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On the interaction between myosin A and F-actin
BIOCHIMICA ET BIOPHYSICA ACTA
507
BBA 4 2 5 3
ON T H E I N T E R A C T I O N B E T W E E N MYOSIN A A N D F-ACTIN* L. B. N A N N I N G A
Department of Physiology, University of Texas Medical Center, Galveston, Texas ( U.S.A.) (Received J u l y i2th, 1963)
SUMMARY
The interaction of myosin and actin was studied by means of two techniques: (I) the inhibition of myosin ATPase (ATP-phosphohydrolase, E.C. 3.6.1.3) by actin addition, and (2) centrifugation experiments, carried out so that only free myosin appears in the supernatant. Association constants are calculated and a binding curve for myosin is obtained. The maximum binding agrees with a weight percentage of 3.7 myosin to I actin. The effects of urea, p-chloromercuribenzoate, CaCI~, temperature and pyrophosphate were studied. It is concluded that ATP and actin bind to the same myosin site and that pyrophosphate binds also to this same site. The association constant for pyrophosphate was calculated from its inhibitory effect on the actin-myosin interaction, while this constant for the actin-myosin interaction was also calculated from experiments by GERGELYet al. on the inhibition of pyrophosphate binding to myosin by actin. The temperature dependence of the binding was found to be quite small and the conclusion was made that the free-energy effect is nearly all due to entropy change.
INTRODUCTION
It has been known for a long time that actin combines with myosin in a weight proportion of I to about 3.5-4. SZENT GY6RGYI2 gave a proportion of 3 myosin to i actin, but this was established later as about 3.7: I for maximum binding (see refs. 3, 4 and this article). A value for the binding constant of actin to myosin derived from light-scattering data had been published in 1957 by GERGELYAND KOHLER4. They concluded to a pK value of IO for the myosin-actin interaction at 23 °, 0.6 M KC1 and pH 7.0. Recently (1962) this value was the subject of an investigation by TONOMURAet al. 3, who found p K 6.3 in 0.6 M KC1, pH 7.5 at 9 ° and about 8 at 17 °, also derived from light-scattering data. In this article the same constant has been determined by means of two different techniques: (1) the inhibition of Ca2+-activated myosin ATPase (ATP-phosphohydrolase, EC 3.6.1.3) by actin and (2) centrifugation of myosin-actin mixtures at 30000 rev./min and determination of free myosin in the supernatant. A b b r e v i a t i o n : PCMB, p - c h l o r o m e r c u r i b e n z o a t e . * An a b s t r a c t of this i n v e s t i g a t i o n was p r e s e n t e d at t h e 66th A n n u a l M e e t i n g of t h e T e x a s A c a d e m y of Science, D e c e m b e r I962 at Austin, T e x a s (see ref. I).
Biochim. Biophys. Acta, 82 (1964) 507 517
5Oc~
L.B.
NANN1NGA
The effects of t e m p e r a t u r e , Ca '~+ concentration, urea, PCMB, a n d t}5 rot)host~hates on the association of aetin to m y o s i n has been studied. During recent years it has been clearly established t h a t the contractile protein called " m y o s i n B " is a c t o m v o s i n (GERGELY AND KOHLER 4, HAYASHI et al. ~, WEBER 6, t-IuYs 7, a n d MA1~roxos~ el al. s) so t h a t the actin--myosin association equilibrium is of considerable t)hysiological importance. The values o b t a i n e d for the b i n d i n g c o n s t a n t a p p l y only to dilute solutions of actin a n d m y o s i n u n d e r the e x p e r i m e n t a l conditions (pH, KCI, t e m p e r a t u r e ) . This " n a t u r a l a c t o m y o s i n " or " m y o s i n B " has a p p r o x i m a t e l ) the same prot)ortion of actin a n d m y o s i n as the m a x i m u m value of b o u n d myosin to actin. MATERIALS
Myosin A was o b t a i n e d from r a b b i t skeletal nmscle according to MO~.L\.IAERrs". i t was dissolved in o.3 M KC1, o.oI M Tris (pH 7) a n d k e p t in ice u n d e r chloroform, with a m i n i m u m of air a b o v e it. I n this w a y it keeps well its e n z y m i c a c t i v i t y a n d also its c a p a b i l i t y to combine with actin. Actin was p r e p a r e d from the mince residue after the myosin e x t r a c t i o n according to MOMMAERTS9,1° t a k i n g into account some recent modifications like the use of ascorbic acid with the A T P in the e x t r a c t of the acetone t)owder a n d the use of I o -a M Mg ~- in the I)olymerization process n. The whole procedure was performed at o ~ which p r e v e n t s e x t r a c t i o n of t r o p o m y o s i n ~, vL The p o l y m e r i z a t i o n was &me for i h at room t e m p e r a t u r e , then overnight in the cold room. T h e F - a c t i n was centrifuged at 3oooo rev./min for 2 h a n d the pellets are taken u t) in o.z M KC1. This was r e p e a t e d 3 - 4 times in order to reduce the a b s o r b a n e v of the s u p e r n a t a n t at 28o m/z to values which arc low enough to be a c c e p t a b l e in the exp e r i m e n t s on t h e actin myosin mixtures. Crystalline d i s o d i m n - A T P of P a b s t which is known to be at least 99 % pure was used a n d set to p H 7. The concentrations of myosin m~d actin solutions were d e t e r m i n e d from their a b s o r b a n c y at z8o a n d 32o m/~, c a l i b r a t e d on t h e K j e l d a h l m e t h o d , e28~j e.320- - o.5() for o . 1 % m y o s i n A a n d 0.965 for o . 1 % F-actin (I cm light path). METHODS
T h e A T P a s e activities were m e a s u r e d in the presence of Ca ~+, final c o n c e n t r a t i o n z" xo -2 M (except for some e x p e r i m e n t s in which the Ca '~+ effect was studied), o.I M NaC1, o.oI M Tris buffer (pH 7) a n d at 25 ~ or other t e m p e r a t u r e s . The l i b e r a t e d ino r g a n i c p h o s p h a t e was d e t e r m i n e d b y m e a n s of m o l y b d a t e sulphuric acid a n d dilute SnC12 ~a a d d e d to trichloroacetic acid filtrates. I n the e x p e r i m e n t s where aetin is a d d e d , t h i s is a d d e d first to the m y o s i n a n d t h e A T P is a d d e d last. In the centrifugation e x p e r i m e n t s the actin a n d myosin were i n c u b a t e d in t h e right ionic e n v i r o n m e n t at different t e m p e r a t u r e s for 3o min, t h e n centrifuged for L5 h at 3oooo rev./min. Afterwards, t h e s u p e r n a t a n t is read at 28o a n d 32o m/x. RESUI, TS
The e x p e r i m e n t s on the A T P a s e a c t i v i t y (v) of m y o s i n a n d its inhibition by actin in t h e presence of o.x M NaC1 a n d o . o i M Tris buffer (pH 7.o) a n d o.o2 M CaC12 show Biochim. Biophys. ,qcta, 82 (I004) 507 517
ON THE INTERACTION BETWEEN MYOSIN A AND F-ACTIN
509
both at 25 ° and at 35 ° t h a t actin gives competitive inhibition, as in a plot of I/V against I / [ A T P ] the line obtained without actin and the one in the presence of actin intersect on the ordinate (see Fig. I). 5
4 ._.q E
-o3
2
1
i
L
i
,
i
1
i
i
cl17xlo
i
,
4
i
>
Fig. i. R e c i p r o c a t e of a c t i v i t y a g a i n s t reciprocate of A T P c o n c e n t r a t i o n w i t h ( O - - O , I.O6°/00) a n d w i t h o u t ( O - - O ) actin. T h e a c t i v i t y is expressed in c h a n g e in a b s o r b a n c y at 61o m/t per m i n (not c o n v e r t e d in m m o l e s P as it cancels o u t in calculations of tl'm a n d Ki). T h e s y s t e m c o n t a i n e d : o . i M NaC1, 0.o2 M CaCli, o.oi M Tris (pH 7), o.o77°/00 m y o s i n . I n c u b a t i o n t i m e 5 rain; A T P a d d e d last; t e m p . 25°; Km 1 . 2 " 1 0 - 5 ; irk'i 1. 5" IO -~.
The Michaelis constant, Km, is calculated from slope ordinate (I/V) as: i i Km i v
v +
(KIn~V) and
intercept on
1,," IS]
IS] ~- ATP concentration.
