1008
Specialia
t h e n e w m e t a b o l i t e . T h i s o b s e r v a t i o n , as well as c h r o m a t o g r a p h i c a l d a t a , i n d i c a t e d t h a t t h e n e w m e t a b o l i t e was acetylated m e t h y l h i s t a m i n e or a n o t h e r h y d r o l y s a b l e conjugate of methylhistamine. The radioactivity remaining in the fraction of the new metabolite after hydrolysis m a y be due to incomplete hydrolysis. However, the possibility also exists that at least part of the activity, but more than 2-3%, m a y stem from histaminol. B y the methods used, quantitation of histaminol was impossible. T o identify the u n k n o w n metabolite, it was necessary to separate it from N-acetylhistamine. The main fractions of the new Inetabolite were rechromatographed on an Aminex column until the metabolite seemed to m o v e as a single compound. The new metabolite is easily extractable from a basic solution with n-butanol and can be reextracted as histamine, from the organic solution with HCI. Butanol extraction followed by reextraction with HCI was combined with repeated column chromatography for isolation and purification. ]By starting with a 24-h urine, enough substance for direct mass-spectrographic analysis was obtained. The mass-spectra (figure 2 a) indicate that the substance is N-acetyl-l.4-methylhistamine. Reference N-acetylmethylhistamine was synthesized by methylation of N-acetylhistamine with dimethylsulfate and purified by means of T L C and butanol-HCl extractions. The purified compound could be visualized neither with ninhydrin nor by diazotation. The mass-spectrum for synthetic N-acetylmethylhistamine HCI (figure 2 b) is nearly identical to that of the new metabolite isolated from the hens' urine, F r o m this evidence, we conclude that N-acetyl-l.4-methyl-histamine is a metabolite of histamine in chickens. About 9 0 % of the radioactivity could be accounted for by identified histamine-metabolites, the remaining 1 0 %
EXPERIENTIA33/8
m a y r e p r e s e n t o t h e r m e t a b o l i t e s s u c h as f r a g m e n t s of t h e h i s t a m i n e - r i n g 6. H o w e v e r , one c a n n o t exclude t h e possib i l i t y t h a t c h r o m a t o g r a p h i c a l effects m a y r e s u l t in u n d e r e s t i m a t i o n of some of t h e m e t a b o l i t e s . T h e t a b l e d e m o n s t r a t e s t h e m e t a b o l i c p a t t e r n of e x o g e n o u s h i s t a m i n e in chickens. T h e m e t a b o l i c p a t t e r n of h i s t a m i n e in c h i c k e n differs largely f r o m t h a t f o u n d in o t h e r species. T h i s difference is first a n d f o r e m o s t a reflection of t h e n e w m e t a b o l i t e , N - a c e t y l m e t h y l h i s t a m i n e , w h i c h acc o u n t e d for a b o u t 2 0 % of t h e e x c r e t e d r a d i o a c t i v i t y , b u t also of t h e g r e a t Q u a n t i t a t i v e i m p o r t a n c e of N - a c e t y l h i s t a m i n e . T h e i d e n t i t y of N - a c e t y l h i s t a m i n e was confirmed with mass-spectrography. T h e p r e s e n t e x p e r i m e n t s s u p p o r t t h e c o n c l u s i o n of S h i f r i n e e t a l ) t h a t a c e t y l a t i o n is of g r e a t i m p o r t a n c e in t h e d e t o x i c a t i o n of h i s t a m i n e in chicken. T h i s m i g h t a p p l y t o fowls g e n e r a l l y since in v i t r o e x p e r i m e n t s w i t h liver slices a n d cell free e x t r a c t s of p i g e o n liver are p o t e n t to conjugate histamine L The fact that Bergmark and Granerus4 found only very low r a d i o a c t i v i t y i n t h e f r a c t i o n s w h e r e N - a c e t y l m e t h y l h i s t a m i n e s h o u l d occur suggests t h a t t h i s m e t a b o l i t e , q u a n t i t a t i v e l y i m p o r t a n t ill chickens, is negligible, if p r e s e n t a t all, in m a n . W h e t h e r a c e t y l a t i o n or m e t h y l a t i o n of h i s t a m i n e is t h e first s t e p in t h e f o r m a t i o n of N - a c e t y l m e t h y l h i s t a m i n e , a n d in w h i c h tissues t h e s e r e a c t i o n s t a k e place, is u n d e r e x a m i n a t i o n . Since n e i t h e r N - a c e t y l h i s t a m i n e n o r 1.4m e t h y l h i s t a m i n e possess biological a c t i v i t y , i t seems likely t h a t t h i s is t r u e also for N - a c e t y l - l . 4 - m e t h y l h i s t a mine. 6 O.V. Sjaastad and R. N. B. Kay, Experientia 26, 1197 (1970). 7 R. C. Millican, N. Sandford, M. Rosenthal and H. Tabor, J. Pharmac. exp. Ther. 97, 4 (1949).
