p e c t r u m l i g h t caused m a r k e d r e d u c t i o n s in t h e level of ~4C-acetate i n c o r p o r a t e d i n t o sterols of h u m a n skin 1. I n c o r p o r a t i o n of a c e t a t e i n t o o t h e r classes of lipids was similarly a f f e c t e d b y light. E x a m i n a t i o n of t h e sterol b i o s y n t h e t i c p a t h w a y r e v e a l e d t h a t light h a d no effect o n t h e i n c o r p o r a t i o n level of m e v a l o n a t e , t h e c o m m i t t e d s t e p in sterol s y n t h e s i s . Nor were t h e r e s i g n i f i c a n t effects u p o n r e s p i r a t o r y r a t e s of i r r a d i a t e d tissues. I t was suggested, f r o m t h e s e o b s e r v a tions, t h a t t h e i n h i b i t o r y effect of light was a t a c o m m o n p o i n t in t h e lipid s y n t h e t i c p a t h w a y s a n d p r o b a b l y inv o l v e d a c e t a t e a c t i v a t i o n or t h e a v a i l a b i l i t y of acetyl C o e n z y m e A pools ot sufficient size to s u s t a i n e n d o g e n o u s lipogenesis. B e c a u s e sufficient q u a n t i t i e s of f r e s h h u m a n skin were n o t available for isolation a n d direct m e a s u r e -

m e n t of a c e t a t e a c t i v a t i n g e n z y m e , i n d i r e c t lines of e v i d e n c e were s o u g h t to elucidate t h e specific site(s) of action of l i g h t u p o n skin lipogenesis a n d to assess t h e i r physiological i m p o r t a n c e . Materials and methods. F r e s h h u m a n skin was o b t a i n e d , i r r a d i a t e d , a n d p r o c e s s e d e x a c t l y as p r e v i o u s l y d e s c r i b e d ~. A f t e r i r r a d i a t i o n t h e skin s p e c i m e n s were placed in 15 ml m a n o m e t r i c flasks c o n t a i n i n g 2.0 rnl of K r e b s - R i n g e r p h o s p h a t e buffer, p H 7.4, 2 m M glucose, a n d t h e app r o p r i a t e r a d i o - l a b e l l e d i n t e r m e d i a t e or precursor. I n

Table I. Incorporation of exogenous aH-acetyl CoA into skin sterols a

Table II. Effects of broad spectrum light on aeetyl Co A incorporation into skin sterols

Incubation (min)

1 H. S. BLACK and E. W. RAUSCHKOLB,J. Invest. Derm. 56, 387 (1971).

cpm/100 mg tissue wt. cpm/100 mg tissue wt.

45 90 180 360

237 261 388 1,394

Free sterols were isolated from total lipid extracts by thin layer chromatography. The chromatograms were developed in a 1,2dichloroethane solvent system.

Control Irradiated

ltC-acetate

aH-acetyl Co A

6,240 (--79%) 1,292

1,075 (--25%) 0,808

Values represent the mean of 3 experiments. Final concentration ol aH-aeetyl Co A was 2.5 • 10-6M.

