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Enzymic formation of choline sulfate
z9o
SHORT COMMUNICATIONS
VOL.
30
(I958)
Enzymic formation of choline sulfate WOOLLEY A N D
PETERSON 1
isolated a n d identified cyclic choline sulfate from t h e m y c e l i u m of
Aspergillus sydowi. The occurrence of t h i s c o m p o u n d in t h e mycelium of a strain of Penicillum chrysogenum h a s also been reported 2. I n t h e present s t u d y a cell-free extract from Aspergillus sydowi h a s b~en obtained which is capable of catalyzing t h e sulfofiation of choline. The mold was grown in modified Fries m e d i u m 8 (sucrose replaced b y glucose) for 3-4 days at 3 °0 with shaking. A crude extract was obtained b y grinding t h e mycelia with a glass homogenizer in o.o2 M p h o s p h a t e buffer (pH 7.2) in t h e presence of cysteine (o.oi M). After centrifugation t h e protein was precipitated b y s a t u r a t i n g t h e s u p e r n a t a n t fluid with a m m o n i u m sulfate. The precipitate was dissolved in p h o s p h a t e buffer (0.02 M, p H 7.2). containing o.oi M cysteine and dialyzed overnight against t h e s a m e buffer at 0-5 ° . TABLE I ENZYMIC FORMATION OF CHOLINE SULFATE
The complete s y s t e m contained 4 ° #moles ATP, io #moles phospho-enol-pyruvate (PEP), 4 ° #moles choline chloride, 12.5 #moles MgC1v IO #moles cysteine, 26 #C of carrier-free sulfate and 14 m g protein in 2.o 5 ml o.oi M p h o s p h a t e buffer (pH 7.2). After 2-h incubation at 37 ° t h e reaction m i x t u r e was h e a t e d for about 2 m i n at IOO°, centrifuged, a n d a z-ml aliquot was applied to a paper c h r o m a t o g r a m . The choline sulfate spot was eluted, lyophilized, and counted. Deletions [tom reaction
Choline sullate Counts~rain
-Choline -ATP -ATP, PEP -PEP* - M g ++ None
1068 273 463 30320 17267 28 379
* I n this t u b e an additional 2o #moles A T P was included to compensate for the omission of P E P . The reaction m i x t u r e required for t h e formation of choline sulfate is shown in Table I. The reaction product was separated b y paper c h r o m a t o g r a p h y~. The radioactive spot corresponding to chemically synthesized4 choline sulfate (~S-labelled) was eluted, lyophilized, a n d counted with a C~M counter. C h r o m a t o g r a p h y of a m i x t u r e of t h e eluted material a n d t h e synthetic choline sulfate gave only one radioactive spot. T h e eluted material was not absorbed b y Dowex-i resin (formate form) a n d did n o t move on paper electrophoresis at p H 5.6. The chenfically synthesized choline sulfate behaved exactly in the s a m e m a n n e r . As shown in Table I, adenosine triphosphate (ATP), Mg++, a n d choline are required for the formation of t h e product. In earlier experiments with crude extract, the addition of p h o s p h o e n o l p y r u v a t e (PEP) stimulated sulfate incorporation. z I0
o
o
~8 & o6 z
~2 0, TIME ( M I N }
Fig. I. Time course of labeled-sulfate incorporation into choline sulfate. The percentage incorporation was calculated by counting radioactivity of the sulfate a n d t h e choline sulfate spot on the chromatogram. The respective radioactive spots were located b y radioautography.
