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Proteolytic fragments from the lobster myosin molecule
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Biochimica et Biophysica Acta, 536 (1978) 142--155 © Elsevier/North-HollandBiomedicalPress
BBA 37981 PROTEOLYTIC FRAGMENTS FROM THE LOBSTER MYOSIN MOLECULE
C. RICHARD ZOBEL Department of Biophysical Sciences, State University of New York, Amherst, N.Y. 14226 (U.S.A.)
(Received January 12th, 1978) Summary The fragments produced by proteolysis of lobster abdominal muscle myosin with trypsin, a-chymotrypsin and papain have been investigated by sodium dodecyl sulfate (SDS) gel electrophoresis. Essentially monodisperse populations of long rods are produced by a-chymotryptic and papain digestion of rabbit myosin but corresponding digestion of lobster myosin yields multicomponent species. Similarly the low ionic strength insoluble fraction from tryptic digestion of lobster myosin is polydisperse in contrast to essentially monodisperse light meromyosin from rabbit myosin. Comparative tryptic digestion of rabbit and lobster myosin papain long rods shows that the latter have five susceptible cleavage sites in the subfragment-2 region while rabbit long rods have only one: both long rods appear to have three cleavage sites in the light meromyosin region. The fragments produced by tryptic digestion of rabbit myosin papain long rods have been tentatively identified by comparison with fragments isolated from papain digests of rabbit heavy meromyosin and tryptic digests of rabbit light meromyosin. The results suggest differences in sensitivity to enzymic proteolysis between the subfragment-2 regions in rabbit and lobster myosin as well as relative differences in proteolytic sensitivity between the subfragment-2 and light meromyosin region within the individual molecules. Partial explanation of the observation is proposed on the basis of differences in heavy chain compositions.
Introduction
Brief tryptic digestion of rabbit skeletal muscle myosin results in cleavage of the molecule into two fragments [1,2]; heavy meromyosin and light meromyosin. Further 'tryptic .digestion produces subfragment-1 from heavy meromyosin [3] and a number of rod-like fragments from light meromyosin [4]. In a number of studies Harrington and coworkers [2] have analyzed the digestion process and concluded that it could be described in terms of a fast and slow
143 reaction. The two different reaction rates reflecting the existence of two different classes of bonds in the molecular structure of myosin. Bonds readily susceptible to cleavage by trypsin are attributed to less stable regions of the molecule while those less susceptible to cleavage are presumably in more stable regions of the molecule [5]. From studies of this type there has emerged a model for the myosin molecule in which two apparently rigid a-helical regions (light meromyosin and subfragment-2) exist separated by a more flexible region, the hinge region. Similarly the globular headpieces are believed to be joined to the rod portion of the molecule through a flexible section [6]. These physical properties of the myosin molecule are presumed to have physiological importance. Thus, in several models for muscle contraction the flexible regions within the myosin rod play prominent roles in mediating the structural interaction between thick and thin filaments [7--9]. The light meromyosin terminus of the rod controls the assembly of myosin into filaments and the long rod into ordered arrays, [10--12] the particular molecular arrangements within these ordered structures depending upon the histological source of the parent myosin [ 13,14]. In lobster fast flexor abdominal and crusher claw muscles the thick myosin filaments are considerably larger than those in vertebrate striated muscle [15]. To determine if these apparent structural differences reflect differences in the intrinsic nature of the constituent myosin molecules we have initiated an investigation of the aggregative properties of lobster myosins (and their fragments) as well as the nature of the molecule, per se, as reflected in its susceptibility to enzymic proteolysis. Our results have show that in contrast to the long (approx. 2 pm) spindle shaped aggregates formed by vertebrate muscle myosin at low ionic strength, lobster abdominal muscle myosin only formed short (approx. 0.3 pm) aggegates [16] and that in tryptic hydrolysis of lobster abdominal myosin, there were 280 bonds in the rapidly cleaved class in contrast to 73 fast bonds in rabbit skeletal myosin [17]. On the basis of this latter observation we proposed that lobster myosin was much more susceptible to proteolysis in the subfragment-2 region than was skeletal muscle myosin. Investigation of the susceptibility of lobster myosin to proteolysis with papain and a-chymotrypsin has provided further support for this hypothesis. Furthermore, it has been found that tryptic digestion of papain long rods from both rabbit and lobster myosin results in somewhat different cleavage products as ascertained by sodium dodecyl sulfate (SDS) gel electrophoresis. The products of digestion of rabbit myosin have been identified by direct comparison with fragments isolated from digests of heavy meromyosin and light meromyosin. To the extent that susceptibility of the myosin molecule to enzymic attack reflects its underlying substructure, these results suggest local compositional differences between the heavy chain in rabbit and lobster myosin. Such local compositional differences may well control the aggregative and structural characteristics of the myosin molecule and result in different molecular packing in the thick filaments in the two different types of muscle. Experimental procedures Protein preparation. Lobster myosin was prepared from abdominal flexor muscle of lobster Homerus americanus as described previously [17]. Myosin
144
from the back and leg muscles of rabbits was prepared essentially by the SzentGyorgyi procedure [18] with slight modifications. General procedures for protein digestion. Myosin that had been dialyzed into 0.12 M phosphate/1 mM EDTA, pH 7, was digested with a-chymotrypsin: digestion was terminated by adding phenylmethylsulfonyl fluoride to a final concentration of 1 mM [19]. Papain digestion was carried out on myosin that had been dialyzed into 0.02 M KC1/0.02 M borate, pH 7, and was terminated with a 50-fold molar excess of iodoacetic acid in 10 mM cysteine/4 mM EDTA (excess iodoacetic acid was removed with a 2-fold weight excess of cysteine). For tryptic digestion myosin was dialyzed into 0.5 M KC1/0.05 M phosphate, pH 7. Digestion was termined with a 2-fold weight excess of soybean trypsin inhibitor. Myosin concentrations of 15--20 mg/ml were used with substrate/enzyme ratios of 300/1, for digestion times of 10 min at 23°C in all digestions. After termination of digestion, samples were titrated to pH 6 and centrifuged for 60--90 min at approx. 