Enhancement of actomyosin ATPase activity by tropomyosin

290

Biochimica etBiophysi¢~ Acta,

788 (1984)290-297 Elsevier

BBA31937

E N H A N C E M E N T OF A C T O M Y O S I N ATPase ACTIVITY BY T R O P O M Y O S I N RECOMBINATION O F MYOSIN AND T R O P O M Y O S I N BETWEEN M U S C L E S AND PLATELET SHUICHI NOSAKA a TOYOSHI ONJI b and NOBUHIKO SHIBATA b Departments of a Anesthesiology and b Internal Medicine, The Center for Adult Diseases, Osaka, 1 - 3 - 3 Nakamichi, Higashinariku Osaka, 537 (Japan)

(Received December20th, 1983)

Key words: Tropomyosin; Actomyosin; ATPase; (Smooth muscle, Platelet)

In skeletal muscle, the physiological role of tropomyosin has been assumed to be the 'blocking' of the actin-myosin interaction. In smooth muscle and platelet, however, tropomyosin was shown to 'enhance' the interaction. To investigate the reason for this apparent contradiction, we carded out recombination experiments using reconstituted actomyosins and different tropomyosins. Tropomyosins from skeletal muscle, arterial smooth muscle and platelet were recombined with skeletal, arterial and platelet myosins. The effects of tropomyosins on the actin-activated ATPase activities of myosins were then examined. The results are as follows. (i) Although tropomyosins from artery and platelet are distinctively different in molecular weight, they are interchangeable in enhancing the ATPase activities of both arterial and platelet actomyosins. The enhancement, however, is reduced by increasing the concentration of M g . ATP and decreasing the concentration of myosin. (ii) Arterial and platelet tropomyosins are not capable of inhibiting the ATPase activity of skeletal actomyosin. (iii) Skeletal tropomyosin enhances arterial and platelet actomyosin ATPase activities in the same way as arterial and platelet tropomyosins. The results indicate that the major determinant of the effect of tropomyosin on the actomyosin-ATPase activity is the state of actomyosin. We suggest that any tropomyosin enhances the actin-activated ATPase activity of myosin recombined with skeletal actin, under the condition where actin and myosin form a 'rigor' (tight) complex.

Introduction

In skeletal muscle, tropomyosin was initially thought to have no or an inhibitory effect on the actomyosin ATPase reaction. Although it was found that tropomyosin enhances the ATPase activity at lower concentration of Mg. ATP, the physiological role of tropomyosin in skeletal muscle was assumed to be inhibitory, as was proposed by the steric blocking model [1]. On the other hand, tropomyosin was found to have a marked potentiating effect on the actomyosin ATPase activity in smooth muscle [2,3]. This finding was concomitant 0167-4838/84/$03.00 © 1984 ElsevierSciencePublishers B.V.

with the discovery that the calcium sensitivity in smooth muscle contraction is mediated by the phosphorylation of the M r 20000 light chain of myosin [2-6]. The physiological role of tropomyosin in this tissue was proposed to be the potentiation of actin-myosin interaction [2,3]. Recently, we found that platelet tropomyosin, which is distinctively different from its muscle counterparts [7,8], also enhances the actomyosin ATPase activity in platelet [9]. The complicated effects of tropomyosin have been studied by several workers from different aspects. In skeletal muscle, tropomyosin was shown not to block the actin-myosin interac-

291 tion, as proposed by the steric blocking model, but was indicated to affect the interaction as an allosteric effector [10-13]. Probably, tropomyosin affects the kinetic step of the actomyosin ATPase reaction at the point of phosphate release from the myosin-products complex [11]. Meanwhile, Sobieszek [10] showed that tropomyosins from smooth muscle and the brain enhance the skeletal actomyosin ATPase activity and indicated that this effect is different from the effect of tropomyosin from skeletal muscle. His results, therefore, seem to indicate that there are two types of tropomyosin; one is 'skeletal' type and the other 'smooth and non-muscle' type. We undertook the present series of experiments to reassess the nature of the effect of this important regulatory protein, i.e., tropomyosin, by extending the objects to non-muscle myosin and tropomyosin. Cross-recombination of three kinds (skeletal muscle, arterial smooth muscle and platelet) of myosin and tropomyosin were performed, using skeletal actin. By employing this method, we can examine the nature of the effect without having a precise knowledge about the reaction at the molecular level. The result indicates that the effect of tropomyosin on the actomyosin ATPase activity is determined more by the state of actomyosin than by the origin of tropomyosin. We suggest that any tropomyosin enhances the ATPase activity of any actomyosin whenever actin and myosin are in the state of 'rigor' complex. Materials and Methods

