- Email: [email protected]
Decoration of actin filaments with skeletal muscle heavy meromyosin containing either phosphorylated or dephosphorylated regulatory light chains
Biochimica etBiophysica Acta 830 (1985) 337-340
337
Elsevier
BBA30103
BBA Report
Decoration of actin filaments with skeletal muscle heavy meromyosin containing either phosphorylated or dephosphorylated regulatory light chains D . S t f p k o w s k i , H . O s i f l s k a , D . S z c z ~ s n a , M . W r o t e k a n d I. K ~ k o l * Department of Cellular Biochemistry, Neneki Institute of Experimental Biology, 3, Pasteur Street, 02-093 Warsaw (Poland)
(ReceivedMay 7th, 1985)
Key words: Regulatorylight chain phosphorylation;Regulatorylight chain dephosphorylation;Decoratedactin filament; (Rabbit skeletal muscle)
Heavy meromyosin containing almost intact regulatory light chains (LC2) was obtained from monomeric phosphorylated and dephosphorylated rabbit fast skeletal muscle myosin by brief chymotryptic digestion in the presence of CaCI 2. Actin filaments, complexed with heavy meromyosin, display two different forms of arrowhead, depending on the form of LC 2.
It is well known that regulatory light chains (LC2) in fast skeletal muscle myosin undergo p h o s p h o r y l a t i o n - d e p h o s p h o r y l a t i o n processes [1,2], but the physiological role of these processes still remain an open question. Experiments on skinned fibres or intact muscle have provided evidence that phosphorylation of LC 2 decreases the crossbridge cycling rate [3-5] and removal of these light chains leads to reduced maximal velocity of shortening [6]. Butler et al. [7] and Barsotti and Butler [8], however, have not obtained significant evidence enabling them to correlate LC 2 phosphorylation with the in vivo response of muscle fibres. Phosphorylation of LC 2 is not essential for actin-activated adenosine triphosphatase activity of skeletal muscle [9]. There are contradictory reports as to whether phosphorylation is a factor influencing actin-activated MgZ+-ATPase of myosin [9-12]. On the other hand, phosphorylation of LC 2 alters the structure of myosin, as evidenced by the * To whom correspondenceshould be addressed. Abbreviations: LC2,19 kDa light chains of fast skeletal muscle; PMSF, phenylmethylsulphonylfluoride.
differentiated susceptibility to limited proteolysis [13], and causes changes of cross-bridge conformation, as shown in crosslinking experiments [14]. The possible role of LC 2 in determining the conformation of myosin heads was postulated by Craig et al. [15] on the basis of the results showing differences in the forms of the pattern of arrowheads obtained by decoration of actin filaments with intact and devoid of LC 2 myosin subfragment 1. The Ca2+-dependent conformational state of F-actin induced by myosin subfragment 1 was found to be related to the presence of LC 2 [161. To obtain new information on the role of phosphorylation and dephosphorylation of LC 2, the complexing of actin filaments with skeletal muscle heavy meromyosin containing regulatory light chains in a phosphorylated or dephosphorylated form was studied. Phosphorylated or dephosphorylated myosin (10 m g / m l ) obtained according to Michnicka et al. [12] was dissolved in 0.5 M KC1/20 mM phosphate buffer (pH 7.0)/2 mM CaC1 z. Digestion was carried out at 22-24°C for 4 min in the presence of trypsin-free a-chymotrypsin (5 m g / m l ) with enzyme/substrate ratio 1:260. PMSF (8.7
0167-4838/85/$03.30 © 1985 ElsevierSciencePublishers B.V. (BiomedicalDivision)