In the presence of actin K I is found from: I
I
Km(I
+
[I]/Ki)
I
in which [I] = actin concentration, Kt inhibition constant or dissociation constant of the enzyme-inhibitor complex, in this case the actomyosin complex. The unit in which the activity is expressed does not m a t t e r since this cancels out in the calculation of Kin. The Kt value depends on the molarity of the inhibitor. For Kin, 1.2" IO -5 is obtained at 25 ° The competitive inhibition proves that the actin binds to the same site on the myosin as the A T P does. For the inhibitor constant is found 1. 5-1o -6, when the actin concentration is expressed in moles based on a molecular weight of 1140o0. This represents a rink of 2 G-actin molecules, with a molecular weight of 57 ooo (compare I~¢[OMMAERTSI°), which binds I myosin molecule. One comes to this conclusion from MO~IMAERTS AND ALDRICH'S value (1958) for Biochim. Biophys. Mcta, 82 (1964) 5o7-517
510
L. B. N A N N I N G A
t h e m o l e c u l a r weight of myosin, 42o ooo (ref. 14) a n d the fact t h a t 42o o o o : I 14 ooo 3.7 : I so t h a t in this w a y the right weight p r o p o r t i o n for m a x i m u m binding is o b t a i n e d . The value of 420 ooo was found to fit the d a t a on A T P binding to myosin resulting in a m a x i m u m b i n d i n g of I mole A T P per mole of myosin, which m e a n s one e n z y m i c site per mole of m y o s i n ~5. This value fitted also the d a t a on the m a x i m u m effect of A T P on a c t o m v o s i n dissociation, i.e. I mole A T P to 504000 g actomyosin'~*, 14. T h e same value was o b t a i n e d in 1958 b y VAN HIPPEL et al. 16. Other a u t h o r s (1959-63) came to 49oooo (ref. 17) , 43oooo (ref. 18), 44ooo0 (ref. 19), 47oooo (ref. 2o), 45oooo (ref. 2I), 426ooo (ref. 22), 5ooooo (ref. 23). The a v e r a g e of these values is 4470oo which is 6.5 °5 higher t h a n 42oooo. W i t h KAYI;'s value of 616oo (ref. 24) for t h e molecular weight of G-actin, which is 8 % higher t h a n MOMMAEI~TS value, this gives for the weight proportion m y o s i n : a c t i n 3.63 : I b a s e d on 447ooo for m y o s i n a n d 3.4: I based on 42oooo; again on the a s s u m p t i o n t h a t each actin d i m e r has one binding site for mw~sin. F r o m t h e generally a c c e p t e d molecular weights a n d the i n a x i m u m binding p r o p o r t i o n , the condition can be d r a w n t h a t a c t o m y o s i n m u s t have a s t r u c t u r e like Fig. 2 a n d t h a t at m a x i m u m b i n d i n g e v e r y second actin m o n o m e r carries I m y o s i n molecule. The reason t h a t the o t h e r m o n o m e r units c a n n o t c a r r y a myosin molecule m a y be steric hindrance. T h e actin c o n c e n t r a t i o n is here expressed in e q u i v a l e n t s of m y o s i n b i n d i n g sites.
j/
o
'
Fig. 2. Supposed structure of actomyosin. Every second G-actin contains t myosin. Explanation in text. In these e x p e r i m e n t s the actin c o n c e n t r a t i o n is a b o u t o.I ° o or 8. 3. lO -8 M, the m y o s i n c o n c e n t r a t i o n 0.005 % or 1.2- lO .7 M a n d t h e A T P c o n c e n t r a t i o n IO-4-IO -3 M. There is therefore a considerable excess of actin over myosin. Since the a c t i v i t y is a b o u t two times lower t h a n w i t h o u t actin at lO -4 M A T P a n d since the inhibition is of c o m p e t i t i v e c h a r a c t e r this m e a n s t h a t half of the A T P b i n d i n g sites are i n o p e r a t i v e because of actin b i n d i n g to these sites. Change in t e m p e r a t u r e to o ° gives still a p p r o x i m a t e l y the same i n h i b i t o r c o n s t a n t (2-1o-6). W h e n i n s t e a d of actin, bovine serum a l b u m i n is a d d e d to the s a m e final c o n c e n t r a t i o n n o t a n y inhibition of A T P a s e a c t i v i t y is obtained. This shows t h a t the actin inhibition is specific. T h e same a m o u n t of actin as a d d e d in Fig. I was ashed a n d t h e ash dissolved in HC1 and neutralized. W h e n this is a d d e d i n s t e a d of t h e actin no inhibition is o b t a i n e d which shows t h a t the inhibition is n o t due to ions like Mg ~+ which m i g h t be a d s o r b e d to t h e actin*. * Moreover actin preparations, polymerized without the addition of ~o 4 M MgCI2, gave the same inhibition effects of myosin ATPase. Biochim. Biophys. ,4cta, 82 (1964) 507-517
ON THE INTERACTION BETWEEN MYOSIN A AND F-ACTIN
511
Addition of urea decreases the inhibitory effect by actin considerably (Fig. 3)This makes it probable that one or more hydrogen bonds are involved in the binding of actin to myosin. Fig. 4 shows the effect of PCMB on the ATPase activity with and without actin. Without actin an increase to about double activity is found as described by KIELLEY 0.6
E ~0.4 c~ fl_
"~0.2 E
"6
o ×
~
3
4
Urea (°/o)
Fig. 3. I n h i b i t i o n of m y o s i n A T P a s e b y actin in urea. R e l a t i v e inhibition e q u a l s t h e a c t i v i t y w i t h u r e a o v e r t h e a c t i v i t y w i t h o u t urea. T h e s y s t e m c o n t a i n e d : o.o51°[00 m y o s i n , i.o5°[0 o actin, o.i M NaC1, 0.02 M CaC12, o.oi M Tris (pH 7), lO-4 M ATP. T e m p . 25 °.
I
1
I
2
I
3
i
4
~_
I
p-Chloromercuribenzoate (x ]O-6M)
Fig. 4. A c i t i v i t y of m y o s i n A T P a s e a g a i n s t conc e n t r a t i o n of PCMB. T h e a c t i v i t y is e x p r e s s e d in m m o l e s p h o s p h a t e per ml f o r m e d p e r min. O - - O , w i t h o u t actin; 0 - - 0 , w i t h actin. 1.o8. io -4 M A T P ; o t h e r conditions as in Fig. 3.
A~D BRADLEY25. In the presence of actin the activity increases or the inhibition disappears with increase of the PCMB concentration. At higher PCMB concentrations the curves intersect which m a y be due to binding of PCMB to the actin. The effects of PCMB suggest that an SH-group is involved in the binding of actin. This is probably an SH-group on the myosin since this is known to have an important 2
U
Concentration of CaCI 2 (x 10-9 I-1)
Fig. 5. I n h i b i t i o n of m y o s i n A T P a s e b y actin as f u n c t i o n of Ca 2+ c o n c e n t r a t i o n . T h e s y s t e m cont a i n e d : o.o68°/00 m y o s i n , i °/0o actin, 0.85" lO -4 M ATP, o. I M NaC1, o.ot M Tris (pH 7). T e m p . 25 °. Biochim. Biophys. Acta, 82 (1964) 5o7-517
512
L.B. NANNINGA
role in A T P h y d r o l y s i s 2a. Also, BAILEY AND PEt{RV found t h a t actin with blocked S H groups still binds to m y o s i n a°. Increase or decrease of the Ca 2+ c o n c e n t r a t i o n affects the inhibition b y actin. At zero Ca 2+ c o n c e n t r a t i o n the a c t i v i t y goes down to zero a n d also the inhibition b y actin decreases to zero (Fig. 5). A t 5' i o -2 M CaCh, the inhibition also decreases considerably. T h e actin c o m p e t i t i o n is o p t i m a l at 2" lO -2 M CaC12, similar to the effect ~f ('a ~+ on A T P a s e a c t i v i t y 26. A t higher p H the inhibition 173' actin decreases: at p H (} actin reduces the a c t i v i t y for lO-4 M A T P only to o.7 i n s t e a d ot to o.5. This results in a higher inhibition c o n s t a n t (4.5" IO~) • Centrifl~gation experime~ts M i x t u r e s of actin (final c o n c e n t r a t i o n 0.028 %) a n d increasing a m o u n t s of myo>in (up to o.2-0.3 %) in a salt milieu of 0.3 M KC1, o . o i M Tris buffer (pH 7) are centrifuged for I. 5 h at 30000 r e v . / m i n . B l a n k runs are m a d e w i t h actin alone a n d with th<, highest concentration of myosin alone. The actin in the b l a n k is found to have a s u p e r n a t a n t with a very low a b s o r b a n c y at 280 mix. The myosin blank is done in order to check t h a t m y o s i n ahme does not give a p r e c i p i t a t e a n d its s u p e r n a t a n t is the sam~. as the original myosin after dilution to the same concentration. The myosin solutions h a v e been centrifuged beforehand for 2 h at 3 ° ooo rev./min a n d are q u i t e clear. After the m i x t u r e s have been centrifuged p a r t of the s u p e m a t a n t is carefully t a k e n off a n d read on its a b s o r b a n c y at 28o a n d 32o m/x. The difference gives the myosin conc e n t r a t i o n in t h e s u p e m a t a n t since actin a n d a c t o m y o s i n b o t h p r e c i p i t a t e under thes~ conditions. The m y o s i n c o n c e n t r a t i o n in the s u p e r n a t a n t is c a l c u l a t e d from the a b s o r b a n c y difference at 280 a n d 320 m ~ as a o.I % solution gives for this difference 0.59. The b o u n d myosin is f o u n d b y s u b t r a c t i o n of the free myosin from the t o t a l nlyosin. In e x p e r i m e n t s in which the myosin b l a n k gives a lower s u p e r n a t a n t t h a n the. c a l c u l a t e d one, correcti(ms are m a d e for myosin in the s u p e r n a t a n t of the actin -myosin mixtures. The a b s o r b a n c y at 280 m/, minus t h a t at 320 in/, for the s u p e r n a t a n t is first corrected for the usually very small a m o u n t given b y two b l a n k s w i t h o u t myosin b u t with actin (aromld o.oi). In presence of increasing a m o u n t s of myosin the abs()rb;, nci,,, increase up to 1.2. It was e s t a b l i s h e d t h a t tip to this value the a b s o r b a n c \ ~f t)m~ ' mvosin is p r o p o r t i o n a l t~) its concentration. W h e n m y o s i n w i t h o u t actin gives a co:> c e n t r a t i o n which is IO % lower t h a n the c a l c u l a t e d one, the values of all the supern a t a n t s were m u l t i p l i e d bv I.IO in order to correct for this. U s u a l l y the figures fi)r c a l c u l a t e d a n d m e a s u r e d myosin in the e x p e r i m e n t w i t h o u t actin agreed within a few percent. Figures were rejected when the myosin found was less t h a n 9 ° 00 of the calc u l a t e d vahle. T h e b i n d i n g is expressed as b o u n d m y o s i n verslls free myosin in Fig. (). I t is clearly seen t h a t a m a x i m u m b i n d i n g a t 25 ° is a p p r o a c h e d at a value of 1.o5°/00 m y o s i n b o u n d to an actin concentration of o.28"/0 o (final c o n c e n t r a t i o n s (Fig. 6)). This gives a p r o p o r t i o n of 3.75:1 which agrees with values published b \ GERGELY AN D N O H I ~ E R 4 a n d TONOMURA et al. a. A t o~ the m a x i m u m binding is found t(, be only slightly less t h a n this (3.4: I). The presence of IO -a M p y r o p h o s p h a t e reduces the binding considerably (see below). Hiochim, Biophys. .!c[a, se (~904} 5o7 51 7
ON THE INTERACTION BETWEEN MYOSIN
A AND F-ACTIN
513
Half of maximum binding occurs at o.o18 % or 4.3" 10-7 M myosin, based on a molecular weight 420000. This gives an association constant of 2.32" 106. The dissociation constant is about three times smaller than the inhibition constant as obtained before (0.43. lO-6 compared to 1.5" lO-6). The difference is probably caused by the different salt conditions and the absence of ATP which acts as a competitor for the binding of actin.