Reduction of adenylylsulfate and 3'-phosphoadenylylsulfate in phototrophic bacteria A. S c h m i d t a n d H. G. T r i i p e r 1
Botanisches Inslitut der Universitiit'M~nchen, Menzinger Strasse 64, D-8000 Mi~nchen; and Inslitut [i~r Mikrobiologie der Universitiit Bonn, Meckenheimer Allee 768, D-5300 Bonn (Federal Republic o[ Germany, BRD), 28 January 1977 Summary. E x t r a c t s of 14 species of p h o t o t r o p h i c b a c t e r i a , p a r t l y g r o w n w i t h d i f f e r e n t sulfur c o m p o u n d s , were t e s t e d for t h e i r a b i l i t y t o f o r m v o l a t i l e sulfur c o m p o u n d s f r o m a d e n y l y l s u l f a t e (APS) a n d 3 ' - p h o s p h o a d e n y l y l s u l f a t e ( P A P S ) . T h e R h o d o s p i r i l l u m species s h o w e d m a r k e d a c t i v i t i e s w i t h b o t h A P S a n d P A P S while t h e R h o d o p s e u d o m o n a s species s e e m t o prefer P A P S . T h e C h r o m a t i a c e a e e x h i b i t e d t h e s t r o n g e s t a c t i v i t i e s w i t h A P S , w h e r e a s C h l o r o b i u m limicola had equally high activity with PAPS. During dissimilatory sulfur metabolism in Chlorobiaceae a n d C h r o m a t i a c e a e , sulfite is o x i d i z e d b y A P S 2 r e d u c t a s e 3. T h e r e l a t i v e l y h i g h levels of A P S r e d u c t a s e f o u n d in Chromatium vinosum and Thiocapsa roseopersicina a f t e r p h o t o h e t e r o t r o p h i c g r o w t h w i t h sulfate, as t h e sole s u l f u r source, led t o t h e a s s u m p t i o n t h a t A P S r e d u c t a s e m i g h t b e i n v o l v e d also in a s s i m i l a t o r y s u l f u r m e t a b o l i s m i n t h e s e b a c t e r i a 4, i.e. in t h e r e d u c t i o n of A P S t o sulfite. O n t h e o t h e r h a n d , n o n e of t h e R h o d o s p i r i l l a c e a e c o n t a i n A P S r e d u c t a s e 4, a n d t h e f o r m a t i o n of P A P S 2 f r o m sulf a t e alld A T P h a s b e e n r e p o r t e d to o c c u r i n c h r o m a t o p h o r e s of R h o d o s p i r i l l u m r u b r u m ~. T h e a i m of t h i s s t u d y was t o t e s t a n u m b e r of r e p r e s e n t a t i v e species of t h e p h o t o t r o p h i c b a c t e r i a as to w h e t h e r t h e y are able t o f o r m v o l a t i l e sulfur c o m p o u n d s f r o m P A P S or A P S (or b o t h ) . Material and methods. T h e R h o d o s p i r i l l a c e a e were g r o w n o n c o n v e n t i o n a l m e d i a 6, 7 w i t h sulfate, or special m e d i a s, 9 with reduced sulfur compounds. The Chlorobium strain a n d t h e C h r o m a t i a c e a e were c u l t i v a t e d in P f e n n i g ' s
m e d i u m 1~ w i t h sulfide, C. v i n o s u m also p h o t o h e t e r o t r o p h i c a l l y . I n c u b a t i o n o c c u r r e d in 500 m l s c r e w - c a p p e d b o t t l e s a t 2 5 - 3 0 ~ a n d a b o u t 2000 lux. F r o z e n cells were t h a w e d a n d s u s p e n d e d in a b u f f e r cont a i n i n g 0.1 m Tris-HC1, p H 8.0, 10 m M MgC12, 0.1 M KC1, 1
We thank J. F. Imhoff for skilful assistance and the Deutsche Forschungsgemeinsehaft for financial support. 2 Abbreviations. APS, Adenylylsulfate ( = adenosine phosphosulfate). DTE, Dithioerythritol. PAPS, 3'-Phosphoadenylylsulfate. 3 H.G. Triiper, P1. Soil 43, 350 (1975). 4 H.G. Triiper and H. D. Peck, Arch. Mikrobiol. 73, 125 (1970). 5 M.L. Ibafiez and E. S. Lindstrom, J. Bact. 84, 451 (1962). 6 H. Gest, M. D. Kamen and H. M. Bregoff, J. biol. Chem. 182, 153 (1950). 7 C.B. van Niel, in: Methods in Enzymology, Vol. 23A, p. 3. Ed. S. P. Colowick and N. O. Kaplan. Academic Press, New York 1971. 8 T.A. Hansen, Doctoral Thesis, Univ. Groningen (1974). 9 N. Pfennig, Arch. Mikrobiol. 700, 197 (1974). 10 N. Pfennig, Zentbl. Bakt. ParasitKde I, Suppl. 7, 179 (1965).
15.8. 1977
Specialia
a n d 5 m M D T E ~. 2 m l of b u f f e r were u s e d for 1.0 g of w e t w e i g h t cells. T h e s e were b r o k e n a t 10,000 psi ( F r e n c h press) a n d c e n t r i f u g e d t o r e m o v e u n b r o k e n m a t e r i a l . P r o t e i n was d e t e r m i n e d b y t h e b i u r e t m e t h o d a f t e r trichloroacetic acid p r e c i p i t a t i o n a n d 2fold a c e t o n e e x t r a c t i o n t o r e m o v e p i g m e n t s n . P r e p a r a t i o n of A P S a n d P A P S was p e r f o r m e d u s i n g t h e s l i g h t l y m o d i f i e d Chlorella s y s t e m 1~, z3.
Table 1. Reduction of sulfonucleotides in crude (chromatophorecontaining) extracts of phototrophic bacteria Species
Strain
PAPS
Sulfate Cysteine Sulfate
0.234 0.168 0.020 0.126 0.206 0.372 0
0.453 0.173 0.066 0.554 0.493 0.468 0
BN982"
Thiosulfate Sulfide Thiosulfate Sulfate Sulfate Sulfate Sulfide Sulfate Cysteine Thiosulfate Sulfate Sulfate Sulfide Sulfide
0 0 0 0.012 0.006 0.099 0.006 0.019 0 0.003 0.005 0 0 0
0.017 0.113 0.261 0.165 0.461 0.145 0.019 0.029 0 0.004 0.009 0.023 0.001 0
DSM219 DSM226
Sulfide Sulfide
4.720 0.700
0.447 0.012
DSM180
Sulfate Sulfide
0.174 0.571
0.074 0.287
DSM249
Sulfide
0.640
0.745
UW DSM123* DSM127
viridis capsulata
BN128" BN170 DSM155
gelatinosa globiformis
DSM149 DSM161
sulfidophila
W4
Thiocapsa roseopersieina pfennigii Chromatium vinosum Chlorobium limicola
nmoles sulfonucleotide reduced per mg protein/h APS
Rhodospirillum rubrum 1761-1a fulvum BN211 (chromatophore fraction alone) (supernatant alone) tenue BN230 photometrieum Rhodopseudomonas palustris
Sulfur source
Sulfate Sulfate
Conditions see table 2. *KC1 omitted from buffer mixture during preparation of extract.
Table 2. Isotope exchange reaction between labelled sulfonucleotides and unlabelled sulfite catalyzed by bacterial extracts
Organism
Formation of radioactive acid volatile sulfur (nmoles]h • protein) from AP85S pAp35s
R. rubrum Chr. vinosum ]'ca. pfennigii Chl. limicola
0.005 0.32 1.16 1,58
0.027 0.013 0.14 1.04
Conditions (in tzmoles) : Tris-HC1, pH 9.0 : 100; MgC12:10;unlabelled sulfite: 100; AP35S: 0.035 (1 nmole ~ 1891 cpm) when indicated; PAP35S: 0.035 (1 nmole ~ 1315 epm) when indicated; bacterial extracts, total volume 1 ml. After 1 h at 37~ under N2, 0.1 ml of 1 M SO~- was added and the acid volatile radioactivity trapped in 1 ml 1 M triethanolamine. The radioactivity was determined by scintillation counting 1. in a Packard Tricarb.