Preparation of myosins Bovine arterial myosin was prepared by the method of Takeuchi and Tonomura [14] with slight modifications. Porcine platelet myosin was prepared by the method previously described [9]. Judging from the electrophoretic pattern in alkaline urea gel, the M r 20 000 light chain of arterial myosin was 80-90% phosphorylated and that of platelet myosin was 40-70% phosphorylated. Rabbit skeletal myosin was prepared by the method of Inoue et al. [15] with slight modifications.

Preparatoin of tropomyosins Bovine arterial tropomyosin was prepared from

native tropomyosin (tropomyosin-kinase) fraction [3] by the following procedure. To native tropomyosin, 1 M MgC12 solution was added to obtain a final concentration of 15 raM, and the precipitates formed were collected by centrifugation at 27 000 × g for 30 rain. To the supernatant, 1 M MgC12 was added again to a final concentration of 30 mM. The precipitates formed were collected by centrifugation, dialyzed against 2 mM NaHCO 3 and used as a tropomyosin preparation. By this method, the myosin light-chain kinase activity in the native tropomyosin fraction was completely eliminated (data not shown). Porcine platelet tropomyosin was prepared as described previously [9]. Rabbit skeletal tropomyosin was prepared by the method of Bailey [16] with slight modifications.

Other methods Rabbit skeletal actin was prepared by the method of Spudich and Watt [17]. ATPase activity was determined using the coupled phosphoenolpyruvate-pyruvate kinase system [14]. SDS-polyacrylamide gel electrophoresis and urea polyacrylamide gel electrophoresis were performed by the methods of Weber and Osborn [18] and Perrie and Perry [19], respectively. Amino-acid composition of tropomyosins were analyzed by the Hitachi automatic amino-acid analyzer No. 835. Protein concentratoin was estimated by the method of Bradford [20] using bovine serum albumin as a standard. Assay conditions are described in the figure legends. All the chemicals used were of the reagent grade. Results

Properties of tropomyosins from bovine carotid artery and porcine platelet Both platelet and arterial tropomyosins migrate in SDS-polyacrylamide gel electrophoresis as a single band (Fig. la,b). The molecular weight of the former is around 29 000, which is distinctively different from the latter and is very similar to that from the human [21] and equine [7,8] platelet. The molecular weight of the latter is around 34000, which is not distinguishable from skeletal tropomyosin. Both tropomyosins from smooth muscle and platelet form so called paracrystals in

292

(i)(a) (b)

(c)

(d)

(ii)

(A)

(B)

Fig. 1. Properties of tropomyosins from bovine carotid artery and porcine platelet. (i) Electrophoretic patterns of the tropomyosins. (a) porcine platelet tropomyosin; (b) bovine arterial tropomyosin; (c) rabbit skeletal tropomyosin; (d) rabbit skeletal actin. 0.1% SDS, 7.5% polyacrylamide gel electrophoresis was performed and the gels were stained by Coomassie brilliant blue. (ii) Paracrystals of the tropomyosins. Paracrystals of either platelet (A) or arterial smooth muscle (B) tropomyosin were formed by dialyzing the proteins against 50 mM MgCI2/1 mM dithiothreitol/50 mM Tris-HC1 (pH 8.0). The precipitates formed were stained with 1% uranyl acetate and observed by a model JEM-100CX electron microscope.

TABLE I AMINO-ACID COMPOSITIONS OF VARIOUS TROPOMYOSINS The amino-acid compositions of bovine arterial and porcine platelet tropomyosins were analyzed. The molecular percentage of amino acid is shown in each column. The data in parentheses are cited from the following papers, a Equine platelet [8]; b chicken gizzard [30]: c rabbit skeletal a-tropomyosin [31]. mol%

Asp Thr Ser Glu Pro Gly Ala Val Cys Met Ile Leu Tyr Phe Lys His Arg

Porcine platelet (equine platelet a)

Bovine carotid artery (chicken gizzard b)

(Rabbit skeletal muscle a-tropomyosin ~)

