- Email: [email protected]
Isolation of acetyl peptides from acetylchymotrypsin
voL. 27 (1958)
SHORT COMMUNICATIONS
423
Similar findings h a v e been r e p o r t e d p r e v i o u s l y for t h e r a t s a n d t h e dog* b u t in r u m i n a n t s (goat TM a n d cow 11) o n l y iodide h a s been o b s e r v e d in t h e m i l k a f t e r lSlI a d m i n i s t r a t i o n . N o m e t a b o l i c a l l y a c t i v e c o m p o u n d s h a v e been c h r o m a t o g r a p h i c a l l y d e t e c t e d in milk. T h i s is in a g r e e m e n t w i t h p r e v i o u s r e s u l t s b a s e d o n biological t e s t i n g 12. T h e relative s c a r c i t y or a b s e n c e of D I T in t h e presence of large a m o u n t s of M I T in m i l k is puzzling; e n z y m i c d e i o d i n a t i o n of D I T in t h e b o d y does n o t a p p e a r to s t o p a t t h e M I T s t a g e 6. F a i l u r e to i o d i n a t e b e y o n d t h e M I T s t a g e a p p e a r s to be a m o r e likely e x p l a n a t i o n a n d m a y be s i m p l y t h e c o n s e q u e n c e of a n excess of t y r o s i n e w i t h r e s p e c t to t h e a v a i l a b l e iodine. H o w e v e r , it is of i n t e r e s t t h a t a n excess of M I T over D I T a p p e a r s to be c h a r a c t e r i s t i c of t h e i o d i n a t e d p r o t e i n s f o r m e d b y s e v e r a l " p r i m i t i v e " or " a b n o r m a l " biological i o d i n a t i n g s y s t e m s s u c h as t h e l a c t a t i n g b r e a s t in vivo, cell-free p r e p a r a t i o n s of f u n c t i o n i n g m a m m a r y t i s s u e or t h y r o i d in vitrol3,X4, t h e d e v e l o p i n g chick t h y r o i d 16, c a r c i n o m a s of t h e t h y r o i d TM,17 a n d c e r t a i n a d e n o m a s of t h e t h y r o i d TM. T h a n k s are d u e to Sir CHARLES HARINGTON, Sc. D., F.R.S., a n d to Dr. R. PITT-RIvERS, F.R.S. for t h e o p p o r t u n i t y afforded to one of u s (K. B.-G.) to s p e n d s o m e t i m e as a g u e s t w o r k e r a t t h e N a t i o n a l I n s t i t u t e for Medical R e s e a r c h . K. BROWN-GRANT* National Institute/or Medical Research, London (England) VALERIE A. GALTON 1 K. BROWN-GRANT, J. Physiol., 135 (1957) 644. :2 G. D. POTTER AND I. L. CHAIKOFF, Biochim. Biophys. Acta, 21 (1956) 400. a j . GROSS, Brit. Med. Bull., i o (1954) 218. 4 j , GROSS AND C. P. LEBLOND, Endocrinology, 48 (I951) 714 . 6 W . TONG, A. TAUROG AND I. L. CHAIKOFF, J. Biol. Chem., 195 (1952) 407 . J. B. STANBURY, A. A. H. KASSENAAR, J. W. A. MEIGER AND J. TERPSTRA, J. Clin. Endocrinol. and Metabolism, 16 (1956) 735. 7 j . ROCHE, R. MICHEL AND E. VOLPEET, Compt. rend. soc. biol., 148 (1954) 21. $ K . BROWN-GRANT A N D J. G. GIBSON, J. Physiol., 127 (1955) 341. 6 L. VAN MIDDLESWORTH, J. Clin. Endocrinol. and Metabolism, 16 (1956) 989. 10 W . E. WRIGHT, J. E. CHRISTIAN AND F. N. ANDREWS, J. Dairy Sci., 38 (1955) 31. n R. F. GLASCOCK, J. Dairy Research, 2I (1954) 318. 13 K. L. BLAXTER, Vitamins and Hormones, IO (1952) 217. is A. TAUROG, G. D. POTTER, W. TONG AND I. L. CHAIKOFF, Endocrinology, 58 (1956) 132. 14 A. TAUROG, G. D. POTTER AND I. L. CHAIKOFF, J. Biol. Chem., 213 (I955) 119. 15 j . B. TRUNNELL AND P. V~rAOE, J. Clin. Endocrinol. and Metabolism, 15 (1955) lO7. 16 j . R. TATA, J. E. RALL AND R. W. RAWSON, J. Clin. Endocrinol. and Metabolism, I6 (1956) 1554. iv p. G. STANLEY, Biochem. J., 63 (1956) 581. 18 R. PITT-RIVERS, D. HUBELE AND W. H. HOATHI~R, J. Clin. Endocrinol. and Metabolism, in t h e press. R e c e i v e d O c t o b e r 29th , I957 * P r e s e n t a d d r e s s : D e p a r t m e n t of P h y s i o l o g y , U n i v e r s i t y of B i r m i n g h a m .