I n s u b s e q u e n t experiments with preparations obtained b y fractionation with a m m o n i u m sulfate and with excess ATP, t h e effect of P E P was not observed. A t i m e course of t h e ~ncorporation of labeled sulfate is shown in Fig. i. It h a s been reported ~'e, t h a t adenosine-3'-phosphate-5'-phosphosulfate(PAPS) is t h e actual sulfate donor in t h e enzymic sulfonation of p-nitrophenol by rat-liver extracts. This reaction
VOT., 30 (1958)
z9z
SHO~T COMMUNICATIONS
iwcolves a c t i v a t i o n of sulfate b y A T P a n d its s u b s e q u e n t t r a n s f e r to p - n i t r o p h e n o l f o r m i n g p - n i t r o p h e n o l sulfate. T h e Aspergillus sydowi e n z y m e in t h e presence of A T P , s u l f a t e a n d p-nitrop h e n o l is n o t capable of f o r m i n g p - n i t r o p h e n o l sulfate unless t h e s u l f a t e - t r a n s f e r r i n g e n z y m e f r o m r a t liver is added. T h e r e s u l t s indicate t h e presence of t h e s u l f a t e - a c t i v a t i n g e n z y m e in t h e Aspergillus sydowi. I n a d d i t i o n it w a s f o u n d t h a t a n i m a l e n z y m e s w h i c h are c a p a b l e of f o r m i n g p - n i t r o p h e n o l s u l f a t e f r o m A T P , S O l - - , a n d p - n i t r o p h e n o l do n o t c a t a l y z e choline s u l f a t e f o r m a tion. However, w h e n t h e s e a n i m a l e n z y m e s were c o m b i n e d w i t h t h e Aspergillus sydowi e n z y m e , t h e i n c o r p o r a t i o n of ~ S O s - - into choline sulfate w a s s t i m u l a t e d . T h e s u l f a t e - t r a n s f e r r i n g e n z y m e f r o m t h e r a t liver ~ did n o t s t i m u l a t e t h e choline sulfate f o r m a t i o n b y t h e Aspergillus sydowi enzyme. I t c a n be concluded t h a t t h e e n z y m e c a t a l y z i n g t h e s u l f o n a t i o n of choline is different f r o m t h a t w h i c h c a t a l y z e s t h e f o r m a t i o n of p - n i t r o p h e n o l sulfate f r o m P A P S . I t is possible t h a t P A P S is t h e i m m e d i a t e sulfate donor for t h e f o r m a t i o n of choline sulfate. However, t h e possibility t h a t a d e n o s i n e - 5 ' - p h o s p h o s u l f a t e (APS) is t h e i m m e d i a t e sulfate d o n o r h a s n o t been eliminated. T h e a u t h o r s wish to t h a n k Dr. ICHIHARA for his cooperation in t h e b e g i n n i n g of this s t u d y , a n d to a c k n o w l e d g e t h e s u p p o r t of t h e Office of N a v a l R e s e a r c h a n d t h e A t o m i c E n e r g y Commission. AKIEA KAJI The McCollum Pratt Institute, Department o/Biology, The Johns Hopkins W . D . MCELROY
University, Baltimore, Md. (U.S.A.) i D. W. WOOLLEY AUI) W. H. PETERSON, J. Biol. Chem., 124 (1937) 213. J. DEFLIN~S, J. Am. Chem. Soc., 77 (1955) 1676. G. W . B~AI)LE AND E. L. TATUM, Am. J. Botany, 32 (1945) 678. E. SCI~MIDT, Ann., 337 (19o4) 54. P. ROBmNS AND F. LIPMANN, J. Biol. Chem., 229 (1957) 837. e j . D. GREGORY AND F. LIPMAN~, J. Biol. Chem., 229 (1957) lO81. R. H. D~MEIo, M. WIZERKAMIUK AND I. SCHREmMAN, J. Biol. Chem., 2i 3 (1955) 439R e c e i v e d April 28th, I958
Relation entre I'activit6 physiologique de I'ocytocine et 1'6tat de son pont disulfure Les r ~ s u l t a t s des t r a v a u x s u r les relations e n t r e l'activit6 p h y s i o l o g i q u e de l'ocytocine et l'6tat d u p o n t disulfure d a n s cette moMcule o n t ~t~ j u s q u ' i c i contradictoires. SEALOCK ET DU V I G ~ A U D 1, e n r ~ d u i s a n t le p o n t disulfure p a r la cyst~ine n ' o n t p a s o b t e n u d ' i n a c t i v a t i o n , s a u f darts le cas o~ les g r o u p e m e n t s - S H f o r m , s 6talent bloqu6s p a r benzylation. Ces r~sultats o n t ~t6 confirm6s r 6 c e m m e n t p a r ACHER et coll. $. D ' a u t r e p a r t , GULLAND I~-TRANDALL3, 4 o n t t r o u v 6 q u e la r~duction de l ' h o r m o n e p r o v o q u a i t u n e i n a c t i v a t i o n de 5 ° % et q u e ceUe-ci ~tait p a r t i e l l e m e n t r~versible p a r r~oxydation. U n a u t r e groupe de c h e r c h e u r s 5 a 6 g a l e m e n t t r o u v ~ u n e i n a c t i v a t i o n de l'ocytocine p a r r~duction du p o n t disulfure. L'activit~ de r o c y t o c i n e r~duite a ~t6 e x a m i n e e d a n s la p l u p a r t des cas, soft s u r l'anirnal v i v a n t , soft s u r l ' u t ~ r u s isol6 en a6robiose. GULLAND ET RANDALL8 a v a i e n t c e p e n d a n t utilis~ u n dosage s u r l ' u t 6 r u s de c o b a y e en ana~robiose. L e s d~tails de ce dosage n ' o n t p a s 6t~ publi~s et la sp6cificit6 des r 6 d u c t e u r s utilis6s