1024

Specialia

some cases t h i s c o n s i s t e d of a H - A c e t y l C o e n z y m e A {1.2 Ci/mmoles) a t a final c o n c e n t r a t i o n of 2.0 • 1 0 - ~ M or i b~C of l~C-acetate (52.9 m C i / m m o l e s ) . O t h e r flasks cont a i n e d n o n - l a b e l l e d a c e t y l Co A a t c o n c e n t r a t i o n s r a n g i n g f r o m 1 • 10 -~ t o 2.5 • 1 0 - 6 M and, in a d d i t i o n , 1 btC of 14C-acetate. Pyruvate-2-*4C was used in some studies (1 b~C; 4.04 mC/m2VI). T h e flasks were s h a k e n (120 s t r o k e s / rain) on a Gilson r e s p i r o n l e t e r for 6 h a t 37~ A f t e r t h e i n c u b a t i o n period t h e r e a c t i o n s were h a l t e d , t h e tissue s e c t i o n e d on a freezing s t a g e m i c r o t o m e , a n d t o t a l lipids e x t r a c t e d 2. F r e e sterols were i s o l a t e d as t h e d i g i t o n i d e s a. A l i q u o t s of a c h l o r o f o r m - m e t h a n o l s o l u t i o n (2:1 v/v) of t h e d i g i t o n i d e s were d r i e d u n d e r n i t r o g e n a n d levels of r a d i o a c t i v i t y d e t e r m i n e d b y liquid s c i n t i l l a t i o n spectrom e t r y . C o u n t i n g efficiencies for 14C a n d aH were 83% a n d 34%, respectively. Results and discussion. T h e s y n t h e s i s of a c e t y l Co A is k n o w n to r e s u l t f r o m t h e d e g r a d a t i o n of c a r b o h y d r a t e s or glucogenic a m i n o acids, v i a p y r u v i c acid, as well as f r o m t h e o x i d a t i o n of f a t t y acids. I n r o d e n t skin t h e p r i n c i p a l sources of s u b s t r a t e s for e n d o g e n o u s o x i d a t i o n are lipids 4. T h e s e o x i d a t i o n s occur i n t r a m i t o c h o n d r i a l l y a n d r e s u l t in f o r m a t i o n of acetyI Co A, t h e p r e c u r s o r for lipogenesis. O n t h e o t h e r h a n d m o s t lipid s y n t h e s i s occurs e x t r a m i t o chondrially and probably requires mitochondrial produced a c e t y l Co A. I t was s h o w n t h a t , in t h e case of r a t liver, a c e t y l Co A diffuses across t h e m i t o c h o n d r i a l m e m b r a n e v e r y slowly a n d t h e r a t e of diffusion of a c e t y l Co A could n o t a c c o u n t for t h e r a t e s of lipogenesis o b s e r v e d . T h e a l t e r n a t i v e possibilities for f o r m a t i o n of lipogenic s u s t a i n ing levels of e x t r a - m i t o c h o n d r i a l a c e t y l Co A h a v e b e e n discussed 5.

Table III. Effects of aeetyl Co A on 14C-acetate incorporation into sterols by irradiated skin cpm/100 mg tissue wt. Treatment

l~C-aeetate

laC-acetate + acetyl Co A

1 Control Irradiated 2 Control Irradiated 3 Control Irradiated

4,811 1,367 (--70%) 5,183 2,204 (--57%) 4,975 1,270 (--75%)

6,251 0,712 (--89%) 4,970 2,743 (--45%} 3,183 0,772 (--76%)

Values are from single experiments. Concentrations of non-labelled acetyl Co A are 2.5 • 10-6M, 5 • 10 SM, and 1 • 10 aM for treatments 1-3, respectively.