Isolation of acetyl peptides from acetylchymotrypsin D u r i n g t h e last few y e a r s several p a p e r s h a v e been p u b l i s h e d on t h e r e a c t i o n of a - c h y m o t r y p s i n w i t h d i i s o p r o p y l p h o s p h o r o f l u o r i d a t e (DFP). T h i s r e a c t i o n resulted in a c o m p l e t e inhibition of t h e e n z y m i c a c t i v i t y a n d w a s a c c o m p a n i e d b y t h e i n t r o d u c t i o n of t h e d i i s o p r o p y l p h o s p h o r y l g r o u p in t h e e n z y m e molecule ( c h y m o t r y p s i n - D P ) . SCHAFFER et al. 1 h a v e d e m o n s t r a t e d t h a t u p o n acid h y d r o l y s i s of c h y m o t r y p s i n - D P , t h e p h o s p h o r u s m o i e t y of D F P w a s p r e s e n t in t h e p e p t i d e c h a i n Gly. A s p . p h o s p h o Ser. Gly. T h i s s e q u e n c e could be e x t e n d e d to Gly. A s p . p h o s p h o S e r . Gly. Glu. T h e s e r e s u l t s were p a r t i a l l y c o n f i r m e d 2. W e f o u n d t h a t u p o n p r o t e o l y t i c digestion of c h y m o t r y p s i n D P a p e p t i d e could be isolated w h i c h c o n t a i n e d a d i i s o p r o p y l p h o s p h o r y l g r o u p s u b s t i t u t e d to t h e h y d r o x y l g r o u p of a serine r e s i d u e 3-6. T h e a m i n o - a c i d s e q u e n c e in t h i s p e p t i d e w a s s h o w n to be Gly. Asp- Ser. Gly. Gly. Pro- Leu. T h e s e r e s u l t s indicate t h a t t h e a t t a c k of D F P on t h e e n z y m e is directed a g a i n s t t h e h y d r o x y l of a serine r e s i d u e w h i c h is o c c u r r i n g in t h e p e p t i d e s e q u e n c e Gly- Asp- Ser. Gly. T h e r e is good r e a s o n to believe t h a t t h e site on t h e e n z y m e w h i c h c o m b i n e s w i t h D F P is identical to t h a t p a r t of t h e e n z y m e w h e r e s u b s t r a t e h y d r o l y s i s is p e r f o r m e d e. Moreover, it is a t t r a c t i v e to consider r e l a t e d m e c h a n i s m s to g o v e r n s u b s t r a t e h y d r o l y s i s a n d i n h i b i t i o n b y D F P . I n t h e f o r m e r case a labile a c e t y l - e n z y m e c o m p l e x is a s s u m e d to be f o r m e d w h i c h b r e a k s d o w n r a p i d l y in w a t e r ; in t h e l a t t e r case a n a n a l o g o u s b u t stable e n z y m e - D P c o m p l e x is formed. T h e s e
424
SHORT COMMUNICATIONS
VOL. 2 7 ( i 9 5 8 )
conclusions, d r a w n f r o m kinetic evidence 7, were s t r o n g l y s u p p o r t e d b y t h e isolation a t low p H of t h e a c e t y l a t e d e n z y m e b y BALLS AND WOODSs. H o w e v e r , t h e isolation of a n a c e t y l - e n z y m e interm e d i a t e is b y itself n o t sufficient evidence for a n a n a l o g o u s m e c h a n i s m , o p e r a t i n g a t one a n d t h e s a m e site of t h e e n z y m e , for b o t h s u b s t r a t e h y d r o l y s i s a n d inhibition b y o r g a n o p h o s p h a t e . It r e m a i n s to be d e m o n s t r a t e d t h a t t h e acetyl g r o u p in t h e a c e t y l a t e d e n z y m e is a c t u a l l y linked to t h e s a m e k n o w n g r o u p as t h e p h o s p h o r y l radical in t h e e n z y m e - D P complex. To e s t a b l i s h t h e p o s i t i o n of t h e acetyl g r o u p in a c e t y l c h y m o t r y p s i n we h a v e followed a similar p r o c e d u r e as w a s u s e d for t h e a n a l y s i s of c h y m o t r y p s i n - D P . A l t h o u g h t h e labile b i n d i n g of t h e a c e t y l g r o u p in a c e t y l c h y m o t r y p s i n a b o v e p H 5.0 m i g h t interfere w i t h s u c h a procedure, t h e possibility t h a t u p o n d e n a t u r a t i o n of a c e t y l c h y m o t r y p s i n a s t a b i l i s a t i o n m i g h t occur did n o t s e e m unlikely*. D i g e s t i o n of acetyl c h y m o t r y p s i n w i t h p e p s i n a t p H 2.0 w a s indeed f o u n d t o stabilise t h e acetyl g r o u p sufficiently, T h i s e n a b l e d u s to c o n t i n u e t h e digestion at p H 7.8 w i t h p a n creatine, a c o m m e r c i a l c r u d e p a n c r e a s e x t r a c t . A c e t y l c h y m o t r y p s i n (IOO mg) labeled w i t h 14C in t h e c a r b o x y l g r o u p of t h e