p a r ces a u t e u r s a 6t~ raise en q u e s t i o n p a r SEALOClC ET DU VIGNEAUD. ]~tant donn~ q u e le p o n t disulfure de l'ocytocine se ref o r m e s p o n t a n 6 m e n t sous l'influence de l'oxyg~ne moMculaire 6, il p o u v a i t p a r a t t r e int~ressant & e x a m i n e r p a r u n e m ~ t h o d e de dosage s t r i c t e m e n t ana6robie !'activit6 p h y s i o l o g i q u e ~ventuelle de l'ocytocine r~duite p a r u n s y s t ~ m e r ~ d u c t e u r sp6cifique des p o n t s S-S, tel q u e l ' o n t e m p l o y 6 SEALOCK ET DU VIGNEAUDI. I1 f a u t d'ailleurs n o t e r q u ' u n e r~activation ~ventuelle de l'ocytocine p a r r ~ o x y d a t i o n d a n s l ' o r g a n i s m e a v a i t ~t6 envisag~e p a r ces a u t e u r s . I1 a 6t6 ~tabli q u e le glucose, qui n ' a a u c u n e influence s u r la respiration de l'ut~rus~, s p e r m e t A cet o r g a n e de se c o n t r a c t e r n o r m a l e m e n t e n pr6sence d ' o c y t o c i n e e n ana~robiose. Cette c o n s t a r a t i o n a p e r m i s de m e t t r e a u p o i n t u n dosage sp~cifique de l'ocytocine en ana~robiose en p r e s e n c e de glucose. U n e ~tude de cet~e m ~ t h o d e de dosage et de sa signification p h y s i o l o g i q u e sera publi~e ult~rieurement. L e s experiences de r~duction de l'ocytocine o n t ~t~ faites d a n s des conditions voisines de ,ceUes de SEALOCK ET DU VIGNEAUD1; l'ocytocine (Pitocine P a r k e - D a v i s & Co, D6troit, Mich.
SHORT COMMUNICATIONS
VOL.
30
(I958)
Enzymic formation of choline sulfate WOOLLEY A N D
PETERSON 1
isolated a n d identified cyclic choline sulfate from t h e m y c e l i u m of
Aspergillus sydowi. The occurrence of t h i s c o m p o u n d in t h e mycelium of a strain of Penicillum chrysogenum h a s also been reported 2. I n t h e present s t u d y a cell-free extract from Aspergillus sydowi h a s b~en obtained which is capable of catalyzing t h e sulfofiation of choline. The mold was grown in modified Fries m e d i u m 8 (sucrose replaced b y glucose) for 3-4 days at 3 °0 with shaking. A crude extract was obtained b y grinding t h e mycelia with a glass homogenizer in o.o2 M p h o s p h a t e buffer (pH 7.2) in t h e presence of cysteine (o.oi M). After centrifugation t h e protein was precipitated b y s a t u r a t i n g t h e s u p e r n a t a n t fluid with a m m o n i u m sulfate. The precipitate was dissolved in p h o s p h a t e buffer (0.02 M, p H 7.2). containing o.oi M cysteine and dialyzed overnight against t h e s a m e buffer at 0-5 ° . TABLE I ENZYMIC FORMATION OF CHOLINE SULFATE
The complete s y s t e m contained 4 ° #moles ATP, io #moles phospho-enol-pyruvate (PEP), 4 ° #moles choline chloride, 12.5 #moles MgC1v IO #moles cysteine, 26 #C of carrier-free sulfate and 14 m g protein in 2.o 5 ml o.oi M p h o s p h a t e buffer (pH 7.2). After 2-h incubation at 37 ° t h e reaction m i x t u r e was h e a t e d for about 2 m i n at IOO°, centrifuged, a n d a z-ml aliquot was applied to a paper c h r o m a t o g r a m . The choline sulfate spot was eluted, lyophilized, and counted. Deletions [tom reaction
Choline sullate Counts~rain
-Choline -ATP -ATP, PEP -PEP* - M g ++ None
1068 273 463 30320 17267 28 379
* I n this t u b e an additional 2o #moles A T P was included to compensate for the omission of P E P . The reaction m i x t u r e required for t h e formation of choline sulfate is shown in Table I. The reaction product was separated b y paper c h r o m a t o g r a p h y~. The radioactive spot corresponding to chemically synthesized4 choline sulfate (~S-labelled) was eluted, lyophilized, a n d counted with a C~M counter. C h r o m a t o g r a p h y of a m i x t u r e of t h e eluted material a n d t h e synthetic choline sulfate gave only one radioactive spot. T h e eluted material was not absorbed b y Dowex-i resin (formate form) a n d did n o t move on paper electrophoresis at p H 5.6. The chenfically synthesized choline sulfate behaved exactly in the s a m e m a n n e r . As shown in Table I, adenosine triphosphate (ATP), Mg++, a n d choline are required for the formation of t h e product. In earlier experiments with crude extract, the addition of p h o s p h o e n o l p y r u v a t e (PEP) stimulated sulfate incorporation. z I0
o
o
~8 & o6 z
~2 0, TIME ( M I N }
Fig. I. Time course of labeled-sulfate incorporation into choline sulfate. The percentage incorporation was calculated by counting radioactivity of the sulfate a n d t h e choline sulfate spot on the chromatogram. The respective radioactive spots were located b y radioautography.
