The optical-rotatory dispersion of myosin A

The optical-rotatory dispersion of myosin A

2~ B I O C H I M I C A E T B I O P H Y S I C A ACTA BBA 3855 THE OPTICAL-ROTATORY II. EFFECT YUJI OF DIOXANE TONOMURA, DISPERSION OF MYOSIN ...

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B I O C H I M I C A E T B I O P H Y S I C A ACTA

BBA 3855

THE

OPTICAL-ROTATORY

II. EFFECT YUJI

OF DIOXANE TONOMURA,

DISPERSION

OF

MYOSIN

A

AND p-CHLOROMERCUI~II~ENZOATE

KAZUKO

SEKIYA

A.'~D K I I C H I

IMAMURA

Research Institute for Catalysis and Chemistry Department, Faculty of .Science. Hokkaido UniversiO,, Sapporo (Japan) C R e c e i v e d J u l y x8th, 196z)

SUMMARY

Effects of d i o x a n e a n d p - c h l o r o m e r c u r i b e n z o a t e (PCMB) on t h e o p t i c a l - r o t a t o r y d i s p e r s i o n of m y o s i n A w e r e m e a s u r e d in 0.6 M KCI a t p H 7.0 a n d c o m p a r e d w i t h t h o s e on t h e A T P a s e a c t i v i t y . T h e a-helical c o n t e n t of m y o s i n A e s t i m a t e d f r o m t h e b 0 t e r m of t h e MOFFI'rT--YA:~G p l o t w a s 5 7 - 6 1 % . O n a d d i n g 8 - i o v o l u m e p e r c e n t of d i o x a n e , i n c r e a s e in t h e helical c o n t e n t b y s e v e r a l p e r c e n t a n d p r o n o u n c e d a c t i v a t i o n of A T P a s e w e r e firstly o b s e r v e d a n d were followed b y g r a d u a l d e c r e a s e s in t h e helical c o n t e n t a n d t h e A T P a s e a c t i v i t y . 2 h a f t e r t h e a d d i t i o n of d i o x a n e , t h e helical c o n t e n t d e c r e a s e d o n l y b y a few p e r c e n t , while t h e A T P a s e a c t i v i t y d i s a p p e a r e d c o m p l e t e l y . I m m e d i a t e l y a f t e r t h e a d d i t i o n of d i o x a n e , specific r o t a t o r y p o w e r a t 50o0 A s h o w e d its m a x i m u m a t a b o u t i o % of d i o x a n e in a c c o r d a n c e w i t h t h e a c t i v a t i o n of A T P a s e . T h e helical c o n t e n t of t h e a l k a l i n e - i n a c t i v a t e d m y o s i n A, h o w e v e r , r e m a i n e d c o n s t a n t on t h e a d d i t i o n of d i o x a n e . O n a d d i n g P P t before a n d a t v a r i o u s t i m e s a f t e r t h e a d d i t i o n of d i o x a n e , shift in t h e helical c o n t e n t c a u s e d b y d i o x a n e w a s d e p r e s s e d completely and the content remained constant during the measurements. On adding 3 - 4 m o l e s P C M B p e r I o ~ g of m y o s i n A, t h e m a x i m u m v e l o c i t y a n d t h e ,,~ich~-I:.s~,. c o n s t a n t of A T P a s e a t 2o ° w e r e i n c r e a s e d , r e s p e c t i v e l y , f r o m o.z2 to o.44 m m o l e s P d m i n / g a n d f r o m 1.3 t o 1 . 5 - I o -4 M, a n d t e m p e r a t u r e d e p e n d e n c e of t h e m a x i m u m v e l o c i t y w a s i n c - e a s e d significantly, while on a d d i n g o° moles P C M B t h e A T P a s c a c t i v i t y w a s c o m p l e t e l y i n h i b i t e d . T h e helical c o n t e n t of m y o s i n A i n c r e a s e d b y s e v e r a l p e r c e n t on a d d i t i o n of 4 moles P C M B a n d d e c r e a s e d b y several p e r c e n t on 8 m o l e s P C M B p e r ~o 5 g. O n a d d i n g P C M B in t h e p r e s e n c e of A T P or P P t , t h e helical c o n t e n t fell in b e t w e e n t h o s e in t h e p r e s e n c e of e i t h e r of t h e two. O n t h e basis of t h e s e a n d o t h e r o b s e r v a t i o n s , it w a s s u g g e s t e d t h a t c o n f o r m a t i o n of t h e a c t i v e site is ver~" s u s c e p t i b l e t o i n f l u e n c e s of d i o x a n e a n d P C M B a n d t h a t , a c c o r d i n g l y , a m i n u t e c h a n g e in t h e helical c o n t e a t i n d u c e s a p r o n o u n c e d c h a n g e in t h e A T P a s e a c t i v i t y .

l NTRODUCTION

S e v e r a l i n v e s t i g a t i o n s h a v e b e e n r e p o r t e d o n t h e c o n n e c t i o n of e n z y m i c a c t i v i t y w i t h m o l e c t f l a r s t r u c t u r e of m y o s i n A. LEvY e t a / . t i n f e r r e d f r o m t e m p e r a t u r e d e p e n d e n c e s Abbreviation:

P C M B , p-chloromercuribenzoate.

Biovhim. Biophys..4~ta, ~9 I J963) 2 9 6 - 3 0 5

O P T I C A L R O T A T I O N O F M Y O S I N . II.