acetyl, w a s p r e p a r e d following t h e p r o c e d u r e of BALLS7, u s i n g p - n i t r o p h e n y l a c e t a t e w i t h a specific radioa c t i v i t y of o.31 m c / m m o l e * * . T h e a c e t y l a t e d e n z y m e w a s dissolved in 2 m l w a t e r a n d d i g e s t e d w i t h 0.25 % p e p s i n a t p H 2.o a n d 2o °. After 16 h t h e p H w a s a d j u s t e d to 7.8 a n d t h e d i g e s t i o n cont i n u e d for 16 h a t 37 ° a f t e r t h e a d d i t i o n of 4 m g p a n c r e a t i n e . T h e 14C-containing d e r i v a t i v e s were f r a c t i o n a t e d a n d purified b y m e a n s of h i g h - v o l t a g e electrophoresis a t p H 6.5 a n d p H 3.5 followed b y p a p e r c h r o m a t o g r a p h y in b u t a n o l - a c e t i c a c i d - w a t e r (BAW) (4 : i : 5)- O n e of t h e p e p t i d e s w a s also c h r o m a t o g r a p h e d in b u t a n o l - w a t e r (BW). T h e s e p a r a t i o n of t h e p e p t i d e s is s h o w n in T a b l e I. T h e purified 14C-containing p e p t i d e s were c o m p l e t e l y h y d r o l y s e d ( 6 N HC1 a t lO5 ° for 24 h) a n d t h e a m i n o acids in t h e h y d r o l y s a t e s were d e t e r m i n e d b y m e a n s of t w o - d i m e n s i o n a l p a p e r c h r o m a t o g r a p h y in p h e n o l - a m m o n i a a n d B A W . TABLE I ELECTROPHORESIS
AND
PAPER
CHROMATOGRAPHY
OF ACETYLPEPTIDES
Paper electrophoresis was performed for 2 h at 3ooo V/4o cm. The figures refer to the migration in m m of the radioactive zones towards the anodic (+I or cathodic (--) site. Electvopkoresis
Peptide
A B C D E
Chromatography (RF)
pH 6. 5
pH3.6
BAW
BW
+ 54 + 71 + 8I + 1°3 + 122
--42 + 35 --32 -- 4 @ 86
0.49 0.77 o.19 °.19 0,44
0.06 -----
T h e c o m p o s i t i o n s of t h e five p e p t i d e s i n v e s t i g a t e d are s u m m a r i z e d in T a b l e II. T h e recorded n u m b e r s of residues are b a s e d o n v i s u a l e s t i m a t i o n of t h e s t r e n g t h of t h e n i n h y d r i n - c o l o u r e d s p o t s w i t h r e s p e c t to t h e 14C c o n t e n t of t h e p e p t i d e s . T h e a c t u a l a m i n o - a c i d s e q u e n c e s of t h e s e five p e p t i d e s h a v e n o t been established. H o w e v e r , it is m o s t i n t e r e s t i n g to o b s e r v e t h a t t h e i r composit i o n is e n t i r e l y c o m p a t i b l e w i t h t h e r e q u i r e m e n t s of t h e s e q u e n c e Gly. Asp. Ser, Gly. Gly, P r o . L e u T A B L E II AMINO-ACID
A Gly Asp Set Gly Gly Pro Leu
COMPOSITION
B
Ser Gly Gly Pro Leu
C Asp Ser Gly Gly
OF ACETYLPEPTIDES
D Gly Asp Ser Gly
E
Set Gly GIy
* T h i s a s s u m p t i o n w a s b a s e d m a i n l y o n t h e o b s e r v a t i o n b y Dr. H. S. JANsz (this laboratory) t h a t t h e possibility of releasing t h e D P - g r o u p f r o m D F P - i n h i b i t e d horse-liver ali-esterase w i t h nueleophilic r e a g e n t s w a s lost a f t e r d e n a t u r a t i o n . ** W e are g r e a t l y i n d e b t e d to Mr. F. BERENDS a n d Miss I. VAN DER SLUYS f r o m t h i s l a b o r a t o r y for t h e p r e p a r a t i o n of t h i s c o m p o u n d .
VOL. 27 (1958)
snoRT COMMUmCATZONS
425
t h a t h a s b e e n d e m o n s t r a t e d in t h e p e p t i d e isolated f r o m c h y m o t r y p s i n - D P S - L A l t h o u g h t h e p o s i t i o n of t h e a c e t y l g r o u p h a s n o t b e e n i n v e s t i g a t e d it s e e m s m o s t likely t h a t it is a s s o c i a t e d in e s t e r l i n k a g e w i t h t h e serine h y d r o x y l . P a r t i c u l a r l y t h e c o m p o s i t i o n of t h e s m a l l a c e t y l p e p t i d e E (Table II) h a r d l y leaves r o o m for o t h e r possibilities. T h e p r e s e n t r e s u l t s p r o v i d e s t r o n g evidence t h a t , in t h e course of t h e h y d r o l y s i s b y c h y m o t r y p s i n of its s u b s t r a t e , t h e h y d r o x y l g r o u p of t h e s a m e serine r e s i d u e is i n v o l v e d as h a s been f o u n d to r e a c t w i t h D F P . T h i s p e p t i d e s t r u c t u r e or a t least p a r t of it m u s t t h e r e f o r e p a r t i c i p a t e in t h e e n z y m i c a l l y - a c t i v e site of c h y m o t r y p s i n .