100 000 × g. Supernatants were then dialyzed overnight vs. 0.05 M phosphate, pH 6, for rabbit myosin and 0.05 M phosphate/0.1 M KC1, pH 6, for lobster myosin. After dialysis protein solutions were centrifuged as before. The resulting supernatants were then either used directly for gel electrophoresis or dialyzed vs. 0.5 M KC1/0.05 M phosphate, pH 7, for fractionation by gel filtration (Sephadex G-200). Pellets from centrifugation were resuspended in 0.5 M KC1/0.05 M phosphate, pH 7, and then denatured with a 10 volume excess of ethanol for 3 h at room temperature. Subsequent steps for the preparation of rod fragments were essentially as described by SzentGyorgyi et al. [20]. The protein, precipitated by ethanol was packed into a pellet by centrifugation and then dialysed against 0.5 M KC1/0.05 M phosphate. R o d fragments were extracted into the solvent. In the discussion that follows the term 'ethanol-resistant components' will refer to those fragments which were resolubilized by extraction of the ethanol-precipitated protein with buffere 0.5 M KC1. Those components originally soluble in 0.5 M KC1 but which could not be resolubilized by 0.5 M KC1 extraction after ethanol precipitation will be referred to as 'ethanol-sensitive components'. SDS gel electrophoresis. Gel electrophoresis in dissociating media was carried o u t essentially by the procedure of Weber and Osborn [21]. Proteins were prepared for gel electrophoresis by adding aliquots to an equal volume of solution containing 2% fi-mercaptoethanol, 2% sodium lauryl sulphate in 0.2 M phosphate buffer, pH 7.3, on a boiling water bath. After 2 min the samples were placed in dialysis bags and dialyzed vs. 0.1% ~-mercaptoethanol/0.1% sodium lauryl sulphate/0.01 M phosphate, pH 7.3, overnight. Sucrose was added to the samples to a final concentration of 10--20% (w/w) prior to their application to 5 or 8% polyacrylamide gels. Following electrophoresis (8 mA/ gel) gels were stained with Coomassie Brilliant Blue and destained by diffusion with a solution of methanol/glacial acetic acid/water (3 : 1 : 6, v/v). For quantitative measurements gels were scanned on a Gilford spectrophotometer and peak areas were measured by planimetry. Molecular weights were determined by using as standards: myosin, J3-galactosidiase, phosphorylase a, bovine serum albumin, actin, tropomyosin and hemoglobin. The mobility of long rods (from rabbit, lobster, and chicken myosins) was consistently less than expected on the basis of previous reports [5,6,12--14] and ultracentrifuge measurements of
145
their molecular weights. Experiments with different gel concentrations and buffers showed that although increasing the gel concentration did have some effect on the relative mobility of the long rods it could not explain the apparent high molecular weights observed. To rule out any anomalies that might arise from using globular protein standards with rod-like unknowns [25], paramyosin was also run as a standard and found to have the mobility expected on the basis of its known molecular weight [16,27]. (The author is indebted to Professors W.H. Johnson and R.W. Cowgill for generously providing specimens of paramyosin for use as gel electrophoresis standards). The mobility of myosin long rods was invariably significantly less than that for paramyosin run concurrently. Thus, the chain molecular weights observed here for rabbit and lobster myosin long rods, approx. 125 000--130 000, although about 10% greater than expected, represent the lower range of observed values. Reasonable agreement between results found here and literature values available for other proteolytic fragments of myosin suggest that no anomalies exist for molecular weights about 90 000 or below. Furthermore, since in most instances direct comparisons have been made between digestion products from rabbit and lobster myosin the conclusions drawn are generally independent of absolute molecular weight values. Ultracen trifugation. Molecular weights of some of the myosin fragments were determined by high speed equilibrium centrifugation [28]. The procedures used were as reported previously [17]. A partial specific volume of 0.720 ml/g was assumed for the various fragments. Results
Digestion with a-chymotrypsin and soluble papain Low ionic strength soluble components. Comparison by SDS gel electrophoresis of a-chymotryptic and papain produced subfragment-1 from rabbit and lobster myosin is shown in Fig. 1. The rabbit a-chymotrypsin subfragment-1 has a major component with molecular weight 94 000, light chains at 26 000 and 15 000 and a minor constituent at 62 000. A component with molecular weight approx. 70 000 reported by Weeds and Pope [29] was not generally observed. Rabbit papain subfragment-1 has major components at 94 000 and 74 000, a trace constituent at 62 000 and three light chains at 26 000, 19 000 and 15 000 mol. wt., essentially in agreement with the results of others [30,31]. The subfragment-1 produced by either a-chymotryptic (Fig. le) or papain (Fig. lf) digestion of lobster myosin appears to contain just one major heavy chain component with molecular weight of 95 000. Although the a-chymotrypsin subfragment-1 from lobster myosin retains both light chains, the presence of low molecular weight digestion products obscures identification of light chain two in papain subfragment-1 from lobster myosin. The two minor components (mol. wt. 100 000 and 105 000) apparent in the lobster papain subfragment were consistently observed. Gel filtration (Sephadex G-200) of the a-chymotrypsin digest supernatants readily separates the low molecular weight digestion products (as well as light chain two) from the heavy chain component. Gel filtration of the supernatant from papain digests was
146
Fig. 1. S D S gel e l e c t r o p h o r e s i s o f t h e l o w i o n i c s t r e n g t h s o l u b l e c o m p o n e n t s f r o m c ~ - c h y m o t r y p t i c a n d p a p a i n d i g e s t i o n o f r a b b i t a n d l o b s t e r m y o s i n , a, r a b b i t m y o s i n : b , l o b s t e r m y o s i n : c, r a b b i t m y o s i n : ~ - c h y m t r y p t i c s u f r a g m e n t - 1 ; d , r a b b i t m y o s i n : p a p a i n s u b f r a g m e n t - 1 ; e, l o b s t e r m y o s i n : ~ - c h y m o t r y p t i c s u b f r a g m e n t - 1 ; f, l o b s t e r m y o s i n : p a p a i n s u b f r a g m e n t - 1 .
never successful since the sample seemed to be degraded during dialysis into the 0.5 M, buffered KC1 solution. Low ionic strength insoluble components. The low ionic strength insoluble fraction from papain or a-chyotryptic digestion of rabbit myosin contains one major homogeneous component of molecular weight 127 000 (Fig. 2, gels a and b). In contrast, the long rods produced by digestion of lobster myosin are polydisperse: papain long rods have two major components and two minor
Fig. 2. S D S gel e l e c t r o p h o r e s i s o f t h e l o w i o n i c s t r e n g t h insoluble c o m p o n e n t s f r o m c ~ - c h y m o t r y p t i c a n d p a p a i n d i g e s t i o n o f r a b b i t a n d l o b s t e r m y o s i n , a, r a b b i t m y o s i n : ~ - c b y m o t r y p t i c l o n g r o d s ; b , r a b b i t m y o s i n : p a p a i n l o n g r o d s : c, l o b s t e r m y o s i n : a - c b y m o t r y p t i c l o n g r o d s : d , l o b s t e r m y o s i n : ~ - c h y m o t r y p t i c l o n g r o d s ; e, l o b s t e r m y o s i n : p a p a i n l o n g r o d s .
147 components as shown in Fig. 2e, while a-chymotrypsin long rods usually have three components as shown in Fig. 2d but occassionally four as seen in Fig. 2c. The molecular weights of the four components are about the same for the two types of long rods (127 000, 113 000, 102 000, and 95 000) however, their relative amounts vary. The three principal components in a-chymotrypsin long rods are usually present in equal amounts although occassionally the intermediate one (113 000) appears as a noticeably less intense band on the gels (compare Figs. 2c and 2d). For the papain long rods the two major components (113 000 and 102 000 mol. wt.) constitute 80% of the sample (as determined by planimetry of gel scans), while the 95 000 mo. wt. component is about 13% of the sample. Assuming that the contribution of the various components observed on the gels to the molecular weight of the parent long rods is porportional to their fractional appearance on the gels, it is estimated that the molecular weight of papain long rods is 216 000, which may be compared with a value of 197 000 from high speed equilibrium centrifugation. To estimate the molecular weight of a-chymotrypsin long rods from the gel data it was assumed that the sample contained equal quantities of the four and three component species (Fig. 2, gels c and d) with all components contributing equally. Thus, the molecular weight estimated for the a-chymotryptic long rods from gel data would be 223 000 as compared with a value of 236 000 from ultracentrifugation.