1
._c .C
g
D
g 2;
2~-
C
o
I
r
i
L
Myosin free in %o
Fig. 6. B o u n d against free m y o s i n from centrifugation experiments. The s y s t e m contained: o.280[00 actin, 0. 3 M KCt, o.oi M Tris (pH 7)O O, temp. 26°; 0 - - 0 , temp. o°; A - - A , temp. o °, IO-a M p y r o p h o s p h a t e added.
I
I
I
Pyrophosphate(l-x 10.4 M)
:
5
Fig. 7. Bound myosin against p y r o p h o s p h a t e concentration. This was done at high myosin concentrations relative to the actin concentrations so t h a t m a x i m u m binding is approached, compare Fig. 6. The s y s t e m contained : o.2 I 0/01~ actin, 0. 3 M KC1, o.oi M Tris (pH 7), 5' IO-4 ~I MgC12; total myosin 1.96°/00; temp. 23 °.
The value for the dissociation constant is an "intrinsic" one since many myosin molecules are bound to I F-actin molecule. The temperature dependence is found to be small; the binding is about IO % less at o ° than at 25 ° (Fig. 6). This means that most of the free energy of formation of the actomyosin is due to an entropy increase which may be related with liberation of bound water. The entropy increase is about 31 cal/degree • mole as calculated from the association constant assuming that the enthalpy change is negligibly small. TONOMURA et al. s, however, found a strong temperature dependence and came to a value of 234 cal/degree, mole for the entropy of formation. The maximum binding of myosin to actin is reduced about 4-fold in the presence of IO -a M pyrophosphate at o ° (Fig. 6). With increasing concentrations of pyrophosphate in the presence of 5" IO-4 M Mg2+ at o °, it is found that the bound myosin strongly decreases (Fig. 7). GERGELYet al. ez had found that pyrophosphate binds to myosin in a I : I molar proportion and that this binding is reduced by actin. In their experiments the myosin concentration was 9.3" I°-n M (based on a molecular weight 500000). At a pyrophosphate concentration of lO -5 M, 0.8 mole pyrophosphate is bound by 500000 g myosin giving 7.4" IO -~ M myosin-pyrophosphate. This reduces to 2.8. IO -6 when actin is present*. The * E s t i m a t e d from Fig. 2 in ref. 22.
Biochim. Biophys. Acla, 82 (i964) 5o7-517
514
L.B. NANNINGA
t o t a l actin c o n c e n t r a t i o n is 8. 7- IO -6 M (based on the d i m e r unit of 114000 as a b o v e {}r on t h e m a x i m u m n u m b e r of m y o s i n b i n d i n g sites). The m y o s i n - p y r o p h o s p h a t e red u c t i o n of 4.6.1o -6 m u s t be due to f o r m a t i o n of a c t o m y o s i n leaving 4.1.1o ~ as free actin. Kap p (apparent
association
constant)
..........
[MA2
[A2] [M .+ MPP! [M,:\2
=
4.0' lo 6
]i2] [Mtot ~MAsi
(4 .I' I°-6}
(4"7'
'0-6}
2 . 3 8 . lO 8
[~'L'\2] "
• \
Ktrue
[M]
/
as [MP P] /~MPP
--
[M] EPP;
M, m y o s i n ; A2, a c t i n ; PP, p y r o p h o s p h a t e ; MA2, a c t o m y o s i n ; MPP, m y o s i n - p y r o phosphate. Hence: Ktrue - - lfm, p (t + /x'Mpp • ~ P P ] ) = 0 . 2 3 8 " tO 6 (I @ KMp P " 10 -5)
W h e n t h e value lO6 is t a k e n for the true association c o n s t a n t of m y o s i n - p y r o p h o s p h a t e 22 : KMA2 = 0.238"106 ( I ~ - 1 0 6 ," I O -5) • 2 . 6 2 " I O 6 (true association c o n s t a n t of actomyosin) which value agrees well with 2.32" lO 6 as d e r i v e d from the e x p e r i m e n t s of this article. T h e association c o n s t a n t of m y o s i n - p y r o p h o s p h a t e under tile conditions of t h e a b o v e m e n t i o n e d e x p e r i m e n t s (no large excess of Mg 2+ like in ref. 22) will be derived from Fig. 7. Here t o t a l m y o s i n concentration is o.196 % or 4.66" IO -6 M a n d the b i n d i n g of m y o s i n to actin is in t h e m a x i m u m range (compare Fig. 6). T o t a l actin c o n c e n t r a t i o n is o.o212 % or 1.87- lO _6 M a n d m a x i m a l m y o s i n b o u n d has the s a m e m o l a r i t y as t h e m o l a r i t y of actin is expressed in m y o s i n binding sites. I n t h e presence of 2.5" IO -5 M p y r o p h o s p h a t e the b o u n d m y o s i n is 4 ° % of the b o u n d m y o s i n in t h e absence of p y r o p h o s p h a t e or o.75" IO -~ M (Fig. 7). Hence Ebound myosin]
Kapp = =
o . 7 5 . l O -6
[free myosin] [free actin] = ((-4.66-°. 75).~ o~-6~ (~I.8 7---0_7;) " 1 o-6j 1.71" Io 5
This is t h e a p p a r e n t association c o n s t a n t in the presence of 2.5" lO -5 ~V[p y r o p h o s p h a t e . I t s relation to the true association c o n s t a n t is: Ktrue = Kapv (I 4- KMpp" [PP]) in which [PP] = free p y r o p h o s p h a t e concentration b u t this can be t a k e n equal to t o t a l p y r o p h o s p h a t e as p y r o p h o s p h a t e is in excess over the myosin. KMpp is t h e association Biochim. Biophys. Acta, S2 (1904) 507 5 1 7
ON THE INTERACTION BETWEEN MYOSIN A AND F-ACTIN
515
constant of myosin and pyrophosphate. Hence 2.32"IO° = 1.71"1o 5 (I + Kin, l, × 2.5" lO-5). Kin,l, = 0.50" lO6. For I. lO -4 M pyrophosphate, myosin bound is 2o % of that without pyrophosphate or o.375.1o -6. A similar calculation gives here for KMp1, 0.40" 106. For 2.5" IO-4 M pyrophosphate, myosin bound is 8.75 % of that without pyrophosphate or o.164-IO -6. This gives for KM1,p 0.43" lO6. For I. lO -5 M pyrophosphate myosin bound is 62.5 % of that without pyrophosphate or 1.16- IO -6 M and Kin, l, ~- 0.39" lO6. Average value of Kraal, = 0.43" lO6. This is lower than the value found by GERGELYet al. 2~ of 10°, probably due to the excess of Mg2+ over pyrophosphate in their experiments. The decrease of bound myosin due to pyrophosphate is stronger in Fig. 7 than in Fig. 6 due to the fact that in Fig. 6 pyrophosphate was added in the absence of Mg2+, while in Fig. 7, 5" IO-4 M Mg2+ was present. It is known that one needs to add more moles of pyrophosphate than ATP in order to dissociate a certain amount of actomyosin, compare Acs et a l Y and MOMMAERTS2s. This is what can be expected since the association constant of ATP to myosin is supposed to be higher than 3.1o 7 (dissociation constant smaller than 3 . I o 4 as derived by NANNINGAAND MOMMAERTS29), while this constant for pyrophosphate is about xo ° with excess Mg2+, or 30 times lower. DISCUSSION B A I L E Y AND P E R R Y 3° a n d . ENGELHARDT AND IAROVAIA 31 s h o w e d t h a t i n a c t i v a t i o n
of
myosin ATPase and loss of actin binding capacity go parallel. In the splitting of myosin in " m e r o m y o s i n s " these two functions go again together in the heavy meromyosin while the light meromyosin lacks both of them, compare SZENT GYORGY1:32.Also the heavy meromyosin contains all free SH-groups of myosin 3.. From the experiments described here it appears that actin binding and ATP splitting occur on the same site, in other words that actin binds to the enzymic site; the actin displaces the ATP partly if its concentration is high enough, i.e. if there is a large excess of actin over myosin. In order to block part of the ATP binding a large excess of actin is needed. This occurs in the inhibition experiments with 1.o60/00 actin and 0.077°/oo myosin, or 9.3.Io-6M actin and 1.8.1o -7 M myosin (a 52-fold molar excess of actin over the myosin). On the other hand ATP displaces actin in the dissociation of actomyosin; here is no molar excess of actin over myosin and small quantities of ATP suffice for dissociation, compare }/~OMMA_ERTS AND HANSON 33 a n d NANNINGA AND ~V[OMMAERTS 34. These quantities are of the same order of molarity as the actomyosin MA2-unit (lO 4 M)* and they suffice due to the strong binding of ATP to the myosin is. That ATP binds to myosin and not to actin was already indicated by YAGIs7 who showed that the ATP concentration is proportional to the amount of myosin and not to the amount of actin in different actomyosins. Also it agrees with the subsequent splitting of the ATP since actin is no ATPase. There is a reasonable agreement in the value of the association constant both with TONOMURAet d. 3 and with conclusions derived in the present article from experiments on the effects of actin on pyrophosphate binding * W i t h 8. 3- io -~ M ATP, 6o ~o of t h e a c t o m y o s i n is dissociated. T h e a c t o m y o s i n c o n c e n t r a t i o n w a s o.5°]00 or lO -6 M, b a s e d on a m o l e c u l a r weight of one MA~-unit of 5ooooo (refs. 33, 34).