Medical Biological Laboratory of the National De]ence Reseamh Council T.N.O., Rijswijk-Z.H. (The Netherlands)
R . A . OOSTERBAAN M . E . VAN ADRICHEM
1 2(. K. SCHAFFER, L. SIMET, S. HARSHMAN, 1{. 1{. ENGLE AND 1{. W. DRISKO, J. Biol. Chem., 225 (1957) 197 . . . . . . . . . 2 F. TURBA AND G. GUNDLACH, Biochem. Z., 327 (1955) 186. 3 1{. A. OOSTERBAAN, P. KUNST, J. VAN 1{OTTERDAM AND J. A. COHEN, Bioehim. Biophys. Aeta, (in t h e press). * R. A. OOSTERBAAN, H. S. JANSZ AND J. A. COHEN, Biochim. Biophys. Acta, 20 (1956) 402. 5 j . A. COHEN, R. A. OOSTERBAAN, M. G. P. J. WARRINGA AND H. S. JANSZ, Discussions Faraday Soc., 20 (1955) 114. 6 I. t3. WILSON, in W. D. MCELRoX, AND ]3. GLASS, The Mechanism o/Enzyme Action, ( S y m p o s i u m ) J o h n s H o p k i n s Press, B a l t i m o r e , 1954. 7 13. S. HARTLEY AND 13. A. KILBEY, Biochem. J., 56 (1954) 288. 8 A. K. BALLS AND H. N. WOOD, J. Biol. Chem., 219 (1956) 245. Received O c t o b e r 3 I s t , 1957
On the enzymic reduction of folic acid by a purified hydrogenase* T h e a c t i v a t i o n of folic acid a s s o c i a t e d w i t h t h e cofactor f u n c t i o n s of this v i t a m i n i n v o l v e s h y d r o g e n a t i o n of P G A * * , p r e s u m a b l y to t h e t e t r a - h y d r o f o r m 1 a n d s u b s e q u e n t formylationZ,8, 4 or hydroxymethylationS, e. T h e h y d r o x y m e t h y l c o m p o u n d c a n be oxidized to t h e c o r r e s p o n d i n g f o r m y l derivative% 8. I n t h e p r e s e n t i n v e s t i g a t i o n , folic acid h y d r o g e n a s e w h i c h c a t a l y z e s t h e r e d u c t i o n of P G A w a s p r e p a r e d f r o m a n a c e t o n e p o w d e r of chicken liver. T h e e n z y m e w a s purified 8-fold b y r e m o v a l of p r o t e i n a t p H - 5 ~ 5 - a t ~ d p H - 4 . 5 foliowed-~bT-fr~tional p r e c i p i t a t i o n of t h e e n z v m e w i t h cold acetone. T h e f r a c t i o n s b e t w e e n 4 1 % a n d 6 0 % a c e t o n e c o n t a i n e d m o s t of t h e a c t i v i t y . B y t h i s p r o c e d u r e folic acid h y d r o g e n a s e w a s s e p a r a t e d f r o m e n z y m e s w h i c h c o n v e r t P G A H 4 to its f o r m y l a t e d derivatives. T h e specific r e q u i r e m e n t for T P N H in t h e r e d u c t i o n of P G A could be s h o w n , s u b s t a n t i a t i n g earlier observationsX, 9. T h e purification of t h e e n z y m e w a s sufficient to p e r m i t s t u d y of t h e s t o i c h i o m e t r i c relationship b e t w e e n t h e r e d u c t i o n of P G A a n d t h e o x i d a t i o n of T P N H . Direct evidence is p r e s e n t e d t h a t P G A H 4 r a t h e r t h a n P G A H 2 is t h e p r o d u c t of t h e e n z y m i c reaction. T h e m e t h o d for t h e d e t e r m i n a t i o n of P G A H i w a s b a s e d on t h e n o n - e n z y m i c d e c o m p o s i t i o n of P G A H 4 w h e n e x p o s e d to airZ°, ix yielding P A B G A q u a n t i t a t i v e l y l ; t h i s p r o d u c t w a s d e t e r m i n e d b y t h e BRATTON-MARSHALL r e a c t i o n 12. T h e m a x i m u m c o n v e r s i o n of P G A to P G A H 4 w a s o b t a i n e d a t p H 4.7 in a c e t a t e buffer a n d p H 5.2 in p h o s p h a t e buffer. As s h o w n in T a b l e I, D P N H w a s o n l y 20% as active as T P N H in t h i s reaction. D p N + a n d T P N + were inactive. T h e reversibility of t h e r e a c t i o n could n o t be d e m o n s t r a t e d ; however, t h e a d d i t i o n of T P N + d u r i n g t h e i n c u b a t i o n i n h i b i t e d t h e f u r t h e r c o n v e r s i o n of P G A to P G A H 4. T h e f o r m a t i o n of diazotizable a m i n e w a s c o r r e l a t e d w i t h t h e o x i d a t i o n of T P N H in t h e e x p e r i m e n t s h o w n in T a b l e II. T h e d a t a i n d i c a t e t h a t t w o m o l e s of T P N H are oxidized for e a c h mole of P A B G A formed, in accord w i t h t h e equations : PGA + 2TPNH + 2H + ~ PGAH 4 + 2TPN+ P G A H 4 non-enzymic ~ P t e r i d i n e + PA13GA A m e t h o p t e r i n ( 4 - a m i n o - i o - m e t h y l PGA) c o m p l e t e l y i n h i b i t e d t h e a c t i v i t y of folic acid h y d r o genase at a c o n c e n t r a t i o n of 4 . 2 - I o - S M in t h e p r e s e n c e of 4.4" I o - S M P G A ; a t a c o n c e n t r a t i o n of 4.2. i o - g M A m e t h o p t e r i n , o n l y io % i n h i b i t i o n w a s observed. T h e colorimetric m e t h o d for t h e d e t e r m i n a t i o n of P G A H 4 is limited to t h o s e a n a l o g u e s of * T h i s w o r k w a s s u p p o r t e d in p a r t b y a r e s e a r c h g r a n t (CY-29o6) f r o m t h e N a t i o n a l I n s t i t u t e s of H e a l t h , U.S. Public H e a l t h Service. ** A b b r e v i a t i o n s : PGA, p t e r o y l g l u t a m i c acid; P G A H ~ , d i h y d r o p t e r o y l g l u t a m i c acid; P G A H I , t e t r a h y d r o p t e r o y l g l u t a m i c acid; T P N +, t r i p h o s p h o p y r i d i n e nucleotide; D P N + , d i p h o s p h o p y r i d i n e n u c l e o t i d e ; TP2ffH, r e d u c e d t r i p h o s p h o p y r i d i n e n u c l e o t i d e ; D P N H , r e d u c e d d i p h o s p h o p y r i d i n e n u c l e o t i d e ; PA13GA, p - a m i n o b e n z o y l g l u t a m i c acid.