1009
I n o r d e r to o b t a i n useful c o n c e n t r a t i o n s for a r e a c t i o n m i x t u r e s u i t a b l e t o s t u d y volatile sulfur f o r m a t i o n f r o m labelled sulfonucleotides, a p i l o t s t u d y w i t h R. r u b r u m was m a d e . The o p t i m a l p H for P A P S r e d u c t i o n was 9.0. The r e l a t i o n s h i p b e t w e e n r e a c t i o n r a t e a n d p r o t e i n conc e n t r a t i o n s w a s linear u p t o a b o u t 25 m g of p r o t e i n p e r t e s t volume. F r o m t h e influence of t h e P A P S c o n c e n t r a tions, a n a p p a r e n t K m - v a l u e of 4 9 10 -s M for P A P S was o b t a i n e d b y L i n e w e a v e r - B u r k plot. A s u i t a b l e r e a c t i o n m i x t u r e was p r e p a r e d f r o m t h e s e d a t a , b u f f e r e d a t p H 9.0, c o n t a i n i n g 35 ~xmoles of labelled sulf o n u c l e o t i d e a n d 1-20 m g of p r o t e i n p e r ml. Results. Table 1 s h o w s t h e result of 54 i n c u b a t i o n s c a r r i e d o u t w i t h e x t r a c t s of 14 d i f f e r e n t species of p h o t o t r o p h i c b a c t e r i a p a r t l y g r o w n on d i f f e r e n t sulfur sources. The h i g h e s t a c t i v i t i e s w i t h A P S were f o u n d in t h o s e b a c t e r i a t h a t are k n o w n to c o n t a i n A P S r e d u c t a s e , t h e C h r o m a t i a c e a e a n d C h l o r o b i u m . B u t t h e s e b a c t e r i a also s h o w e d r e l a t i v e l y h i g h a c t i v i t y w i t h P A P S . A n o t h e r g r o u p exh i b i t i n g fairly h i g h a c t i v i t y w i t h A P S is f o u n d in t h e R h o d o s p i r i l l u m species (except R. p h o t o m e t r i c u m ) , w h e r e r e a c t i v i t y w i t h P A P S was o n l y s l i g h t l y higher. The l o w e s t a c t i v i t i e s - o f t e n d o w n to zero - for A P S occur in t h e R h o d o p s e u d o m o n a s species t e s t e d , w h e r e t h e a c t i v i t y w i t h P A P S was always higher, especially w h e n t h e cells h a d b e e n g r o w n w i t h sulfate. The e x p e r i m e n t s were carried o u t w i t h c h r o m a t o p h o r e c o n t a i n i n g c r u d e e x t r a c t s t h a t were o n l y freed f r o m unb r o k e n cells, large f r a g m e n t s a n d sulfur globules b y low s p e e d c e n t r i f u g a t i o n . I t is, h o w e v e r , m o s t u n l i k e l y t h a t t h e e x t r a c t s still c o n t a i n e d or p r o d u c e d (by p h o t o p h o s p h o r y l a t i o n ) sufficient A T P to allow a p h o s p h o r y l a t i o n of A P S to P A P S b y A P S kinase. This is also s u p p o r t e d b y the comparison made with chromatophore-free fractions (table 1) a n d c o n t r o l s in t h e d a r k ( d a t a n o t shown). On t h e c o n t r a r y , t h e a p p a r e n t P A P S r e d u c t i o n also o c c u r r i n g in t h e C h r o m a t i a c e a e a n d in C h l o r o b i u m is m o s t likely due t o t h e p r e s e n c e of a 3 ' - n u c l e o t i d a s e in t h e e x t r a c t s , leading t o a n i n t e r m e d i a r y A P S f o r m a t i o n f r o m P A P S . This a s s u m p t i o n is s u p p o r t e d b y t h e isotope exc h a n g e r e a c t i o n b e t w e e n labelled A P S (or P A P S ) a n d u n l a b e l l e d sulfite (table 2). If a regular A P S r e d u c t a s e is p r e s e n t , t o g e t h e r w i t h a small a m o u n t of A M P , one w o u l d e x p e c t r e a c t i v i t y in b o t h directions15, l~: I n r e a c t i o n m i x t u r e s c o n t a i n i n g 35S-sulfonucleotide a n d u n l a b e l l e d sulfite, t h e l a t t e r t o g e t h e r w i t h A M P w o u l d f o r m unlabelled A P S . This w o u l d m i x w i t h labelled A P S a n d r e a c t b a c k f o r m i n g labelled sulfite. Thus, label in sulfite (i.e., v o l a t i l e sulfur) will increase. Again, a 3 ' - n u c l e o t i d a s e w o u l d facilitate P A P S t o p a r t i c i p a t e via A P S . This has t o be p o s t u l a t e d , b e c a u s e A P S r e d u c t a s e s h a v e b e e n s h o w n to be specific for A P S 16. T a b l e 2 s h o w s t h e results of s u c h i s o t o p e e x c h a n g e e x p e r i m e n t s . The organisms known to contain A P S reductase show r e a s o n a b l e e x c h a n g e rates, while in R. r u b r u m no subs t a n t i a l e x c h a n g e t a k e s place. This p r o v e s t h a t a different, n o n - r e v e r s i b l e m e c h a n i s m , s u c h as occurs in all assimilat o r y s u l f a t e r e d u c e r s so far s t u d i e d , is p r e s e n t also in t h i s species.
11 12 13 14 15 16
K. Schmidt, S. Liaaen-Jensen and H. G. Schlegel, Arch. Mikrobiol. J6, 117 (1963). R.C. Hodson and J. A. Schiff, Archs. ]3ioehem. Biophys. J32, 151 (1969). A. Schmidt, Planta 724, 267 (1975). M.S. Patterson and R. C, Greene, Analyt. Chem. 37, 854 (1965). H.D. Peck, Biochim. biophys. Acta ,/9, 621 (1961). H . D . Peck, T. E. Deacon and J. T. Davidson, Biochim. biophys. Acta 96, 429 (1965).
1010
Specialia
T h e r e s u l t s w i t h R. r u b r u m , a n d p r o b a b l y t h e o t h e r 1Rhodospirillum species (except R. p h o t o m e t r i c u m ) , p o i n t to t h e p o s s i b i l i t y t h a t in t h e s e b a c t e r i a a n A P S sulfot r a n s f e r a s e like t h a t f o u n d in algae a n d h i g h e r p l a n t s la, ~7, is is active. W e k n o w t h a t o u r r e s u l t s do n o t p r e s e n t final p r o o f for t h i s possibility, a n d also t h a t we
EXPERIENTIA 33/8
c a n n o t e x c l u d e t h e p r e s e n c e of A P S s u l f o t r a n s f e r a s e besides A P S r e d u c t a s e in t h e p h o t o t r o p h i c sulfur b a c t e r i a . F u r t h e r s t u d i e s w i t h p u r i f i e d e n z y m e s are u n d e r way, 17 A. Schmidt, Z. Naturf. 27b, 183 (1972). 18 A. Schmidt, Planta 130, 257 (1976).