I n s u b s e q u e n t experiments with preparations obtained b y fractionation with a m m o n i u m sulfate and with excess ATP, t h e effect of P E P was not observed. A t i m e course of t h e ~ncorporation of labeled sulfate is shown in Fig. i. It h a s been reported ~'e, t h a t adenosine-3'-phosphate-5'-phosphosulfate(PAPS) is t h e actual sulfate donor in t h e enzymic sulfonation of p-nitrophenol by rat-liver extracts. This reaction
VOT., 30 (1958)
z9z
SHO~T COMMUNICATIONS
iwcolves a c t i v a t i o n of sulfate b y A T P a n d its s u b s e q u e n t t r a n s f e r to p - n i t r o p h e n o l f o r m i n g p - n i t r o p h e n o l sulfate. T h e Aspergillus sydowi e n z y m e in t h e presence of A T P , s u l f a t e a n d p-nitrop h e n o l is n o t capable of f o r m i n g p - n i t r o p h e n o l sulfate unless t h e s u l f a t e - t r a n s f e r r i n g e n z y m e f r o m r a t liver is added. T h e r e s u l t s indicate t h e presence of t h e s u l f a t e - a c t i v a t i n g e n z y m e in t h e Aspergillus sydowi. I n a d d i t i o n it w a s f o u n d t h a t a n i m a l e n z y m e s w h i c h are c a p a b l e of f o r m i n g p - n i t r o p h e n o l s u l f a t e f r o m A T P , S O l - - , a n d p - n i t r o p h e n o l do n o t c a t a l y z e choline s u l f a t e f o r m a tion. However, w h e n t h e s e a n i m a l e n z y m e s were c o m b i n e d w i t h t h e Aspergillus sydowi e n z y m e , t h e i n c o r p o r a t i o n of ~ S O s - - into choline sulfate w a s s t i m u l a t e d . T h e s u l f a t e - t r a n s f e r r i n g e n z y m e f r o m t h e r a t liver ~ did n o t s t i m u l a t e t h e choline sulfate f o r m a t i o n b y t h e Aspergillus sydowi enzyme. I t c a n be concluded t h a t t h e e n z y m e c a t a l y z i n g t h e s u l f o n a t i o n of choline is different f r o m t h a t w h i c h c a t a l y z e s t h e f o r m a t i o n of p - n i t r o p h e n o l sulfate f r o m P A P S . I t is possible t h a t P A P S is t h e i m m e d i a t e sulfate donor for t h e f o r m a t i o n of choline sulfate. However, t h e possibility t h a t a d e n o s i n e - 5 ' - p h o s p h o s u l f a t e (APS) is t h e i m m e d i a t e sulfate d o n o r h a s n o t been eliminated. T h e a u t h o r s wish to t h a n k Dr. ICHIHARA for his cooperation in t h e b e g i n n i n g of this s t u d y , a n d to a c k n o w l e d g e t h e s u p p o r t of t h e Office of N a v a l R e s e a r c h a n d t h e A t o m i c E n e r g y Commission. AKIEA KAJI The McCollum Pratt Institute, Department o/Biology, The Johns Hopkins W . D . MCELROY
University, Baltimore, Md. (U.S.A.) i D. W. WOOLLEY AUI) W. H. PETERSON, J. Biol. Chem., 124 (1937) 213. J. DEFLIN~S, J. Am. Chem. Soc., 77 (1955) 1676. G. W . B~AI)LE AND E. L. TATUM, Am. J. Botany, 32 (1945) 678. E. SCI~MIDT, Ann., 337 (19o4) 54. P. ROBmNS AND F. LIPMANN, J. Biol. Chem., 229 (1957) 837. e j . D. GREGORY AND F. LIPMAN~, J. Biol. Chem., 229 (1957) lO81. R. H. D~MEIo, M. WIZERKAMIUK AND I. SCHREmMAN, J. Biol. Chem., 2i 3 (1955) 439R e c e i v e d April 28th, I958