SHORT COMMUNICATIONS
423
Similar findings h a v e been r e p o r t e d p r e v i o u s l y for t h e r a t s a n d t h e dog* b u t in r u m i n a n t s (goat TM a n d cow 11) o n l y iodide h a s been o b s e r v e d in t h e m i l k a f t e r lSlI a d m i n i s t r a t i o n . N o m e t a b o l i c a l l y a c t i v e c o m p o u n d s h a v e been c h r o m a t o g r a p h i c a l l y d e t e c t e d in milk. T h i s is in a g r e e m e n t w i t h p r e v i o u s r e s u l t s b a s e d o n biological t e s t i n g 12. T h e relative s c a r c i t y or a b s e n c e of D I T in t h e presence of large a m o u n t s of M I T in m i l k is puzzling; e n z y m i c d e i o d i n a t i o n of D I T in t h e b o d y does n o t a p p e a r to s t o p a t t h e M I T s t a g e 6. F a i l u r e to i o d i n a t e b e y o n d t h e M I T s t a g e a p p e a r s to be a m o r e likely e x p l a n a t i o n a n d m a y be s i m p l y t h e c o n s e q u e n c e of a n excess of t y r o s i n e w i t h r e s p e c t to t h e a v a i l a b l e iodine. H o w e v e r , it is of i n t e r e s t t h a t a n excess of M I T over D I T a p p e a r s to be c h a r a c t e r i s t i c of t h e i o d i n a t e d p r o t e i n s f o r m e d b y s e v e r a l " p r i m i t i v e " or " a b n o r m a l " biological i o d i n a t i n g s y s t e m s s u c h as t h e l a c t a t i n g b r e a s t in vivo, cell-free p r e p a r a t i o n s of f u n c t i o n i n g m a m m a r y t i s s u e or t h y r o i d in vitrol3,X4, t h e d e v e l o p i n g chick t h y r o i d 16, c a r c i n o m a s of t h e t h y r o i d TM,17 a n d c e r t a i n a d e n o m a s of t h e t h y r o i d TM. T h a n k s are d u e to Sir CHARLES HARINGTON, Sc. D., F.R.S., a n d to Dr. R. PITT-RIvERS, F.R.S. for t h e o p p o r t u n i t y afforded to one of u s (K. B.-G.) to s p e n d s o m e t i m e as a g u e s t w o r k e r a t t h e N a t i o n a l I n s t i t u t e for Medical R e s e a r c h . K. BROWN-GRANT* National Institute/or Medical Research, London (England) VALERIE A. GALTON 1 K. BROWN-GRANT, J. Physiol., 135 (1957) 644. :2 G. D. POTTER AND I. L. CHAIKOFF, Biochim. Biophys. Acta, 21 (1956) 400. a j . GROSS, Brit. Med. Bull., i o (1954) 218. 4 j , GROSS AND C. P. LEBLOND, Endocrinology, 48 (I951) 714 . 6 W . TONG, A. TAUROG AND I. L. CHAIKOFF, J. Biol. Chem., 195 (1952) 407 . J. B. STANBURY, A. A. H. KASSENAAR, J. W. A. MEIGER AND J. TERPSTRA, J. Clin. Endocrinol. and Metabolism, 16 (1956) 735. 7 j . ROCHE, R. MICHEL AND E. VOLPEET, Compt. rend. soc. biol., 148 (1954) 21. $ K . BROWN-GRANT A N D J. G. GIBSON, J. Physiol., 127 (1955) 341. 6 L. VAN MIDDLESWORTH, J. Clin. Endocrinol. and Metabolism, 16 (1956) 989. 10 W . E. WRIGHT, J. E. CHRISTIAN AND F. N. ANDREWS, J. Dairy Sci., 38 (1955) 31. n R. F. GLASCOCK, J. Dairy Research, 2I (1954) 318. 13 K. L. BLAXTER, Vitamins and Hormones, IO (1952) 217. is A. TAUROG, G. D. POTTER, W. TONG AND I. L. CHAIKOFF, Endocrinology, 58 (1956) 132. 14 A. TAUROG, G. D. POTTER AND I. L. CHAIKOFF, J. Biol. Chem., 213 (I955) 119. 15 j . B. TRUNNELL AND P. V~rAOE, J. Clin. Endocrinol. and Metabolism, 15 (1955) lO7. 16 j . R. TATA, J. E. RALL AND R. W. RAWSON, J. Clin. Endocrinol. and Metabolism, I6 (1956) 1554. iv p. G. STANLEY, Biochem. J., 63 (1956) 581. 18 R. PITT-RIVERS, D. HUBELE AND W. H. HOATHI~R, J. Clin. Endocrinol. and Metabolism, in t h e press. R e c e i v e d O c t o b e r 29th , I957 * P r e s e n t a d d r e s s : D e p a r t m e n t of P h y s i o l o g y , U n i v e r s i t y of B i r m i n g h a m .