338 m g / m l in ethanol) was added to the final concentration 0.36 mM to stop the reaction [17]. The solution was dialyzed against 20 mM phosphate buffer (pH 7.0) to precipitate undigested myosin and all fragments insoluble at low ionic strength. The precipitate was removed by centrifugation. Heavy meromyosin was purified by standard methods. Typical yield for phosphorylated heavy meromyosin was 22-26% and for dephosphorylated 14-18%. Heavy meromyosins were examined on electrophoretic gels containing SDS [18] and urea [19] stained by Coomassie brillant blue. Both types of electrophoresis indicate that light chains of heavy meromyosin remain mostly intact (Fig. 1A1,2, B1. 2 ). The staining intensities of the bands of light chains were measured with a Vitatron densitometer equipped with an automatic integrator. It was assumed that staining intensities are proportional to the amount of light chains. The distribution of light-chain bands shows that proteolysis does not influence the ratio L C 2 : (LC 1 + LC3), which is the same as in native myosin and equals almost 1. Phosphorylated LC 2 retains after digestion its higher mobility in urea gel than dephosphorylated LC 2 and the amount of phosphorylated LC 2 remains about 95-100% of total LC 2 and lower than 1% of that of dephosphorylated one. The enzymatic properties of phosphorylated and dephosphorylated heavy meromyosin were controlled by determination of K +-, Ca 2+-, Mg 2+and actin-stimulated ATPase activities. ATPase activities were determined at 25°C by the rate of Pi released as a function of time [12]. All assays were done in 20 mM Tris-acetate (pH 7.0) with the following specific additions. For Ca 2+ ATPase: 50 mM KCI, 10 mM CaC12, 1 mM ATP. For K + ATPase: 500 mM KC1, 2.0 mM dithiothreitol, 5 mM EDTA, 2 mM ATP. For actin-stimulated Mg 2+ ATPase: 50 mM KC1, 1 mM MgC12, 2 mM MgATP; actin concentration 0.05 mg/ml. The concentration of enzyme was 0.1-0.2 m g / m l in a final volume of 2 ml. The values of ATPase activities, expressed as mol ATP hydrolyzed per mol of heavy meromyosin per s, were as follows: K+-stimulated ATPase, 12.64 and 14.16; Ca2+-ATPase, 12.0 and 12.32; Mg 2+- and actin-stimulated ATPase, 6.13 and 7.5, of phosphorylated and dephosphorylated heavy
meromyosin, respectively. The similarity of Ca 2+and K+-activated ATPase activities of both heavy meromyosin preparations and the corresponding electrophoretic patterns lead us to assume that our two kinds of heavy meromyosin preparation differ from each other only in the form of LC 2. Pure skeletal muscle actin obtained according to Strzelecka-Gotaszewska [20] was decorated with heavy meromyosin in media containing 20 mM phosphate buffer (pH 7.0) and 25 mM KC1. Concentration of heavy meromyosin and actin was 0.180 m g / m l and 0.050 mg/ml, respectively. Samples of actin-heavy meromyosin complexes for electron microscopy were negatively stained with 1% uranyl acetate on grids coated with formvar and carbon and examined in a JEOL JEM 100B electron microscope. The magnification of electron micrographs was calibrated with smooth muscle tropomyosin paracrystals, assuming 40 nm regular repeat [21]. The arrowheads obtained with heavy meromyosin containing LC 2 in the phosphorylated form are shown in Fig. 1A3_5, and with dephosphorylated heavy meromyosin in Fig. 1B3, 134.5. The measured maximum width of the phosphorylated arrowhead is 19 + 1 nm (mean + S.D. of 30 measurements taken from different complexed filaments). The analogous maximum width of dephosphorylated arrowhead is 24 + 1 nm. The observed profiles of complexation seem very similar to those described by Craig et al. [15]: arrowheads obtained with skeletal muscle myosin subfragment 1 - with LC 2 present - termed by them the 'barbed' form, and without LC 2, termed 'blunted' form. The latter form was earlier demonstrated by Moore et al. [22]. Phosphorylated arrowheads correspond to the blunted form, the dephosphorylated ones to the barbed form. Barbed arrowheads formed from dephosphorylated heavy meromyosin are less regular than those obtained by Craig and co-workers with myosin subfragment 1 [15]. Maximum widths of the described two types of arrowhead are comparable with those measured by Craig for barbed and blunted patterns obtained with scallop myosin subfragment 1 containing or not containing the regulatory light chain, respectively. Complexation of actin with intact phosphorylated myosin yields an occasionally clean blunted
A1 w m
A2
i
BI
v
i
LC 1 LC 2 LC 3
LC 1 ~i!