~'97

of t h e r a t e s of hydrolysis of A T P a n d I T P t h a t c o n f o r m a t i o n of t h e a c t i v e site changes on its b i n d i n g w i t h t h e s u b s t r a t e . -The p r e s e n t a'uthors 2 h a v e r e c e n t l y o b s e r v e d t h e c h a n g e s in t h e n u m b e r of " a b n o r m a l " t y r o s i n e a n d in t h e excess r i g h t - h a n d e d helical c o n t e n t of myosin A on its b i n d i n g w i t h P P i a n d ATP. T h e a c t i v a t i o n a n d inhibition of A T P a s e b y PCMB was firstly observed b y KIELLEY AND. BRADLEY a. BLUM 4 d e d u c e d from his kinetic studies on t h e a c t i v a t i o n of A T P a s e b y PCMB t h a t t h e b i n d i n g of PCMB i n d u c e s s t r u c t u r a l c h a n g e s in t h e a c t i v e site of myosin A. KOMINZ 5 h a s observed a r e m a r k a b l e c h a n g e in t h e s e d i m e n t a t i o n coefficient of m y o s i n A on its t r e a t m e n t w i t h m e t h y l m e r c u r i c hydroxide. I n t h e previous paper* t h e effects of d i o x a n e on t h e molecular s h a p e a n d t h e A T P a s e a c t i v i t y of m y o s i n A were reported. Soon after t h e a d d i t i o n of IO volume p e r c e n t d i o x a n e to o.6 M KC1 solution of m y o s i n A, an e n h a n c e m e n t of A T P a s e a c t i v i t y , a n increase in t h e r a d i u s of g y r a t i o n , a n d a decrease in t h e viscosity were observed c o n c o m i t a n t l y . T h e s e c h a n g e s were followed b y a g r a d u a l decrease in t h e r a d i u s of g y r a t i o n , an increase in t h e viscosity, a n d i n h i b i t i o n of A T P a s e a c t i v i t y . F u r t h e r m o r e , it was found t h a t t h e p r o p e r t i e s of e n h a n c e m e n t of A T P a s e by d i o x a n e are v e r y similar to those b y PCMB. On t h e basis of these results, c h a n g e s in t h e A T P a s e a c t i v i t y a n d t h e o p t i c a l - r o t a t o r y dispersion of m y o s i n A solution on a d d i n g d i o x a n e a n d PCMB were i n v e s t i g a t e d to clarify t h e r e l a t i o n b e t w e e n t h e A T P a s e a c t i v i t y a n d t h e s e c o n d a r y s t r u c t u r e of m y o s i n A. EXPERIMENTAL

PROCEDURE

Myosin A solutions were o b t a i n e d by t h e m e t h o d of PERRY 7 a f t e r m i n o r modifications 6. T h e solutions were clarified b y c e n t r i f u g a t i o n a t lO 5 × g for 2 h before use. Crystalline d i s o d i u m salt of A T P was o b t a i n e d from Sigma Chemical C o m p a n y . PCMB a n d o t h e r chemicals were c o m m e r c i a l p r o d u c t s of g u a r a n t e e d grade. Since MORALES AND HOTTAa h a v e p o i n t e d o u t t h a t dioxane c o n t a i n s a small a m o u n t of peroxide a n d t h e d i o x a n e effect on A T P a s e is m i m i c k e d b y h y d r o g e n peroxide, g u a r a n t e e d g r a d e d i o x a n e was purified b y t h e following proceduresg: 3oo ml of d i o x a n e was boiled in t h e presence of 3 - 4 g of Ag~O for 2.5 h, a n d distilled a f t e r a d d i t i o n of fused K O H . A fraction w a s ~ s t i ! l e d again in t h e presence of N a m e t a l . 2o g of iron p o w d e r was a d d e d to 250 ml of t h e distillate, a n d distilled after boiling for 2 h. Purified d i o x a n e was k e p t as 8 o % a q u e o u s solution in a refrigerator. B y t h e s e procedures t h e c o n t e n t of peroxide w a s r e d u c e d from I. 5 to lower t h a n o.25/~moles/ml. T h e r e a c t i o n nMxture for m e a s u r e m e n t of A T P a s e a c t i v i t y c o n t a i n e d 0.6 M KCI, 7 mM CaClz as an a c t i v a t o r , I o mM T r i s - m a l e a t e buffer (pH 7.o) a n d 0. 5 mM A T P a n d o.2-0.3 m g / m l of m y o s i n A. M e a s u r e m e n t s were u s u a l l y carried o u t a t 20 °. T h e reaction w a s s t o p p e d b y a d d i n g trichloroacetic acid a t m e a s u r e d i n t e r v a l s of t i m e a n d Pt liberated w a s d e t e r m i n e d b y t h e MARTXN-DoTY m e t h o d 1°. O p t i c a l - r o t a t o r y dispersion of o.6 M KC1 solution of m y o s i n A was m e a s u r e d by m e a n s of a model 2ooS-8o photoelectric s p e c t r o p o l a r i m e t e r w i t h an oscillating polarizer prism (O. C. R u d o l p h a n d Sons (U.S.A.)) in t h e r a n g e of w a v e - l e n g t h from 32oo to 55oo A. A x e n o n c o m p a c t arc l a m p was used as t h e source for c o n t i n u o u s spectra. T h e p o l a r i m e t e r can be set to ~ o.oox5 °, if t h e s a m e e n d plates are used a t fixed positions t h r o u g h o u t one series of e x p e r i m e n t s . T h e c o n c e n t r a t i o n of m y o s i n A was a b o u t 5 m g / m l a n d t h e angle of r o t a t i o n was from - - o . x 5 to --x.5o. Polaximeter t u b e was xo cm in length. I t s t e m p e r a t u r e was m a i n t a i n e d u s u a l l y a t 2o i o,2 ° b y Biockim. Biophys. Acta, 69 (x963) 2 9 6 - - 3 0 5