Effect of t r y p s i n , S - a d e n o s y l m e t h i o n i n e a n d e t h i o n i n e on L - s e r i n e s u l f h y d r a s e a c t i v i t y ~ Darinka Kora6evid and Vidosava Djordjevi6
Department o[ Biochemistry, Faculty o/Medicine, Ni~ (Yougoslavia), 16 September 7976 Summary. T r y p s i n causes a n a c t i v a t i o n of serine s u l f h y d r a s e in t h e l i v e r e x t r a c t s f r o m i n t a c t a n i m a l s , b u t i n h i b i t s e n z y m e a c t i v i t y in t h e l i v e r of e t h i o n i n e t r e a t e d rats. T r y p s i n also decreases a n e l e v a t i o n of serine s u l t h y d r a s e a c t i v i t y caused by S-adenosylmethionine. D u r i n g t h e l a s t few years, s e v e r a l s t u d i e s h a v e i n d i c a t e d t h a t b o t h serene s u l f h y d r a s e (EC 4.2.1.22} a n d c y s t a t h i o n i n e /~-synthetase a c t i v i t i e s are d u e to a single e n z y m e ~-4. R e c e n t l y , i t h a s b e e n d e m o n s t r a t e d t h a t ethionine administration produces a significant activation of c y s t a t h i o n i n e f l - s y n t h e t a s e a n d serine s u l f h y d r a s e in t h e r a t tissues ~-e. I t is also f o u n d t h a t S - a d e n o s y l m e t h i o n i n e a f f e c t s as t h e a c t i v a t o r of c y s t a t h i 0 n i n e f l - s y n t h e t a s e 4,~. M u d d a n d his coworkers 7 h a v e s h o w n t h a t t r y p s i n i n c r e a s e s t h e a c t i v i t y of c y s t a t h i o n i n e s y n t h e t a s e f r o m a n e x t r a c t of h u m a n liver. As t h e c o n t r o l m e c h a n i s m of serine s u l f h y d r a s e a c t i o n h a s n o t b e e n k n o w n of sufficiently, we e x a m i n e d t h e i n t e r a c t i o n of t r y p s i n , e t h i o n i n e a n d S - a d e n o s y l m e t h i o n i n e in r e l a t i o n t o t h e a c t i v i t y of t h i s e n z y m e .
Effects of S-adenosylmethionine (SAM) and trypsin on L-serine sulfhydrase activity in vitro (nmoles cysteine]mg protein h) No. of experiments
Medium for incubation (20 min at 37 ~
Enzyme activity
9 9 9 9
Enzyme + SAM Enzyme + SAM + trypsin Enzyme + trypsin Enzyme
99.6 81.5 97.3 76.1
~--.....
-V 1.8 -t- 1.4 ~ 2.1 -t- 0.9
Ethionine Ethionine+trypsin
Control
400
Material and methods. A l b i n o r a t s w e i g h i n g 160-220 g were used. D L - e t h i o n i n e w a s dissolved i n 0.9% NaC1 a n d i n j e c t e d i.p. in a dose of 200 m g / k g . T h e c o n t r o l g r o u p r e c e i v e d t h e c o r r e s p o n d i n g v o l u m e of 0.9% NaC1. T h e a n i m a l s were killed 3 h a f t e r t h e i n j e c t i o n . Serine suEh y d r a s e was e x t r a c t e d a n d s e p a r a t e d f r o m serine dehydratase according to the Kashiwamata and Greenberg m e t h o d 8. T r y p s i n was dissolved i n 0.1 M tris-HC1 b u f f e r p H 8.3 a n d a d d e d to t h e a l i q u o t s of e n z y m e e x t r a c t in t h e a m o u n t of 0.5 y / m g p r o t e i n . S - a d e n o s y l m e t h i o n i n e was dissolved in I-I~O a n d a d d e d to t h e e n z y m e of c o n t r o l r a t l i v e r i n t h e a m o u n t of 0.1 m i c r o m o l p e r m g p r o t e i n . Enzyme had been preincubated with S-adenosylmethion i n e 5 m i n a t 37 ~ before a d d i n g t r y p s i n . A f t e r i n c u b a t i o n a t 37~ t h e a c t i v i t y of serine s u l f h y d r a s e was d e t e r m i n e d b y t h e p r o c e d u r e of S t e p i e n a n d P i e n i a z e k 9. E n z y m e a c t i v i t y was e x p r e s s e d i n n m o l e s c y s t e i n e p e r m g p r o t e i n p e r h. P r o t e i n was e s t i m a t e d b y t h e m e t h o d of L o w r y e t al. t0. D L - e t h i o n i n e a n d S - a d e n o s y l m e t h i o n i n e were o b t a i n e d f r o m C a l b i o c h e m while t r y p s i n w a s p u r c h a s e d f r o m Merck. Results and discussion. T h e effect of t r y p s i n t r e a t m e n t o n L - s e r i n e s u l f h y d r a s e a c t i v i t y is s h o w n in t h e figure. T r y p s i n causes a n i n c r e a s e in serine s u l f h y d r a s e a c t i v i t y in t h e l i v e r e x t r a c t s f r o m i n t a c t a n i m a l s . O n t h e c o n t r a r y , w h e n t r y p s i n was i n c u b a t e d w i t h e n z y m e e x t r a c t s prep a r e d f r o m t h e liver of t h e r a t s w h i c h h a d r e c e i v e d D L - e t h i o n i n e , a successive d i m i n u t i o n in serine sulfh y d r a s e a c t i v i t y was o b s e r v e d . T h e a c t i o n of S - a d e n o s y l m e t h i o n i n e a n d t r y p s i n u p o n serine s u l f h y d r a s e levels is p r e s e n t e d in t h e t a b l e . I t is e v i d e n t f r o m t h e s e d a t a t h a t S - a d e n o s y l m e t h i o n i n e as well as t r y p s i n p r o d u c e a r e m a r k a b l e rise in serine s u l f h y d r a s e . H o w e v e r , a d d i t i o n
........ Control § ~'2.~
300
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In'vitro effect of trypsin on L-serine sulfhydrase activity in the liver of rats treated with ethionine. Vertical bars indicate standard errors of the mean.
This work was supported by the Serbian Medical Research Foundation. 2 N . J . Pieniazek, P. P. Stepien and A. Paszewski, Biochim. biophys. Aeta 297, 37 (1973). 3 L . L . Jefremova, V'. L. Florentjev and E. V. Gorjangenkova, Molec. Biol. 8, 154 (1974). 4 D. Koradevid, Thesis 1976. 5 D. Koradevid, Experientia 31, 26 (1975). 6 J.D. Finkelstein, W. E. Kyle, J. J. Martin and Ann-Marie Pick, Biochem. biophys. Res. Commun. 66, 81 (1975). 7 S . H . Mudd, W. A. Edwards, P. M. Loeb, M. S. Brown and L, Laster, J. clin. Invest. 49, 1762 (1970). 8 S. Kashiwamata and D. M. Greenberg, Bioohim. biophys. Aeta 212, 488 (1970}. 9 P . P . Stepien and N. J. Pieniazek, Analyt. Biochem. 54, 294 (1973). 10 H.O. Lowry, N. J. Rosebrough, A. L. Farr and J. R. Randall, J. biol. Chem. 193, 265 (1951).