Isolation of acetyl peptides from acetylchymotrypsin D u r i n g t h e last few y e a r s several p a p e r s h a v e been p u b l i s h e d on t h e r e a c t i o n of a - c h y m o t r y p s i n w i t h d i i s o p r o p y l p h o s p h o r o f l u o r i d a t e (DFP). T h i s r e a c t i o n resulted in a c o m p l e t e inhibition of t h e e n z y m i c a c t i v i t y a n d w a s a c c o m p a n i e d b y t h e i n t r o d u c t i o n of t h e d i i s o p r o p y l p h o s p h o r y l g r o u p in t h e e n z y m e molecule ( c h y m o t r y p s i n - D P ) . SCHAFFER et al. 1 h a v e d e m o n s t r a t e d t h a t u p o n acid h y d r o l y s i s of c h y m o t r y p s i n - D P , t h e p h o s p h o r u s m o i e t y of D F P w a s p r e s e n t in t h e p e p t i d e c h a i n Gly. A s p . p h o s p h o Ser. Gly. T h i s s e q u e n c e could be e x t e n d e d to Gly. A s p . p h o s p h o S e r . Gly. Glu. T h e s e r e s u l t s were p a r t i a l l y c o n f i r m e d 2. W e f o u n d t h a t u p o n p r o t e o l y t i c digestion of c h y m o t r y p s i n D P a p e p t i d e could be isolated w h i c h c o n t a i n e d a d i i s o p r o p y l p h o s p h o r y l g r o u p s u b s t i t u t e d to t h e h y d r o x y l g r o u p of a serine r e s i d u e 3-6. T h e a m i n o - a c i d s e q u e n c e in t h i s p e p t i d e w a s s h o w n to be Gly. Asp- Ser. Gly. Gly. Pro- Leu. T h e s e r e s u l t s indicate t h a t t h e a t t a c k of D F P on t h e e n z y m e is directed a g a i n s t t h e h y d r o x y l of a serine r e s i d u e w h i c h is o c c u r r i n g in t h e p e p t i d e s e q u e n c e Gly- Asp- Ser. Gly. T h e r e is good r e a s o n to believe t h a t t h e site on t h e e n z y m e w h i c h c o m b i n e s w i t h D F P is identical to t h a t p a r t of t h e e n z y m e w h e r e s u b s t r a t e h y d r o l y s i s is p e r f o r m e d e. Moreover, it is a t t r a c t i v e to consider r e l a t e d m e c h a n i s m s to g o v e r n s u b s t r a t e h y d r o l y s i s a n d i n h i b i t i o n b y D F P . I n t h e f o r m e r case a labile a c e t y l - e n z y m e c o m p l e x is a s s u m e d to be f o r m e d w h i c h b r e a k s d o w n r a p i d l y in w a t e r ; in t h e l a t t e r case a n a n a l o g o u s b u t stable e n z y m e - D P c o m p l e x is formed. T h e s e
424
SHORT COMMUNICATIONS
VOL. 2 7 ( i 9 5 8 )
conclusions, d r a w n f r o m kinetic evidence 7, were s t r o n g l y s u p p o r t e d b y t h e isolation a t low p H of t h e a c e t y l a t e d e n z y m e b y BALLS AND WOODSs. H o w e v e r , t h e isolation of a n a c e t y l - e n z y m e interm e d i a t e is b y itself n o t sufficient evidence for a n a n a l o g o u s m e c h a n i s m , o p e r a t i n g a t one a n d t h e s a m e site of t h e e n z y m e , for b o t h s u b s t r a t e h y d r o l y s i s a n d inhibition b y o r g a n o p h o s p h a t e . It r e m a i n s to be d e m o n s t r a t e d t h a t t h e acetyl g r o u p in t h e a c e t y l a t e d e n z y m e is a c t u a l l y linked to t h e s a m e k n o w n g r o u p as t h e p h o s p h o r y l radical in t h e e n z y m e - D P complex. To e s t a b l i s h t h e p o s i t i o n of t h e acetyl g r o u p in a c e t y l c h y m o t r y p s i n we h a v e followed a similar p r o c e d u r e as w a s u s e d for t h e a n a l y s i s of c h y m o t r y p s i n - D P . A l t h o u g h t h e labile b i n d i n g of t h e a c e t y l g r o u p in a c e t y l c h y m o t r y p s i n a b o v e p H 5.0 m i g h t interfere w i t h s u c h a procedure, t h e possibility t h a t u p o n d e n a t u r a t i o n of a c e t y l c h y m o t r y p s i n a s t a b i l i s a t i o n m i g h t occur did n o t s e e m unlikely*. D i g e s t i o n of acetyl c h y m o t r y p s i n w i t h p e p s i n a t p H 2.0 w a s indeed f o u n d t o stabilise t h e acetyl g r o u p sufficiently, T h i s e n a b l e d u s to c o n t i n u e t h e digestion at p H 7.8 w i t h p a n creatine, a c o m m e r c i a l c r u d e p a n c r e a s e x t r a c t . A c e t y l c h y m o t r y p s i n (IOO mg) labeled w i t h 14C in t h e c a r b o x y l g r o u p of t h e