The action of pyrophosphate on adenosinetriphosphatase activity of myosin

644

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VOL. 2 4

The action of pyrophosphate on adenosinetriphosphatase activity of myosin* R e c e n t i n v e s t i g a t i o n s on m y o s i n a d e n o s i n e t r i p h o s p h a t a s e (ATPase) h a v e s h o w n t h a t p r o n o u n c e d e n z y m i c a c t i v a t i o n c a n be a c c o m p l i s h e d b y e t h y l e n e d i a m i n e t e t r a a c e t i c acid (EDTA) in t h e presence of a m m o n i u m ion or p o t a s s i u m ion z, 2,3. T h e p r e s e n t c o m m u n i c a t i o n r e p o r t s t h e a c t i v a t i o n of m y o s i n A T P a s e b y inorganic s o d i u m p y r o p h o s p h a t e (P-P) in t h e presence of a m m o n i u m or p o t a s s i u m ion, a n d t h e effect of P - P on a c t i v a t i o n b y E D T A - a m m o n i u m . T h e m y o s i n w a s p r e p a r e d from r a b b i t skeletal muscle b y a m e t h o d similar to t h a t of SZENTGY6RG¥Z4 as modified b y PERRY 5. A l t h o u g h slight m y o k i n a s e a c t i v i t y a n d adenylic d e a m i n a s e activities were present, b o t h e n z y m i c activities were negligible u n d e r t h e conditions described below, n o t a b l y t h e lack of m a g n e s i u m a n d t h e relatively high p H . T h e a m o u n t of p h o s p h a t e liberated b y t h e action of m y o s i n on A T P was m e a s u r e d b y t h e m e t h o d of FISKE AND SUBBAROW 6. I t was f o u n d essential to r e m o v e t h e P - P a f t e r s t o p p i n g t h e reaction, before t h e a n a l y s i s for o r t h o p h o s p h a t e , because c o n c e n t r a t i o n s of P - P g r e a t e r t h a n O . o l M interfere w i t h t h e d e t e r m i n a t i o n of o r t h o p h o s p h a t e . T h e P - P w a s r e m o v e d as follows: To 2 m l of t h e reaction m i x t u r e s described below, i m l 15 % trichloroacetic acid (TCA) was added, followed i m m e d i a t e l y b y i m l o . 3 M M n S O 4 in I M s o d i u m acetate, r e s u l t i n g in a final p H b e t w e e n 3 a n d 4. U n d e r t h e s e conditions 7, m o s t of t h e P - P p r e c i p i t a t e d as t h e m a n g a n o u s salt, leaving inorganic o r t h o p h o s p h a t e , A D P a n d A T P in solution. T h e solutions were filtered a n d i m l of filtrate was u s e d for o r t h o p h o s p h a t e d e t e r m i n a t i o n . T h e effect of P - P as a n a c t i v a t o r of m y o s i n A T P a s e w a s m e a s u r e d as follows: To i ml of Tris buffer, p H 9.1, c o n t a i n i n g NH4C1 a n d v a r y i n g a m o u n t s of P - P , 0. 3 ml of A T P w a s a d d e d ; t h e v o l u m e w a s b r o u g h t to 1.8 m l with water, a n d 0.2 m l of m y o s i n in o . 5 M KC1 w a s a d d e d ; t h e final p H w a s 9.1. T h e final c o n c e n t r a t i o n s in 2 m l of reaction m i x t u r e were: A T P (o.oi M), NH4C1 (o.25M), Tris (o.o5M), a n d m y o s i n r a n g e d f r o m 2 5 - 8 0 / z g / m l . T h e reactions were r u n a t 250 C for 15 rain a n d s t o p p e d w i t h T C A as described above. To s h o w t h e effect of P - P on t h e E D T A - a m m o n i u m s y s t e m , t h e i n c u b a t i o n m i x t u r e used w a s t h e s a m e as t h a t described a b o v e e x c e p t t h a t E D T A w a s i n c o r p o r a t e d to a final c o n c e n t r a t i o n Of O.OOI M . I n Fig. 1 t h e lower c u r v e (O) illustrates t h e P - P - N H 4 + s y s t e m w i t h o u t E D T A . T h i s s y s t e m s h o w s o p t i m u m a c t i v i t y a t a b o u t o.025 to o . o 3 o M P-P, a p p r o x i m a t e l y 3o % of t h a t o b t a i n a b l e u s i n g E D T A ( o . o o I - o . o 5 M ) a n d o . 2 5 M NH4+ w i t h o u t P-P. T h e P - P - N H 4 + a c t i v a t i o n o b t a i n e d w a s g r e a t e r t h a n t h a t f o u n d u s i n g O . o l M CaCI~ a t p H 9.1 in t h e u s u a l m y o s i n A T P a s e assay. I t should be m e n t i o n e d t h a t P - P w i t h o u t NH4+ p r e s e n t does n o t a c t i v a t e t h e e n z y m e , a n d t h i s is a n a l o g o u s to t h e E D T A s y s t e m 3. K +, replacing NH4+, showed similar b u t lesser a c t i v a t i o n , a n d N a + h a d no effect, w h i c h is also t r u e for t h e E D T A s y s t e m 3. I n t h e report of KmLLEY, K A L C K A R AND B R A D L E Y 8, o . 2 5 M NH4 + w a s described as t h e c o n c e n t r a t i o n n e c e s s a r y for o p t i m a l a c t i v i t y . O u r o w n e x p e r i m e n t s confirm t h e s e results. However, w i t h o.o25dl// P - P in t h e a b s e n c e of E D T A , no o p t i m u m was f o u n d u s i n g v a r y i n g NH4+ c o n c e n t r a t i o n s u p to as h i g h as o.5M; 0.0