R e g a r d l e s s of t h e m e c h a n i s m i n v o l v e d for t h e t r a n s f e r of m i t o c h o n d r i a l a c e t y l Co A to t h e sites of sterologenesis, we h a v e s h o w n t h a t e x o g e n o u s a c e t y l Co A does diffuse i n t o h u m a n skin slices a n d is i n c o r p o r a t e d i n t o sterols (Table I). W h e n i r r a d i a t e d skin s p e c i m e n s were i n c u b a t e d in t h e p r e s e n c e of t r i t i a t e d a c e t y l Co A o n l y 25 % i n h i b i t i o n of isotope i n c o r p o r a t i o n occured as c o m p a r e d w i t h 79% inh i b i t i o n o b t a i n e d w i t h labelled a c e t a t e (Table II). If a v a i l a b i l i t y of a c e t y l Co A is t h e r a t e - l i m i t i n g step for sterologenesis in i r r a d i a t e d skin t h e n t h e s l i g h t i n h i b i t i o n ( a b o u t 30% of t h a t o b t a i n e d w i t h a c e t a t e ) o b s e r v e d w i t h labelled a c e t y l Co A could r e p r e s e n t i n a b i l i t y to o v e r c o m e t h i s step due to low d i f f u s i o n r a t e s of e x o g e n o u s a c e t y l Co A i n t o t h e tissue. A l t e r n a t i v e l y t h e r e s i d u a l i n h i b i t i o n could rep r e s e n t a second m i n o r site of i n h i b i t i o n f r o m a c e t y l Co A to m e v a l o n a t e . T h e fact, however, t h a t 7 0 % of t h e u s u a l i n h i b i t o r y effect was n o t p r e s e n t w h e n sterologenesis was t r a c e d w i t h a c e t y l Co A, i n d i c a t e s t h a t t h e p r i n c i p a l i n h i b i t o r y site(s) of l i g h t o n sterologenesis is p r i o r t o t h e f o r m a t i o n of a c e t y l Co A. W i t h t h e k n o w l e d g e t h a t 7 0 % of t h e i n h i b i t o r y effects of l i g h t were n e g a t e d w h e n sterologenesis was followed b y i n c o r p o r a t i o n of e x o g e n o u s a c e t y l Co A, skin slices were i n c u b a t e d in t h e p r e s e n c e of several c o n c e n t r a t i o n s of n o n - l a b e l l e d a c e t y l Co A in a d d i t i o n t o 14C-acetate. As s h o w n in T a b l e I I I t h e i n h i b i t o r y effect of l i g h t on ~4C-acetate i n c o r p o r a t i o n i n t o t h e sterol fract i o n was n o t affected b y a n y of t h e c o n c e n t r a t i o n s of a c e t y l Co A tested. T h e s e d a t a i n d i c a t e t h a t a c e t a t e a c t i v a t i o n is t h e r a t e - l i m i t i n g s t e p of sterologenesis in i r r a d i a t e d skin r a t h e r t h a n a c e t y l Co A pool size. T h e i n c o r p o r a t i o n r a t e s of 14C-acetate s h o u l d parallel, b u t n o t necessarily reflect, t h e a c t u a l r a t e of lipogenesis ~, v. I t h a s b e e n a r g u e d t h a t glucose is t h e p r i m a r y s u b s t r a t e for lipogenesis in skin s. I n o u r earlier s t u d i e s t h e experim e n t a l c o n d i t i o n s e x c l u d e d t h e a d d i t i o n of glucose t o t h e i n c u b a t i o n m e d i u m of t h e skin slices a n d h e n c e s h o u l d reflect, a f t e r 1 t o 2 h i n c u b a t i o n w h e n e n d o g e n o u s glucose w o u l d b e e x h a u s t e d , lipogenic levels s u p p o r t e d f r o m t h e o x i d a t i o n of lipids. W e h a v e found, as h a v e others, t h a t t h e a d d i t i o n of glucose t o t h e i n c u b a t i o n m e d i u m of excised skin g r e a t l y e n h a n c e s t h e i n c o r p o r a t i o n levels of a c e t a t e . H o w e v e r , t h e a d d i t i o n of glucose does n o t a l t e r t h e effect of l i g h t u p o n a c e t a t e i n c o r p o r a t i o n . F u r t h e r more, w h e n p y r u v i c acid is used to t r a c e sterologenesis, t h e effects of l i g h t u p o n i n c o r p o r a t i o n of t h i s glycolytic e n d - p r o d u c t is v e r y s i m i l a r t o t h a t of a c e t a t e (Table IV). T h e effect of l i g h t u p o n p y r u v i c acid i n c o r p o r a t i o n i n t o sterols could r e s u l t f r o m i n h i b i t i o n of p y r u v i c acid dec a r b o x y l a t i o n . A l t e r n a t i v e l y , b e i n g o n l y a few steps rem o v e d f r o m a c e t y l Co A it m i g h t reflect t h e i m p o r t a n c e of a c e t a t e as a n i n t e r m e d i a t e in sterologenesis. I n skin, if e x t r a m i t o c h o n d r i a l a c e t y l Co A is f o r m e d b y t h e a c t i v a t i o n of a c e t a t e d e r i v e d f r o m m i t o c h o n d r i a l a c e t y l Co A, t h e i n h i b i t o r y effect of l i g h t o n a c e t a t e a c t i v a t i o n could

Table IV. A comparison of the effects of broad spectrum light on incorporation of 14C-acetate and 14C-pyruvate into human skin sterols a

cpm/100 mg tissue wt.

Control 2844 (--76.6%)

Acetate

Pyruvate

Irradiated 667

Control 587 (--69.8%)

5 6 Irradiated 194

s 9

Values represent the mean of 3 experiments.

EXPERIENTIA 28/9

j. FOLCH, M. LEES and G. H. SLOANE STANLEY, .[. biol. Chem. 226, 497 (1957). W.M. SPERRY and M. WEBB, J. biol. Chem. 187, 97 (1950). C. N. D. CRUIKSHA~K,M. D. TmOTTmR and J. R. CooPEG J. Invest. Derm. 39, 175 (1962). j. M. LowElvsrxI>r, Biochem. Soc. Symp. 24, 57 (1963). R.J. E~aERSONand J. T. VA~ BRUGGEN,Arch. Bioehem. Biophys. 77, 467 (1958). W. G. DUNCO=BE, Biochem. J. 106, 179 (1968). V. R. WHEATLEY, L. T. HODGINS and W. M. CooN, J. Invest. Derm. 54, 288 (1970). Supported in part by a grant from the Morrison Trust of San Antonio (Texas, USA).

