The initial phase of actomyosin-adenosinetriphosphatase

BIOCHIMICA ET BIOPHYSICA ACTA

135

THE INITIAL PHASE OF ACTOMYOSIN-ADENOSINETRIPHOSPHATASE * II. FACTORS INFLUENCING ACTIVITY Y U J I TONOMURA AND SHOTARO KITAGAWA

Research Institute ]or Catalysis, and Chemistry Department, Faculty o] Science, University o/ Hokkaido, Sapporo (Japan) (Received August 6th, 1959)

SUMMARY

I. The same initial rapid splitting of ATP by myosin B is repeatedly obtained upon adding fresh ATP, if the ATP added previously has been completely hydrolyzed. 2. The quantity of the initial rapid splitting of ITP is very small, as compared with that of ATP. 3. The initial ATP hydrolysis by non-dialysed myosin B preparations is only slightly influenced by the addition of Ca ++ or Mg ++. 4. The initial burst of P-liberation is annihilated by dialysis of the myosin. It is restored by adding Mg++ or Mn++, but not by adding Ca ++. 5. It is also abolished by adding small amounts of EDTA, capable of chelating only a negligible part of the Mg ++ present. It is also decreased by the addition of p-chloromercuribenzoate. 6. When sufficient Mg++ is added to a reaction mixture containing ATP and EDTA, a burst of P-liberation is observed.

INTRODUCTION

In a previous paper I we described an investigation into the initial rapid liberation of P from ATP b y myosin B. The phenomenon was first observed by WEBER AND HASSELBACH2. By means of a special apparatus constructed for the measurement of a rapid reaction, it has been revealed that the inorganic phosphate liberated in the initial rapid phase originates from the terminal phosphate group of the ATP added and that the addition of EDTA leads to complete suppression of the initial phase. In subsequent experiments an attempt has been made to explore the reaction mechanism of the initial phase of the myosin B ATPase action more extensively, and it has been found that Mg++ and the SH groups of myosin B are essential for the initial burst of P-liberation. Abbreviations: ATP, adenosine triphosphate; ATPase, adenosine triphosphatase; ITP, inosine triphosphate; P, inorganic orthophosphate; EDTA, ethylenediaminetetraacetic acid; CyDTA, 1,2-diaminocyclohexane-N,N'-tetraacetic acid; PCMB, p-chloromercuribenzoate.

Biochim. Biophys. Acta, 4 ° (196o) 135-14o

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Y. TONOMIRA, s. NITAGA\VA MATERIALS AND METHODS

Myosin B was prepared from rabbit skeletal muscle according to the standard practice used in this laboratory. /\TP and ITP were Sigma preparations. EDTA, PCMB and other reagents were commercial products of the best reagent R-rades available. The dephosphorylation rate was measured either by using the apparatus described in detail in the previous paper ~ or by the conventional method, as occasion demanded. The intercept of the ordinate obtained by extrapolating the linear P-liberation to zero time was defined as the quantity of the initial burst. The spectrophotometric method of BOYERa was employed to follow the reaction o[ protein-SH with PCMB, assuming the molecular change of optical density at 255 mF as 0,ooo. The contents of Mg, ~ and Ca+-" in the myosin B preparations were determined colorimetrically by a slight modification of the YANA(;ISAWA methodL The dye Plasma Colinth B was purchased from Sumitomo ('heroical Co. The protein denatured in the presence of 2o ')(, of HCIO~ was removed by filtration through ash-free filter paper. The filtrate was neutralized with KOH. The precipitated K(]()~ was also removed by filtration. The determinations were further performed exactly according t o YANAGISAWA'S procedure. Other methods used were the same as described in the previous paper unless otherwise stated. RE f-;ULTS

The relationship between the rate of the liberation of P from ATP by myosin B and the amount of the enzyme is shown in Fig. i. It is clearly seen in this figure that the rates of P-liberation in both the steady and the initial phases are proportional to the concentration of the enzyme. a

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Fig. I. Dependence of rate of liberation of P on a m o u n t of enzyme. ©, i.,~ rag; ;., t.2 nag; %, o.6 mg dry-weight m y o s i n B/ml. Reaction m i x t u r e : I m?¢I ATP, 0.6 AI KC1, 7 i n M Ca ++, p H 6. 7, 23 °. (a) initiaI phase; (b) steady phase.