Biochim. Biophys. Acta, 82 (i964) 5o7-517
516
I.. B. NANN1NGA
by GERGELYet al. 'a2. However, the temperature effects obtained bv TONOMt:ICAet al. in their light-scattering experiments were neither ohvious in the actin inhibition experiments n o r in t h e c e n t r i f u g a t i o n e x p e r i m e n t s . T h e c o n c l u s i o n is t h a t t h e freee n e r g y effects are m o s t l y due to e n t r o p y effects a n d v a l u e s in t h e o r d e r of 30 e.u. are obtained. T h e effects of u r e a a n d S H r e a g e n t s suggest t h a t a c t i n binds to an S H - g r o u p on t h e m y o s i n , s u p p o s e d l y t h e s a m e w h i c h is i n w ) l v e d in t h e A T P b i n d i n g . T h e effect of p y r o p h o s p h a t e s u g g e s t s t h a t p y r o p h o s p h a t e displaces a c t i n likc A T P does a n d is in a g r e e m e n t w i t h t h e s t u d i e s m a d e h v GER(;EI.V el al. 22 a n d MAt¢TONOSI et al. 8. T h e effects of u r e a arc in a g r e e m e n t w i t h e x p e r i m e n t s h v BA~¢ANv ~t a/. aa. T h e i n t e r a c t i o n is n o t m u c h i n h i b i t e d b y u r e a in t h e at)sencc of A T I ' . w h i c h is s i m i l a r to findings b y t h e s e a u t h o r s . T h e y f o u n d t h a t u r e a alone c a n n o t relax a cont r a c t e d fiber b u t u r e a plus A T P can. I t m a y be t h a t u r e a shows m a x i m a l effect o n l y in t h e p r e s e n c e of A T P b e c a u s e ot t h e a l r e a d y r e d u c e d i n t e r a c t i o n of actin t . m y o s i n b y t h e A T P . I n t h e a h s e n c e of A T P this i n t e r a c t i o n is so m u c h h i g h e r t h a t urea is h a r d l y able to s h o w i n h i b i t i o n . I t is also f o u n d t h a t P C M B r e d u c e s t h e b i n d i n g c o n s i d e r a b l y in the c e n t r i f u g a t i o n e x p e r i m e n t s , a l t h o u g h at h i g h e r c o n c e n t r a t i o n s of m \ o s i n t h e effect (tccrcascs. "l'hi~ s u g g e s t s b l o c k i n g of a S H - g r o u l) on the m y o s i n a n d n o t on the actin which agrees w i t h BAILEY AND PERRY'S c(mclusion a°. It b e c o l n e s clearer in t h e light , f findings by BARANY AND ]))AI{ANY a~i t h a t e v e n w h e n t w o t h i r d of its S H - g r o u p s are b l o c k e d the m y o s i n still has the s a m e A T P a s c a c t i v i t y a n d a c t i n b i n d i n g caI)acity. O n l y below this S H level t h e s e effects decrease. T h e conclusi(ms w a c h c d in t h e p r e s e n t article on t h e c o m p e t i t i o n betweci1 A T P or p y r o p h o s p h a t c a n d a c t i u for tho s a m e site are g e n e r a l l y in a g r e e m e n t w i t h conclusions o b t a i n e d b y ]3a~Axv AN~ ]),AI~AX'~"a~. Also in t h e n e w concct)t of F - a c t i n as a d o u b l e helix, one mx,,~sin I)imls i)cr tw(~ G - a c t i n glohulcs, c o m p a r e D a w E s Fig. I (rcf. 38). ACKNOV~'LEI)GEMENTS This i n v e s t i g a t i o n was s u t ) p o r t c d b y the L i b e r t y M u s c u l a r D y s t r o t ) h y R e s e a r c h F o u n d a t i o n , L i b e r t y , "l'exa>. T h a n k s are due to Dr. W. F. H. M. MOMMAEWrS for t h e use of Fig. 2. T h e t e c h nical a s s i s t a n c e of Mr. A. WHAICTON a n d Mrs. M. L:XDAY is gratefully" a c k n o w l e d g e d . 1,~EI;E [IEN( I~;S 1 L. I3. NANNINGA, Texas J. ~bci., I 4 (19(~2) 4332 A. SZENT (;YORGYI, Chemistry of Muscle Conlraction, Academic Press, Nc~ York, ~O.~t, p. 73. a y. TONOMURA,S. TOKURA AND 1"~.SEKIY.k, .]. Biol. (?hem., 237 (I9(~2) io74. 4 j . GERGELY AND H. tXOHLER, CO@ Chem. M u s c u l a r ('ontraction, Tokyo, z957, l g a k t t Shoin, Tokyo, 1958, p. 14. ST. HAYASHL R. I~OSENBLU'rH,1L SATIR AND M. VOZE(-K, Hiochim. Biophys. -tcla, 28 (i95 b) ~. 6 A. ~,VEBER, Biochim. Biophys. ,4eta, t9 (~956) 345. 7 j . H u Y s , Arch. lnler~. Physiol. Biochim., 68 (196o) 445s A. MARTONOSL M. A. GOUVEAAND J. (;ERGEL'Z, J. Biol. (;hem.. 235 (Iq{~oi 3tt,o. 9 W. F. H. M. MOM.~IAVRTS,3/Iethods Med. Res., 7 (195()) ,. 10 \V. F. H. M. MO.XIMA~ZR'rS,.J. Biol. Chem., I98 (I952) 4t5. Biochim. Biophy~..-I~la, ,~2 (I0o 4 ) 5o7 5 ~7
ON THE INTERACTION BETWEEN MYOSIN A AND F-ACTIN
517
21 M. E. CARSTEZ~ AND W. F. H. M. MOMMAERTS, Biochemistry, 2 (1963) 28. 12 W. DRABIKOWSKI AND J. GERGELY, J. Biol. Chem., 237 (1962) 3412. 13 L. B. 1NTANNINGA,Arch. Biochem. Biophys., 96 (1962) 51. 14 W. F. H. M. MOMMAERTS AND B. BLANKENHORN ALDRICH, Biochim. Biophys. Acta, 28 (1958) 627. 15 L . B . IN]-ANNINGA AND W. V. H. M. MOMMAERTS, Proc. Natl. Acad. Sci. U.S., 46 (196o) 1155. 16 p. H. VAN t-IIpPEL, H. K. SCHACHMAN, P. APPEL AND M. MORALES, Biochim. Biophys. Acta, 28 (1958 ) 504 • 17 A. HOLZER AND S. LOWEY, J. Am. Chem. Soc., 81 (1959) 137o. xs j . BRAHMS, J. Am. Chem. Soc., 81 (1959) 4997. 19 j . BRAHMS AND J. BREZNER, Arch. Biochem. Biophys., 95 (1961) 219. 2o S. LowE Y AND C. COttEN, J. Mol. Biol., 4 (1962) 293. ~1 S. LowE Y AND A. HOLTZER, Biochim. Biophys. Acta, 34 (1959) 47 °. 22 j . QERGELY, A. MARTONOSI AND M. A. GOUVEA, in Sulfur in Proteins, A c a d e m i c Press, N e w York, 1959, p- 297. 23 M. F. GELLI~RT AND S. W. ENGLANDER, Biochemistry, 2 (1963) 39. 24 C. M. KAY, Biochim. Biophys. Acta, 43 (196°) 259. 25 W. W. KIELLEY AND L. B. BRADLEY, J. Biol. Chem., 218 (1956) 153. 26 L. B. NANNINGA, Biochim. Biophys. Acta, 34 (1961) 338. 27 G. Acs, K. S. BIRD AND F. B. STRAUB, Hung. Acla Physiol., 2 (1949) 84. 28 W. F. 1~I. M. MOMMAERTS, J. Gen. Physiol,, 31 (1948) 361. 29 L. B. NANNINGA AND W. F. H. M. MOMMAI~RTS, Proc. Natl. Acad. Sci. U.S., 46 (196o) 1166. 30 K. BAILEY AND S. V. PERRY, Biochim. Biophys. Acta, i (1947) 506. 31 W. A. ENGI~LHARDT, Proc. Intern. Syrup. Enzyme Chem. Tokyo-Kyoto, I957, Maruzen, Tokyo, 1958, P. 34. 32 A. G. SZENT GY6RGYI, Arch. Biochem. Biophys., 42 (1953) 305 • 33 W. F. l-~. M. MOMMAERTS AND J. HANSON, J. Gen. Physiol., 39 (1956) 831. 34 L. n. NANNINGA AND W. V. H. 1V[. MOMMAERTS, Proc. Natl. Acad. Sci. U.S., 43 (1957) 54 TM a5 M. BARANY, K. BARAlqY AND W. TRAUTWEIN, Biochim. Biophys. Acta, 45 (196o) 317 . 36 M, BARANY AND K. BARANY, Biochim. Biophys. Acla, 35 (1959) 393. 37 j . YAGt, J. Biochem. Tokyo, 44 (1957) 337. a8 R. E. DAVIES, Nature, 199 (1963) lO68.