9.5 (8.8) 2.9 (3.0) 2.9 (2.9) 26.5(27.9) 0 (0) 3.3 (2.9) 12.4(11.6) 4.1 (4.0) 0.4 (0.8) 2.1 (1.8) 3.7 (3.7) 12.0(11.6) 1.2 (1.3) 0.4 (0.5) 10.0(10.8) 0.8 1.0) 7.5 (7.6)

9.5 (9.6) 2.8 (3.0) 4.2 (4.7) 26.5(30.2) 0 (0) 1.4 (1.7) 13.1(12.3) 3.5 (4.3) 0.4 (0.3) 2.8 (2.7) 3.5 (3.0) 11.7(11.0) 1.1 (1.0) 0.4 (0.3) 12.0(10.0) 1.1 (1.0) 6.0 (5.0)

(10.2) (2.8) (5.3) (24.6) (0) (1.1) 12.7) (3.2) (0.4) (2.1) (4.2) (11.6) (2.1) (0.4) (13.7) (0.7) (5.0)

293

the presence of 50 mM MgC12. The electron micrographs of the paracrystals show the striped pattern that characterizes tropomyosins. The paracrystal of tropomyosin from arterial smooth muscle (Fig. 1B) is very similar to that of skeletal muscle. Both have the same periodic pitch of 40 nm. The paracrystal of tropomyosin from platelet, however, is quite different from that of muscles. It has a different pattern and shorter pitch (34 nm; Fig. 1A), as was already reported for human platelet tropomyosin [21]. The amino-acid compositions of arterial and platelet tropomyosins are shown in comparison to skeletal tropomyosin (Table I). The absence of proline is common to all three. Arterial tropomyosin is very similar to skeletal tropomyosin with the only significant difference of the small er content of tyrosine. It is also very similar to gizzard tropomyosin. Porcine platelet tropomyosin is very similar to the equine platelet tropomyosin. Both are somewhat different from the muscle counterparts, having a lower content of serine and a higher content of glycine and probably of arginine. The difference gives a marked difference in the lysine/arginine ratio (twice in muscle as compared to that in platelet). However, the sum of arginine and lysine residues are roughly the same for all three tropomyosins. They have, therefore, the same net positive charge. Recombination experiments of myosin and tropomyosin between arterial smooth muscle and platelet It has been shown that tropomyosin enhances the actomyosin ATPase activity in smooth muscle [2,3] and platelet [9]. In the first stage of the series, the hypothesis was tested that arterial ('large') tropomyosin and platelet (' small') tropomyosin are indistinguishable in their capacity of enhancement. As seen in Fig. 2a, tropomyosins from artery and platelet enhance the ATPase activity of skeletal acto-arterial myosin. The data demonstrate that 'small' tropomyosin is not different from 'large' tropomyosin in its effect on the actomyosin ATPase activity of arterial smooth muscle. The hypothesis was also tested by substituting tropomyosin in platelet actomyosin. We have already shown that the ATPase activity of platelet actomyosin is enhanced by platelet tropomyosin [9]. In this report, it is shown that the 'large' tropomyosin is capable

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Fig. 2. Effects of arterial or platelet tropomyosin on the ATPase activity of reconstituted skeletal actoarterial myosin. (a) Arterial or platelet tropomyosin was recombined with reconstituted actomyosin at different concentrations of actin (sk-Ac) and the ATPase activity was determined. Assay condition: arterial myosin, 100 kLg/ml; skeletal actin, 0-400 # g / m l ; platelet tropomyosin, 100 # g / m l ; arterial tropomyosin, 100 ~ g / m l . Incubation was carried out in 50 m M K C I / 5 m M MgC12/10 m M Tris-maleate (pH 6.8)/0.1 m M CaCl 2 and 1 / ~ g / m l pyruvate kinase (Boehringer) at 25°C. The reaction was started by adding 1 m M ATP and 2 m M phosphoenolpyruvate. The a m o u n t of pyruvate produced at the steady state (1-16 min) was determined [14]. O- . . . . . O, control. ,x A, arterial tropomyosin. O O, platelet tropomyosin. (b) Arterial tropomyosin was recombined with reconstituted actomyosin and the ATPase activity was determined. The assay condition was the same as in (a) except that the concentration of arterial tropomyosin was varied at 100 # g / m l actin, 10 # g / m l myosin in 10 m M MgCl 2 and 9 m M ATP in the reaction mixture.

of enhancing the activity (Fig. 3a). It is noted that the titration curve of both tropomyosins on the ATPase activity (Fig. 3b, solid lines) are very similar. These results support the hypothesis that the large and small tropomyosins are interchangeable in enhancing the actomyosin ATPase activities of both artery and platelet. In skeletal muscle, it was shown that tropo-