Specialia
t h e n e w m e t a b o l i t e . T h i s o b s e r v a t i o n , as well as c h r o m a t o g r a p h i c a l d a t a , i n d i c a t e d t h a t t h e n e w m e t a b o l i t e was acetylated m e t h y l h i s t a m i n e or a n o t h e r h y d r o l y s a b l e conjugate of methylhistamine. The radioactivity remaining in the fraction of the new metabolite after hydrolysis m a y be due to incomplete hydrolysis. However, the possibility also exists that at least part of the activity, but more than 2-3%, m a y stem from histaminol. B y the methods used, quantitation of histaminol was impossible. T o identify the u n k n o w n metabolite, it was necessary to separate it from N-acetylhistamine. The main fractions of the new Inetabolite were rechromatographed on an Aminex column until the metabolite seemed to m o v e as a single compound. The new metabolite is easily extractable from a basic solution with n-butanol and can be reextracted as histamine, from the organic solution with HCI. Butanol extraction followed by reextraction with HCI was combined with repeated column chromatography for isolation and purification. ]By starting with a 24-h urine, enough substance for direct mass-spectrographic analysis was obtained. The mass-spectra (figure 2 a) indicate that the substance is N-acetyl-l.4-methylhistamine. Reference N-acetylmethylhistamine was synthesized by methylation of N-acetylhistamine with dimethylsulfate and purified by means of T L C and butanol-HCl extractions. The purified compound could be visualized neither with ninhydrin nor by diazotation. The mass-spectrum for synthetic N-acetylmethylhistamine HCI (figure 2 b) is nearly identical to that of the new metabolite isolated from the hens' urine, F r o m this evidence, we conclude that N-acetyl-l.4-methyl-histamine is a metabolite of histamine in chickens. About 9 0 % of the radioactivity could be accounted for by identified histamine-metabolites, the remaining 1 0 %
EXPERIENTIA33/8
m a y r e p r e s e n t o t h e r m e t a b o l i t e s s u c h as f r a g m e n t s of t h e h i s t a m i n e - r i n g 6. H o w e v e r , one c a n n o t exclude t h e possib i l i t y t h a t c h r o m a t o g r a p h i c a l effects m a y r e s u l t in u n d e r e s t i m a t i o n of some of t h e m e t a b o l i t e s . T h e t a b l e d e m o n s t r a t e s t h e m e t a b o l i c p a t t e r n of e x o g e n o u s h i s t a m i n e in chickens. T h e m e t a b o l i c p a t t e r n of h i s t a m i n e in c h i c k e n differs largely f r o m t h a t f o u n d in o t h e r species. T h i s difference is first a n d f o r e m o s t a reflection of t h e n e w m e t a b o l i t e , N - a c e t y l m e t h y l h i s t a m i n e , w h i c h acc o u n t e d for a b o u t 2 0 % of t h e e x c r e t e d r a d i o a c t i v i t y , b u t also of t h e g r e a t Q u a n t i t a t i v e i m p o r t a n c e of N - a c e t y l h i s t a m i n e . T h e i d e n t i t y of N - a c e t y l h i s t a m i n e was confirmed with mass-spectrography. T h e p r e s e n t e x p e r i m e n t s s u p p o r t t h e c o n c l u s i o n of S h i f r i n e e t a l ) t h a t a c e t y l a t i o n is of g r e a t i m p o r t a n c e in t h e d e t o x i c a t i o n of h i s t a m i n e in chicken. T h i s m i g h t a p p l y t o fowls g e n e r a l l y since in v i t r o e x p e r i m e n t s w i t h liver slices a n d cell free e x t r a c t s of p i g e o n liver are p o t e n t to conjugate histamine L The fact that Bergmark and Granerus4 found only very low r a d i o a c t i v i t y i n t h e f r a c t i o n s w h e r e N - a c e t y l m e t h y l h i s t a m i n e s h o u l d occur suggests t h a t t h i s m e t a b o l i t e , q u a n t i t a t i v e l y i m p o r t a n t ill chickens, is negligible, if p r e s e n t a t all, in m a n . W h e t h e r a c e t y l a t i o n or m e t h y l a t i o n of h i s t a m i n e is t h e first s t e p in t h e f o r m a t i o n of N - a c e t y l m e t h y l h i s t a m i n e , a n d in w h i c h tissues t h e s e r e a c t i o n s t a k e place, is u n d e r e x a m i n a t i o n . Since n e i t h e r N - a c e t y l h i s t a m i n e n o r 1.4m e t h y l h i s t a m i n e possess biological a c t i v i t y , i t seems likely t h a t t h i s is t r u e also for N - a c e t y l - l . 4 - m e t h y l h i s t a mine. 6 O.V. Sjaastad and R. N. B. Kay, Experientia 26, 1197 (1970). 7 R. C. Millican, N. Sandford, M. Rosenthal and H. Tabor, J. Pharmac. exp. Ther. 97, 4 (1949).