A repeated rapid splitting of ATP was found upon adding fresh ATP to the reaction mixture whenever the previously added ATP was hydrolyzed completely. Similar observations have already been made by WEBER AND HASSELBACtt2. However, the phenomenon remained to be studied quantitatively. Therefore fresh ATP was also added before the ATP added previously had completely disappeared. At various intervals after adding 5o/xM ATP to a series of identical reaction mixtures I m M fresh ATP was added. The result is illustrated in Fig. 2. No initial burst was observed when the remaining concentration of ATP was 25 ~M (arrow a), and the t3iochb,, lfMpAys.. I cla, 4o (196o) i 35--I 40

137

ACTIVITY IN INITIAL PHASE OF ACTOMYOSIN-ATPASE

burst was about 25 % of the control value when the concentration of remaining ATP was 18/~M (arrow b), while the burst had a normal value again as soon as the ATP was exhausted (arrow c). From these results it is evident that during hydrolysis of ATP, the enzyme is quickly restored to its original state. When I T P was the substrate in the presence of IO m M Mg ++ the initial burst was very small as compared with ATP hydrolysis in the presence of I m M Mg++ (see Fig. 3). E m

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Fig. 3. I n i t i a l phase of hydrolysis of ITP. Reaction m i x t u r e : i m M ITP, o.6 M KCI, i o m M Mg ++, p H 6. 7, 22 °. Thin line, I m M ATP, 0.6 M KC1, I m M M g ++, p H 6 . 7 , 22 ° .

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Fig. 2. Initial phase a t various residues ATP concentrations. D o t t e d line, hydrolysis of 5 ° / ~ M ATP. Arrows, additions of I m M ATP w h e n concentrations of residual ATP are (a) 25 # M , (b) 18 # M and (c) o/~M. Reaction m i x t u r e : o.6 M KC1, I m M Mg ++, p H 6. 7, 22 °.

E~ect of divalent cations A typical example of the effect of the addition of Ca++ or Mg ++ on ATP-hydrolysis is illustrated in Fig. 4. The activation b y Ca ++ and inhibition by Mg ÷+, which together form one of the characteristic properties of the steady phase ATPase action 5, are absent in the initial phase. The variation of the KC1 concentration produced a comparable degree of effect on the initial and the steady ATPase: in the presence of

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Fig. 4. Effects of Mg ++ a n d Ca ++ on initial phase. O, 7 m M Ca ++; &, 4.5 m M Mg ++; x , no divalent cation, i m M ATP, 0.6 M KC1, p H 6.7, 22 °. I n s e r t e d figure, (a) 7 m M Ca++; (b) no divalent cation; (c) 4.5 m M Mg ++.

Biochim.Biophys.Acta, 4 °

(196o) 135-14o

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Y. T O N O M U R A , S. KITAGAV~'A

I m M Mg ++ a n d 0.6 M KC1 the velocity in the initial phase was 3 times as high as in the presence of I m M Mg ++ a n d o.o55 M KC1, a n d in the steady phase 4 times. The c o n t e n t s of Ca +~ a n d Mg ++ in the myosin B preparations used were ~.2--x.8 moles a n d 1.4-1.6 moles per IO5 g of protein, respectively. The differences of these values from HASSELBACH'S6 (0.85 mole Ca ++ a n d o.52 mole Mg~+/Ioa g protein) will be due to the difference in the procedure of myosin B preparation. After 43 h of exhaustive dialysis of myosin B solutions against o.6 3.1 KC1 at o ~, the a m o u n t of Ca ++ a n d Mg ++ decreased to o.28-o.34 mole a n d o.27 o.32 mole per IOa g, respectiv(ly. As shown in Fig. 5, no appreciable change of the velocity in the steady phase was observed, b u t the initial burst was abolished almost completely b y dialysis. The restoring effect of several divalent cations on the initial burst, whose concentrations had been reduced b y dialysis, is illustrated by Figs. 5 a n d 6. 5 t~M Mg++ had no effect, while 5o/xM Mg ~+ restored the b u r s t to ahnost the level observed before dialysis. W h e n z m M Mg ~ was added to the dialysed preparation, the initial burst ranged from 15o to 2oo % of the control value in the presence of non-dialvsed myosin B. I m M of Mn ~+ or Sr ~ + produced the same restoring effect as 5o t,M Mg ++, while concentrations of from 5o /xM to I m M ('a -~ ~ were ineffective. Obivouslv it is the presence of Mg ++ t h a t is responsible tor the initial phase. The burst was almost unaffected b y three to eight repetitions of purification b y the dilution precipitation procedure. This result seems to indicate t h a t Mg~ ~ necessary to the initial phase is not l e m o v e d b y this procedure. 2 fl_