298

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c i r c u l a t i n g w a t e r from a n u l t r a t h e r m o s t a t H z a k {Gern~_y.} t h r o u g h t h e j a c k e t s u r r o u n d i n g t h e t u b e . All t h e results axe e ~ i n t e r m s off t h e e q u a t i o n of M O F F I T T A N D Y A N G 11"

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w h e r e [m'j is t h e so-called effective r e s i d u e r o t a t i o n a t a n y ~ r ~ v e - ~ , 2, this being t h e o b s e r v e d specific r o t a t i o n , [~], c o r r e c t e d for t h e e~itet~ off t h e refiractive index, n. m e a s u r e d b y a n A b b e t y p e r e f r a c t o m e t e r , a n d t h e a~nerage ~ w e ; g h t of t h e single residue, M 0, c a l c u l a t e d to be 117 (see t e l ~t2). T h e z a i ] ~ ~ p a r a m e t e r , ~-o, w a s t a k e n as c o n s t a n t a n d e q u a l t o 214 ° A. E x c e s s r i g ~ - ~ ~eK,eat c o n t e n t was e s t i m a t e d b y d i v i d i n g t h e --bo t e r m b y 580 ( ~ e reff. x3}, ~ t h e a~ t e r m varies not o n l y w i t h t h e helical c o n t e n t b u t also w i t h chaaages off fl~e ~ _ C o n c e n t r a t i o n of t h e p r o t e i n w a s c a l c u l a t e d b y m ~ - ~ the tdtrogen content, d e t e r m i n e d b y t h e m i c r o - K j e l d a h l m e t h o d or b y t h e m i e r o - ~ m e t h o d (Coleman's n i t r o g e n a n a l y z e r ) , b y a f a c t o r of 6. RESULTS

Effect of dioxane D i s p e r s i o n d a t a a t v a r i o u s t i m e s a f t e r t h e a d d i t i o n ~f 8 ~ - ~ p e r c e n t of purified d i o x a n e a r e p r e s e n t e d in Fig. x in t h e forms of t h e ..plot r e o ~ m ~ x K l e d b y MOFFITT A~D YAN6 u . As clearly seen in t h e figure, slight ~ m t h e ~Q® t e r m a n d slight i n c r e a s e in t h e ~ b 0 t e r m were o b s e r v e d i m p , after ~ a d d i t i o n of dioxane, a n d t h e n a g r a d u a l i n c r e a s e in - - a 0 a n d a d e c r e a s e ha --gPe ~ t i m e w e r e observed. S i m i l a r r e s u l t s were also o b t a i n e d b y a d d i n g ~ ~ d~x~ L,lto m y o s i n A solut_;on. T h e s e c h a n g e s in a 0 a n d b 0 w e r e obser~'ed ~ exxon for all e i g h t preparations tested. I n Fig. z a r e s h o w n t h e t i m e r a t e of t h e ~ in ex~ ~t-handed helical c o n t e n t of m y o s i n A a f t e r t h e a d d i t i o n of 8-I.o v o l u m e p e t r _ e ~ dio.~me t o g e t h e r w i t h -5

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F i g . x. M o x ~ n ~ r - - Y a ~ G p l o t s o f t y p i c a l e ~ . m p l e s o~ ~ ~ ~ ~ times after addition o f 8: v o l u m e per_cent p u r i f i e d d i o x a n e , o . 6 M K C l , " m y ~ m A . : ~ _ ~7~...:~25 ~ , a t z o ° ( p H 7.o). • 'I:~.X--×, c o n t r o i ; 0 ~ 0 , 3o rain" ~--D, 12o ~ ; . A:---A;:~ m m ~ . ~ of dioxane.

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OPTICAL ROTATION OF MYOSIN.

299

II.

those of the ATPase activity. The excess right-handed helical content of myosin A in 0.6 M KCI "fluctuated from one preparation to another in the range from 57 to 61%. Comparing the results in Fig. 2 with those presented in the previous paper a (see especially Fig. 8 of ref. 6), it is clear t h a t the helical content changes parallel to the ATPase activity and the radius of gyration but inversely to the reduced viscosity.

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InculDotion time (rain) Fig. z. T i m e - c o u r s e s of c h a n g e s in h e l i c a l c o n t e n t ( O - - O , 0--0) and ATPase activity (zX--A A - - A ) a f t e r a d d i t i o n of d i o x a n e . 0 . 6 M KC1, a t 2o ° ( p H 7.0). H e l i c a l c o n t e n t w a s e s t i m a t e d f r o m b o t e r m a n d A T P a s e a c t i v i t y m e a s u r e d in 7 m M C a 2+, 0 . 5 m M A T P a n d lO m M T r i s - m a l e a t e b u f f e r . H e l i c a l c o n t e n t : O - - C ) , t o v o l u m e p e r c e n t d i o x a n e , m y o s i n A N o . 7 z, 4 . 6 5 m g / m l ; 0 - - 0 , S v o l u m e p e r c e n t p u r i f i e d d i o x a n e , m y o s i n A N o . 90, 3.62 m g / m l . A T P a s e a c t i v i t y : z X - - / ~ , ~o v o l u m e p e r c e n t d i o x a n e , m y o s i u A N o . 72, o . 2 m g / m l ; ~ k - - A , 8 v o l u m e p e r c e n t p u r i f i e d dioxa~ae, m y o s i n A No. 89, o , z m g / m l .