15.9. 1972

Specialia

r e s u l t i n a s u b s t a n t i a l effect u p o n Iipogenesis. A l t h o u g h t h e p r e s e n t d a t a i n d i c a t e t h a t one i n h i b i t o r y site of a c t i o n of l i g h t is u p o n a c e t a t e a c t i v a t i o n t h e y do n o t p r e c l u d e a n effect o n o t h e r s u s c e p t i b l e sites p r i o r t o t h e f o r m a t i o n of a c e t y l Co Ag.

Zusammen/assu~g. Die S y n t h e s e y o n S t e r o l e n in m e n s c h l i c h e r H a u t , welche i m l a n g w e l l i g e n S p e k t r a l b e r e i c h b e s t r a h l t w o r d e n war, w u r d e m i t r a d i o a k t i v m a r k i e r t e m Acetyl-Co A, A c e t a t a n d P y r u v a t geprilft. V o n Acetyl-Co A k o n n t e n u r geringe H e m m u n g n a c h g e w i e s e n werden, w~thrend L i c h t eine m e r k b a r e H e m m u n g y o n A c e t a t u n d P y r u v a t i n k o r p o r a t i o n v e r u r s a c h t e . Die Re-

1025

s u l f a t e lassen v e r m u t e n , dass die A c e t a t a k t i v i e r u n g die y o n L i c h t b e e i n f l u s s b a r e Stufe in d e r B i o s y n t h e s e y o n S t e r o l e n ist. H. S. BLACK, J. D. SMITH, B. J. CUMBUS a n d W. t3. L o

Veterans Administration Hospital and Departments of Dermatology and Biochemistry, Baylor College o/ Medicine, Houston (Texas 77037, USA), 6 March 1972.