Digestion with trypsin Low ionic strength soluble components. Heavy meromyosin produced by tryptic digestion of soluble rabbit skeletal muscle myosin has three principal components on SDS gels: 76 000, 65 000 (frequently resolved into two bands at 66 000 and 61 000) and 52 000 (Fig. 3a), essentially in agreement with Balint et al. [32]. Additional minor components at 147 000, 136 000, 128 000 and 84 000 mol. wt. are frequently seen as well. Lower molecular weight species occur at 26 000 and 16 000. Equivalent tryptic digestion of lobster myosin (10 min, 300/1) followed by dialysis of the digest against 0.1 M KCI, pH 6 and centrifugation yields a supernatant fraction that gives rise to the gel pattern shown in Fig. 3b. Three principal components are apparent in this fraction also: 83 000, 71 000 and 55 000. If digestion time is decreased, a band (faintly appearing in Fig. 3b) corresponding to 94 000 mol. wt. has increased in intensity. Longer digestion periods are accompanied by decreasing intensity of the 83 000 band and increasing intensity of bands corresponding to molecular weights of 55 000, 50 000 and 40 000. Low molecular weight species are apparent at 26 000 and 20 000. If this lobster heavy meromyosin is treated with ethanol and the precipitate extracted with 0.5 M KC1, it is found that the bands corresponding to molecular weights of 83 000 and 55 000 are due to ethanol-resistant components (Fig. 3c). Furthermore, if this ethanol-resistant fraction is dialyzed against 0.1 M KC1, pH 4.5, and the precipitate separated by centrifugation, these two components appear in the pellet fraction (Fig. 3d). Alternatively these two components can be separated directly from the tryptic digest by dialysis against pH 4.5 buffered 0.1 M KC1 and subsequent treatment of the precipitate with ethanol. Further discussion of these two components is presented in the following section.
148 A
76-65-52~
--69 "~55
26--~ 16-~ a MxlO. 3 ~ Fig. 3. SDS gel e l e c t r o p h o r e s i s of f r a g m e n t s o b t a i n e d f r o m t r y p t i c d i g e s t i o n o f r a b b i t a n d l o b s t e r m y o s i n ( d i g e s t i o n c o n d i t i o n s p H 7, 2 3 ° C , 0.5 M KCI, 30011 s u b s t r a t e p e r e n z y m e ) , a, r a b b i t h e a v y m e r o m y o s i n : soluble in 0 . 0 5 M p h o s p h a t e , p H 6, b - - e , l o b s t e r m y o s i n : l o w ionic s t r e n g t h soluble f r a c t i o n f r o m t r y p t i c digests; b , t r y p t i c digest f r a c t i o n soluble at p H 6 in 0.1 M KCl. c, c o m p o n e n t s o b t a i n e d w h e n t h e f r a c t i o n s h o w n in (b) ( a b o v e ) w a s t r e a t e d w i t h e t h a n o l a n d t h e p r e c i p i t a t e e x t r a c t e d w i t h 0.5 M KCl ( p r i m a r i l y s h o w i n g t h a t the 83 0 0 0 a n d 55 0 0 0 tool. w t . c o m p o n e n t s are e t h a n o l - r e s i s t a n t ) ; d, c o m p o n e n t s precipit a t e d w h e n t h e f r a c t i o n s h o w n in (b) ( a b o v e ) w a s d i a l y z e d vs. 0.1 M KC1, pH 4.5 ( p r i m a r i l y s h o w i n g t h a t t h e 83 0 0 0 a n d 55 0 0 0 tool. w t , c o m p o n e n t s are insoluble a t p H 4.5 in 0.1 M KCl)-" e, t r y p t i c digest fract i o n soluble at p H 4.5 in 0.1 M KC1 ( c o m p a r i s o n w i t h b a n d c s h o w s t h a t t h e 71 0 0 0 m o l . w t . c o m p o n e n t is e t h a n o l - s e n s i t i v e ) ; f, r a b b i t light m e r o m y o s i n p r e p a r e d b y t r y p t i c d i g e s t i o n ; g - - h , l o b s t e r m y o s i n t r y p t i c digest f r a c t i o n s insoluble in 0.1 M KC1 a n d r e s i s t a n t to e t h a n o l ; g, f r a c t i o n p r e c i p i t a t e d at p H 6 ( n o t e t h e h e a v y b a n d c o r r e s p o n d i n g to m o l . wt. 83 0 0 0 ) . h, f r a c t i o n p r e c i p i t a t e d at p H 4.5 ( n o t e t h a t the 83 0 0 0 m o l . w t . b a n d is r e l a t i v e l y less i n t e n s e t h a n in gel 3 g a n d t h a t an a d d i t i o n b a n d a p p e a r s at 55 0 0 0 mol. wt.).
Of the three principal heavy molecular weight components in lobster heavy meromyosin (83 000, 71 000, and 55 000, Fig. 3, gel b) only the 71 000 mol. wt. constituent is sensitive to ethanol and soluble at pH 4.5 in 0.1 M KC1 (Fig. 3, gel e). In rabbit heavy meromyosin, of the three heavy molecular weight components (76 000. 65 000, 52 000, Fig. 3a) all three appear to be ethanol sensitive although with further digestion the 65 000 mol. wt. component breaks down to 37 000 and 21 000 fragments that are ethanol resistant [32]. Since the 64 000 c o m p o n e n t observed by Balint et al. [32] was derived from an 81 000 mol. wt. constituent, the latter might be somewhat analogous to the ethanol-resistant 83 000 c o m p o n e n t observed here in lobster myosin. Gel filtration removed some low molecular weight constituents from the heavy meromyosin fraction but caused no significant change in the gel electrophoretic pattern. Low ionic strength insoluble components. SDS gel electrophoresis of the insoluble fraction obtained by dialysis of a 10 min tryptic digest of lobster myosin against 0.1 M KC1, pH 6, solution followed by ethanol treatment of the precipitate is shown in Fig. 3g. Components are present with bands at 96 000, 83 000 and 69 000. The band at 83 000 is noticeably less intense than those at 96 000 and 69 000. Occassionally traces of a 55 000 band are seen as well. This is in marked contrast to the gel pattern for rabbit myosin light meromyosin fraction i (10 min digest) which has just one major c o m p o n e n t with a chain weight of 75 000 (Fig. 3f), although traces of three other bands are frequently discernable at 68 000, 65 000, and 60 000. If, on the other hand, the lobster myosin digest is dialyzed vs. 0.1 M KC1, pH 4.5, instead of pH 6, the insoluble
149 fraction contains, in addition to the bands at 96 000, 83 000, and 69 000, a strong component at 55 000. Furthermore, the relative intensity of the band corresponding to a polypeptide chain weight of 83 000 is very noticeably increased (Fig. 3h). Thus, it is clear that the 55 000 mol. wt. species is soluble in 0.1 M KC1, pH 6, but not at pH 4.5; hence when the digest is dialyzed at pH 6 this component appears in the soluble fraction (i.e. note the absence of this band in Fig. 3g). But if the digest is dialyzed at pH 4.5 this component appears in the insoluble fraction {Fig. 3h). The 83 000 mol. wt. constituent is partially soluble at pH 6; hence it appears in both the soluble phase (Fig. 3b) and insoluble phase (Fig. 3g) when the digestion products are dialyzed at this pH but only the insoluble phase (Fig. 3h) when the digest is dialyzed at pH 4.5. Both component~ are ethanol resistant and therefore are presumed to have a rod-like structure. More limited digestion gives rise to an additional ethanolresistant component with mass 111 000 and the band corresponding to the component with chain weight of 96 000 is relatively more intense in the gel patterns. These results indicate that a simple mono-disperse light meromyosinlike species is not produced by tryptic digestion of lobster myosin and that the molecule has more sites susceptible to tryptic proteolysis than does rabbit myosin.