294

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60

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Fig. 3. Effectsof arterial or platelet tropomyosinon the ATPase activity of reconstituted skeletal actoplatelet myosin.(a) Arterial tropomyosin was recombined with reconstituted actomyosinat different concentrations of actin and the ATPase activity was determined. The assay condition was the same as in Fig. 2a, except that platelet myosin (100/~g/ml) was used. • ..... -'•, control. © O, arterial tropomyosin. (b) Arterial or platelet tropomyosin was recombined with reconstituted actomyosin and the ATPase activity was determined. The assay condition for the solid line was the same as in Fig. 2a, except that the concentration of tropomyosinwas varied at 100/~g/ml of actin. The assay condition for the broken line was the same as in Fig. 2b. O, •, arterial tropomyosin, zx, A, platelet tropomyosin.

myosin enhances the actomyosin ATPase activity under conditions where the concentratoin of Mg. ATP is less than 10 -4 M [22], or the concentration of heavy meromyosin [12] or subfragrnent-1 [13] is very high. We hypothesized that the enhancement of actomyosin ATPase by tropomyosin takes place by the common mechanism in any actomyosin. If this assumption is correct, it is expected that the enhancement observed in smooth muscle and platelet is reduced when the concentration of Mg. ATP is increased a n d / o r the concentration of

myosin is reduced. This hypothesis was tested in the next series of experiments. As seen in Fig. 2b, the hypothesis was correct in smooth muscle. Tropomyosin shows only insignificant enhancement when the concentration of mg. ATP is increased (10-times that in Fig. 2a) and the concentration of myosin is reduced (one-tenth of that in Fig. 2a). The hypothesis was also correct in platelet. As seen in Fig. 3b (dotted line) the enhancement caused by platelet tropomyosin is abolished under the same condition as in smooth muscle. It should be noted that in this experiment the arterial and platelet tropomyosins are again indistinguishable. It is also emphasized that we could not find any condition where tropomyosin inhibits the ATPase activities more than by 20%. In this respect, the effect of tropomyosin in artery and platelet makes a sharp contrast to that in skeletal muscle. From these series of experiments, it is concluded that: (i) tropomyosins from platelet and artery are not distinguishable in their effects on the actin-activated ATPase activities of both arterial and platelet myosins; (ii) the enhancement caused by either tropomyosin is reduced by increasing the concentration of Mg. ATP and decreasing the concentration of myosin.

Recombination experiments of myosin and tropomyosin: exchange of tropomyosins between skeletal and non-skeletal myosins The enhancing effect of gizzard tropomyosin on the ATPase activity of gizzard actomyosin was first reported by Sobieszek [3]. Subsequently, he showed that tropomyosins from both smooth muscle and the brain enhance the acto-heavy meromyosin ATPase activity of skeletal muscle under the condition in which skeletal tropomyosin inhibits the activity [10]. The results imply that skeletal tropomyosin is unique in its inhibitory effect on the actomyosin ATPase activity. The results, however, raise the question of what is the major determinant of the effect of tropomyosin. This question should be answered if tropomyosins are exchanged between different actomyosins reconstituted from different myosins and same actin. If the type of tropomyosin is the major determinant, changing tropomyosin in the same actomyosin system should change the effect of tropomyo-

295 sin. O n the other h a n d , if the t y p e of m y o s i n is the m a j o r d e t e r m i n a n t , c h a n g i n g m y o s i n for the s a m e a c t i n / t r o p o m y o s i n c o m p l e x should change the effect of t r o p o m y o s i n . T h e first a s s u m p t i o n is n o t shown to be the case, as d e s c r i b e d below: (i) The A T P a s e activity of p l a t e l e t a c t o m y o s i n is e n h a n c e d b y p l a t e l e t t r o p o m y o s i n [9]. The activity of the same a c t o m y o s i n is also e n h a n c e d when t r o p o m y o s i n is c h a n g e d ; arterial t r o p o m y o s i n (Fig. 3a), as well as skeletal t r o p o m y o s i n (Fig. 5, solid line with o p e n triangle), enhances the activity. (ii) T h e A T P a s e activity of arterial a c t o m y o s i n is e n h a n c e d b y arterial t r o p o m y o s i n (Fig. 2a). It is also e n h a n c e d when the t r o p o m y o s i n is changed;