LC2
~
I_.C3
Fig. 1. Electron micrographs of F-actin filaments complexed with phosphorylated (A3_5) and dephosphorylated (B3_5) heavy meromyosin and respective electrophoretic patterns (A1,2) and (B1.2) of heavy meromyosins used for decoration. Magnification, 96000 (A4,5, B4,5) and 240000 (A3, B3). Electrophoresis was performed in 8 M urea gel (A1, B1) and SDS gel (A 2, B2). p a t t e r n , while in the case of d e p h o s p h o r y l a t e d m y o s i n we d o n o t observe regular arrowheads. A difference in w i d t h of filaments c o m p l e x e d with p h o s p h o r y l a t e d a n d d e p h o s p h o r y l a t e d m y o s i n was
also o b s e r v e d a n d r e s e m b l e d that o b t a i n e d when actin was c o m p l e x e d with m y o s i n p r o t e o l y t i c fragments. T h e o b s e r v a t i o n s presented a b o v e c o n c e r n i n g
340 the rigor linkage between actin a n d myosin or its soluble fragments lead to two m a i n conclusions: (A) P h o s p h o r y l a t e d and dephosphorylated arrowhead patterns are different a n d resemble those o b t a i n e d with skeletal muscle m y o s i n s u b f r a g m e n t 1 devoid of LC 2 a n d c o n t a i n i n g LC 2, respectively. Therefore, it m a y be assumed that the consequences of conformational changes of m y o s i n a c c o m p a n y i n g phosp h o r y l a t i o n of LC 2 can be similar to those i n d u c e d b y removal of this light chain. (B) The arrowhead p a t t e r n representing rigor a c t i n - m y o s i n linkage is modified b y phosp h o r y l a t i o n of LC 2. One could expect that p h o s p h o r y l a t i o n of LC 2 could modify the mechanical properties of working skeletal muscle by i n f l u e n c i n g the t r a n s i t i o n from the actomyosin A D P P i state to rigor state. The authors would like to t h a n k Professor Ferenc G u b a for s t i m u l a t i n g discussion.
References 1 B~ir~my,K., B~ir~iny,M., Gillis, J.M. and Kushmerick, M.J. (1979) J. Biol. Chem. 254, 3617-3623 2 Manning, D.R. and Stull, J.T. (1979) Biochem. Biophys. Res. Commun. 90, 164-170 3 Cook, R., Franks, K. and Stull, J.T. (1982) FEBS Lett. 144, 33-37
4 Crow, M.T. and Kushmerick, M.J. (1982) Science 217, 835-837 5 Crow, M.T. and Kushmerick, M.J. (1982) J. Biol. Chem. 257, 2121-2124 6 Moss, R.L., Giulian, G.G. and Greaser, M.L. (1982) J. Biol. Chem. 257, 8588-8591 7 Butler, T.M., Siegman, M.J., Mooers, S.U. and Barsotti, R.J. (1983) Science 220, 1167-1169 8 Barsotti, R.J. and Butler, T.M. (1984) J. Muscle Res. Cell Mot. 5, 45-64 9 Morgan, M., Perry, S.V. and Ottaway, J. (1976) Biochem. J. 157, 687-697 10 Pemrick, S.M. (1980) J. Biol. Chem. 255, 8836-8841 11 K~kol, I., Kasman, K. and Michnicka, M. (1982) Biochim. Biophys. Acta 704, 437-443 12 Michnicka, M., Kasman, K. and K~kol, I. (1982) Biochim. Biophys. Acta 704, 470-475 13 Ritz-Gold, C.J., Cooke, R., Blumenthal, D.K. and Stull, J.T. (1980) Biochem. Biophys. Res. Commun. 93, 209-214 14 Mrakov~-Zenic, A. and Reisler, E. (1983) Biochemistry 22, 525-530 15 Craig, R., Szent-Gyorgyi, A.G., Beese, L., Flicker, P., Vibert, P. and Cohen, C. (1980) J. Mol. Biol. 140, 35-55 16 Borovikov, Y.S., Levitskii, D.I., Kirillina, V.P. and Poglazov, B.F. (1982) Eur. J. Biochem. 125, 343-347 17 Cardinaud, R. (1980) Biochimie62, 135-145 18 Weber, K. and Osborn, M. (1969) J. Biol. Chem. 224, 4406-4412 19 Perrie, W.T. and Perry, S.V. (1970) Biochem. J. 119, 31-38 20 Strzelecka-Go/aszewska, H., Jakubiak, M. and Drabikowski, W. (1975) Eur. J. Biochem. 55, 221-230 21 D~browska, R., Nowak, E. and Drabikowski, W. (1980) Comp. Biochem. Physiol. 65B, 75-83 22 Moore, P.B., Huxley, H.E. and De Rosier, D.J. (1970) J. Mol. Biol. 50, 279-295
337
Elsevier
BBA30103
BBA Report
Decoration of actin filaments with skeletal muscle heavy meromyosin containing either phosphorylated or dephosphorylated regulatory light chains D . S t f p k o w s k i , H . O s i f l s k a , D . S z c z ~ s n a , M . W r o t e k a n d I. K ~ k o l * Department of Cellular Biochemistry, Neneki Institute of Experimental Biology, 3, Pasteur Street, 02-093 Warsaw (Poland)
(ReceivedMay 7th, 1985)
Key words: Regulatorylight chain phosphorylation;Regulatorylight chain dephosphorylation;Decoratedactin filament; (Rabbit skeletal muscle)
Heavy meromyosin containing almost intact regulatory light chains (LC2) was obtained from monomeric phosphorylated and dephosphorylated rabbit fast skeletal muscle myosin by brief chymotryptic digestion in the presence of CaCI 2. Actin filaments, complexed with heavy meromyosin, display two different forms of arrowhead, depending on the form of LC 2.