Biochim. Biophys. Acta, 82 (i964) 5o7-517
507
BBA 4 2 5 3
ON T H E I N T E R A C T I O N B E T W E E N MYOSIN A A N D F-ACTIN* L. B. N A N N I N G A
Department of Physiology, University of Texas Medical Center, Galveston, Texas ( U.S.A.) (Received J u l y i2th, 1963)
SUMMARY
The interaction of myosin and actin was studied by means of two techniques: (I) the inhibition of myosin ATPase (ATP-phosphohydrolase, E.C. 3.6.1.3) by actin addition, and (2) centrifugation experiments, carried out so that only free myosin appears in the supernatant. Association constants are calculated and a binding curve for myosin is obtained. The maximum binding agrees with a weight percentage of 3.7 myosin to I actin. The effects of urea, p-chloromercuribenzoate, CaCI~, temperature and pyrophosphate were studied. It is concluded that ATP and actin bind to the same myosin site and that pyrophosphate binds also to this same site. The association constant for pyrophosphate was calculated from its inhibitory effect on the actin-myosin interaction, while this constant for the actin-myosin interaction was also calculated from experiments by GERGELYet al. on the inhibition of pyrophosphate binding to myosin by actin. The temperature dependence of the binding was found to be quite small and the conclusion was made that the free-energy effect is nearly all due to entropy change.
INTRODUCTION
It has been known for a long time that actin combines with myosin in a weight proportion of I to about 3.5-4. SZENT GY6RGYI2 gave a proportion of 3 myosin to i actin, but this was established later as about 3.7: I for maximum binding (see refs. 3, 4 and this article). A value for the binding constant of actin to myosin derived from light-scattering data had been published in 1957 by GERGELYAND KOHLER4. They concluded to a pK value of IO for the myosin-actin interaction at 23 °, 0.6 M KC1 and pH 7.0. Recently (1962) this value was the subject of an investigation by TONOMURAet al. 3, who found p K 6.3 in 0.6 M KC1, pH 7.5 at 9 ° and about 8 at 17 °, also derived from light-scattering data. In this article the same constant has been determined by means of two different techniques: (1) the inhibition of Ca2+-activated myosin ATPase (ATP-phosphohydrolase, EC 3.6.1.3) by actin and (2) centrifugation of myosin-actin mixtures at 30000 rev./min and determination of free myosin in the supernatant. A b b r e v i a t i o n : PCMB, p - c h l o r o m e r c u r i b e n z o a t e . * An a b s t r a c t of this i n v e s t i g a t i o n was p r e s e n t e d at t h e 66th A n n u a l M e e t i n g of t h e T e x a s A c a d e m y of Science, D e c e m b e r I962 at Austin, T e x a s (see ref. I).
Biochim. Biophys. Acta, 82 (1964) 507 517
5Oc~
L.B.
NANN1NGA
The effects of t e m p e r a t u r e , Ca '~+ concentration, urea, PCMB, a n d t}5 rot)host~hates on the association of aetin to m y o s i n has been studied. During recent years it has been clearly established t h a t the contractile protein called " m y o s i n B " is a c t o m v o s i n (GERGELY AND KOHLER 4, HAYASHI et al. ~, WEBER 6, t-IuYs 7, a n d MA1~roxos~ el al. s) so t h a t the actin--myosin association equilibrium is of considerable t)hysiological importance. The values o b t a i n e d for the b i n d i n g c o n s t a n t a p p l y only to dilute solutions of actin a n d m y o s i n u n d e r the e x p e r i m e n t a l conditions (pH, KCI, t e m p e r a t u r e ) . This " n a t u r a l a c t o m y o s i n " or " m y o s i n B " has a p p r o x i m a t e l ) the same prot)ortion of actin a n d m y o s i n as the m a x i m u m value of b o u n d myosin to actin. MATERIALS
Myosin A was o b t a i n e d from r a b b i t skeletal nmscle according to MO~.L\.IAERrs". i t was dissolved in o.3 M KC1, o.oI M Tris (pH 7) a n d k e p t in ice u n d e r chloroform, with a m i n i m u m of air a b o v e it. I n this w a y it keeps well its e n z y m i c a c t i v i t y a n d also its c a p a b i l i t y to combine with actin. Actin was p r e p a r e d from the mince residue after the myosin e x t r a c t i o n according to MOMMAERTS9,1° t a k i n g into account some recent modifications like the use of ascorbic acid with the A T P in the e x t r a c t of the acetone t)owder a n d the use of I o -a M Mg ~- in the I)olymerization process n. The whole procedure was performed at o ~ which p r e v e n t s e x t r a c t i o n of t r o p o m y o s i n ~, vL The p o l y m e r i z a t i o n was &me for i h at room t e m p e r a t u r e , then overnight in the cold room. T h e F - a c t i n was centrifuged at 3oooo rev./min for 2 h a n d the pellets are taken u t) in o.z M KC1. This was r e p e a t e d 3 - 4 times in order to reduce the a b s o r b a n e v of the s u p e r n a t a n t at 28o m/z to values which arc low enough to be a c c e p t a b l e in the exp e r i m e n t s on t h e actin myosin mixtures. Crystalline d i s o d i m n - A T P of P a b s t which is known to be at least 99 % pure was used a n d set to p H 7. The concentrations of myosin m~d actin solutions were d e t e r m i n e d from their a b s o r b a n c y at z8o a n d 32o m/~, c a l i b r a t e d on t h e K j e l d a h l m e t h o d , e28~j e.320- - o.5() for o . 1 % m y o s i n A a n d 0.965 for o . 1 % F-actin (I cm light path). METHODS
T h e A T P a s e activities were m e a s u r e d in the presence of Ca ~+, final c o n c e n t r a t i o n z" xo -2 M (except for some e x p e r i m e n t s in which the Ca '~+ effect was studied), o.I M NaC1, o.oI M Tris buffer (pH 7) a n d at 25 ~ or other t e m p e r a t u r e s . The l i b e r a t e d ino r g a n i c p h o s p h a t e was d e t e r m i n e d b y m e a n s of m o l y b d a t e sulphuric acid a n d dilute SnC12 ~a a d d e d to trichloroacetic acid filtrates. I n the e x p e r i m e n t s where aetin is a d d e d , t h i s is a d d e d first to the m y o s i n a n d t h e A T P is a d d e d last. In the centrifugation e x p e r i m e n t s the actin a n d myosin were i n c u b a t e d in t h e right ionic e n v i r o n m e n t at different t e m p e r a t u r e s for 3o min, t h e n centrifuged for L5 h at 3oooo rev./min. Afterwards, t h e s u p e r n a t a n t is read at 28o a n d 32o m/x. RESUI, TS
The e x p e r i m e n t s on the A T P a s e a c t i v i t y (v) of m y o s i n a n d its inhibition by actin in t h e presence of o.x M NaC1 a n d o . o i M Tris buffer (pH 7.o) a n d o.o2 M CaC12 show Biochim. Biophys. ,qcta, 82 (I004) 507 517
ON THE INTERACTION BETWEEN MYOSIN A AND F-ACTIN
509
both at 25 ° and at 35 ° t h a t actin gives competitive inhibition, as in a plot of I/V against I / [ A T P ] the line obtained without actin and the one in the presence of actin intersect on the ordinate (see Fig. I). 5
4 ._.q E
-o3
2
1
i
L
i
,
i
1
i
i
cl17xlo
i
,
4
i
>
Fig. i. R e c i p r o c a t e of a c t i v i t y a g a i n s t reciprocate of A T P c o n c e n t r a t i o n w i t h ( O - - O , I.O6°/00) a n d w i t h o u t ( O - - O ) actin. T h e a c t i v i t y is expressed in c h a n g e in a b s o r b a n c y at 61o m/t per m i n (not c o n v e r t e d in m m o l e s P as it cancels o u t in calculations of tl'm a n d Ki). T h e s y s t e m c o n t a i n e d : o . i M NaC1, 0.o2 M CaCli, o.oi M Tris (pH 7), o.o77°/00 m y o s i n . I n c u b a t i o n t i m e 5 rain; A T P a d d e d last; t e m p . 25°; Km 1 . 2 " 1 0 - 5 ; irk'i 1. 5" IO -~.