The magnitude of changes iu the helical content was, however, remarkably smaller than in the ATPase activity, the radius of gyration, and the viscosity: Immediateh" after the addition of 8 - 1 o % dioxane tile helical content increased only b y 5-1o % of the control value b u t the ATPase activity increased to 13o-17o% of the original. and Ioo-2oo min after the addition of dioxane the helical content decreased only by 4-5 %, though the ATPase activity disappeared almost completely. As sl:own in this figure, the effect of dioxane on the helical content and on the ATPase activity of myosin A did not change substantially before and after purification of dioxane. The effect of purified dioxane on ATPase was unaffected by the addition of I mM KCN, which completely inhibits the effect of peroxide x4. Therefore, the effect of dioxane must substantially l~e a t t r i b u t e d to dioxane itself but not to peroxide contained in dioxane. However, the time rate of the decrease in ATPase activity by unpurified dioxane was somewhat higher t h a n t h a t by purified dioxane and was retarded by the addition of I mM KCN. When myosin A was incubated for xo h at p H xo. 3, the helical content measured at p H 7.0 decreased from 60 to 50.4% and the enz3anic activity disappeared. The addition of 8 volume percent dioxane to the alkaline-inactivated myosin A caused a slight decrease in the --ao term but no change in the - - b0 term (Fig. 3)- The addition of x mM Mg=+-PPt decreased +ho helical content by several percent 2. When 8 o, dioxane was added to myosin A after addition of I mM Mg=+-PPt, the - - a o term .

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~t first mad then returned gradually to the original v,xlue, whereas constant as shown in Fig. 3. Fig. 4 shows the results obtained ~ [ x m ~ ~gz+-PPt at various times after the addition of 8 % dioxane. ~ facto myosin A solution -5 min after the addition of dioxane, ~ ha the helical content was observed but the helical content o,~er the period of measurements. The helical content was not ~1~o~ of Mg~+-PP~ I5o and 35o rain after the addition of dioxane ~m~t thereafter. ~ ~ependence of the specific rotatory power at 5000 A, [~5ooo,

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F i g . $ . ~J'f~'It ~ d t i e a ~ a e , o t t h e l i c a l c o n t e n t o f a l k a l i n e - d e n a t u r e d m y o s i n A a n d m y o s i n A - P P ~ c ~ . ,et.~ ~ K(L-I],.l~1~sixx .3L .No. IO3, 4 . 2 6 m g / m l , a t 2o ° ( p H 7.o)- 8 v o l u m e p e r c e n t p u r i f i e d diommae ~ ~ a~ ~i,,~::e o. O - - O , control; ×--×, myosin A denatured beforehand by incnba~fiom t[6~r a,~ th am Tp~Ik~Eo. 3 ; O - - L O , m y o s i n A in p r e s e n c e o f z m M MgZ+-PPi. ~ - - - A , A T P a s e a c t i v i t y in 7 m ~ [ C a s+.

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F i g . 5- D e p e n d e n c e o n d i o x a n e c o n c e n t r a t i o n ( v o l u m e p e r c e n t ) of specific rotatory power of m y o s i n A a t 5 o o o .~. o . 6 M KCI, m y o s i n A N o . 8z, 4 . 7 z m g / m l , a t 2o ~ ( p H 7.0). 0 ~ 0 , 5-Io rain ; x -- ×, 5 h after adding dioxane.

B i o c h i m . B i o p h y s . A c t a , 69 ( z 9 6 3 ) 290--2}05

OPTICAL R o T A T I o N OF MYOSIN. II.

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on the concentration of dioxane. I m m e d i a t e l y after the addition of dioxap.e, F~.]~oe increased with increase in t h e concentration of dioxane before it reached t h e m a x i m u m at zo%, a n d t h e n it decreased g r a d u a l l y withdncrease in the concentration. A similar dependence on t h e concentration of dioxane has already ~eeen reported on t h e ATPase activity s. 5 h after t h e addition of dioxane, no increase in ~J~oogF ~ was observed, and it decreased with increase in t h e c o n c e n t r a t i o n especially above i o % .

Effect of PCMB Since the effect of P,CMB on myosin A ATPase is known to depend on temperature, t h e helical c o n t e n t was measured as a function of t e m p e r a t u r e (Fig. 6). T h e helical c o n t e n t shown in Fig. 6 was m e a s u r e d after 2o rain incubation to equilibrate the solution at required t e m p e r a t u r e . T h e helical c o n t e n t decreased significantly w i t h increase in t e m p e r a t u r e above 25 °. 60

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×103

Fig. 7- M a x i m u m v e l o c i t i e s of A T P a s e (Vm) a s f u n c t i o n s of t e m p e r a t u r e i n p r e s e n c e a n d a b s e n c e of 3 m o l e s P C M B / I o ~ g p r o t e i n . A T P a s e a c t i v i t y w a s m e a s u r e d in 7 rnM Ca t+, 2 m M A T P a n d xo m M T r i s - m a l e a t e buffer ( p H 7.o)M y o s i n A No. 96, o . x - o . 4 m g / m l . O - - O , c o n t r o l ; O - - O , 3 m o l e s P C M B / x o s g.