acetyl, w a s p r e p a r e d following t h e p r o c e d u r e of BALLS7, u s i n g p - n i t r o p h e n y l a c e t a t e w i t h a specific radioa c t i v i t y of o.31 m c / m m o l e * * . T h e a c e t y l a t e d e n z y m e w a s dissolved in 2 m l w a t e r a n d d i g e s t e d w i t h 0.25 % p e p s i n a t p H 2.o a n d 2o °. After 16 h t h e p H w a s a d j u s t e d to 7.8 a n d t h e d i g e s t i o n cont i n u e d for 16 h a t 37 ° a f t e r t h e a d d i t i o n of 4 m g p a n c r e a t i n e . T h e 14C-containing d e r i v a t i v e s were f r a c t i o n a t e d a n d purified b y m e a n s of h i g h - v o l t a g e electrophoresis a t p H 6.5 a n d p H 3.5 followed b y p a p e r c h r o m a t o g r a p h y in b u t a n o l - a c e t i c a c i d - w a t e r (BAW) (4 : i : 5)- O n e of t h e p e p t i d e s w a s also c h r o m a t o g r a p h e d in b u t a n o l - w a t e r (BW). T h e s e p a r a t i o n of t h e p e p t i d e s is s h o w n in T a b l e I. T h e purified 14C-containing p e p t i d e s were c o m p l e t e l y h y d r o l y s e d ( 6 N HC1 a t lO5 ° for 24 h) a n d t h e a m i n o acids in t h e h y d r o l y s a t e s were d e t e r m i n e d b y m e a n s of t w o - d i m e n s i o n a l p a p e r c h r o m a t o g r a p h y in p h e n o l - a m m o n i a a n d B A W . TABLE I ELECTROPHORESIS
AND
PAPER
CHROMATOGRAPHY
OF ACETYLPEPTIDES
Paper electrophoresis was performed for 2 h at 3ooo V/4o cm. The figures refer to the migration in m m of the radioactive zones towards the anodic (+I or cathodic (--) site. Electvopkoresis
Peptide
A B C D E
Chromatography (RF)
pH 6. 5
pH3.6
BAW
BW
+ 54 + 71 + 8I + 1°3 + 122
--42 + 35 --32 -- 4 @ 86
0.49 0.77 o.19 °.19 0,44
0.06 -----
T h e c o m p o s i t i o n s of t h e five p e p t i d e s i n v e s t i g a t e d are s u m m a r i z e d in T a b l e II. T h e recorded n u m b e r s of residues are b a s e d o n v i s u a l e s t i m a t i o n of t h e s t r e n g t h of t h e n i n h y d r i n - c o l o u r e d s p o t s w i t h r e s p e c t to t h e 14C c o n t e n t of t h e p e p t i d e s . T h e a c t u a l a m i n o - a c i d s e q u e n c e s of t h e s e five p e p t i d e s h a v e n o t been established. H o w e v e r , it is m o s t i n t e r e s t i n g to o b s e r v e t h a t t h e i r composit i o n is e n t i r e l y c o m p a t i b l e w i t h t h e r e q u i r e m e n t s of t h e s e q u e n c e Gly. Asp. Ser, Gly. Gly, P r o . L e u T A B L E II AMINO-ACID
A Gly Asp Set Gly Gly Pro Leu
COMPOSITION
B
Ser Gly Gly Pro Leu
C Asp Ser Gly Gly
OF ACETYLPEPTIDES
D Gly Asp Ser Gly
E
Set Gly GIy
* T h i s a s s u m p t i o n w a s b a s e d m a i n l y o n t h e o b s e r v a t i o n b y Dr. H. S. JANsz (this laboratory) t h a t t h e possibility of releasing t h e D P - g r o u p f r o m D F P - i n h i b i t e d horse-liver ali-esterase w i t h nueleophilic r e a g e n t s w a s lost a f t e r d e n a t u r a t i o n . ** W e are g r e a t l y i n d e b t e d to Mr. F. BERENDS a n d Miss I. VAN DER SLUYS f r o m t h i s l a b o r a t o r y for t h e p r e p a r a t i o n of t h i s c o m p o u n d .
VOL. 27 (1958)
snoRT COMMUmCATZONS
425
t h a t h a s b e e n d e m o n s t r a t e d in t h e p e p t i d e isolated f r o m c h y m o t r y p s i n - D P S - L A l t h o u g h t h e p o s i t i o n of t h e a c e t y l g r o u p h a s n o t b e e n i n v e s t i g a t e d it s e e m s m o s t likely t h a t it is a s s o c i a t e d in e s t e r l i n k a g e w i t h t h e serine h y d r o x y l . P a r t i c u l a r l y t h e c o m p o s i t i o n of t h e s m a l l a c e t y l p e p t i d e E (Table II) h a r d l y leaves r o o m for o t h e r possibilities. T h e p r e s e n t r e s u l t s p r o v i d e s t r o n g evidence t h a t , in t h e course of t h e h y d r o l y s i s b y c h y m o t r y p s i n of its s u b s t r a t e , t h e h y d r o x y l g r o u p of t h e s a m e serine r e s i d u e is i n v o l v e d as h a s been f o u n d to r e a c t w i t h D F P . T h i s p e p t i d e s t r u c t u r e or a t least p a r t of it m u s t t h e r e f o r e p a r t i c i p a t e in t h e e n z y m i c a l l y - a c t i v e site of c h y m o t r y p s i n .