It is well known that regulatory light chains (LC2) in fast skeletal muscle myosin undergo p h o s p h o r y l a t i o n - d e p h o s p h o r y l a t i o n processes [1,2], but the physiological role of these processes still remain an open question. Experiments on skinned fibres or intact muscle have provided evidence that phosphorylation of LC 2 decreases the crossbridge cycling rate [3-5] and removal of these light chains leads to reduced maximal velocity of shortening [6]. Butler et al. [7] and Barsotti and Butler [8], however, have not obtained significant evidence enabling them to correlate LC 2 phosphorylation with the in vivo response of muscle fibres. Phosphorylation of LC 2 is not essential for actin-activated adenosine triphosphatase activity of skeletal muscle [9]. There are contradictory reports as to whether phosphorylation is a factor influencing actin-activated MgZ+-ATPase of myosin [9-12]. On the other hand, phosphorylation of LC 2 alters the structure of myosin, as evidenced by the * To whom correspondenceshould be addressed. Abbreviations: LC2,19 kDa light chains of fast skeletal muscle; PMSF, phenylmethylsulphonylfluoride.
differentiated susceptibility to limited proteolysis [13], and causes changes of cross-bridge conformation, as shown in crosslinking experiments [14]. The possible role of LC 2 in determining the conformation of myosin heads was postulated by Craig et al. [15] on the basis of the results showing differences in the forms of the pattern of arrowheads obtained by decoration of actin filaments with intact and devoid of LC 2 myosin subfragment 1. The Ca2+-dependent conformational state of F-actin induced by myosin subfragment 1 was found to be related to the presence of LC 2 [161. To obtain new information on the role of phosphorylation and dephosphorylation of LC 2, the complexing of actin filaments with skeletal muscle heavy meromyosin containing regulatory light chains in a phosphorylated or dephosphorylated form was studied. Phosphorylated or dephosphorylated myosin (10 m g / m l ) obtained according to Michnicka et al. [12] was dissolved in 0.5 M KC1/20 mM phosphate buffer (pH 7.0)/2 mM CaC1 z. Digestion was carried out at 22-24°C for 4 min in the presence of trypsin-free a-chymotrypsin (5 m g / m l ) with enzyme/substrate ratio 1:260. PMSF (8.7
0167-4838/85/$03.30 © 1985 ElsevierSciencePublishers B.V. (BiomedicalDivision)
338 m g / m l in ethanol) was added to the final concentration 0.36 mM to stop the reaction [17]. The solution was dialyzed against 20 mM phosphate buffer (pH 7.0) to precipitate undigested myosin and all fragments insoluble at low ionic strength. The precipitate was removed by centrifugation. Heavy meromyosin was purified by standard methods. Typical yield for phosphorylated heavy meromyosin was 22-26% and for dephosphorylated 14-18%. Heavy meromyosins were examined on electrophoretic gels containing SDS [18] and urea [19] stained by Coomassie brillant blue. Both types of electrophoresis indicate that light chains of heavy meromyosin remain mostly intact (Fig. 1A1,2, B1. 