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Fig. 5. Effect of dialysis o11 initial phase. ;4, before dialysis; A, after dialysis; O, 5° I~M Ca++ added after dialysis, i m3I ATP, o.6 ~;'14 t(CI, ?H 6.7, _'t'. Effects of E D T A

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Fig. 6. Restoration of imtial lmrst by adding divalent cations to reaction mixture cor~tairting dialysed myosin B, ×, 5° It,l/Mg+*; ,~,., r m3I Sr++; O, I 1113lMn++. I mM ATP, o.6 3l KCI, pH 0.7, -'J •

a¢zd C y l ) J ) l

The effect of E D T A already observed in our previous paper 1 was studied more thoroughly. As can be seen from the example represented in Fig. 7, the burst was reduced even at I / x M E D T A a n d was almost a n n i h i l a t e d at I o / x M E D T A , while the steady phase was almost unaffected. The concentration of the protein in these experim e n t s was I. 4 mg/ml, a n d consequently the concentrations of the total a n d the dialysable Mg ++ were a b o u t 2o/xM a n d x6/xM, respectively. The i n h i b i t o r y action of CyDTA was inferior to t h a t of E D T A . xoo/zM CyDTA c o n c e n t r a t i o n was required for the disappearance of the initial phase. It is evident from Fig. 8 t h a t the initial phase occurs as soon as the Mg ++ c o n c e n t r a t i o n is sufficiently raised in a reaction m i x t u r e c o n t a i n i n g E D T A . F u r t h e r m o r e , on addition of I m M Mg ~ : I m M ATP Hiochim. Biophys..t~tu, ~<~(19oo) 135 -14°

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ACTIVITY IN INITIAL PHASE OF A C T O M Y O S I N - A T P A s E

to a reaction mixture containing 5o FM EDTA the initial burst, was identical to that observed when I m M ATP was added to the reaction mixture containing I m M Mg ++ + 50 FM EDTA. This means that the Mg++-myosin B equilibrium is reached instantaneously.

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Fig. 7. Effect of c h e l a t i n g r e a g e n t s on initial b u r s t of ]?-liberation. x , i o / , M E D T A ; O, i o o # M C y D T A ; d a s h e d line, control. I m M AT]?, 0.6 M KC1, p H 6.7, 22 °.

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Fig. 8. Initial b u r s t of ]?-liberation b y a d d i n g Mg ++ to a r e a c t i o n m i x t u r e c o n t a i n i n g E D T A a n d AT]?. × , 50 /~M E D T A a n d i m M AT]? a t zero t i m e ; arrow, i m M Mg ++ added. &, 5o # M E D T A a n d I m M AT]?, i m M Mg ++ a d d e d a t zero t i m e . 0.6 M KC1, p H 6. 7, I °.

Fig. 9 shows the relationship between the quantity of PCMB bound to the SH groups of myosin B, the quantity of the burst, and the rate in the steady phase. In accordance with the results of KIELLEY AND BRADLEY7 on myosin A, about 8 moles of PCMB were bound to lO5 g of myosin B.

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Fig. 9. Decrease of q u a n t i t y of initial b u r s t w i t h b i n d i n g of P C M B to m y o s i n 13. × , increase of e x t i n c t i o n a t 255 m # ; O, r a t e in s t e a d y p h a s e ; &, q u a n t i t y of initial b u r s t , i m M AT]?, 0.6 M KC1, 5 m M Ca ++, p H 6.7, 2o °.