Reduction of adenylylsulfate and 3'-phosphoadenylylsulfate in phototrophic bacteria A. S c h m i d t a n d H. G. T r i i p e r 1
Botanisches Inslitut der Universitiit'M~nchen, Menzinger Strasse 64, D-8000 Mi~nchen; and Inslitut [i~r Mikrobiologie der Universitiit Bonn, Meckenheimer Allee 768, D-5300 Bonn (Federal Republic o[ Germany, BRD), 28 January 1977 Summary. E x t r a c t s of 14 species of p h o t o t r o p h i c b a c t e r i a , p a r t l y g r o w n w i t h d i f f e r e n t sulfur c o m p o u n d s , were t e s t e d for t h e i r a b i l i t y t o f o r m v o l a t i l e sulfur c o m p o u n d s f r o m a d e n y l y l s u l f a t e (APS) a n d 3 ' - p h o s p h o a d e n y l y l s u l f a t e ( P A P S ) . T h e R h o d o s p i r i l l u m species s h o w e d m a r k e d a c t i v i t i e s w i t h b o t h A P S a n d P A P S while t h e R h o d o p s e u d o m o n a s species s e e m t o prefer P A P S . T h e C h r o m a t i a c e a e e x h i b i t e d t h e s t r o n g e s t a c t i v i t i e s w i t h A P S , w h e r e a s C h l o r o b i u m limicola had equally high activity with PAPS. During dissimilatory sulfur metabolism in Chlorobiaceae a n d C h r o m a t i a c e a e , sulfite is o x i d i z e d b y A P S 2 r e d u c t a s e 3. T h e r e l a t i v e l y h i g h levels of A P S r e d u c t a s e f o u n d in Chromatium vinosum and Thiocapsa roseopersicina a f t e r p h o t o h e t e r o t r o p h i c g r o w t h w i t h sulfate, as t h e sole s u l f u r source, led t o t h e a s s u m p t i o n t h a t A P S r e d u c t a s e m i g h t b e i n v o l v e d also in a s s i m i l a t o r y s u l f u r m e t a b o l i s m i n t h e s e b a c t e r i a 4, i.e. in t h e r e d u c t i o n of A P S t o sulfite. O n t h e o t h e r h a n d , n o n e of t h e R h o d o s p i r i l l a c e a e c o n t a i n A P S r e d u c t a s e 4, a n d t h e f o r m a t i o n of P A P S 2 f r o m sulf a t e alld A T P h a s b e e n r e p o r t e d to o c c u r i n c h r o m a t o p h o r e s of R h o d o s p i r i l l u m r u b r u m ~. T h e a i m of t h i s s t u d y was t o t e s t a n u m b e r of r e p r e s e n t a t i v e species of t h e p h o t o t r o p h i c b a c t e r i a as to w h e t h e r t h e y are able t o f o r m v o l a t i l e sulfur c o m p o u n d s f r o m P A P S or A P S (or b o t h ) . Material and methods. T h e R h o d o s p i r i l l a c e a e were g r o w n o n c o n v e n t i o n a l m e d i a 6, 7 w i t h sulfate, or special m e d i a s, 9 with reduced sulfur compounds. The Chlorobium strain a n d t h e C h r o m a t i a c e a e were c u l t i v a t e d in P f e n n i g ' s
m e d i u m 1~ w i t h sulfide, C. v i n o s u m also p h o t o h e t e r o t r o p h i c a l l y . I n c u b a t i o n o c c u r r e d in 500 m l s c r e w - c a p p e d b o t t l e s a t 2 5 - 3 0 ~ a n d a b o u t 2000 lux. F r o z e n cells were t h a w e d a n d s u s p e n d e d in a b u f f e r cont a i n i n g 0.1 m Tris-HC1, p H 8.0, 10 m M MgC12, 0.1 M KC1, 1
We thank J. F. Imhoff for skilful assistance and the Deutsche Forschungsgemeinsehaft for financial support. 2 Abbreviations. APS, Adenylylsulfate ( = adenosine phosphosulfate). DTE, Dithioerythritol. PAPS, 3'-Phosphoadenylylsulfate. 3 H.G. Triiper, P1. Soil 43, 350 (1975). 4 H.G. Triiper and H. D. Peck, Arch. Mikrobiol. 73, 125 (1970). 5 M.L. Ibafiez and E. S. Lindstrom, J. Bact. 84, 451 (1962). 6 H. Gest, M. D. Kamen and H. M. Bregoff, J. biol. Chem. 182, 153 (1950). 7 C.B. van Niel, in: Methods in Enzymology, Vol. 23A, p. 3. Ed. S. P. Colowick and N. O. Kaplan. Academic Press, New York 1971. 8 T.A. Hansen, Doctoral Thesis, Univ. Groningen (1974). 9 N. Pfennig, Arch. Mikrobiol. 700, 197 (1974). 10 N. Pfennig, Zentbl. Bakt. ParasitKde I, Suppl. 7, 179 (1965).
15.8. 1977
Specialia
a n d 5 m M D T E ~. 2 m l of b u f f e r were u s e d for 1.0 g of w e t w e i g h t cells. T h e s e were b r o k e n a t 10,000 psi ( F r e n c h press) a n d c e n t r i f u g e d t o r e m o v e u n b r o k e n m a t e r i a l . P r o t e i n was d e t e r m i n e d b y t h e b i u r e t m e t h o d a f t e r trichloroacetic acid p r e c i p i t a t i o n a n d 2fold a c e t o n e e x t r a c t i o n t o r e m o v e p i g m e n t s n . P r e p a r a t i o n of A P S a n d P A P S was p e r f o r m e d u s i n g t h e s l i g h t l y m o d i f i e d Chlorella s y s t e m 1~, z3.
Table 1. Reduction of sulfonucleotides in crude (chromatophorecontaining) extracts of phototrophic bacteria Species
Strain
PAPS
Sulfate Cysteine Sulfate
0.234 0.168 0.020 0.126 0.206 0.372 0
0.453 0.173 0.066 0.554 0.493 0.468 0
BN982"
Thiosulfate Sulfide Thiosulfate Sulfate Sulfate Sulfate Sulfide Sulfate Cysteine Thiosulfate Sulfate Sulfate Sulfide Sulfide
0 0 0 0.012 0.006 0.099 0.006 0.019 0 0.003 0.005 0 0 0
0.017 0.113 0.261 0.165 0.461 0.145 0.019 0.029 0 0.004 0.009 0.023 0.001 0
DSM219 DSM226
Sulfide Sulfide
4.720 0.700
0.447 0.012
DSM180
Sulfate Sulfide
0.174 0.571
0.074 0.287
DSM249
Sulfide
0.640
0.745
UW DSM123* DSM127
viridis capsulata
BN128" BN170 DSM155
gelatinosa globiformis
DSM149 DSM161
sulfidophila
W4
Thiocapsa roseopersieina pfennigii Chromatium vinosum Chlorobium limicola
nmoles sulfonucleotide reduced per mg protein/h APS
Rhodospirillum rubrum 1761-1a fulvum BN211 (chromatophore fraction alone) (supernatant alone) tenue BN230 photometrieum Rhodopseudomonas palustris
Sulfur source
Sulfate Sulfate
Conditions see table 2. *KC1 omitted from buffer mixture during preparation of extract.
Table 2. Isotope exchange reaction between labelled sulfonucleotides and unlabelled sulfite catalyzed by bacterial extracts
Organism
Formation of radioactive acid volatile sulfur (nmoles]h • protein) from AP85S pAp35s
R. rubrum Chr. vinosum ]'ca. pfennigii Chl. limicola
0.005 0.32 1.16 1,58
0.027 0.013 0.14 1.04
Conditions (in tzmoles) : Tris-HC1, pH 9.0 : 100; MgC12:10;unlabelled sulfite: 100; AP35S: 0.035 (1 nmole ~ 1891 cpm) when indicated; PAP35S: 0.035 (1 nmole ~ 1315 epm) when indicated; bacterial extracts, total volume 1 ml. After 1 h at 37~ under N2, 0.1 ml of 1 M SO~- was added and the acid volatile radioactivity trapped in 1 ml 1 M triethanolamine. The radioactivity was determined by scintillation counting 1. in a Packard Tricarb.