Medical Biological Laboratory of the National De]ence Reseamh Council T.N.O., Rijswijk-Z.H. (The Netherlands)
R . A . OOSTERBAAN M . E . VAN ADRICHEM
1 2(. K. SCHAFFER, L. SIMET, S. HARSHMAN, 1{. 1{. ENGLE AND 1{. W. DRISKO, J. Biol. Chem., 225 (1957) 197 . . . . . . . . . 2 F. TURBA AND G. GUNDLACH, Biochem. Z., 327 (1955) 186. 3 1{. A. OOSTERBAAN, P. KUNST, J. VAN 1{OTTERDAM AND J. A. COHEN, Bioehim. Biophys. Aeta, (in t h e press). * R. A. OOSTERBAAN, H. S. JANSZ AND J. A. COHEN, Biochim. Biophys. Acta, 20 (1956) 402. 5 j . A. COHEN, R. A. OOSTERBAAN, M. G. P. J. WARRINGA AND H. S. JANSZ, Discussions Faraday Soc., 20 (1955) 114. 6 I. t3. WILSON, in W. D. MCELRoX, AND ]3. GLASS, The Mechanism o/Enzyme Action, ( S y m p o s i u m ) J o h n s H o p k i n s Press, B a l t i m o r e , 1954. 7 13. S. HARTLEY AND 13. A. KILBEY, Biochem. J., 56 (1954) 288. 8 A. K. BALLS AND H. N. WOOD, J. Biol. Chem., 219 (1956) 245. Received O c t o b e r 3 I s t , 1957
On the enzymic reduction of folic acid by a purified hydrogenase* T h e a c t i v a t i o n of folic acid a s s o c i a t e d w i t h t h e cofactor f u n c t i o n s of this v i t a m i n i n v o l v e s h y d r o g e n a t i o n of P G A * * , p r e s u m a b l y to t h e t e t r a - h y d r o f o r m 1 a n d s u b s e q u e n t formylationZ,8, 4 or hydroxymethylationS, e. T h e h y d r o x y m e t h y l c o m p o u n d c a n be oxidized to t h e c o r r e s p o n d i n g f o r m y l derivative% 8. I n t h e p r e s e n t i n v e s t i g a t i o n , folic acid h y d r o g e n a s e w h i c h c a t a l y z e s t h e r e d u c t i o n of P G A w a s p r e p a r e d f r o m a n a c e t o n e p o w d e r of chicken liver. T h e e n z y m e w a s purified 8-fold b y r e m o v a l of p r o t e i n a t p H - 5 ~ 5 - a t ~ d p H - 4 . 5 foliowed-~bT-fr~tional p r e c i p i t a t i o n of t h e e n z v m e w i t h cold acetone. T h e f r a c t i o n s b e t w e e n 4 1 % a n d 6 0 % a c e t o n e c o n t a i n e d m o s t of t h e a c t i v i t y . B y t h i s p r o c e d u r e folic acid h y d r o g e n a s e w a s s e p a r a t e d f r o m e n z y m e s w h i c h c o n v e r t P G A H 4 to its f o r m y l a t e d derivatives. T h e specific r e q u i r e m e n t for T P N H in t h e r e d u c t i o n of P G A could be s h o w n , s u b s t a n t i a t i n g earlier observationsX, 9. T h e purification of t h e e n z y m e w a s sufficient to p e r m i t s t u d y of t h e s t o i c h i o m e t r i c relationship b e t w e e n t h e r e d u c t i o n of P G A a n d t h e o x i d a t i o n of T P N H . Direct evidence is p r e s e n t e d t h a t P G A H 4 r a t h e r t h a n P G A H 2 is t h e p r o d u c t of t h e e n z y m i c reaction. T h e m e t h o d for t h e d e t e r m i n a t i o n of P G A H i w a s b a s e d on t h e n o n - e n z y m i c d e c o m p o s i t i o n of P G A H 4 w h e n e x p o s e d to airZ°, ix yielding P A B G A q u a n t i t a t i v e l y l ; t h i s p r o d u c t w a s d e t e r m i n e d b y t h e BRATTON-MARSHALL r e a c t i o n 12. T h e m a x i m u m c o n v e r s i o n of P G A to P G A H 4 w a s o b t a i n e d a t p H 4.7 in a c e t a t e buffer a n d p H 5.2 in p h o s p h a t e buffer. As s h o w n in T a b l e I, D P N H w a s o n l y 20% as active as T P N H in t h i s reaction. D p N + a n d T P N + were inactive. T h e reversibility of t h e r e a c t i o n could n o t be d e m o n s t r a t e d ; however, t h e a d d i t i o n of T P N + d u r i n g t h e i n c u b a t i o n i n h i b i t e d t h e f u r t h e r c o n v e r s i o n of P G A to P G A H 4. T h e f o r m a t i o n of diazotizable a m i n e w a s c o r r e l a t e d w i t h t h e o x i d a t i o n of T P N H in t h e e x p e r i m e n t s h o w n in T a b l e II. T h e d a t a i n d i c a t e t h a t t w o m o l e s of T P N H are oxidized for e a c h mole of P A B G A formed, in accord w i t h t h e equations : PGA + 2TPNH + 2H + ~ PGAH 4 + 2TPN+ P G A H 4 non-enzymic ~ P t e r i d i n e + PA13GA A m e t h o p t e r i n ( 4 - a m i n o - i o - m e t h y l PGA) c o m p l e t e l y i n h i b i t e d t h e a c t i v i t y of folic acid h y d r o genase at a c o n c e n t r a t i o n of 4 . 2 - I o - S M in t h e p r e s e n c e of 4.4" I o - S M P G A ; a t a c o n c e n t r a t i o n of 4.2. i o - g M A m e t h o p t e r i n , o n l y io % i n h i b i t i o n w a s observed. T h e colorimetric m e t h o d for t h e d e t e r m i n a t i o n of P G A H 4 is limited to t h o s e a n a l o g u e s of * T h i s w o r k w a s s u p p o r t e d in p a r t b y a r e s e a r c h g r a n t (CY-29o6) f r o m t h e N a t i o n a l I n s t i t u t e s of H e a l t h , U.S. Public H e a l t h Service. ** A b b r e v i a t i o n s : PGA, p t e r o y l g l u t a m i c acid; P G A H ~ , d i h y d r o p t e r o y l g l u t a m i c acid; P G A H I , t e t r a h y d r o p t e r o y l g l u t a m i c acid; T P N +, t r i p h o s p h o p y r i d i n e nucleotide; D P N + , d i p h o s p h o p y r i d i n e n u c l e o t i d e ; TP2ffH, r e d u c e d t r i p h o s p h o p y r i d i n e n u c l e o t i d e ; D P N H , r e d u c e d d i p h o s p h o p y r i d i n e n u c l e o t i d e ; PA13GA, p - a m i n o b e n z o y l g l u t a m i c acid.