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p l a t e l e t t r o p o m y o s i n (Fig. 2a), as well as skeletal t r o p o m y o s i n (Fig. 5, solid line with closed circle), enhances the activity. (iii) T h e A T P a s e activity of skeletal a c t o m y o s i n is not e n h a n c e d b y skeletal t r o p o m y o s i n at the m i l l i m o l a r c o n c e n t r a t i o n of M g - A T P [22]. U n d e r the same c o n d i t i o n , the A T P a s e activity of the s a m e a c t o m y o s i n is n o t e n h a n c e d when the t r o p o m y o s i n is changed; arterial t r o p o m y o s i n (Fig. 4a, o p e n triangle), as well as platelet t r o p o m y o s i n (Fig. 4a, o p e n circle), fails to e n h a n c e the activity. Thus, the effect of t r o p o m y o s i n is qualitatively u n a l t e r e d as long as a c t o m y o s i n is the same. T h e second a s s u m p t i o n is shown to be the case, as d e s c r i b e d in the following: (iv) Platelet t r o p o m y o s i n enhances the A T P a s e activity of p l a t e l e t a c t o m y o s i n [9]. The s a m e t r o p o m y o s i n does n o t e n h a n c e the A T P a s e activity

300

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200

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(b)

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0--

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A

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i 100 (SK-Ac) (JJg/ml)

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Fig. 4. Effects of arterial or platelet tropomyosin on the ATPase activity of reconstituted skeletal acto-skeletal myosin. (a); Arterial or platelet tropomyosin was recombined with reconstituted actomyosin at different concentrations of actin (sk-Ac). The assay condition was the same as in Fig. 2a, except that the reaction time was 5 min. • . . . . . -e, control, zx zx, arterial tropomyosin. © O, platelet tropomyosin. (b) Arterial or platelet tropomyosin was recombined with skeletal actomyosin and the ATPase activity was determined. The assay condition was the same as in Fig. 3a, except that the concentration of ATP was 50 #M and the concentration of MgCI2 was 1 raM.

o

I

I

100

I

200

(sk-Tm) (jdg/m[ ) Fig. 5. Effects of skeletal tropomyosin on the ATPase activity of reconstituted skeletal acto-platelet or arterial myosin. The assay condition for the solid line was the same as in Fig. 2a, except that skeletal tropomyosin was used (0-200 #g/ml) at 100 #g/ml of actin. The assay condition for the broken line was the same as in Fig. 2b, except that skeletal tropomyosin was used (0-200 #g/m1). O, O, arterial myosin. /, platelet myosin.

296 when myosin is changed (Fig. 4a, open circle). (v) Arterial tropomyosin enhances the ATPase activity of arterial actomyosin (Fig. 2a). The same tropomyosin does not enhance the ATPase activity when myosin is changed (Fig. 4a, open triangle). (vi) Skeletal tropomyosin inhibits the ATPase activity of skeletal actomyosin [22]. The same tropomyosin enhances the ATPase activity when myosin is changed (Fig. 5, solid lines). Thus, the effect of tropomyosin is altered when actomyosin is different. Therefore, it is indicated that the major determinant of the effect of tropomyosin is myosin. To be more precise, the data indicate that the state of the interaction between actin and myosin determines the effect of tropomyosin, because of the following reason: (vii) The actomyosin ATPase activity of ptatelet is enhanced either by platelet tropomyosin [9], arterial tropomyosin (Fig. 3a) or skeletal tropomyosin (Fig. 5, open triangle with solid line). The enhancement, however, is abolished by increasing the concentration of Mg. ATP and decreasing the mysoin/actin ratio (Fig. 3b, dotted line; Fig. 5, open triangle with dotted line). (viii) The actomyosin ATPase activity of arterial smooth muscle is enhanced either by arterial tropomyosin (Fig. 2a), platelet tropomyosin (Fig. 2a) or skeletal tropomyosin (Fig. 5, closed circle with solid line). The enhancement, however, is abolished by increasing the concentration of Mg. ATP and decreasing the myosin/actin ratio (Fig. 2b, Fig. 5, open circle with dotted line). (ix) The ATPase activity of skeletal actomyosin is not enhanced either by skeletal tropomyosin [22], arterial tropomyosin or platelet tropomyosin (Fig. 4a). It is, however, enhanced when the concentration of Mg. ATP is decreased or the myosin/actin ratio is increased (Fig. 4b, and Ref. 10) by any tropomyosin. Under these conditions, the protein components are not altered. The concentration of Mg. ATP and myosin/ actin ratio should affect the actin/myosin interaction, but should not affect actin/tropomyosin interaction, primarily. Therefore, the interaction between actin and myosin determines the effect of tropomyosin.