Keratin Decomposition by Dermatophytes: Evidence Of the Sulphitolysis of the Protein D e r m a t o p h y t e s are c a p a b l e of d e c o m p o s i n g k e r a t i n , s u l p h i t e is also p r o d u c e d f r o m p r o t e i n - i n c o r p o r a t e d r e s i s t a n t s c l e r o p r o t e i n v e r y rich in cystine. N e v e r t h e l e s s , cystine, t h e f u n g u s could use it for s p l i t t i n g d i s u l p h i d e t h e m e t a b o l i s m of s u l p h u r in t h e s e f u n g i h a s b e e n g i v e n b o n d s of k e r a t i n . K e r a t i n d e n a t u r e d b y ' s u l p h i t o l y s i s ' s m a l l a t t e n t i o n so i a r 1-~. Therefore, we e x a m i n e d t h e w o u l d t h e n b e easily accessible to t h e p r o t e i n a s e s of t h e g r o w t h of t h e d e r m a t o p h y t e Microsporum gypseum o n fungus. m e d i a c o n t a i n i n g 0.1% cystine. As b a s i c n u t r i e n t s , t h e T h e r e s u l t s c o r r o b o r a t i n g t h i s h y p o t h e s i s were o b t a i n e d m e d i a c o n t a i n e d 1 % gelatin, s e r u m a l b u m i n or casein or b y t h e a n a l y s i s of c u l t u r e f i l t r a t e s of Microsporum gypseum 0 . 8 - 1 . 0 % glucose c o m b i n e d w i t h 0 . 1 - 0 . 4 % p e p t o n e , growing on h u m a n h a i r s in m i n e r a l m e d i u m (Table). I n g l u t a m i n , u r e a or (NH4)2HPO ~. On all media, c y s t i n e was s t a t i o n a r y culture, t h e f u n g u s m a n a g e d t o digest a b o u t i n t e n s i v e l y m e t a b o l i z e d a n d its s u l p h u r a l m o s t q u a n 3 2 % of t h e s u b s t r a t e in 60 days. E v e n here, t h e m a i n titatively converted to sulphate excreted into the culture p r o d u c t of c y s t i n e o x i d a t i o n was s u l p h a t e , w h o s e concenfluid. A p a r t f r o m s u l p h a t e as f i n a l o x i d a t i o n p r o d u c t , we t r a t i o n in t h e m e d i u m a m o u n t e d to 1.6 m g / m l . F r e e h a v e also f o u n d s u l p h i t e in some c u l t u r e filtrates. I t used s u l p h i t e was n o t p r e s e n t in t h e filtrate. I t could h o w e v e r t o a p p e a r in a m o u n t s of several t e n s u p to h u n d r e d s b e d e m o n s t r a t e d , t h a t S - t h i o s u l p h a t e esters ( ' b o u n d # g / m l . I n m o s t of t h e media, no free s u l p h i t e could b e s u l p h i t e ' ) were p r e s e n t . T h e gel f i l t r a t i o n o n S e p h a d e x d e t e c t e d i n a n y p h a s e of t h e g r o w t h . H o w e v e r , considG-50 a n d G-10 p r o v e d t h a t t h i o s u l p h a t e e s t e r g r o u p s are e r a b l e a m o u n t of t h i s c o m p o u n d was f o u n d a f t e r t r e a t b o u n d t o c o m p o u n d s of m o l e c u l a r w e i g h t a m o u n t i n g t o m e n t b y a l k a l i n e c y a n i d e (NaCN, f i n a l c o n c e n t r a t i o n several t h o u s a n d s , i.e. obviously, t o p o l y p e p t i d e s . T h i s 0 . 5 % ; E D T A 10-~M; p H 10-11, 50~ 30 rain). T h i s is i n a g r e e m e n t w i t h t h e p r e s u m p t i o n t h a t t h e c o m p o u n d s ' b o u n d s u l p h i t e ' o b v i o u s l y r e p r e s e n t s t h e S-sulphocysu n d e r s t u d y are p e p t i d e s c o n t a i n i n g c o m b i n e d S-sulphoteine, w h i c h o r i g i n a t e d b y a n o n e n z y m i c r e a c t i o n becysteine. T h e q u a n t i t y f o u n d ( c o r r e s p o n d i n g t o 15-20 t w e e n c y s t i n e a n d sulphiteS: # g / m l of S - s u l p h o c y s t e i n e ) is, t a k e n a b s o l u t e l y , n o t large. R-S.S-1R + H S O ' ~ .~ R - S H + R-S.SO'~. H o w e v e r , it r e p r e s e n t s 2 5 - 6 0 % of all c o m b i n e d c y s t e i n e S - s u l p h o c y a t e i n e (as well as o t h e r S - t h i o s n l p h a t e esters) a n d its d e r i v a t i v e s in t h e m e d i u m . T h i s d e m o n s t r a t e s t h a t c a n b e split b y c y a n i d e to t h i o c y a n a t e a n d s u l p h i t e S : R-S.SOat-I + C N ' -~ R - S C N + HSO'~. T h e r e f o r e we c o n c l u d e d t h a t on o u r m e d i a it was a l w a y s 1 W. I-I. STAHL, B. McQuE, G. R. ~V~ANDELSand G. H. SIu, Arch. also s u l p h i t e t h a t was p r o d u c e d besides s u l p h a t e . I n m o s t Bioehem. 20, 422 (1949). cases t h i s s u l p h i t e d i s a p p e a r e d due t o t h e r e a c t i o n w i t h 2 G.E. MATmSON, Mycopath. 1Vfycol. appl. 27, 225 (1965). excess cystine. T h e c a p a c i t y of p r o d u c i n g s u l p h i t e b y 3 H. ZIEGLER and G. I~EICHMANN,Mykosen 7 I, 903 (1968). o x i d a t i o n of c y s t i n e was l a t e r p r o v e d b y us also in o t h e r " 4 H. G. SCItAPER nnd H. ZI:EGLER, Vortrag, 5. Tagung der Gesellspecies of d e r m a t o p h y t e s . sehaft flit Medizinische Mykologie der DDR, Leipzig, 7.-10.5.1970. T h e f i n d i n g s d e s c r i b e d m a y b e of i m p o r t a n c e for eluciKurzreferat: Mykosen 14, 589 (1971). d a t i n g k e r a t i n d e c o m p o s i t i o n b y d e r m a t o p h y t e s . Since a B. MILLIOANand J. M. SwA~r Rev. pure appl. Chem. 12, 72 (1962}.

Keratin decomposition by Microsporum gypseum.Analysis of the culture/luid Days Substrate digestion (%) pH Proteins (~tg/m~) Sulphate (ixg/ml) Bound sulphite Qzg/mi)

3

ll

17

24

32

40

50

60

0 6.4 110 18 0

4 8.0 560 147 4.1

16 8.3 365 490 10.0

18 8.4 310 538 13.4

25 8.2 465 842 16.7

26 8.1 440 1187 19.0

29 8.2 440 1499 19.0

32 8.1 380 1641 18.5

Cultures with 400 mg of ethylene oxide-sterilized human hairs in 20 ml of simple mineral solution, 29 ~ Substrate digestion was calculated from total dry weight (substratc + mycelium). Proteins (Lowry method) are expressed as p.g/ml bovine serum albumin, sulphate and sulphite as [xg/ml of anhydrous natrium salts. No free sulphite was found; 'bound sulphite' was determined after treatment with cyanide {see text).