The effect of PCMB on the steady ATPase was also quite compatible with Kielley's observations on myosin A. The most interesting point in this experiment was that a linear decrease of the burst was paraleled by a linear increase of the binding of PCMB. The burst had completely disappeared at about 80 % occupation of the SH groups by PCMB. Biochim. Biophys. Acta, 4 ° (196o) i 3 5 - I 4 O

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Y. T O N O M U R A , S. K I T A G A \ V A

DISCUSSION

In the present observations tile most striking feature is tile effect of d i v a l e n t cations on the initial phase of A T P h y d r o l y s i s : (I) using n o n - d i a l y s e d myosin B the r a t e is h a r d l y affected b y a d d i n g Mg ++ or Ca -+, (2) the decrease of the b u r s t p r o d u c e d hv e x h a u s t i v e dialysis is restored to the original level b y the a d d i t i o n of a r a t h e r m i n u t e a m o u n t of Mg ++, which cannot be replaced b y Ca ~-'. I t is e v i d e n t t h a t the i n h i b i t o r y effect of E D T A on the initial phase is not merely due to the r e m o v a l of free Mg ++ bv its chelation. U n d e r our e x p e r i m e n t a l conditions, the a m o u n t of dialysable Mg++ did not change appreciably* on tile a d d i t i o n of I O / , M E D T A , a c o n c e n t r a t i o n t h a t c o m p l e t e l y abolishes the initial burst. Moreover, the i n h i b i t o r y effect of C y I ) T A on the initial b u r s t is much smaller t h a n t h a t of E D T A in spite of the fact t h a t the binding c o n s t a n t of C y D T A 8 to Mg ++ is larger t h a n t h a t of E D T A . The nfinimum c o n c e n t r a t i o n of E D T A (I--IO it,ll) needed for suppressing the initial burst is of the same order of m a g n i t u d e as the c o n c e n t r a t i o n of A T P - ( o r P P - ) b i n d i n g - s i t e s of myosin B in the reaction m i x t u r e r e p o r t e d above, viz. 1.4,/5" IO s M 2.8 I~M (@ Ref. 9). S u b s e q u e n t l y it seems reasonable to assume t h a t the initial A T P a s e is suppressed b y some complex f o r m a t i o n between the p r o t e i n a n d E D T A , p r o b a b l y m e d i a t e d b y MK= ~ firmly b o u n d to the protein. The d i s a p p e a r a n c e of the initial phase, w i t h o u t appreciable decrease of the rate in the s t e a d y phase, brought a b o u t b y dialysis a n d b y a d d i t i o n of E D T A or C y l ) T A is not in accordance with the p r e s u m p t i o n t h a t the initial burst is caused b y formation of an e n z y m e - A D P complex as an i n t e r i n e d i a t e of a c o n v e n t i o n a l Michaelis-Menten t y p e , because the a b o v e supposition would u n e q u i v o c a l l y d e m a n d t h a t the kinetic c o n s t a n t s of the s t e a d y s t a t e m u s t be c h a n g e d whenever tile initial r a t e is altered considerably. As r e p o r t e d p r e v i o u s l y ~, this conclusion is also s u p p o r t e d b y the consideration on the q u a n t i t y of the initial burst. ACKNOWLEDGEMENT

This w o r k was aided b y a g r a n t from the Ministry of E d u c a t i o n of J a p a n given to tile Research G r o u p on the m e c h a n i s m of e n z y m e action. REFERENCES I y. TONOMURAAND S. •ITAGAWA, Biochim. Biophys. Acta, 27 (1957) 15. 2 A. \'VEBER AND W'. HASSELBACH,Bioehim. Biophys. Aeta, 15 (1954) 237. 3 p. D. BOYER, J. Am. Chem. Soc., 76 (1954) 4331. 4 F. YANAGISAWA,J. Biochem. (Tokyo), 42 (1955) 3. 5 H. H. WEBER AND U. PORTZEHL,Advances in Protein Chem., 7 (I952) 16I. 6 x~V"HASSELBACH,Biochim. Biophys. Acta, 25 (1957) 562. 7 ~V. ~vV.KIELLEY AND L. ]3. ]3RADLEY,J. Biol. Chem., 218 (1956) 653. 8 A. E. MARTELL ANn M. CALCIN, Chemistry o[ the Metal Chelate Compounds, P r i n c e - H a l l , Inc., New York, 1952. 9 y. TONOMURAAND F. MORITA,J. Biochem. (Tokyo), 46 (1959) 1367. * I n t h e p r e s e n c e of i inM ATP and per c e n t b y t h e a d d i t i o n of io #M EDTA.

16/zM Ca ++ , 1 6 / z M Mg ++ is r e d u c e d b y o n l y a f e w

Biochim. Biophys. Acta, 4° (196o) ~35-14 °