Tryptic digestion of papain long rods from lobster and rabbit myosin In view of the complexity of the lobster heavy meromyosin and light meromyosin-like fractions, and the presence in the soluble fraction of the myosin digests, of fragments from the globular part of the molecule, efforts to analyze further the proteolysis of the rod portion of the lobster myosin molecule were directed to tryptic digestion of long rods produced by papain digestion. The results of tryptic digestion of lobster myosin papain long rods for intervals ranging from 0 to 120 min are shown in Fig. 4a. Nine bands corresponding to fragments with chain molecular weights of 127 000, 113 000, 102 000, 95 000, 83 000, 68 000, 55 000, 45 000, and 38 000 are apparent, along with additional weak, diffuse bands at 29 000. Bands at 21 000 (due to trypsin inhibitor) and 15 000--17 000 mol. wt. (which appears after about 15 min digestion and is attributed to smaller, heterogeneous degradation products) are also apparent. The band pattern illustrated was reproduced with several different papain long rod samples. The order of appearance of the rod fragments was approximately as expected for sequential degradation of the rod except that the 38 000 fragment appeared before the 45 000 fragment. Except for the 55 000 mol. wt. component (which accounted for approx. 38% of the total sample in digests from 45 to 90 min) none of the various fragments seemed to accumulate preferentially during the course of digestion. Simultaneous gel electrophoresis of the pH 4.5, low ionic strength insoluble fraction from tryptic digestion of lobster myosin (Fig. 3h) with the papain long rod digests showed that the bands in the light meromyosin-like fraction corresponded to the long rod fragments with molecular weights of 95 000, 83 000, 68 000, and 55 000. Concurrent tryptic digestion and gel electrophoresis of both lobster and rabbit papain long rods revealed differences both in the number and molecular weights of the components produced from the two long rod samples (compare Figs. 4a and 4b). Six principal bands (corresponding to chain weights of
150
!
8o
Fig. 4. S D S gel e l e c t r o p h o r e s i s o f t r y p t i c d i g e s t s o f papain long r o d s f r o m lobster and r a b b i t m y o s i n . D i g e s t i o n c o n d i t i o n s as d e s c r i b e d in p r o c e d u r e s , t i m e s as s h o w n o n gels (in r a i n ) , a, t r y p t i c d i g e s t i o n o f l o b s t e r m y o s i n p a p a i n l o n g r o d ; b , tryptic digestion o f r a b b i t m y o s i n p a p a i n l o n g r o d .
127 000, 76 000, 62 000, 56 000, 45 000, and 40 000) are apparent in the gel electrophoresis pattern of extended digests of rabbit papain long rods. In addition a trace band is apparent at 29 000, as well as trypsin inhibitor at 21 000. Digestion of rabbit papain long rods did not proceed quite as had been anticipated on the basis of previous work by others: apart from the minor contamination of the initial papain long rod sample with light meromyosin it is apparent that thte initial stages of digestion were accompanied by the appearance of a component with chain weight of 59 000 rather than 30 000--40 000 as anticipated on the basis of the rods being cleaved directly to light meromyosin and subfragment-2. As digestion proceeded this component gradually disappeared with the concurrent appearance of bands at 62 000, 56 000, 45 000, and 40 000 mol. wt. In contrast to digestion of lobster papain long rods, none of the components in the rabbit papain long rod digest seemed to accumulate preferentially. Tentative identification of the various components in the gel patterns of the rabbit long rod digests was facilitated by comparison
151 with rod fragments prepared from papain digestion of rabbit tryptic heavy meromyosin and tryptic digestion of rabbit tryptic light meromyosin. Brief digestion of rabbit tryptic heavy meromyosin (6 min, 100/1 substate per enzyme, pH 7.8, 0.5 M KC1) followed by passage of the digest mixture through gel filtration columns yielded two fractions. The faster moving fraction was ethanol resistant, appeared as either two bands when electrophoresed on nondissociating gels, or two doublet bands when run on SDS gels (mol. wt. 59 000 and 44 000, Fig. 5a) and ran as a single component in the centrifuge with a sedimentation coefficient of 2.64 S. High speed equilibrium centrifugation showed this fraction to be heterogeneous with a cell average molecular weight of 107 000. More extended papain digestion of rabbit tryptic heavy meromyosin (15 min, 15/1 substrate per enzyme) followed by the same procedure yielded subfragment-2 (ethanol resistant, high speed equilibrium mol. wt = 76 000, SDS chain weight 38 000, Fig. 5b). Subfragment-1 was also produced in this digestion but was not of interest here. Comparison of these results with the tryptic proteolysis of rabbit papain long rods suggests that the initial products of digestion were light meromyosin (76 000) and a rod-like precursor of subfragment-2 with chain weight 59 000. Upon further digestion, this component was degraded to subfragment-2 which appears as a band at 40 000 in the extended digest (Fig. 4b, 120 min digest gel). Controlled tryptic digestion of light meromyosin fraction 1 followed by gel filtration (Sephadex G-200) was used to produce the LF-1, LF-2 and LF-3 fragments with molecular weights of 112 000, 86 000, and 57 000 determined from high speed equilibrium ultracentrifugation and chain weights of 59 000, 44 000, and 30 000 from SDS gels (Figs. 5c--5e). Thus bands in the extended tryptic digestion of rabbit papain longs rods (Fig. 4b, 120 min digest gel) corre-
59~
i
44~
• ~
Hi
--59
~"~ ~IHDP --
a MxlO. 3 ~
~.b ~L..__~-J~
d
--44
-111' - 3 0
e
m.~.,..~.-~-:=,,~
Fig. 5. SDS gel e l e c t r o p h o r e s i s of f r a g m e n t s isolated f r o m p a p a i n d i g e s t i o n o f r a b b i t tryptic heavy m e r o m y o s i n (a a n d b) a n d txyptic digestion o f r a b b i t t r y p t i c light m e r o m y o s i n (c--e). a, subfragment-2 precursor (59 0 0 0 a n d 4 4 0 0 0 ) ; b , s u b f r a g m e n t - 2 (38 0 0 0 ) ; c, LF-1 (59 0 0 0 ) ; d, LF-2 (44 0 0 0 ) ; e, L F - 3 (30 000).