As has been already reported 3, 4,za t h e ATPase a c t i v i t y exhibits a m~ke-.l i n c ~ ' e ~ when a b o u t one half t h e sulfhydryl grouFs h a v e been t i t r a t e d with PCMB a n d completely ~ p p e a r s ,:;hen all t h e SH groups h a v e reacted. Fig. 7 represents tezaperature dependence of t h e m a x i m u m velocity, Vm, of A T P a s e in t h e presence a n d t h e absence of 3 moles P C M B / I o 5 g; a t 20 ° Vm increased from 0.22 to 0.44 moles Pl]min/g. T h e Michaelis c o n s t a n t , Kin, increased from x.3" xo -4 to 1.5" xo -4 M on t h e addition of 4 moles P C M B / I o 5 g. The a p p a r e n t a c t i v a t i o n energies of Vm a n d Km in t h e absence of PCMB were 8.2 a n d 5.o kcal/mole, respectively, a n d those in t h e presence of PCMB were 23.2 a n d 6.2 kcal]mole, respectively. Thus, dioxane e a n d PCMB increased Vm, Kin, a n d especially t h e t e m p e r a t u r e dependence of Vm. A typical example of t h e effect of PCMB on t h e optical-rotatory dispersion of myosin A is shown in Fig. 8 a n d several results obtained are listed in Table L ' [ h e helical c o n t e n t was calculated from t h e b 0 t e r m measured 3o-6o rain after t h e addition of PCMB. A t all t h e t e m p e r a t u r e s m e a s u r e d t h e helical c o n t e n t increased b y x - 7 % Biochim. Biopkys. Acta, 69 ( I ~ 3 ) 2~--..]o~

302

Y. T O N O M U R A ,

K. S E K I Y A ,

K. I M A M U R A

o n a d d i n g 4 m o l e s P C M B / I o -~g, while in t h e p r e s e n c e of 8 - 9 ra~Aes P C M B / z o s g it w a s lower t h a n in t h e p r e s e n c e of 4 m o l e s ; C M B a n d m o s t l y l o w e r b y 2 - 4 % t h a n t h a t of t h e control. S e v e r a l p e r c e n t increase in t h e helical c o n t e n t b y 4 m o l e s P C M B [ I o 5 g w a s also o b s e r v e d a t p H 5.6 a n d 1o.6.

C~

r~

-20

~0

15

20

F i g . 8. MOFFn-r--YA,~6 p l o t s o f d i s p e r s i o n d a t a in p r e s e n c e o f v a r i o u s & m o u n t s o f P C M B , 0 , 6 M KCI. m y o s i n A N o . 8 4 , 5.1 m g / m l , a t zo ° ( p H 7, I ) . × - - × , control; O~O, 4 moles PCMB/[o 5 g; 7 _ l ~ Z 1 . 8 m o l e s P C M B / I o s g.

I n t h e p r e s e n c e of 4 m o l e s P C M B / z o 5 g t h e o p t i c a l p o w e r r e m a i n e d u n c h a n g e d w i t h t i m e , b u t in t h e p r e s e n c e of 8 m o l e s P C M B t h e - - b o t e r m d e c r e a s e d g r a d u a l l y . F u r t h e r m o r e , it w a s f o u n d t_b.at t h e o p t i c a l r o t a t o r y d i s p e r s i o n c u r v e of m y o s i n A r e c o v e r s t o t h e original o n e b y t h e t r e a t m e n t w i t h 14o m o l e s f l - m e r c a p t o e t h a n o l / i o 5 g a f t e r t h e t r e a t m e n t w i t h 7 m o l e s P C M B / I o 5 g*. A f t e r s u c c e s s i v e a d d i t i o n of x m M Mg~+-PPt a n d 4 moles P C M B / I O s g, t h e helical c o n t e n t of m y o s i n A w a s o b s e r v e d t o be n e a r l y e q u a l t o t h a t of t h e orig;nal m y o s i n A, while it d e c r e a s e d b y s e v e r a l p e r c e n t on a d d i n g P P I a l o n e a. T h e helical c o n t e n t of myGzin A incre~_sed b y se~-era| p e r c e n t on a d d i n g 3 m M MgZ+-ATP (see ref. 2), while on a d d i n g 8 m o l e s P C M B / z o 5 g in t h e p r e s e n c e of A T P it fell in b e t w e e n t h o s e in t h e p r e s e n c e of e i t h e r of t h e two. S e v e r a l p e r c e n t i n c r e a s e in t h e helical c o n t e n t w a s also o b s e r v e d in o.6 M NaCl e i t h e r b y A T P or b y 4 moles P C M B / I o 5 g. Contraryt o t h e case of t h e a b s e n c e of A T P , t h e helical c o n t e n t in t h e p r e s e n c e of A T P d e c r e a s e d b y s e v e r a l p e r c e n t on a d d i n g 4 moles P C M B / I o ~ g. " O n e o f t h e p r e s e n t a u t h o r s ~* h a s d e m o n s t r a t e d t h a t t h e " i n t r i n s i c " C a z+ is r e m o v e d b y t h e treatment of myosin A with PCMB and then with cysteine or/$-mereaptoethanol. Actomyosin. w h i c h w a s c o m p o s e d o f C a f + - f r e e m y o s i n A a n d F - a c t a, s u p e r p r e c i p i t a t e d w i t h o u t s h o w i n g t h e "'clearing response" immediately after the addition of a high concentration of ATP, though the ATPase activity of this modified myosin A was the same as that of the original myosin A and this modification produced no change in the viscosity, the sedimentation, and the extent of binding to F-actin. These results, together with the one described here, suggest that the molecular structure of myosin A does not change on this modification and that the combination of a relaxing factor ( h i g h A T P e t c . ) t o t h e " ' i n t r i n s i c " C a ffi+ is n e c e s s a r y t o t h e " ' c l e a r i n g r e s p o n s e " o f a c t o m y o s i n .