2 ). The staining intensities of the bands of light chains were measured with a Vitatron densitometer equipped with an automatic integrator. It was assumed that staining intensities are proportional to the amount of light chains. The distribution of light-chain bands shows that proteolysis does not influence the ratio L C 2 : (LC 1 + LC3), which is the same as in native myosin and equals almost 1. Phosphorylated LC 2 retains after digestion its higher mobility in urea gel than dephosphorylated LC 2 and the amount of phosphorylated LC 2 remains about 95-100% of total LC 2 and lower than 1% of that of dephosphorylated one. The enzymatic properties of phosphorylated and dephosphorylated heavy meromyosin were controlled by determination of K +-, Ca 2+-, Mg 2+and actin-stimulated ATPase activities. ATPase activities were determined at 25°C by the rate of Pi released as a function of time [12]. All assays were done in 20 mM Tris-acetate (pH 7.0) with the following specific additions. For Ca 2+ ATPase: 50 mM KCI, 10 mM CaC12, 1 mM ATP. For K + ATPase: 500 mM KC1, 2.0 mM dithiothreitol, 5 mM EDTA, 2 mM ATP. For actin-stimulated Mg 2+ ATPase: 50 mM KC1, 1 mM MgC12, 2 mM MgATP; actin concentration 0.05 mg/ml. The concentration of enzyme was 0.1-0.2 m g / m l in a final volume of 2 ml. The values of ATPase activities, expressed as mol ATP hydrolyzed per mol of heavy meromyosin per s, were as follows: K+-stimulated ATPase, 12.64 and 14.16; Ca2+-ATPase, 12.0 and 12.32; Mg 2+- and actin-stimulated ATPase, 6.13 and 7.5, of phosphorylated and dephosphorylated heavy
meromyosin, respectively. The similarity of Ca 2+and K+-activated ATPase activities of both heavy meromyosin preparations and the corresponding electrophoretic patterns lead us to assume that our two kinds of heavy meromyosin preparation differ from each other only in the form of LC 2. Pure skeletal muscle actin obtained according to Strzelecka-Gotaszewska [20] was decorated with heavy meromyosin in media containing 20 mM phosphate buffer (pH 7.0) and 25 mM KC1. Concentration of heavy meromyosin and actin was 0.180 m g / m l and 0.050 mg/ml, respectively. Samples of actin-heavy meromyosin complexes for electron microscopy were negatively stained with 1% uranyl acetate on grids coated with formvar and carbon and examined in a JEOL JEM 100B electron microscope. The magnification of electron micrographs was calibrated with smooth muscle tropomyosin paracrystals, assuming 40 nm regular repeat [21]. The arrowheads obtained with heavy meromyosin containing LC 2 in the phosphorylated form are shown in Fig. 1A3_5, and with dephosphorylated heavy meromyosin in Fig. 1B3, 134.5. The measured maximum width of the phosphorylated arrowhead is 19 + 1 nm (mean + S.D. of 30 measurements taken from different complexed filaments). The analogous maximum width of dephosphorylated arrowhead is 24 + 1 nm. The observed profiles of complexation seem very similar to those described by Craig et al. [15]: arrowheads obtained with skeletal muscle myosin subfragment 1 - with LC 2 present - termed by them the 'barbed' form, and without LC 2, termed 'blunted' form. The latter form was earlier demonstrated by Moore et al. [22]. Phosphorylated arrowheads correspond to the blunted form, the dephosphorylated ones to the barbed form. Barbed arrowheads formed from dephosphorylated heavy meromyosin are less regular than those obtained by Craig and co-workers with myosin subfragment 1 [15]. Maximum widths of the described two types of arrowhead are comparable with those measured by Craig for barbed and blunted patterns obtained with scallop myosin subfragment 1 containing or not containing the regulatory light chain, respectively. Complexation of actin with intact phosphorylated myosin yields an occasionally clean blunted
A1 w m
A2
i
BI
v
i
LC 1 LC 2 LC 3
LC 1 ~i!