152
sponding to molecular weights of 56 000, 45 000, and 29 000 were identified as LF-1, LF-2, and LF-3, respectively. The remaining c o m p o n e n t at 62 000 has not been specifically identified but is assumed to be partially degraded light meromyosin. Discussion The polydispersity evident in the long rod preparations made by a-chymotryptic or papain proteolysis of lobster myosin suggests that there are several sites susceptible to enzymic cleavage in the region of the molecule where the globular head units join the rod. The molecular weight differences between the largest and smallest components in these fractions indicate that the cleavage sites could be as much as 4 0 0 / ~ apart along the myosin rod (if the relative proportions of the components are taken into consideration it appears that the principal cleavage sites occur with a maximum spread of about 300 A). In contrast, rabbit myosin long rod preparations are monodisperse with extended digestion yielding light meromyosin in addition to the long rods. These results provide additional support for the conclusion drawn from previous kinetic studies on tryptic hydrolysis of lobster myosin [17]; namely, that the lobster myosin molecule is much more susceptible to enzymic cleavage in the subfragment-2 region than is rabbit myosin. Although the long rods produced from lobster myosin are polydisperse, the corresponding subfragment-1 preparations have only one principal heavy chain c o m p o n e n t in contrast to the two or three major heavy chain components observed in SDS gel electrophoresis of rabbit myosin subfragment-1. The enzymatic and molecular weight (ultracentrifuge) characteristics of the low ionic strength soluble fragments will be discussed elsewhere. Although the low ionic strength soluble and insoluble fractions obtained after dialysis of tryptic digests of lobster myosin may have one or more c o m m o n components (i.e. the 83 000 and 55 000 mol. wt. components) and thus not be completely analogous to rabbit meromyosins, it is nevertheless instructive to compare the fractions obtained from lobster myosin with the rabbit meromyosins as well as the fragments produced by tryptic digestion of long rods from the two species of myosin. The major ethanol-sensitive component (presumably derived from subfragment-1) of lobster heavy meromyosin has a molecular weight of 71 000 as compared with the 76 000 component in rabbit heavy meromyosin. Comparison of the results obtained here with those of Balint et al. [32] suggests that the ethanol-resistant components of 83 000 and 55 000 mol. wt. might be related to the 84 000 c o m p o n e n t in rabbit heavy meromyosin which on further proteolysis yields a 64 000 fragment that in turn is split to produce ethanol-resistant fragments of 37 000 and 21 00. If the 83 000 mol. wt. c o m p o n e n t is essentially a-helical as implied by its resistance to ethanol, it would certainly be the maximum size rod fragment that could be accomodated in heavy meromyosin. Weeds and Pope [29] have recently suggested that subfragment-2 may be as large as 73 000 when produced from rabbit myosin by limited a-chymotryptic digestion and observed on dissociating gels. Alternatively, the limited low ionic strength solubility of the 83 000 c o m p o n e n t suggests that it may relate more directly to
153 light meromyosin. That this component does come from the myosin rod and does not include part of the globular head is indicated by its appearance in tryptic digests of lobster papain long rods. The low ionic strength precipitable components {ethanol resistant) from tryptic digests of lobster myosin are rod fragments that can be compared directly with those observed by SDS gel electrophoresis of tryptic digests of the papain long rods. The lobster light meromyosin fraction always contains several major components in contrast to rabbit light meromyosin which has just one principal constituent (76 000 chain weight) although trace bands for minor components are frequently observed also. From direct comparison of SDS gel electrophoretic patterns of tryptic digests of lobster and rabbit papain long rods, it is evident that under comparable digestion conditions lobster long rods yield more fragments than do the rabbit long rods. The large component chain weights {>70 000) and low ionic strength insolubility near neutral pH of these fragments, indicates that five cleavage sites occur in the subfragment-2 region of the lobster myosin rod; whereas, for rabbit myosin there is only one cleavage locus (producing long rods) in the corresponding region. Weeds and Pope [29] have suggested that light meromyosin and subfragment-2 produced by a-chymotryptic digestion of rabbit myosin are multicomponent species having fragments with chain weights of 77 000, 69 000, 66 000, and 61 000 for light meromyosin and 73 000, 59 000, and 46 000 for subfragment-2. Rabbit light meromyosin produced here by tryptic digestion had one major component (76 000) with trace bands at 68 000, 65 000, and 60 000, while the subfragment produced by limited papain digestion of rabbit heavy meromyosin had components of 59 000 and 44 000 similar to the subfragment-2 of Weeds and Pope. A 73 000 mol. wt. component for subfragment-2 was not detected in these preparations. The molecular weight of the subfragment-2 (76 000 from equilibrium centrifugation and SDS gels) isolated after more extended papain digestion of heavy meromyosin is in good agreement with the results of Biro et al. [4] but somewhat larger than the value of 60 000 frequently reported [5,6]. The molecular weights of the three rabbit light meromyosin fragments isolated here are in good agreement with those reported by Biro et al. [4] and correspond well with the bands observed in SDS gel electrophoresis of tryptic digests of the rabbit myosin long rods {Fig. 4b). In tryptic digests of lobster long rods two bands were clearly observed (45 000 and 38 000 mol. wt.} that would indicate cleavage of the light meromyosin portion of the molecule. A third band {29 000) was faintly observed and increased in intensity somewhat with further digestion. Thus, both rabbit and lobster myosin have three principal tryptic sensitive sites in the light meromyosin region but whereas rabbit myosin has only one sensitive locus in the subfragment-2 region lobster myosin has five. Although the chain weights of two of the three light meromyosin-like lobster subfragments (45 000, 38 000, and 29 000) were the same as those for light fragments from rabbit light meromyosin proteolysis (56 000, 45 000, and 29 000), further information concerning their composition and position in the myosin rod will be required to ascertain if they do indeed correspond to each other in the rod structure. If susceptibility to proteolysis is interpreted as a measure of molecular
154
stability [5], these results suggest that the subfragment-2 region of lobster myosin is less stable than the corresponding region in rabbit myosin. Similarly, the results suggest significant differences in residue composition for the heavy chains of the two myosins in these regions. Amino acid composition data for rabbit myosin show that the number of lysine and arginine residues (per l 0 s g protein, [6] ) is approximately the same in the light meromyosin and subfragment-2 regions of the molecule. However, preliminary amino acid analysis results for lobster myosin indicate an excess of lysine residues i n t h e subfragment-2 region of the molecule relative to the light meromyosin region. These data are consistent with the relative susceptibilities of the molecular rods to tryptic proteolysis but leave open the question of their sensitivity to papain or chymotryptic cleavage. Isolation and analysis of the various fragments produced in the digestion process can be expected to yield further insight into the relative substructure of both the various regions of the lobster myosin molecule and differences between lobster and rabbit myosin that have a bearing on their assembly into thick filaments and functional role in muscle contraction. Acknowledgements The author is indebted to C. Brexel and E. Edwards for technical assistance during part of this work and to H. Manuel for the ultracentrifuge molecular weight determinations for the ~-chymotrypsin and papain lobster long rods. Extensive helpful discussions with R. Siemankowski concerning various aspects of this work are also gratefully acknowledged. References 1 2 3 4 5 6 7 8 9 10 11 12 3 14 15 16 17 18 19 20 21 22 23 24
Szent-GyorgyL A.G. (1953) A r c h . B i o c h e m . B i o p h y s . 42. 305--320 Mihalyi, E. and Harrington, W.F. (1959) Biochirn. B i o p h y s . A c t a 36, 447--466 Young, D.M., Himmelfarb. S. and HHarrington, W.F. (1965) J. Biol. C h e m . 240, 2428--2436 Bixo, N.A.0 Szflagyi, L. and Ballnt, M. (1972) Cold Spring I-Iarb. Syrup. Quant. Biol. 37, 55--63 Burke, M., Himmelfarb. S. and Harrington, W.F. (1973) Biochemistry 12, 701--710 Lowey , S. (1971) in Subunits in Biological S y s t e m s ( T i m a s h e f f , S . N . and F a s m a n . C.D., eds.), pp. 201--259, Marcel Dekker, New Y o r k Pepe. F.A. (1971) in Progress in B i o p h y s i c s and Molecular B i o l o g y (Butler, J . A . V . and N o b l e , D., eds.), pp. 75--96, P e r g a m o n Press, N e w York Harrington, W.F. (1971) Proc. Natl. A c a d . Sci. U.S. 68, 685--689 Huxley, H.E. (1969) Science 164, 1356--1366 H u x l e y , H.E. ( 1 9 6 3 ) J. Mol. Biol. 7 , 2 8 1 - - 3 0 8 Kaminer. B. and Bell, A.L. (1966) J. Mol. Biol. 20, 391--401 Harrison, R.G., Lowey, S. and C o h e n , C. (1971) J. Mol. Biol. 59, 531--535 Kaminer, B. (1969) J. Mol. Biol. 39, 257--264 Nakamura, A., Sreter0 F. and Gergely, J. (1971) J. Cell Biol. 49. 883--898 Hayes, D., Huang, M. and Zobel, C.R. (1971) J. Ultrastruct. Res. 3 7 . 1 7 - - 3 0 Siemankowski. R.F. and Zobel, C.R. (1976) B i o c h i m . B i o p h y s . A c t a 420, 406--416 Siemankowski. R.F. and Zobel, C.R. (1976) J. M e c h a n o c h e m . Cell Motility 3 , 1 7 1 - - 1 8 4 Szent-Gyorgyi, A. (1951) Chemistry of Muscular C o n t r a c t i o n . 2nd e d n . , A c a d e m i c Press, N e w Y ork Weeds. A.G. and Taylor, R.S. (1975) Nature 257, 54--56 Szent-Gyorgyi, A.G., Cohen, C. a n d Philpott, D.E. (1960) J. Mol. Biol. 2, 133--142 Weber, K. and Osborn, M. (1969) J. Biol. C h e m . 244, 44 06--4412 Gazith, J., Himmelfarh, S. and Harrington, W.F. (1970) J. Biol. C h e m . 245, 15--22 L o w e y , S., Slayter, H.S., Weeds, A . G . and Baker, H. (1969) J. Mol. Biol. 42, 1--29 Szuchet. S. and Zohel. C.R. (1974) Biochemistry 13, 1482--1491
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25 26 27 28 29 30 31 32
Mattice, W.L., Riser, J.M. and Clark, D.S. (1976) Biochemistry 15. 4 2 6 4 - - 4 2 7 2 Halsey, J.F. and Harrington, W.F. (1973) Biochemistry 12, 693--701 Cowgill, R.W. (1975) Biochemistry 14, 503--509 Yphantis, D.A. (1964) Biochemistry 3. 297--317 Weeds, A.G. and Pope, B. (1977) J. Mol. Biol. 1 1 1 , 1 2 9 - - 1 5 7 Balint, M., Sreter, F.A. and Gergely, J. (1975) Arch. Biochem. Biophys. 1 6 8 , 5 5 7 - - 5 6 6 Margossian, S.S. and Lowey, S. (1973) J. Mol. Biol. 74. 301--311 Balint, M., Sreter, F.A.0 Wolf, I., Nagy, B. and Gergely, J. (1975) J. Biol. Chem. 250, 6168--6177
Biochimica et Biophysica Acta, 536 (1978) 142--155 © Elsevier/North-HollandBiomedicalPress