Discussion

Our observations described in this report are discussed with the related findings previously reported by others. (1) When skeletal actin is used to reconstitute actomyosin, any tropomyosin enhances the ATPase activity of actomyosin except that skeletal tropomyosin is recombined with skeletal actomyosin (this report and Refs. 2, 3, 6, 10-13, 22, 23). (2) The inhibition of the ATPase activity of skeletal actomyosin by skeletal tropomyosin is changed to enhancement when the concentration of Mg. ATP is lowered or the myosin/actin ratio is raised [10-13,22,23]. (3) The enhancement of the ATPase activity of non-skeletal actomyosin is abolished when the concentration of M g - A T P is raised and the myosin/actin ratio is lowered (this report). From these observations, it is concluded that: (i) Any tropomyosin enhances the actomyosin ATPase activity of any myosin under appropriate conditions of Mg. ATP and myosin/actin ratio. (ii) Only the skeletal tropomyosin is capable of inhibiting the ATPase activity of the skeletal actomyosin under specific conditions of the concentration of Mg. ATP and the myosin/actin ratio. These two conclusions are discussed below: (A) First, the condition for enhancement is discussed. Bremel et al. [23] postulated that the enhanced ATPase activity by skeletal tropomyosin is a result of 'rigor' interaction between myosin and actin. The molecular mechanism by which tropomyosin enhances the ATPase activity of 'rigor' actomyosin is still obscure. However, it is evident that tropomyosin does not take a 'blocking' position on actin filaments [24,25] when actin and myosin form a 'rigor' complex. It is speculated by analogy that in smooth muscle and platelet, actin and myosin form a 'rigor'-like complex even at the higher concentration of Mg • ATP. This assumption is supported by the direct evidence that after the addition of ATP, the rate of dissociation and reassociation of actin and smooth muscle myosin is lower than that of skeletal muscle [26-28]. Myosins from smooth muscle and platelet are distinctively different from skeletal myosin in their property that their actin-activated ATPase activities are regulated by the phosphorylation of the 20 kDa light chain. Therefore, it is likely that the 'phosphorylation-regulated' myosin is easier

297

than skeletal myosin to form 'rigor' complex with skeletal actin. It is shown that both skeletal and gizzard tropomyosins enhance the actomyosin ATPase activity of gizzard myosin recombined with either skeletal or gizzard actin [2]. We have also observed that platelet tropomyosin enhances the ATPase activity of platelet myosin recombined either with gizzard or non-muscle actin (unpublished observations). These data indicate that it is easier for the 'phosphorylation-regulated' myosin to have a 'rigor' interaction with any actin. On the other hand, Yang et al. [29] reported that skeletal tropomyosin enhances the ATPase activity of skeletal myosin recombined with Acanthamoeba actin under the condition where it inhibits the activity if the same myosin is recombined with skeletal actin. This observation seems to indicate that even skeletal mysoin is capable of forming a 'rigor' complex with specific actin at millimolar concentration of Mg. ATP. In general, therefore, the enhancement, not the inhibition, by tropomyosin is the rule in any actin-myosin combination under physiological conditions. (B) Secondly, the condition for inhibition is discussed. As summarized above, the only condition under which inhibition takes place is that where skeletal actomyosin is recombined with skeletal tropomyosin at millimolar concentratoin of Mg. ATP and over a limited range of the myosin/actin ratio. Any substitution of the protein and any change in the concentration of Mg. ATP and myosin/actin ratio would change the effect from inhibition to enhancement. This means that there may be a unique interaction among skeletal myosin, skeletal actin and skeletal tropomyosin. It should be pointed out that this inhibitory condition is that under which the troponin complex exerts its effect of Ca 2 +-sensitive regulation. It is likely that the 'primary' role of tropomyosin was to enhance the actomyosin ATPase activity. Only after the evolution of the troponin system in skeletal muscle did the inhibition by tropomyosin evolve. The effect of the troponin complex is regarded as restoration of the enhancement of tropomyosin when the concentration of Ca 2. is increased. Acknowledgements We thank Mrs. Michiko Takagi for her excel-

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