1009
I n o r d e r to o b t a i n useful c o n c e n t r a t i o n s for a r e a c t i o n m i x t u r e s u i t a b l e t o s t u d y volatile sulfur f o r m a t i o n f r o m labelled sulfonucleotides, a p i l o t s t u d y w i t h R. r u b r u m was m a d e . The o p t i m a l p H for P A P S r e d u c t i o n was 9.0. The r e l a t i o n s h i p b e t w e e n r e a c t i o n r a t e a n d p r o t e i n conc e n t r a t i o n s w a s linear u p t o a b o u t 25 m g of p r o t e i n p e r t e s t volume. F r o m t h e influence of t h e P A P S c o n c e n t r a tions, a n a p p a r e n t K m - v a l u e of 4 9 10 -s M for P A P S was o b t a i n e d b y L i n e w e a v e r - B u r k plot. A s u i t a b l e r e a c t i o n m i x t u r e was p r e p a r e d f r o m t h e s e d a t a , b u f f e r e d a t p H 9.0, c o n t a i n i n g 35 ~xmoles of labelled sulf o n u c l e o t i d e a n d 1-20 m g of p r o t e i n p e r ml. Results. Table 1 s h o w s t h e result of 54 i n c u b a t i o n s c a r r i e d o u t w i t h e x t r a c t s of 14 d i f f e r e n t species of p h o t o t r o p h i c b a c t e r i a p a r t l y g r o w n on d i f f e r e n t sulfur sources. The h i g h e s t a c t i v i t i e s w i t h A P S were f o u n d in t h o s e b a c t e r i a t h a t are k n o w n to c o n t a i n A P S r e d u c t a s e , t h e C h r o m a t i a c e a e a n d C h l o r o b i u m . B u t t h e s e b a c t e r i a also s h o w e d r e l a t i v e l y h i g h a c t i v i t y w i t h P A P S . A n o t h e r g r o u p exh i b i t i n g fairly h i g h a c t i v i t y w i t h A P S is f o u n d in t h e R h o d o s p i r i l l u m species (except R. p h o t o m e t r i c u m ) , w h e r e r e a c t i v i t y w i t h P A P S was o n l y s l i g h t l y higher. The l o w e s t a c t i v i t i e s - o f t e n d o w n to zero - for A P S occur in t h e R h o d o p s e u d o m o n a s species t e s t e d , w h e r e t h e a c t i v i t y w i t h P A P S was always higher, especially w h e n t h e cells h a d b e e n g r o w n w i t h sulfate. The e x p e r i m e n t s were carried o u t w i t h c h r o m a t o p h o r e c o n t a i n i n g c r u d e e x t r a c t s t h a t were o n l y freed f r o m unb r o k e n cells, large f r a g m e n t s a n d sulfur globules b y low s p e e d c e n t r i f u g a t i o n . I t is, h o w e v e r , m o s t u n l i k e l y t h a t t h e e x t r a c t s still c o n t a i n e d or p r o d u c e d (by p h o t o p h o s p h o r y l a t i o n ) sufficient A T P to allow a p h o s p h o r y l a t i o n of A P S to P A P S b y A P S kinase. This is also s u p p o r t e d b y the comparison made with chromatophore-free fractions (table 1) a n d c o n t r o l s in t h e d a r k ( d a t a n o t shown). On t h e c o n t r a r y , t h e a p p a r e n t P A P S r e d u c t i o n also o c c u r r i n g in t h e C h r o m a t i a c e a e a n d in C h l o r o b i u m is m o s t likely due t o t h e p r e s e n c e of a 3 ' - n u c l e o t i d a s e in t h e e x t r a c t s , leading t o a n i n t e r m e d i a r y A P S f o r m a t i o n f r o m P A P S . This a s s u m p t i o n is s u p p o r t e d b y t h e isotope exc h a n g e r e a c t i o n b e t w e e n labelled A P S (or P A P S ) a n d u n l a b e l l e d sulfite (table 2). If a regular A P S r e d u c t a s e is p r e s e n t , t o g e t h e r w i t h a small a m o u n t of A M P , one w o u l d e x p e c t r e a c t i v i t y in b o t h directions15, l~: I n r e a c t i o n m i x t u r e s c o n t a i n i n g 35S-sulfonucleotide a n d u n l a b e l l e d sulfite, t h e l a t t e r t o g e t h e r w i t h A M P w o u l d f o r m unlabelled A P S . This w o u l d m i x w i t h labelled A P S a n d r e a c t b a c k f o r m i n g labelled sulfite. Thus, label in sulfite (i.e., v o l a t i l e sulfur) will increase. Again, a 3 ' - n u c l e o t i d a s e w o u l d facilitate P A P S t o p a r t i c i p a t e via A P S . This has t o be p o s t u l a t e d , b e c a u s e A P S r e d u c t a s e s h a v e b e e n s h o w n to be specific for A P S 16. T a b l e 2 s h o w s t h e results of s u c h i s o t o p e e x c h a n g e e x p e r i m e n t s . The organisms known to contain A P S reductase show r e a s o n a b l e e x c h a n g e rates, while in R. r u b r u m no subs t a n t i a l e x c h a n g e t a k e s place. This p r o v e s t h a t a different, n o n - r e v e r s i b l e m e c h a n i s m , s u c h as occurs in all assimilat o r y s u l f a t e r e d u c e r s so far s t u d i e d , is p r e s e n t also in t h i s species.
11 12 13 14 15 16
K. Schmidt, S. Liaaen-Jensen and H. G. Schlegel, Arch. Mikrobiol. J6, 117 (1963). R.C. Hodson and J. A. Schiff, Archs. ]3ioehem. Biophys. J32, 151 (1969). A. Schmidt, Planta 724, 267 (1975). M.S. Patterson and R. C, Greene, Analyt. Chem. 37, 854 (1965). H.D. Peck, Biochim. biophys. Acta ,/9, 621 (1961). H . D . Peck, T. E. Deacon and J. T. Davidson, Biochim. biophys. Acta 96, 429 (1965).
1010
Specialia
T h e r e s u l t s w i t h R. r u b r u m , a n d p r o b a b l y t h e o t h e r 1Rhodospirillum species (except R. p h o t o m e t r i c u m ) , p o i n t to t h e p o s s i b i l i t y t h a t in t h e s e b a c t e r i a a n A P S sulfot r a n s f e r a s e like t h a t f o u n d in algae a n d h i g h e r p l a n t s la, ~7, is is active. W e k n o w t h a t o u r r e s u l t s do n o t p r e s e n t final p r o o f for t h i s possibility, a n d also t h a t we
EXPERIENTIA 33/8
c a n n o t e x c l u d e t h e p r e s e n c e of A P S s u l f o t r a n s f e r a s e besides A P S r e d u c t a s e in t h e p h o t o t r o p h i c sulfur b a c t e r i a . F u r t h e r s t u d i e s w i t h p u r i f i e d e n z y m e s are u n d e r way, 17 A. Schmidt, Z. Naturf. 27b, 183 (1972). 18 A. Schmidt, Planta 130, 257 (1976).