BBA 37981 PROTEOLYTIC FRAGMENTS FROM THE LOBSTER MYOSIN MOLECULE
C. RICHARD ZOBEL Department of Biophysical Sciences, State University of New York, Amherst, N.Y. 14226 (U.S.A.)
(Received January 12th, 1978) Summary The fragments produced by proteolysis of lobster abdominal muscle myosin with trypsin, a-chymotrypsin and papain have been investigated by sodium dodecyl sulfate (SDS) gel electrophoresis. Essentially monodisperse populations of long rods are produced by a-chymotryptic and papain digestion of rabbit myosin but corresponding digestion of lobster myosin yields multicomponent species. Similarly the low ionic strength insoluble fraction from tryptic digestion of lobster myosin is polydisperse in contrast to essentially monodisperse light meromyosin from rabbit myosin. Comparative tryptic digestion of rabbit and lobster myosin papain long rods shows that the latter have five susceptible cleavage sites in the subfragment-2 region while rabbit long rods have only one: both long rods appear to have three cleavage sites in the light meromyosin region. The fragments produced by tryptic digestion of rabbit myosin papain long rods have been tentatively identified by comparison with fragments isolated from papain digests of rabbit heavy meromyosin and tryptic digests of rabbit light meromyosin. The results suggest differences in sensitivity to enzymic proteolysis between the subfragment-2 regions in rabbit and lobster myosin as well as relative differences in proteolytic sensitivity between the subfragment-2 and light meromyosin region within the individual molecules. Partial explanation of the observation is proposed on the basis of differences in heavy chain compositions.
Introduction
Brief tryptic digestion of rabbit skeletal muscle myosin results in cleavage of the molecule into two fragments [1,2]; heavy meromyosin and light meromyosin. Further 'tryptic .digestion produces subfragment-1 from heavy meromyosin [3] and a number of rod-like fragments from light meromyosin [4]. In a number of studies Harrington and coworkers [2] have analyzed the digestion process and concluded that it could be described in terms of a fast and slow
143 reaction. The two different reaction rates reflecting the existence of two different classes of bonds in the molecular structure of myosin. Bonds readily susceptible to cleavage by trypsin are attributed to less stable regions of the molecule while those less susceptible to cleavage are presumably in more stable regions of the molecule [5]. From studies of this type there has emerged a model for the myosin molecule in which two apparently rigid a-helical regions (light meromyosin and subfragment-2) exist separated by a more flexible region, the hinge region. Similarly the globular headpieces are believed to be joined to the rod portion of the molecule through a flexible section [6]. These physical properties of the myosin molecule are presumed to have physiological importance. Thus, in several models for muscle contraction the flexible regions within the myosin rod play prominent roles in mediating the structural interaction between thick and thin filaments [7--9]. The light meromyosin terminus of the rod controls the assembly of myosin into filaments and the long rod into ordered arrays, [10--12] the particular molecular arrangements within these ordered structures depending upon the histological source of the parent myosin [ 13,14]. In lobster fast flexor abdominal and crusher claw muscles the thick myosin filaments are considerably larger than those in vertebrate striated muscle [15]. To determine if these apparent structural differences reflect differences in the intrinsic nature of the constituent myosin molecules we have initiated an investigation of the aggregative properties of lobster myosins (and their fragments) as well as the nature of the molecule, per se, as reflected in its susceptibility to enzymic proteolysis. Our results have show that in contrast to the long (approx. 2 pm) spindle shaped aggregates formed by vertebrate muscle myosin at low ionic strength, lobster abdominal muscle myosin only formed short (approx. 0.3 pm) aggegates [16] and that in tryptic hydrolysis of lobster abdominal myosin, there were 280 bonds in the rapidly cleaved class in contrast to 73 fast bonds in rabbit skeletal myosin [17]. On the basis of this latter observation we proposed that lobster myosin was much more susceptible to proteolysis in the subfragment-2 region than was skeletal muscle myosin. Investigation of the susceptibility of lobster myosin to proteolysis with papain and a-chymotrypsin has provided further support for this hypothesis. Furthermore, it has been found that tryptic digestion of papain long rods from both rabbit and lobster myosin results in somewhat different cleavage products as ascertained by sodium dodecyl sulfate (SDS) gel electrophoresis. The products of digestion of rabbit myosin have been identified by direct comparison with fragments isolated from digests of heavy meromyosin and light meromyosin. To the extent that susceptibility of the myosin molecule to enzymic attack reflects its underlying substructure, these results suggest local compositional differences between the heavy chain in rabbit and lobster myosin. Such local compositional differences may well control the aggregative and structural characteristics of the myosin molecule and result in different molecular packing in the thick filaments in the two different types of muscle. Experimental procedures Protein preparation. Lobster myosin was prepared from abdominal flexor muscle of lobster Homerus americanus as described previously [17]. Myosin
144
from the back and leg muscles of rabbits was prepared essentially by the SzentGyorgyi procedure [18] with slight modifications. General procedures for protein digestion. Myosin that had been dialyzed into 0.12 M phosphate/1 mM EDTA, pH 7, was digested with a-chymotrypsin: digestion was terminated by adding phenylmethylsulfonyl fluoride to a final concentration of 1 mM [19]. Papain digestion was carried out on myosin that had been dialyzed into 0.02 M KC1/0.02 M borate, pH 7, and was terminated with a 50-fold molar excess of iodoacetic acid in 10 mM cysteine/4 mM EDTA (excess iodoacetic acid was removed with a 2-fold weight excess of cysteine). For tryptic digestion myosin was dialyzed into 0.5 M KC1/0.05 M phosphate, pH 7. Digestion was termined with a 2-fold weight excess of soybean trypsin inhibitor. Myosin concentrations of 15--20 mg/ml were used with substrate/enzyme ratios of 300/1, for digestion times of 10 min at 23°C in all digestions. After termination of digestion, samples were titrated to pH 6 and centrifuged for 60--90 min at approx. 