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Fig. i, T h e effect of increasing t h e inorganic p y r o p h o s p h a t e c o n c e n t r a t i o n is s h o w n in t h e s y s t e m c o n t a i n i n g no E D T A ( 0 ) a n d t h e s y s t e m c o n t a i n i n g o . o o l M E D T A (A). Full a c t i v i t y (io0 %) is e q u i v a l e n t to t h e liberation of 5 . 2 / t m o l e s inorganic o r t h o p h o s p h a t e .

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Fig. 2. A c o m p a r i s o n is s h o w n of t h e effects on A T P a s e a c t i v i t y u p o n a d d i n g inorganic p y r o p h o s p h a t e ( × ) or a d d i t i o n a l A T P (O) to t h e optimal system containing o.oozM EDTA, o . o o 8 M ATP, o . 2 5 M Ntt4C1. T h e s y s t e m cont a i n e d 60/zg m y o s i n / m l . T h e abscissa (logarithm scale) r e p r e s e n t s t h e m o l a r i t y of a d d e d p y r o p h o s p h a t e or of additional A T P .

* T h i s s t u d y h a s been s u p p o r t e d b y a g r a n t from t h e A m e r i c a n H e a r t Association.

voL. 24 (1957)

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645

t h e a c t i v i t y c o n t i n u e d to rise linearly. I n addition, A D P could n o t replace P - P as a n a c t i v a t o r , n o r could A T P a c t as its o w n a c t i v a t o r . T h e u p p e r c u r v e (~) of Fig. i illustrates t h e r e s u l t s of a d d i n g P - P to t h e E D T A - N H 4 + s y s t e m : P - P did n o t increase t h e a c t i v a t i o n , b u t a t h i g h e r c o n c e n t r a t i o n p r e v e n t e d m a x i m a l a c t i v a t i o n . T h e t w o c u r v e s a p p r o a c h e a c h o t h e r a t a p p r o x i m a t e l y o.o 4 M P - P . I n t h e E D T A - N H 4 + s y s t e m c o n t a i n i n g no P - P , t h e m a x i m u m velocity w a s o b t a i n e d w i t h o.oo8 M A T P . Fig. 2 s h o w s t h e effect of A T P or i n o r g a n i c p y r o p h o s p h a t e a d d e d to t h e o p t i m a l s y s t e m . I t is clearly seen t h a t t h e s u b s t r a t e inhibition arising f r o m o v e r o p t i m a l a m o u n t s of A T P is e x a c t l y t h e s a m e as t h a t o b t a i n e d b y a d d i t i o n of e q u i v a l e n t q u a n t i t i e s of P - P . A D P did n o t s h o w t h i s effect. I n h i b i t i o n b y A T P w a s n o t d e t e c t a b l e in t h e P-P-NH4C1 s y s t e m lacking EDTA. No simple e x p l a n a t i o n of t h e effects recorded a b o v e c a n be offered. A t present, t h e effects of o t h e r t r i p h o s p h o r y l a t e d n u c l e o t i d e s a n d o t h e r c h e l a t i n g a g e n t s are b e i n g i n v e s t i g a t e d ; a n d all of t h e a g e n t s are being f u r t h e r s t u d i e d u s i n g H - m e r o m y o s i n as t h e e n z y m e .

Dept. of Medicine, Long Island Jewish Hospital, New Hyde Park, L.I., N.Y. (U.S.A.)