Biochim. Biophys. Mcta, 69 (x963) 2 9 6 - 3 0 5

O P T I C A L R O T A T I O ~ O F M'YOSIN. I1.

EFFECT

Pt,eparation N o .

OF

PCMB

oN

TABLE

I

HELICAL

CO~'E~T

84

.$$

MYOSIN

~7

PCI~IB

(moles/z°6 g)

OF

r

303

A*

86

~

a.

4-5

x~

~

~

4.5

61.3 62.5

57-3 6~.6

56-5 6t_6

60-4

56-5 58.2

57-6 61.2

53-5

59-7

55- 8

....

o 4.0 4" 7 8.o 9.4

6L2 54. t 56-8

55.3

" H e l i c a l c o n t e n t w a s 6 s t i m a t e d f r o m t h e be tcnrm i n 0 . 6 M K ( I a n d a t p H 7 - 7 - 3 .

DISCUSSION

O n adding I o % d i o x a n e t h e helical cont¢ ~t of myos~a A e s t i m a t e d from t h e b0 t e r m increased firstly a n d t h e n decreased g r a d u a l l y . T h e effect of dioxane on t h e optical r o t a t i o n of myosin A could n o t be ascribed t o t h e c h a n g e in t h e e n v i r o n m e n t of a m i n o acid residues b y t h e a b s o r p t i o n of d i o x a n e t o m y o s i n A, since n o t only t h e a 0 t e r m b u t also t h e b 0 t e r m c h a n g e d in a similar w a y t o t h e A T P a s e a c t i v i t y , t h e r a d i u s of g y r a t i o n , a n d t h e viscosity of myosin A. F u r t h e r m o r e , w m a r k a b l e a c t i v a t i o n a n d inhibition of A T P a s e b y dio x a n e were observed c o n c o m i t a n t l y w i t h slight increase a n d decrease in helical c o n t e n t , respectively. EL6DfS r e c e n t investigations~7, is h a v e also s h o w n t h a t organic solvents produce significant a c t i v a t i o n a n d inhibition of t h e activities a n d m a r k e d increases in t h e viscosity w i t h o u t p r o n o u n c e d c h a n g e s in o p t i c a l - r o t a t o r y power of p h o s p h o g l y c e r a l d e h y d e d e h y ~ l ~ a n d nqbonuclease. BLUM4 h a s suggested from his k i n e t i c s t u d y o n t h e P C M B - a c t i v a t i o n a s t r u c t u r a l c h a n g e in t h e myosin A molecule b y its b i n d i n g w i t h I ~ M B . We/s h a v e also d e d u c e d the same conclusion from t h e o b s e r v a t i o n s t h a t A T P a s e is m o r e readily i n a c t i v a t e d b y u r e a in t h e presence t h a n in t h e a b s e n c e of I ~ M B . I n t h e p r e s e n t paper, it h a s been d e m o n s t r a t e d t h a t 4 moles PCMB/xo ~ g i n d u c e s a n increase in the helical c o n t e n t by a few p e r c e n t a n d a r e m a r k a b l e a c t i v a t i o n of A T P a s e , while 8 moles PCMB/xo s g induce a decrease in t h e helical c o n t e n t b y a few p e r c e n t a n d a complete inhibition of ATPase*. F u r t h e r m o r e , b o t h PCMB a n d dioxmae i n c r e a s e d Vm, Km a n d t h e temp e r a t u r e d e p e n d e n c e of Vm a n d r e m o v e d t h e n e u t r a l depression of t h e p H a c t i v i t y c u r v e 6 : L T h e A T P a s e a c t i v i t y was gener~Uy mea.~ated a t zo ° a n d in t h e presence of Ca 2+, while t h e optical r o t a t o r y m e a s u r e u ~ n t s w e r e m a d e in t h e absence of Ca ~+. T h e n it m u s t be said t h a t t h e c h a n g e in helical c o n t e n t is closely r e l a t e d to t h e c h a n g e in properties of A T P a s e a n d n o t to t h e A T P a s e a c t i v i t y itself. I t seems, however, to be v e r y difficult t o d a r i f v t h e relationship b e t w e e n e n z y m i c * Optical-rotatory dispersion curve was ~ in the albsence of ATP, while the ATPase a c t i v i t y w a s m e a s u r e d o f c o u r s e i n t h e p ~ o ~ [ ATIP. ~ a r e , i t m a y b e a e a i r a b l e t o d e t e r m i n e effects of medium and reagent on the optical rotatizm in the ~ of ATP. Such measurements are, however, very difficult and remain to a furtlh~ r~earch~ since both ATP and ADP are optically a c t i v e l e , a n d s i n c e A T P is h y d r o l y z e d r a p i d l y L e c m a ~ o f h i g h c o ~ e n t r a t i o n o f m y o s i n A. Biocki~. •ii:

Biopkys.