The Michaelis constant, Km, is calculated from slope ordinate (I/V) as: i i Km i v
v +
(KIn~V) and
intercept on
1,," IS]
IS] ~- ATP concentration.
In the presence of actin K I is found from: I
I
Km(I
+
[I]/Ki)
I
in which [I] = actin concentration, Kt inhibition constant or dissociation constant of the enzyme-inhibitor complex, in this case the actomyosin complex. The unit in which the activity is expressed does not m a t t e r since this cancels out in the calculation of Kin. The Kt value depends on the molarity of the inhibitor. For Kin, 1.2" IO -5 is obtained at 25 ° The competitive inhibition proves that the actin binds to the same site on the myosin as the A T P does. For the inhibitor constant is found 1. 5-1o -6, when the actin concentration is expressed in moles based on a molecular weight of 1140o0. This represents a rink of 2 G-actin molecules, with a molecular weight of 57 ooo (compare I~¢[OMMAERTSI°), which binds I myosin molecule. One comes to this conclusion from MO~IMAERTS AND ALDRICH'S value (1958) for Biochim. Biophys. Mcta, 82 (1964) 5o7-517
510
L. B. N A N N I N G A
t h e m o l e c u l a r weight of myosin, 42o ooo (ref. 14) a n d the fact t h a t 42o o o o : I 14 ooo 3.7 : I so t h a t in this w a y the right weight p r o p o r t i o n for m a x i m u m binding is o b t a i n e d . The value of 420 ooo was found to fit the d a t a on A T P binding to myosin resulting in a m a x i m u m b i n d i n g of I mole A T P per mole of myosin, which m e a n s one e n z y m i c site per mole of m y o s i n ~5. This value fitted also the d a t a on the m a x i m u m effect of A T P on a c t o m v o s i n dissociation, i.e. I mole A T P to 504000 g actomyosin'~*, 14. T h e same value was o b t a i n e d in 1958 b y VAN HIPPEL et al. 16. Other a u t h o r s (1959-63) came to 49oooo (ref. 17) , 43oooo (ref. 18), 44ooo0 (ref. 19), 47oooo (ref. 2o), 45oooo (ref. 2I), 426ooo (ref. 22), 5ooooo (ref. 23). The a v e r a g e of these values is 4470oo which is 6.5 °5 higher t h a n 42oooo. W i t h KAYI;'s value of 616oo (ref. 24) for t h e molecular weight of G-actin, which is 8 % higher t h a n MOMMAEI~TS value, this gives for the weight proportion m y o s i n : a c t i n 3.63 : I b a s e d on 447ooo for m y o s i n a n d 3.4: I based on 42oooo; again on the a s s u m p t i o n t h a t each actin d i m e r has one binding site for mw~sin. F r o m t h e generally a c c e p t e d molecular weights a n d the i n a x i m u m binding p r o p o r t i o n , the condition can be d r a w n t h a t a c t o m y o s i n m u s t have a s t r u c t u r e like Fig. 2 a n d t h a t at m a x i m u m b i n d i n g e v e r y second actin m o n o m e r carries I m y o s i n molecule. The reason t h a t the o t h e r m o n o m e r units c a n n o t c a r r y a myosin molecule m a y be steric hindrance. T h e actin c o n c e n t r a t i o n is here expressed in e q u i v a l e n t s of m y o s i n b i n d i n g sites.
j/
o
'
Fig. 2. Supposed structure of actomyosin. Every second G-actin contains t myosin. Explanation in text. In these e x p e r i m e n t s the actin c o n c e n t r a t i o n is a b o u t o.I ° o or 8. 3. lO -8 M, the m y o s i n c o n c e n t r a t i o n 0.005 % or 1.2- lO .7 M a n d t h e A T P c o n c e n t r a t i o n IO-4-IO -3 M. There is therefore a considerable excess of actin over myosin. Since the a c t i v i t y is a b o u t two times lower t h a n w i t h o u t actin at lO -4 M A T P a n d since the inhibition is of c o m p e t i t i v e c h a r a c t e r this m e a n s t h a t half of the A T P b i n d i n g sites are i n o p e r a t i v e because of actin b i n d i n g to these sites. Change in t e m p e r a t u r e to o ° gives still a p p r o x i m a t e l y the same i n h i b i t o r c o n s t a n t (2-1o-6). W h e n i n s t e a d of actin, bovine serum a l b u m i n is a d d e d to the s a m e final c o n c e n t r a t i o n n o t a n y inhibition of A T P a s e a c t i v i t y is obtained. This shows t h a t the actin inhibition is specific. T h e same a m o u n t of actin as a d d e d in Fig. I was ashed a n d t h e ash dissolved in HC1 and neutralized. W h e n this is a d d e d i n s t e a d of t h e actin no inhibition is o b t a i n e d which shows t h a t the inhibition is n o t due to ions like Mg ~+ which m i g h t be a d s o r b e d to t h e actin*. * Moreover actin preparations, polymerized without the addition of ~o 4 M MgCI2, gave the same inhibition effects of myosin ATPase. Biochim. Biophys. ,4cta, 82 (1964) 507-517
ON THE INTERACTION BETWEEN MYOSIN A AND F-ACTIN
511
Addition of urea decreases the inhibitory effect by actin considerably (Fig. 3)This makes it probable that one or more hydrogen bonds are involved in the binding of actin to myosin. Fig. 4 shows the effect of PCMB on the ATPase activity with and without actin. Without actin an increase to about double activity is found as described by KIELLEY 0.6
E ~0.4 c~ fl_
"~0.2 E
"6
o ×
~
3
4
Urea (°/o)
Fig. 3. I n h i b i t i o n of m y o s i n A T P a s e b y actin in urea. R e l a t i v e inhibition e q u a l s t h e a c t i v i t y w i t h u r e a o v e r t h e a c t i v i t y w i t h o u t urea. T h e s y s t e m c o n t a i n e d : o.o51°[00 m y o s i n , i.o5°[0 o actin, o.i M NaC1, 0.02 M CaC12, o.oi M Tris (pH 7), lO-4 M ATP. T e m p . 25 °.
I
1
I
2
I
3
i
4
~_
I
p-Chloromercuribenzoate (x ]O-6M)
Fig. 4. A c i t i v i t y of m y o s i n A T P a s e a g a i n s t conc e n t r a t i o n of PCMB. T h e a c t i v i t y is e x p r e s s e d in m m o l e s p h o s p h a t e per ml f o r m e d p e r min. O - - O , w i t h o u t actin; 0 - - 0 , w i t h actin. 1.o8. io -4 M A T P ; o t h e r conditions as in Fig. 3.