PAUL M. GALLOP CARL FRANZBLAU EDWARD MEILMAN

X W . J. BowEN AND T. D. KERWlN, J. Biol. Chem., 211 (1954) 237. 2 W . W . KIELLEY AND L. ]3. BRADLEY, J. Biol. Chem., 218 (1956) 653. a W . W . KIELLEY, H. M. KALCKAR AND L. B. BRADLEY, J. Biol. Chem,, 219 (1956) 95. 4 A. SZENT-GY6RGYI, Muscular Contraction, A c a d e m i c P r e s s Inc., N e w York, I947, 5 S. V. PERRY, in S. P. COLOWICK AND •. O. KAPLAN, Methods in Enzymology, Vol. II, A c a d e m i c P r e s s Inc,, N e w York, 1955. 6 C. H. FISKE AND Y. SUBBAROW,J, Biol. Chem., 66 (1925) 375A. KORNBERG, J . Biol. Chem., 182 (195 o) 779. Received M a r c h i g t h , 1957

Esterification of protein and amino acid carboxyl groups by mustard gas and related compounds F r o m c h e m i c a l c o n s i d e r a t i o n s m u s t a r d gas a n d related s u l p h u r c o m p o u n d s (S-mustards) c a n r e a c t w i t h a n y of t h e p o l a r g r o u p s o c c u r r i n g in p r o t e i n s a n d a m i n o acids p r o v i d e d t h e s e g r o u p s are p r e s e n t in t h e n e c e s s a r y ionic s t a t e . A t physiological p H v a l u e s t h e c a r b o x y l g r o u p s of p r o t e i n s a n d a m i n o acids w o u l d be in t h e ionised s t a t e a n d t h u s w o u l d be able to c o m b i n e w i t h t h e s e c o m p o u n d s . A l t h o u g h esterification of t h e a n i o n s of v a r i o u s organic acids b y S - m u s t a r d s a n d s o m e of t h e related n i t r o g e n c o m p o u n d s ( N - m u s t a r d s ) h a s b e e n conclusively d e m o n s t r a t e d 1, z, evidence for t h i s reaction w i t h p r o t e i n c a r b o x y l g r o u p s is indirect in n a t u r e , b e i n g o b t a i n e d either f r o m c h a n g e s in t h e t i t r a t i o n c u r v e s of p r o t e i n s following t r e a t m e n t w i t h S-mustards3, ~ or f r o m e x a m i n a t i o n of t h e p H s t a b i l i t y of b o n d s f o r m e d b e t w e e n S - m u s t a r d s a n d proteinsS, s. T h e nucleophilic reagent, h y d r o x y l a m i n e , reacts v e r y readily w i t h esters to liberate a n alcohol molecule a n d f o r m a h y d r o x a m i c acid w h i c h c a n be d e t e r m i n e d b y t h e coloured c o m p l e x it f o r m s w i t h F e +++ ions. T h e possibility of u s i n g t h i s r e a g e n t to follow O - a l k y l a t i o n r e a c t i o n s of S - m u s t a r d s w i t h p r o t e i n a n d a m i n o acid c a r b o x y l g r o u p s h a s t h e r e f o r e b e e n i n v e s t i g a t e d . H i d e p o w d e r collagen w a s t r e a t e d w i t h r a d i o a c t i v e (35S) di-(2-chloroethyl) s u l p h i d e (H) e s s e n t i a l l y as described b y PIRIE 5. T h e t r e a t e d collagen w a s e x h a u s t i v e l y S o x h l e t - e x t r a c t e d w i t h a c e t o n e a n d e t h e r to r e m o v e a n y u n b o u n d radio~active material. S a m p l e s of ±he t r e a t e d collagen were t h e n i n c u b a t e d for 24 h a t pFI 7.5 a n d 38 ° in t h e presence of 1 . 2 M h y d r o x y l a m i n e . T h i s t r e a t m e n t r e s u l t e d in t h e loss of 56 % of t h e b o u n d H as d e t e r m i n e d b y r a d i o a c t i v i t y m e a s u r e m e n t s w h e r e a s i n c u b a t i o n u n d e r t h e s a m e c o n d i t i o n s b u t in t h e a b s e n c e of h y d r o x y l a m i n e r e s u l t e d in t h e liberation of only 3 % of t h e b o u n d H. W h e n i n c u b a t i o n w a s carried o u t a t p H i i .9, h o w e v e r , 6o % of t h e b o u n d H w a s liberated in t h e a b s e n c e of h y d r o x y l a m i n e . T h e r e is t h u s close a g r e e m e n t b e t w e e n t h e b o u n d H w h i c h is alkali-labile a n d t h a t w h i c h c a n be liberated b y h y d r o x y l a m i n e t r e a t m e n t , as w o u l d be e x p e c t e d if h y d r o x y l a m i n e were a t t a c k i n g e s t e r - t y p e linkages b e t w e e n H a n d collagen. A f u r t h e r series of e x p e r i m e n t s w a s carried o u t u s i n g glycine a n d several S - m u s t a r d s . 2 m m o l e s m u s t a r d were allowed to r e a c t w i t h 24o m m o l e s glycine a t r o o m t e m p e r a t u r e in 20o m l w a t e r . T h e p H w a s m a i n t a i n e d a t 6.0 to a v o i d t h e possibility of a n y N - a l k y l a t i o n reactions. T h e large excess of glycine w a s e m p l o y e d in a n a t t e m p t to e l i m i n a t e loss of t h e r e a g e n t s b y 42