100 000 × g. Supernatants were then dialyzed overnight vs. 0.05 M phosphate, pH 6, for rabbit myosin and 0.05 M phosphate/0.1 M KC1, pH 6, for lobster myosin. After dialysis protein solutions were centrifuged as before. The resulting supernatants were then either used directly for gel electrophoresis or dialyzed vs. 0.5 M KC1/0.05 M phosphate, pH 7, for fractionation by gel filtration (Sephadex G-200). Pellets from centrifugation were resuspended in 0.5 M KC1/0.05 M phosphate, pH 7, and then denatured with a 10 volume excess of ethanol for 3 h at room temperature. Subsequent steps for the preparation of rod fragments were essentially as described by SzentGyorgyi et al. [20]. The protein, precipitated by ethanol was packed into a pellet by centrifugation and then dialysed against 0.5 M KC1/0.05 M phosphate. R o d fragments were extracted into the solvent. In the discussion that follows the term 'ethanol-resistant components' will refer to those fragments which were resolubilized by extraction of the ethanol-precipitated protein with buffere 0.5 M KC1. Those components originally soluble in 0.5 M KC1 but which could not be resolubilized by 0.5 M KC1 extraction after ethanol precipitation will be referred to as 'ethanol-sensitive components'. SDS gel electrophoresis. Gel electrophoresis in dissociating media was carried o u t essentially by the procedure of Weber and Osborn [21]. Proteins were prepared for gel electrophoresis by adding aliquots to an equal volume of solution containing 2% fi-mercaptoethanol, 2% sodium lauryl sulphate in 0.2 M phosphate buffer, pH 7.3, on a boiling water bath. After 2 min the samples were placed in dialysis bags and dialyzed vs. 0.1% ~-mercaptoethanol/0.1% sodium lauryl sulphate/0.01 M phosphate, pH 7.3, overnight. Sucrose was added to the samples to a final concentration of 10--20% (w/w) prior to their application to 5 or 8% polyacrylamide gels. Following electrophoresis (8 mA/ gel) gels were stained with Coomassie Brilliant Blue and destained by diffusion with a solution of methanol/glacial acetic acid/water (3 : 1 : 6, v/v). For quantitative measurements gels were scanned on a Gilford spectrophotometer and peak areas were measured by planimetry. Molecular weights were determined by using as standards: myosin, J3-galactosidiase, phosphorylase a, bovine serum albumin, actin, tropomyosin and hemoglobin. The mobility of long rods (from rabbit, lobster, and chicken myosins) was consistently less than expected on the basis of previous reports [5,6,12--14] and ultracentrifuge measurements of
145
their molecular weights. Experiments with different gel concentrations and buffers showed that although increasing the gel concentration did have some effect on the relative mobility of the long rods it could not explain the apparent high molecular weights observed. To rule out any anomalies that might arise from using globular protein standards with rod-like unknowns [25], paramyosin was also run as a standard and found to have the mobility expected on the basis of its known molecular weight [16,27]. (The author is indebted to Professors W.H. Johnson and R.W. Cowgill for generously providing specimens of paramyosin for use as gel electrophoresis standards). The mobility of myosin long rods was invariably significantly less than that for paramyosin run concurrently. Thus, the chain molecular weights observed here for rabbit and lobster myosin long rods, approx. 125 000--130 000, although about 10% greater than expected, represent the lower range of observed values. Reasonable agreement between results found here and literature values available for other proteolytic fragments of myosin suggest that no anomalies exist for molecular weights about 90 000 or below. Furthermore, since in most instances direct comparisons have been made between digestion products from rabbit and lobster myosin the conclusions drawn are generally independent of absolute molecular weight values. Ultracen trifugation. Molecular weights of some of the myosin fragments were determined by high speed equilibrium centrifugation [28]. The procedures used were as reported previously [17]. A partial specific volume of 0.720 ml/g was assumed for the various fragments. Results
Digestion with a-chymotrypsin and soluble papain Low ionic strength soluble components. Comparison by SDS gel electrophoresis of a-chymotryptic and papain produced subfragment-1 from rabbit and lobster myosin is shown in Fig. 1. The rabbit a-chymotrypsin subfragment-1 has a major component with molecular weight 94 000, light chains at 26 000 and 15 000 and a minor constituent at 62 000. A component with molecular weight approx. 70 000 reported by Weeds and Pope [29] was not generally observed. Rabbit papain subfragment-1 has major components at 94 000 and 74 000, a trace constituent at 62 000 and three light chains at 26 000, 19 000 and 15 000 mol. wt., essentially in agreement with the results of others [30,31]. The subfragment-1 produced by either a-chymotryptic (Fig. le) or papain (Fig. lf) digestion of lobster myosin appears to contain just one major heavy chain component with molecular weight of 95 000. Although the a-chymotrypsin subfragment-1 from lobster myosin retains both light chains, the presence of low molecular weight digestion products obscures identification of light chain two in papain subfragment-1 from lobster myosin. The two minor components (mol. wt. 100 000 and 105 000) apparent in the lobster papain subfragment were consistently observed. Gel filtration (Sephadex G-200) of the a-chymotrypsin digest supernatants readily separates the low molecular weight digestion products (as well as light chain two) from the heavy chain component. Gel filtration of the supernatant from papain digests was
146
Fig. 1. S D S gel e l e c t r o p h o r e s i s o f t h e l o w i o n i c s t r e n g t h s o l u b l e c o m p o n e n t s f r o m c ~ - c h y m o t r y p t i c a n d p a p a i n d i g e s t i o n o f r a b b i t a n d l o b s t e r m y o s i n , a, r a b b i t m y o s i n : b , l o b s t e r m y o s i n : c, r a b b i t m y o s i n : ~ - c h y m t r y p t i c s u f r a g m e n t - 1 ; d , r a b b i t m y o s i n : p a p a i n s u b f r a g m e n t - 1 ; e, l o b s t e r m y o s i n : ~ - c h y m o t r y p t i c s u b f r a g m e n t - 1 ; f, l o b s t e r m y o s i n : p a p a i n s u b f r a g m e n t - 1 .
never successful since the sample seemed to be degraded during dialysis into the 0.5 M, buffered KC1 solution. Low ionic strength insoluble components. The low ionic strength insoluble fraction from papain or a-chyotryptic digestion of rabbit myosin contains one major homogeneous component of molecular weight 127 000 (Fig. 2, gels a and b). In contrast, the long rods produced by digestion of lobster myosin are polydisperse: papain long rods have two major components and two minor
Fig. 2. S D S gel e l e c t r o p h o r e s i s o f t h e l o w i o n i c s t r e n g t h insoluble c o m p o n e n t s f r o m c ~ - c h y m o t r y p t i c a n d p a p a i n d i g e s t i o n o f r a b b i t a n d l o b s t e r m y o s i n , a, r a b b i t m y o s i n : ~ - c b y m o t r y p t i c l o n g r o d s ; b , r a b b i t m y o s i n : p a p a i n l o n g r o d s : c, l o b s t e r m y o s i n : a - c b y m o t r y p t i c l o n g r o d s : d , l o b s t e r m y o s i n : ~ - c h y m o t r y p t i c l o n g r o d s ; e, l o b s t e r m y o s i n : p a p a i n l o n g r o d s .