A eta, 6 9 (x963) 2 9 6 - 3 o $

304

Y. TONOMURA, K. SEKIYA, K. IMAMURA

a c t i v i t y a n d helical s t r u c t u r e , as t h e a c t i v e site m a k e s u p a v e r y s m a l l f r a c t i o n of t h e e n z y m e m o l e c u l e a n d o p t i c a l r o t a t i o n c a n s h o w o n l y a n e t increase or d e c r e a s e of helical c o n t e n t b u t does n o t reveal o t h e r c o n f o r m a t i o n c h a n g e s . T h e a p p a r e n t l y close r e l a t i o n b e t w e e n r e m a r k a b l e a c t i v a t i o n a n d i n h i b i t i o n in A T P a s e a c t i v i t y b y d i o x a n e a n d P C M B a n d s l i g h t increase a n d d e c r e a s e in helical c o n t e n t m a y be d u e t o a coinc i d e n t c h a n g e in A T P a s e a c t i v i t y a n d t h e net c h a n g e in helical c o n t e n t of m y o s i n A m o l e c u l e as a whole. H o w e v e r , a possible e x p l a n a t i o n of t h e p r e s e n t results i:; as follows; t h e s t r u c t u r e of t h e a c t i v e site is helical, t h e s t r u c t u r e a r o u n d t h e a c t i v e site is v e r y s u s c e p t i b l e to the influence of m e d i u m a n d r e a g e n t , a n d n e t c h a n g e s i.n t h e helical c o n t e n t of p o l y p e p t i d ~ c h a i n of o t h e r p a r t t h a n t h e a c t i v e site is not large*. T h i s a s s u m p t i o n seems to be s u p p o r t e d b y t h e r e s u l t s t h a t on a d d i n g d i o x a n e to t h e a l k a l i n e - i n a c t i v a t e d m y o s i n A -~°no c h a n g e in t h e helical c o n t e n t w a s o b s e r v e d a n d t h a t in t h e p r e s e n c e of P P t , w h i c h b i n d s t o t h e a c t i v e site of m y o s i n A A T P a s e , t h e shift in t h e helical c o n t e n t w i t h t i m e on a d d i n g d i o x a n e w a s n o t o b s e r v e d a n y more. T h e r e s u l t t h a t t h e helical c o n t e n t a f t e r a d d i t i o n of A T P or P P t a n d P C M B fell in b e t w e e n t h e v a l u e s in t h e p r e s e n c e of e i t h e r of t h e t w o s e e m s also t o s u p p o r t t h e a b o v e a s s u m p t i o n , since P C M B a n d A T P or P P i a r e k n o w n t o b i n d t o m y o s i n A c o m p e t i t i v e l y 22. O u r investigations2°, 23 on t h e h e a t , acid, a l k a l i n e a n d s a l t i n a c t i v a t i o n s of A T P a s e h a v e also r e v e a l e d t h a t t h e s e c o n d a r y s t r u c t u r e of t h e a c t i v e site is p a r t i c u larly sensitive to these treatments. ACKNO~,VLEDGEMENTS T h e a u t h o r s wish to t h a n k P r o f e s s o r M. F. MORALES a n d Dr. K. HOTTA of California U n i v e r s i t y , for t h e i r k i n d n e s s in c o m m u n i c a t i n g t h e i r r e s u l t s to us b e f o r e publication. T h i s i n v e s t i g a t i o n w a s s u p p o r t e d b y R e s e a r c h G r a n t A-4233 f r o m t h e U.S. P u b l i c H e a l t h Service, a n d b y g r a n t s f r o m t h e M i n i s t r y of E d u c a t i o n of j a p a n a n d f r o m t h e T o y o R a y o n F o u n d a t i o n t o t h e R e s e a r c h G r o u p on " M o l e c u l a r M e c h a n i s m of Muscle C o n t r a c t i o n " . REFERENCES H. M. LEVY, N. SHARON, E, M. RAY'AN AND D. E. KOSItI,AND, JR., Biochim. Bioph3,s...|cta, .Sb (t9oz) ~ 8 . Y. TONOStURA, K. SEKtYA, K. [MAMUnAAND T. TOKIXVA, Biochim. Biophy¢..4eta. (;9 (t963) 305. s W. V¢. KIELLEV AND L. B. BnADLEY, J. Biol. Chem,, 2[8 (1956) 6.53. 4 j. j, ELuM, Arch. Biochem. Biophys., 87 (I96o) To4. D. R. KOMINz, Biochim. Biophys. Acla, 5 t (x96x) 456. 6 y . TO~OMURA, S. TOKURA, K. SEKIYA AND K. IMAMURA,Arch. Biochem. Biophys., 95 (1962) z29. S. V. l~nnY, in S. P Co~ OW~CK AND N. O. KAPt,AN, IVlethods in Enzvmoi~gy. Vol. 2, Academic Press, Inc., New York, ~955, P. 582. s M. F , MORm-~S A~D K. HOTT^, personal communication. • H . HEPWERTH, J. Chem. Sot., If9 (192t) t256. to j . B . MARTIN AND D . M. DOTY, A n a l . Chem., 2 I (I949) 965U Wr. MOFFITT AND J. T. YANG, Proc. Natl. Acad. Sci. U.S., 42 (I956) .596. * T h e c h a n g e i n h e l i c a l c o n t e n t r e p o r t e d in t h i s p a p e r c a n n o t b e a t t r i b u t e d t o i m p u r i t i e s p r e s e n t i n o u r m y o s i n A p r e p a r a t i o n , s i n c e t h e change i n h e l i c a l c o n t e n t w a s n o t a f f e c t e d by further purification of myosin A by the DEAE-cellulose column chromatography, and since the a m o u n t o f N - t e r m i n a l a m i n o a c i d w a s o . o 6 2 m o l e s p e r l o 5 g p r o t e i n , w h i c h is m u c h s m a l l e r t h a n t h a t r e p o r t e d b y B^ILEY sl ( o I3 m o l e / l o ~ g p r o t e i n ) .