LC2
~
I_.C3
Fig. 1. Electron micrographs of F-actin filaments complexed with phosphorylated (A3_5) and dephosphorylated (B3_5) heavy meromyosin and respective electrophoretic patterns (A1,2) and (B1.2) of heavy meromyosins used for decoration. Magnification, 96000 (A4,5, B4,5) and 240000 (A3, B3). Electrophoresis was performed in 8 M urea gel (A1, B1) and SDS gel (A 2, B2). p a t t e r n , while in the case of d e p h o s p h o r y l a t e d m y o s i n we d o n o t observe regular arrowheads. A difference in w i d t h of filaments c o m p l e x e d with p h o s p h o r y l a t e d a n d d e p h o s p h o r y l a t e d m y o s i n was
also o b s e r v e d a n d r e s e m b l e d that o b t a i n e d when actin was c o m p l e x e d with m y o s i n p r o t e o l y t i c fragments. T h e o b s e r v a t i o n s presented a b o v e c o n c e r n i n g
340 the rigor linkage between actin a n d myosin or its soluble fragments lead to two m a i n conclusions: (A) P h o s p h o r y l a t e d and dephosphorylated arrowhead patterns are different a n d resemble those o b t a i n e d with skeletal muscle m y o s i n s u b f r a g m e n t 1 devoid of LC 2 a n d c o n t a i n i n g LC 2, respectively. Therefore, it m a y be assumed that the consequences of conformational changes of m y o s i n a c c o m p a n y i n g phosp h o r y l a t i o n of LC 2 can be similar to those i n d u c e d b y removal of this light chain. (B) The arrowhead p a t t e r n representing rigor a c t i n - m y o s i n linkage is modified b y phosp h o r y l a t i o n of LC 2. One could expect that p h o s p h o r y l a t i o n of LC 2 could modify the mechanical properties of working skeletal muscle by i n f l u e n c i n g the t r a n s i t i o n from the actomyosin A D P P i state to rigor state. The authors would like to t h a n k Professor Ferenc G u b a for s t i m u l a t i n g discussion.
References 1 B~ir~my,K., B~ir~iny,M., Gillis, J.M. and Kushmerick, M.J. (1979) J. Biol. Chem. 254, 3617-3623 2 Manning, D.R. and Stull, J.T. (1979) Biochem. Biophys. Res. Commun. 90, 164-170 3 Cook, R., Franks, K. and Stull, J.T. (1982) FEBS Lett. 144, 33-37
4 Crow, M.T. and Kushmerick, M.J. (1982) Science 217, 835-837 5 Crow, M.T. and Kushmerick, M.J. (1982) J. Biol. Chem. 257, 2121-2124 6 Moss, R.L., Giulian, G.G. and Greaser, M.L. (1982) J. Biol. Chem. 257, 8588-8591 7 Butler, T.M., Siegman, M.J., Mooers, S.U. and Barsotti, R.J. (1983) Science 220, 1167-1169 8 Barsotti, R.J. and Butler, T.M. (1984) J. Muscle Res. Cell Mot. 5, 45-64 9 Morgan, M., Perry, S.V. and Ottaway, J. (1976) Biochem. J. 157, 687-697 10 Pemrick, S.M. (1980) J. Biol. Chem. 255, 8836-8841 11 K~kol, I., Kasman, K. and Michnicka, M. (1982) Biochim. Biophys. Acta 704, 437-443 12 Michnicka, M., Kasman, K. and K~kol, I. (1982) Biochim. Biophys. Acta 704, 470-475 13 Ritz-Gold, C.J., Cooke, R., Blumenthal, D.K. and Stull, J.T. (1980) Biochem. Biophys. Res. Commun. 93, 209-214 14 Mrakov~-Zenic, A. and Reisler, E. (1983) Biochemistry 22, 525-530 15 Craig, R., Szent-Gyorgyi, A.G., Beese, L., Flicker, P., Vibert, P. and Cohen, C. (1980) J. Mol. Biol. 140, 35-55 16 Borovikov, Y.S., Levitskii, D.I., Kirillina, V.P. and Poglazov, B.F. (1982) Eur. J. Biochem. 125, 343-347 17 Cardinaud, R. (1980) Biochimie62, 135-145 18 Weber, K. and Osborn, M. (1969) J. Biol. Chem. 224, 4406-4412 19 Perrie, W.T. and Perry, S.V. (1970) Biochem. J. 119, 31-38 20 Strzelecka-Go/aszewska, H., Jakubiak, M. and Drabikowski, W. (1975) Eur. J. Biochem. 55, 221-230 21 D~browska, R., Nowak, E. and Drabikowski, W. (1980) Comp. Biochem. Physiol. 65B, 75-83 22 Moore, P.B., Huxley, H.E. and De Rosier, D.J. (1970) J. Mol. Biol. 50, 279-295