A~D BRADLEY25. In the presence of actin the activity increases or the inhibition disappears with increase of the PCMB concentration. At higher PCMB concentrations the curves intersect which m a y be due to binding of PCMB to the actin. The effects of PCMB suggest that an SH-group is involved in the binding of actin. This is probably an SH-group on the myosin since this is known to have an important 2
U
Concentration of CaCI 2 (x 10-9 I-1)
Fig. 5. I n h i b i t i o n of m y o s i n A T P a s e b y actin as f u n c t i o n of Ca 2+ c o n c e n t r a t i o n . T h e s y s t e m cont a i n e d : o.o68°/00 m y o s i n , i °/0o actin, 0.85" lO -4 M ATP, o. I M NaC1, o.ot M Tris (pH 7). T e m p . 25 °. Biochim. Biophys. Acta, 82 (1964) 5o7-517
512
L.B. NANNINGA
role in A T P h y d r o l y s i s 2a. Also, BAILEY AND PEt{RV found t h a t actin with blocked S H groups still binds to m y o s i n a°. Increase or decrease of the Ca 2+ c o n c e n t r a t i o n affects the inhibition b y actin. At zero Ca 2+ c o n c e n t r a t i o n the a c t i v i t y goes down to zero a n d also the inhibition b y actin decreases to zero (Fig. 5). A t 5' i o -2 M CaCh, the inhibition also decreases considerably. T h e actin c o m p e t i t i o n is o p t i m a l at 2" lO -2 M CaC12, similar to the effect ~f ('a ~+ on A T P a s e a c t i v i t y 26. A t higher p H the inhibition 173' actin decreases: at p H (} actin reduces the a c t i v i t y for lO-4 M A T P only to o.7 i n s t e a d ot to o.5. This results in a higher inhibition c o n s t a n t (4.5" IO~) • Centrifl~gation experime~ts M i x t u r e s of actin (final c o n c e n t r a t i o n 0.028 %) a n d increasing a m o u n t s of myo>in (up to o.2-0.3 %) in a salt milieu of 0.3 M KC1, o . o i M Tris buffer (pH 7) are centrifuged for I. 5 h at 30000 r e v . / m i n . B l a n k runs are m a d e w i t h actin alone a n d with th<, highest concentration of myosin alone. The actin in the b l a n k is found to have a s u p e r n a t a n t with a very low a b s o r b a n c y at 280 mix. The myosin blank is done in order to check t h a t m y o s i n ahme does not give a p r e c i p i t a t e a n d its s u p e r n a t a n t is the sam~. as the original myosin after dilution to the same concentration. The myosin solutions h a v e been centrifuged beforehand for 2 h at 3 ° ooo rev./min a n d are q u i t e clear. After the m i x t u r e s have been centrifuged p a r t of the s u p e m a t a n t is carefully t a k e n off a n d read on its a b s o r b a n c y at 28o a n d 32o m/x. The difference gives the myosin conc e n t r a t i o n in t h e s u p e m a t a n t since actin a n d a c t o m y o s i n b o t h p r e c i p i t a t e under thes~ conditions. The m y o s i n c o n c e n t r a t i o n in the s u p e r n a t a n t is c a l c u l a t e d from the a b s o r b a n c y difference at 280 a n d 320 m ~ as a o.I % solution gives for this difference 0.59. The b o u n d myosin is f o u n d b y s u b t r a c t i o n of the free myosin from the t o t a l nlyosin. In e x p e r i m e n t s in which the myosin b l a n k gives a lower s u p e r n a t a n t t h a n the. c a l c u l a t e d one, correcti(ms are m a d e for myosin in the s u p e r n a t a n t of the actin -myosin mixtures. The a b s o r b a n c y at 280 m/, minus t h a t at 320 in/, for the s u p e r n a t a n t is first corrected for the usually very small a m o u n t given b y two b l a n k s w i t h o u t myosin b u t with actin (aromld o.oi). In presence of increasing a m o u n t s of myosin the abs()rb;, nci,,, increase up to 1.2. It was e s t a b l i s h e d t h a t tip to this value the a b s o r b a n c \ ~f t)m~ ' mvosin is p r o p o r t i o n a l t~) its concentration. W h e n m y o s i n w i t h o u t actin gives a co:> c e n t r a t i o n which is IO % lower t h a n the c a l c u l a t e d one, the values of all the supern a t a n t s were m u l t i p l i e d bv I.IO in order to correct for this. U s u a l l y the figures fi)r c a l c u l a t e d a n d m e a s u r e d myosin in the e x p e r i m e n t w i t h o u t actin agreed within a few percent. Figures were rejected when the myosin found was less t h a n 9 ° 00 of the calc u l a t e d vahle. T h e b i n d i n g is expressed as b o u n d m y o s i n verslls free myosin in Fig. (). I t is clearly seen t h a t a m a x i m u m b i n d i n g a t 25 ° is a p p r o a c h e d at a value of 1.o5°/00 m y o s i n b o u n d to an actin concentration of o.28"/0 o (final c o n c e n t r a t i o n s (Fig. 6)). This gives a p r o p o r t i o n of 3.75:1 which agrees with values published b \ GERGELY AN D N O H I ~ E R 4 a n d TONOMURA et al. a. A t o~ the m a x i m u m binding is found t(, be only slightly less t h a n this (3.4: I). The presence of IO -a M p y r o p h o s p h a t e reduces the binding considerably (see below). Hiochim, Biophys. .!c[a, se (~904} 5o7 51 7
ON THE INTERACTION BETWEEN MYOSIN
A AND F-ACTIN
513
Half of maximum binding occurs at o.o18 % or 4.3" 10-7 M myosin, based on a molecular weight 420000. This gives an association constant of 2.32" 106. The dissociation constant is about three times smaller than the inhibition constant as obtained before (0.43. lO-6 compared to 1.5" lO-6). The difference is probably caused by the different salt conditions and the absence of ATP which acts as a competitor for the binding of actin.
1
._c .C
g
D
g 2;
2~-
C
o
I
r
i
L
Myosin free in %o
Fig. 6. B o u n d against free m y o s i n from centrifugation experiments. The s y s t e m contained: o.280[00 actin, 0. 3 M KCt, o.oi M Tris (pH 7)O O, temp. 26°; 0 - - 0 , temp. o°; A - - A , temp. o °, IO-a M p y r o p h o s p h a t e added.
I
I
I
Pyrophosphate(l-x 10.4 M)
:
5
Fig. 7. Bound myosin against p y r o p h o s p h a t e concentration. This was done at high myosin concentrations relative to the actin concentrations so t h a t m a x i m u m binding is approached, compare Fig. 6. The s y s t e m contained : o.2 I 0/01~ actin, 0. 3 M KC1, o.oi M Tris (pH 7), 5' IO-4 ~I MgC12; total myosin 1.96°/00; temp. 23 °.
The value for the dissociation constant is an "intrinsic" one since many myosin molecules are bound to I F-actin molecule. The temperature dependence is found to be small; the binding is about IO % less at o ° than at 25 ° (Fig. 6). This means that most of the free energy of formation of the actomyosin is due to an entropy increase which may be related with liberation of bound water. The entropy increase is about 31 cal/degree • mole as calculated from the association constant assuming that the enthalpy change is negligibly small. TONOMURA et al. s, however, found a strong temperature dependence and came to a value of 234 cal/degree, mole for the entropy of formation. The maximum binding of myosin to actin is reduced about 4-fold in the presence of IO -a M pyrophosphate at o ° (Fig. 6). With increasing concentrations of pyrophosphate in the presence of 5" IO-4 M Mg2+ at o °, it is found that the bound myosin strongly decreases (Fig. 7). GERGELYet al. ez had found that pyrophosphate binds to myosin in a I : I molar proportion and that this binding is reduced by actin. In their experiments the myosin concentration was 9.3" I°-n M (based on a molecular weight 500000). At a pyrophosphate concentration of lO -5 M, 0.8 mole pyrophosphate is bound by 500000 g myosin giving 7.4" IO -~ M myosin-pyrophosphate. This reduces to 2.8. IO -6 when actin is present*. The * E s t i m a t e d from Fig. 2 in ref. 22.
Biochim. Biophys. Acla, 82 (i964) 5o7-517
514
L.B. NANNINGA
t o t a l actin c o n c e n t r a t i o n is 8. 7- IO -6 M (based on the d i m e r unit of 114000 as a b o v e {}r on t h e m a x i m u m n u m b e r of m y o s i n b i n d i n g sites). The m y o s i n - p y r o p h o s p h a t e red u c t i o n of 4.6.1o -6 m u s t be due to f o r m a t i o n of a c t o m y o s i n leaving 4.1.1o ~ as free actin. Kap p (apparent
association
constant)
..........
[MA2
[A2] [M .+ MPP! [M,:\2
=
4.0' lo 6
]i2] [Mtot ~MAsi
(4 .I' I°-6}
(4"7'
'0-6}
2 . 3 8 . lO 8
[~'L'\2] "
• \
Ktrue
[M]
/
as [MP P] /~MPP
--
[M] EPP;
M, m y o s i n ; A2, a c t i n ; PP, p y r o p h o s p h a t e ; MA2, a c t o m y o s i n ; MPP, m y o s i n - p y r o phosphate. Hence: Ktrue - - lfm, p (t + /x'Mpp • ~ P P ] ) = 0 . 2 3 8 " tO 6 (I @ KMp P " 10 -5)
W h e n t h e value lO6 is t a k e n for the true association c o n s t a n t of m y o s i n - p y r o p h o s p h a t e 22 : KMA2 = 0.238"106 ( I ~ - 1 0 6 ," I O -5) • 2 . 6 2 " I O 6 (true association c o n s t a n t of actomyosin) which value agrees well with 2.32" lO 6 as d e r i v e d from the e x p e r i m e n t s of this article. T h e association c o n s t a n t of m y o s i n - p y r o p h o s p h a t e under tile conditions of t h e a b o v e m e n t i o n e d e x p e r i m e n t s (no large excess of Mg 2+ like in ref. 22) will be derived from Fig. 7. Here t o t a l m y o s i n concentration is o.196 % or 4.66" IO -6 M a n d the b i n d i n g of m y o s i n to actin is in t h e m a x i m u m range (compare Fig. 6). T o t a l actin c o n c e n t r a t i o n is o.o212 % or 1.87- lO _6 M a n d m a x i m a l m y o s i n b o u n d has the s a m e m o l a r i t y as t h e m o l a r i t y of actin is expressed in m y o s i n binding sites. I n t h e presence of 2.5" IO -5 M p y r o p h o s p h a t e the b o u n d m y o s i n is 4 ° % of the b o u n d m y o s i n in t h e absence of p y r o p h o s p h a t e or o.75" IO -~ M (Fig. 7). Hence Ebound myosin]