Biockim. B i o p h y s . . 4 a a , 69 ¢1963) 296-3c.3

OPTICAL ROTATION OF MYOSIN. II.

305

~ L ~ . R~ K~MtNZ, A. t t o u G ~ , P. SV,~tONDS A N b K . LAKI, .4rch. Biochem. Biophys., ,50 ( I 9 5 4 ) t4S. tt~si~.. I~ttr~;. Pf'oceedings of the Intern. 5~vmp. on ~I4acromolecular Chem., Prague, Pergamon P r e s s , FLtmdbn~ t 9 5 Z , p. 5. Ji4 []".'. [])ltlXI4,1~N~q-A.ND (~.. E . GLOCK, B i o c h i m . B i o p h y s . . 4 e t a , 7 ( I 9 5 t) 578. tt~"~:.'[]~l~MtrRA AND K. FURUYA, J . Biochem. (Tokyo}, 48 (196o) 899. 10;,4~. K~III-I.~A~.$VA, j . YOSHIMURA AND Y. TONOMURA, J . Biol. Chem., 236 (1961) 902. ,r~ ~'..IibM~DI~ Biochim. B i o p h y s . . 4 e t a , 44 ( t 9 6 o ) 6 1 o . tes l~>..iEaa6Ol'~ l~tochim. 13ioph3,s. Acta, 53 (1961) 2~8. llt~ [~. I~{. I~HX~EDAHL AND m. XV. JAMES, B i o c h i m . B i o p h y s . . 4 c t a , z I (19.-36) 298. 2~)I~. "II~A'iiASHI, T , YASUI, Y, HASHIMOTO AND Y . TONOMURA, .4rch. 13iochem. 13iophys.,

~ IlK. B~.'~t~v, Biochem. J . , 40 (1951~ 23. "~'_~.(~HitG~!r-Y, ;'1. MARTONOSI AND M. m. GOUVEA, i n R . BENESCH, R . E . BlZNESCH, P . l). BOYER, [].. X'I]. [{'~LIOTZ, ~V. R . MIDDLEBROOK AND A. G . SZENT-GYORGYI, S u l f u r in Proteins, A c a d e m i c l ~ / x ~ Iht~., N e w Y o r k , 1959, p. z~ 7. "-~''X~:.'[]~IICOMURA, l(. SEKIYA AZ~D K. |~MAMURA0 J . Biol. Chem.. in t h e press.

B i o c h i m . ~ i o p h y s . Acta, 09 (x903~ z96--3o5

• ~E

OPTICAL-ROTATORY

DISPERSION

[II. E F F E C T OF A D E N O S I N E

OF MYOSIN

TRIPHOSPHATE

A

AND

I N O R G A N IC PY R O P H O S P H A T E "A3r~[]II I]0)N,O M U R A ,

KAZUKO

SEKIYA.

KIICHI

IMAMURA

AND T O M O N O B U

TOKIXVA

ltesearch Institute f o r Catalysis and Chemistry Deparlnwnt, F a c u l t y o f Science, H o k k a i d o UniversiO,, Sapporo {Japan) (Received J u l y tSth, x962)

SUMMARY

~ ~ t ~ of: PPi and ATP on the spectrophotometric titration curve and the optical~tttmui~ dispersion curve of myosin A were investigated. In o. 5 M KC1 the number ~fff"Mh~x~md" tyrosine increased from 3 . 6 - 3 . 7 moles to 6 . 2 - 7 . 0 moles and 5-5 moles m~'g~ protein on adding PPa and ATP, respectively, while it did not change on ~--'~SEA. in 0.5 M NaCI "normal" and "'abnormal" tyrosine could not be , t | ~ d ~ and no significant change in the dissociation state of tyrosine could be ,dban~emlt on~ adding ATP. The electrostatic interaction factor, w, of the dissociation ,,ff"immm£" tyrosine was measured under various conditions. l]mo~6,~l~ KCI, PPt decreases the helical content of myosin A, while A T P increases ~ content b y several percent. However, the content does not change on adlEmg~A~?~ • i n o.6 M NaC1 A T P increases the helical content of myosin A b y several ~mm ina t h e presence and absence of Mg 1+ and even in the presence of E D T A , wdtm~iII]?' was not decomposed b y myosin A.

Biochim. B i o p h y s . Acta, 69 ( x 9 6 3 ) 3o5--3x:~