Relation entre I'activit6 physiologique de I'ocytocine et 1'6tat de son pont disulfure Les r ~ s u l t a t s des t r a v a u x s u r les relations e n t r e l'activit6 p h y s i o l o g i q u e de l'ocytocine et l'6tat d u p o n t disulfure d a n s cette moMcule o n t ~t~ j u s q u ' i c i contradictoires. SEALOCK ET DU V I G ~ A U D 1, e n r ~ d u i s a n t le p o n t disulfure p a r la cyst~ine n ' o n t p a s o b t e n u d ' i n a c t i v a t i o n , s a u f darts le cas o~ les g r o u p e m e n t s - S H f o r m , s 6talent bloqu6s p a r benzylation. Ces r~sultats o n t ~t6 confirm6s r 6 c e m m e n t p a r ACHER et coll. $. D ' a u t r e p a r t , GULLAND I~-TRANDALL3, 4 o n t t r o u v 6 q u e la r~duction de l ' h o r m o n e p r o v o q u a i t u n e i n a c t i v a t i o n de 5 ° % et q u e ceUe-ci ~tait p a r t i e l l e m e n t r~versible p a r r~oxydation. U n a u t r e groupe de c h e r c h e u r s 5 a 6 g a l e m e n t t r o u v ~ u n e i n a c t i v a t i o n de l'ocytocine p a r r~duction du p o n t disulfure. L'activit~ de r o c y t o c i n e r~duite a ~t6 e x a m i n e e d a n s la p l u p a r t des cas, soft s u r l'anirnal v i v a n t , soft s u r l ' u t ~ r u s isol6 en a6robiose. GULLAND ET RANDALL8 a v a i e n t c e p e n d a n t utilis~ u n dosage s u r l ' u t 6 r u s de c o b a y e en ana~robiose. L e s d~tails de ce dosage n ' o n t p a s 6t~ publi~s et la sp6cificit6 des r 6 d u c t e u r s utilis6s p a r ces a u t e u r s a 6t~ raise en q u e s t i o n p a r SEALOClC ET DU VIGNEAUD. ]~tant donn~ q u e le p o n t disulfure de l'ocytocine se ref o r m e s p o n t a n 6 m e n t sous l'influence de l'oxyg~ne moMculaire 6, il p o u v a i t p a r a t t r e int~ressant & e x a m i n e r p a r u n e m ~ t h o d e de dosage s t r i c t e m e n t ana6robie !'activit6 p h y s i o l o g i q u e ~ventuelle de l'ocytocine r~duite p a r u n s y s t ~ m e r ~ d u c t e u r sp6cifique des p o n t s S-S, tel q u e l ' o n t e m p l o y 6 SEALOCK ET DU VIGNEAUDI. I1 f a u t d'ailleurs n o t e r q u ' u n e r~activation ~ventuelle de l'ocytocine p a r r ~ o x y d a t i o n d a n s l ' o r g a n i s m e a v a i t ~t6 envisag~e p a r ces a u t e u r s . I1 a 6t6 ~tabli q u e le glucose, qui n ' a a u c u n e influence s u r la respiration de l'ut~rus~, s p e r m e t A cet o r g a n e de se c o n t r a c t e r n o r m a l e m e n t e n pr6sence d ' o c y t o c i n e e n ana~robiose. Cette c o n s t a r a t i o n a p e r m i s de m e t t r e a u p o i n t u n dosage sp~cifique de l'ocytocine en ana~robiose en p r e s e n c e de glucose. U n e ~tude de cet~e m ~ t h o d e de dosage et de sa signification p h y s i o l o g i q u e sera publi~e ult~rieurement. L e s experiences de r~duction de l'ocytocine o n t ~t~ faites d a n s des conditions voisines de ,ceUes de SEALOCK ET DU VIGNEAUD1; l'ocytocine (Pitocine P a r k e - D a v i s & Co, D6troit, Mich.