Kapp = =
o . 7 5 . l O -6
[free myosin] [free actin] = ((-4.66-°. 75).~ o~-6~ (~I.8 7---0_7;) " 1 o-6j 1.71" Io 5
This is t h e a p p a r e n t association c o n s t a n t in the presence of 2.5" lO -5 ~V[p y r o p h o s p h a t e . I t s relation to the true association c o n s t a n t is: Ktrue = Kapv (I 4- KMpp" [PP]) in which [PP] = free p y r o p h o s p h a t e concentration b u t this can be t a k e n equal to t o t a l p y r o p h o s p h a t e as p y r o p h o s p h a t e is in excess over the myosin. KMpp is t h e association Biochim. Biophys. Acta, S2 (1904) 507 5 1 7
ON THE INTERACTION BETWEEN MYOSIN A AND F-ACTIN
515
constant of myosin and pyrophosphate. Hence 2.32"IO° = 1.71"1o 5 (I + Kin, l, × 2.5" lO-5). Kin,l, = 0.50" lO6. For I. lO -4 M pyrophosphate, myosin bound is 2o % of that without pyrophosphate or o.375.1o -6. A similar calculation gives here for KMp1, 0.40" 106. For 2.5" IO-4 M pyrophosphate, myosin bound is 8.75 % of that without pyrophosphate or o.164-IO -6. This gives for KM1,p 0.43" lO6. For I. lO -5 M pyrophosphate myosin bound is 62.5 % of that without pyrophosphate or 1.16- IO -6 M and Kin, l, ~- 0.39" lO6. Average value of Kraal, = 0.43" lO6. This is lower than the value found by GERGELYet al. 2~ of 10°, probably due to the excess of Mg2+ over pyrophosphate in their experiments. The decrease of bound myosin due to pyrophosphate is stronger in Fig. 7 than in Fig. 6 due to the fact that in Fig. 6 pyrophosphate was added in the absence of Mg2+, while in Fig. 7, 5" IO-4 M Mg2+ was present. It is known that one needs to add more moles of pyrophosphate than ATP in order to dissociate a certain amount of actomyosin, compare Acs et a l Y and MOMMAERTS2s. This is what can be expected since the association constant of ATP to myosin is supposed to be higher than 3.1o 7 (dissociation constant smaller than 3 . I o 4 as derived by NANNINGAAND MOMMAERTS29), while this constant for pyrophosphate is about xo ° with excess Mg2+, or 30 times lower. DISCUSSION B A I L E Y AND P E R R Y 3° a n d . ENGELHARDT AND IAROVAIA 31 s h o w e d t h a t i n a c t i v a t i o n
of
myosin ATPase and loss of actin binding capacity go parallel. In the splitting of myosin in " m e r o m y o s i n s " these two functions go again together in the heavy meromyosin while the light meromyosin lacks both of them, compare SZENT GYORGY1:32.Also the heavy meromyosin contains all free SH-groups of myosin 3.. From the experiments described here it appears that actin binding and ATP splitting occur on the same site, in other words that actin binds to the enzymic site; the actin displaces the ATP partly if its concentration is high enough, i.e. if there is a large excess of actin over myosin. In order to block part of the ATP binding a large excess of actin is needed. This occurs in the inhibition experiments with 1.o60/00 actin and 0.077°/oo myosin, or 9.3.Io-6M actin and 1.8.1o -7 M myosin (a 52-fold molar excess of actin over the myosin). On the other hand ATP displaces actin in the dissociation of actomyosin; here is no molar excess of actin over myosin and small quantities of ATP suffice for dissociation, compare }/~OMMA_ERTS AND HANSON 33 a n d NANNINGA AND ~V[OMMAERTS 34. These quantities are of the same order of molarity as the actomyosin MA2-unit (lO 4 M)* and they suffice due to the strong binding of ATP to the myosin is. That ATP binds to myosin and not to actin was already indicated by YAGIs7 who showed that the ATP concentration is proportional to the amount of myosin and not to the amount of actin in different actomyosins. Also it agrees with the subsequent splitting of the ATP since actin is no ATPase. There is a reasonable agreement in the value of the association constant both with TONOMURAet d. 3 and with conclusions derived in the present article from experiments on the effects of actin on pyrophosphate binding * W i t h 8. 3- io -~ M ATP, 6o ~o of t h e a c t o m y o s i n is dissociated. T h e a c t o m y o s i n c o n c e n t r a t i o n w a s o.5°]00 or lO -6 M, b a s e d on a m o l e c u l a r weight of one MA~-unit of 5ooooo (refs. 33, 34).
Biochim. Biophys. Acta, 82 (i964) 5o7-517
516
I.. B. NANN1NGA
by GERGELYet al. 'a2. However, the temperature effects obtained bv TONOMt:ICAet al. in their light-scattering experiments were neither ohvious in the actin inhibition experiments n o r in t h e c e n t r i f u g a t i o n e x p e r i m e n t s . T h e c o n c l u s i o n is t h a t t h e freee n e r g y effects are m o s t l y due to e n t r o p y effects a n d v a l u e s in t h e o r d e r of 30 e.u. are obtained. T h e effects of u r e a a n d S H r e a g e n t s suggest t h a t a c t i n binds to an S H - g r o u p on t h e m y o s i n , s u p p o s e d l y t h e s a m e w h i c h is i n w ) l v e d in t h e A T P b i n d i n g . T h e effect of p y r o p h o s p h a t e s u g g e s t s t h a t p y r o p h o s p h a t e displaces a c t i n likc A T P does a n d is in a g r e e m e n t w i t h t h e s t u d i e s m a d e h v GER(;EI.V el al. 22 a n d MAt¢TONOSI et al. 8. T h e effects of u r e a arc in a g r e e m e n t w i t h e x p e r i m e n t s h v BA~¢ANv ~t a/. aa. T h e i n t e r a c t i o n is n o t m u c h i n h i b i t e d b y u r e a in t h e at)sencc of A T I ' . w h i c h is s i m i l a r to findings b y t h e s e a u t h o r s . T h e y f o u n d t h a t u r e a alone c a n n o t relax a cont r a c t e d fiber b u t u r e a plus A T P can. I t m a y be t h a t u r e a shows m a x i m a l effect o n l y in t h e p r e s e n c e of A T P b e c a u s e ot t h e a l r e a d y r e d u c e d i n t e r a c t i o n of actin t . m y o s i n b y t h e A T P . I n t h e a h s e n c e of A T P this i n t e r a c t i o n is so m u c h h i g h e r t h a t urea is h a r d l y able to s h o w i n h i b i t i o n . I t is also f o u n d t h a t P C M B r e d u c e s t h e b i n d i n g c o n s i d e r a b l y in the c e n t r i f u g a t i o n e x p e r i m e n t s , a l t h o u g h at h i g h e r c o n c e n t r a t i o n s of m \ o s i n t h e effect (tccrcascs. "l'hi~ s u g g e s t s b l o c k i n g of a S H - g r o u l) on the m y o s i n a n d n o t on the actin which agrees w i t h BAILEY AND PERRY'S c(mclusion a°. It b e c o l n e s clearer in t h e light , f findings by BARANY AND ]))AI{ANY a~i t h a t e v e n w h e n t w o t h i r d of its S H - g r o u p s are b l o c k e d the m y o s i n still has the s a m e A T P a s c a c t i v i t y a n d a c t i n b i n d i n g caI)acity. O n l y below this S H level t h e s e effects decrease. T h e conclusi(ms w a c h c d in t h e p r e s e n t article on t h e c o m p e t i t i o n betweci1 A T P or p y r o p h o s p h a t c a n d a c t i u for tho s a m e site are g e n e r a l l y in a g r e e m e n t w i t h conclusions o b t a i n e d b y ]3a~Axv AN~ ]),AI~AX'~"a~. Also in t h e n e w concct)t of F - a c t i n as a d o u b l e helix, one mx,,~sin I)imls i)cr tw(~ G - a c t i n glohulcs, c o m p a r e D a w E s Fig. I (rcf. 38). ACKNOV~'LEI)GEMENTS This i n v e s t i g a t i o n was s u t ) p o r t c d b y the L i b e r t y M u s c u l a r D y s t r o t ) h y R e s e a r c h F o u n d a t i o n , L i b e r t y , "l'exa>. T h a n k s are due to Dr. W. F. H. M. MOMMAEWrS for t h e use of Fig. 2. T h e t e c h nical a s s i s t a n c e of Mr. A. WHAICTON a n d Mrs. M. L:XDAY is gratefully" a c k n o w l e d g e d . 1,~EI;E [IEN( I~;S 1 L. I3. NANNINGA, Texas J. ~bci., I 4 (19(~2) 4332 A. SZENT (;YORGYI, Chemistry of Muscle Conlraction, Academic Press, Nc~ York, ~O.~t, p. 73. a y. TONOMURA,S. TOKURA AND 1"~.SEKIY.k, .]. Biol. (?hem., 237 (I9(~2) io74. 4 j . GERGELY AND H. tXOHLER, CO@ Chem. M u s c u l a r ('ontraction, Tokyo, z957, l g a k t t Shoin, Tokyo, 1958, p. 14. ST. HAYASHL R. I~OSENBLU'rH,1L SATIR AND M. VOZE(-K, Hiochim. Biophys. -tcla, 28 (i95 b) ~. 6 A. ~,VEBER, Biochim. Biophys. ,4eta, t9 (~956) 345. 7 j . H u Y s , Arch. lnler~. Physiol. Biochim., 68 (196o) 445s A. MARTONOSL M. A. GOUVEAAND J. (;ERGEL'Z, J. Biol. (;hem.. 235 (Iq{~oi 3tt,o. 9 W. F. H. M. MOM.~IAVRTS,3/Iethods Med. Res., 7 (195()) ,. 10 \V. F. H. M. MO.XIMA~ZR'rS,.J. Biol. Chem., I98 (I952) 4t5. Biochim. Biophy~..-I~la, ,~2 (I0o 4 ) 5o7 5 ~7
ON THE INTERACTION BETWEEN MYOSIN A AND F-ACTIN
517
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