Effect of t r y p s i n , S - a d e n o s y l m e t h i o n i n e a n d e t h i o n i n e on L - s e r i n e s u l f h y d r a s e a c t i v i t y ~ Darinka Kora6evid and Vidosava Djordjevi6
Department o[ Biochemistry, Faculty o/Medicine, Ni~ (Yougoslavia), 16 September 7976 Summary. T r y p s i n causes a n a c t i v a t i o n of serine s u l f h y d r a s e in t h e l i v e r e x t r a c t s f r o m i n t a c t a n i m a l s , b u t i n h i b i t s e n z y m e a c t i v i t y in t h e l i v e r of e t h i o n i n e t r e a t e d rats. T r y p s i n also decreases a n e l e v a t i o n of serine s u l t h y d r a s e a c t i v i t y caused by S-adenosylmethionine. D u r i n g t h e l a s t few years, s e v e r a l s t u d i e s h a v e i n d i c a t e d t h a t b o t h serene s u l f h y d r a s e (EC 4.2.1.22} a n d c y s t a t h i o n i n e /~-synthetase a c t i v i t i e s are d u e to a single e n z y m e ~-4. R e c e n t l y , i t h a s b e e n d e m o n s t r a t e d t h a t ethionine administration produces a significant activation of c y s t a t h i o n i n e f l - s y n t h e t a s e a n d serine s u l f h y d r a s e in t h e r a t tissues ~-e. I t is also f o u n d t h a t S - a d e n o s y l m e t h i o n i n e a f f e c t s as t h e a c t i v a t o r of c y s t a t h i 0 n i n e f l - s y n t h e t a s e 4,~. M u d d a n d his coworkers 7 h a v e s h o w n t h a t t r y p s i n i n c r e a s e s t h e a c t i v i t y of c y s t a t h i o n i n e s y n t h e t a s e f r o m a n e x t r a c t of h u m a n liver. As t h e c o n t r o l m e c h a n i s m of serine s u l f h y d r a s e a c t i o n h a s n o t b e e n k n o w n of sufficiently, we e x a m i n e d t h e i n t e r a c t i o n of t r y p s i n , e t h i o n i n e a n d S - a d e n o s y l m e t h i o n i n e in r e l a t i o n t o t h e a c t i v i t y of t h i s e n z y m e .
Effects of S-adenosylmethionine (SAM) and trypsin on L-serine sulfhydrase activity in vitro (nmoles cysteine]mg protein h) No. of experiments
Medium for incubation (20 min at 37 ~
Enzyme activity
9 9 9 9
Enzyme + SAM Enzyme + SAM + trypsin Enzyme + trypsin Enzyme
99.6 81.5 97.3 76.1
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-V 1.8 -t- 1.4 ~ 2.1 -t- 0.9
Ethionine Ethionine+trypsin
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400
Material and methods. A l b i n o r a t s w e i g h i n g 160-220 g were used. D L - e t h i o n i n e w a s dissolved i n 0.9% NaC1 a n d i n j e c t e d i.p. in a dose of 200 m g / k g . T h e c o n t r o l g r o u p r e c e i v e d t h e c o r r e s p o n d i n g v o l u m e of 0.9% NaC1. T h e a n i m a l s were killed 3 h a f t e r t h e i n j e c t i o n . Serine suEh y d r a s e was e x t r a c t e d a n d s e p a r a t e d f r o m serine dehydratase according to the Kashiwamata and Greenberg m e t h o d 8. T r y p s i n was dissolved i n 0.1 M tris-HC1 b u f f e r p H 8.3 a n d a d d e d to t h e a l i q u o t s of e n z y m e e x t r a c t in t h e a m o u n t of 0.5 y / m g p r o t e i n . S - a d e n o s y l m e t h i o n i n e was dissolved in I-I~O a n d a d d e d to t h e e n z y m e of c o n t r o l r a t l i v e r i n t h e a m o u n t of 0.1 m i c r o m o l p e r m g p r o t e i n . Enzyme had been preincubated with S-adenosylmethion i n e 5 m i n a t 37 ~ before a d d i n g t r y p s i n . A f t e r i n c u b a t i o n a t 37~ t h e a c t i v i t y of serine s u l f h y d r a s e was d e t e r m i n e d b y t h e p r o c e d u r e of S t e p i e n a n d P i e n i a z e k 9. E n z y m e a c t i v i t y was e x p r e s s e d i n n m o l e s c y s t e i n e p e r m g p r o t e i n p e r h. P r o t e i n was e s t i m a t e d b y t h e m e t h o d of L o w r y e t al. t0. D L - e t h i o n i n e a n d S - a d e n o s y l m e t h i o n i n e were o b t a i n e d f r o m C a l b i o c h e m while t r y p s i n w a s p u r c h a s e d f r o m Merck. Results and discussion. T h e effect of t r y p s i n t r e a t m e n t o n L - s e r i n e s u l f h y d r a s e a c t i v i t y is s h o w n in t h e figure. T r y p s i n causes a n i n c r e a s e in serine s u l f h y d r a s e a c t i v i t y in t h e l i v e r e x t r a c t s f r o m i n t a c t a n i m a l s . O n t h e c o n t r a r y , w h e n t r y p s i n was i n c u b a t e d w i t h e n z y m e e x t r a c t s prep a r e d f r o m t h e liver of t h e r a t s w h i c h h a d r e c e i v e d D L - e t h i o n i n e , a successive d i m i n u t i o n in serine sulfh y d r a s e a c t i v i t y was o b s e r v e d . T h e a c t i o n of S - a d e n o s y l m e t h i o n i n e a n d t r y p s i n u p o n serine s u l f h y d r a s e levels is p r e s e n t e d in t h e t a b l e . I t is e v i d e n t f r o m t h e s e d a t a t h a t S - a d e n o s y l m e t h i o n i n e as well as t r y p s i n p r o d u c e a r e m a r k a b l e rise in serine s u l f h y d r a s e . H o w e v e r , a d d i t i o n
........ Control § ~'2.~
300
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In'vitro effect of trypsin on L-serine sulfhydrase activity in the liver of rats treated with ethionine. Vertical bars indicate standard errors of the mean.
This work was supported by the Serbian Medical Research Foundation. 2 N . J . Pieniazek, P. P. Stepien and A. Paszewski, Biochim. biophys. Aeta 297, 37 (1973). 3 L . L . Jefremova, V'. L. Florentjev and E. V. Gorjangenkova, Molec. Biol. 8, 154 (1974). 4 D. Koradevid, Thesis 1976. 5 D. Koradevid, Experientia 31, 26 (1975). 6 J.D. Finkelstein, W. E. Kyle, J. J. Martin and Ann-Marie Pick, Biochem. biophys. Res. Commun. 66, 81 (1975). 7 S . H . Mudd, W. A. Edwards, P. M. Loeb, M. S. Brown and L, Laster, J. clin. Invest. 49, 1762 (1970). 8 S. Kashiwamata and D. M. Greenberg, Bioohim. biophys. Aeta 212, 488 (1970}. 9 P . P . Stepien and N. J. Pieniazek, Analyt. Biochem. 54, 294 (1973). 10 H.O. Lowry, N. J. Rosebrough, A. L. Farr and J. R. Randall, J. biol. Chem. 193, 265 (1951).