147 components as shown in Fig. 2e, while a-chymotrypsin long rods usually have three components as shown in Fig. 2d but occassionally four as seen in Fig. 2c. The molecular weights of the four components are about the same for the two types of long rods (127 000, 113 000, 102 000, and 95 000) however, their relative amounts vary. The three principal components in a-chymotrypsin long rods are usually present in equal amounts although occassionally the intermediate one (113 000) appears as a noticeably less intense band on the gels (compare Figs. 2c and 2d). For the papain long rods the two major components (113 000 and 102 000 mol. wt.) constitute 80% of the sample (as determined by planimetry of gel scans), while the 95 000 mo. wt. component is about 13% of the sample. Assuming that the contribution of the various components observed on the gels to the molecular weight of the parent long rods is porportional to their fractional appearance on the gels, it is estimated that the molecular weight of papain long rods is 216 000, which may be compared with a value of 197 000 from high speed equilibrium centrifugation. To estimate the molecular weight of a-chymotrypsin long rods from the gel data it was assumed that the sample contained equal quantities of the four and three component species (Fig. 2, gels c and d) with all components contributing equally. Thus, the molecular weight estimated for the a-chymotryptic long rods from gel data would be 223 000 as compared with a value of 236 000 from ultracentrifugation.
Digestion with trypsin Low ionic strength soluble components. Heavy meromyosin produced by tryptic digestion of soluble rabbit skeletal muscle myosin has three principal components on SDS gels: 76 000, 65 000 (frequently resolved into two bands at 66 000 and 61 000) and 52 000 (Fig. 3a), essentially in agreement with Balint et al. [32]. Additional minor components at 147 000, 136 000, 128 000 and 84 000 mol. wt. are frequently seen as well. Lower molecular weight species occur at 26 000 and 16 000. Equivalent tryptic digestion of lobster myosin (10 min, 300/1) followed by dialysis of the digest against 0.1 M KCI, pH 6 and centrifugation yields a supernatant fraction that gives rise to the gel pattern shown in Fig. 3b. Three principal components are apparent in this fraction also: 83 000, 71 000 and 55 000. If digestion time is decreased, a band (faintly appearing in Fig. 3b) corresponding to 94 000 mol. wt. has increased in intensity. Longer digestion periods are accompanied by decreasing intensity of the 83 000 band and increasing intensity of bands corresponding to molecular weights of 55 000, 50 000 and 40 000. Low molecular weight species are apparent at 26 000 and 20 000. If this lobster heavy meromyosin is treated with ethanol and the precipitate extracted with 0.5 M KC1, it is found that the bands corresponding to molecular weights of 83 000 and 55 000 are due to ethanol-resistant components (Fig. 3c). Furthermore, if this ethanol-resistant fraction is dialyzed against 0.1 M KC1, pH 4.5, and the precipitate separated by centrifugation, these two components appear in the pellet fraction (Fig. 3d). Alternatively these two components can be separated directly from the tryptic digest by dialysis against pH 4.5 buffered 0.1 M KC1 and subsequent treatment of the precipitate with ethanol. Further discussion of these two components is presented in the following section.
148 A
76-65-52~
--69 "~55
26--~ 16-~ a MxlO. 3 ~ Fig. 3. SDS gel e l e c t r o p h o r e s i s of f r a g m e n t s o b t a i n e d f r o m t r y p t i c d i g e s t i o n o f r a b b i t a n d l o b s t e r m y o s i n ( d i g e s t i o n c o n d i t i o n s p H 7, 2 3 ° C , 0.5 M KCI, 30011 s u b s t r a t e p e r e n z y m e ) , a, r a b b i t h e a v y m e r o m y o s i n : soluble in 0 . 0 5 M p h o s p h a t e , p H 6, b - - e , l o b s t e r m y o s i n : l o w ionic s t r e n g t h soluble f r a c t i o n f r o m t r y p t i c digests; b , t r y p t i c digest f r a c t i o n soluble at p H 6 in 0.1 M KCl. c, c o m p o n e n t s o b t a i n e d w h e n t h e f r a c t i o n s h o w n in (b) ( a b o v e ) w a s t r e a t e d w i t h e t h a n o l a n d t h e p r e c i p i t a t e e x t r a c t e d w i t h 0.5 M KCl ( p r i m a r i l y s h o w i n g t h a t the 83 0 0 0 a n d 55 0 0 0 tool. w t . c o m p o n e n t s are e t h a n o l - r e s i s t a n t ) ; d, c o m p o n e n t s precipit a t e d w h e n t h e f r a c t i o n s h o w n in (b) ( a b o v e ) w a s d i a l y z e d vs. 0.1 M KC1, pH 4.5 ( p r i m a r i l y s h o w i n g t h a t t h e 83 0 0 0 a n d 55 0 0 0 tool. w t , c o m p o n e n t s are insoluble a t p H 4.5 in 0.1 M KCl)-" e, t r y p t i c digest fract i o n soluble at p H 4.5 in 0.1 M KC1 ( c o m p a r i s o n w i t h b a n d c s h o w s t h a t t h e 71 0 0 0 m o l . w t . c o m p o n e n t is e t h a n o l - s e n s i t i v e ) ; f, r a b b i t light m e r o m y o s i n p r e p a r e d b y t r y p t i c d i g e s t i o n ; g - - h , l o b s t e r m y o s i n t r y p t i c digest f r a c t i o n s insoluble in 0.1 M KC1 a n d r e s i s t a n t to e t h a n o l ; g, f r a c t i o n p r e c i p i t a t e d at p H 6 ( n o t e t h e h e a v y b a n d c o r r e s p o n d i n g to m o l . wt. 83 0 0 0 ) . h, f r a c t i o n p r e c i p i t a t e d at p H 4.5 ( n o t e t h a t the 83 0 0 0 m o l . w t . b a n d is r e l a t i v e l y less i n t e n s e t h a n in gel 3 g a n d t h a t an a d d i t i o n b a n d a p p e a r s at 55 0 0 0 mol. wt.).
Of the three principal heavy molecular weight components in lobster heavy meromyosin (83 000, 71 000, and 55 000, Fig. 3, gel b) only the 71 000 mol. wt. constituent is sensitive to ethanol and soluble at pH 4.5 in 0.1 M KC1 (Fig. 3, gel e). In rabbit heavy meromyosin, of the three heavy molecular weight components (76 000. 65 000, 52 000, Fig. 3a) all three appear to be ethanol sensitive although with further digestion the 65 000 mol. wt. component breaks down to 37 000 and 21 000 fragments that are ethanol resistant [32]. Since the 64 000 c o m p o n e n t observed by Balint et al. [32] was derived from an 81 000 mol. wt. constituent, the latter might be somewhat analogous to the ethanol-resistant 83 000 c o m p o n e n t observed here in lobster myosin. Gel filtration removed some low molecular weight constituents from the heavy meromyosin fraction but caused no significant change in the gel electrophoretic pattern. Low ionic strength insoluble components. SDS gel electrophoresis of the insoluble fraction obtained by dialysis of a 10 min tryptic digest of lobster myosin against 0.1 M KC1, pH 6, solution followed by ethanol treatment of the precipitate is shown in Fig. 3g. Components are present with bands at 96 000, 83 000 and 69 000. The band at 83 000 is noticeably less intense than those at 96 000 and 69 000. Occassionally traces of a 55 000 band are seen as well. This is in marked contrast to the gel pattern for rabbit myosin light meromyosin fraction i (10 min digest) which has just one major c o m p o n e n t with a chain weight of 75 000 (Fig. 3f), although traces of three other bands are frequently discernable at 68 000, 65 000, and 60 000. If, on the other hand, the lobster myosin digest is dialyzed vs. 0.1 M KC1, pH 4.5, instead of pH 6, the insoluble
149 fraction contains, in addition to the bands at 96 000, 83 000, and 69 000, a strong component at 55 000. Furthermore, the relative intensity of the band corresponding to a polypeptide chain weight of 83 000 is very noticeably increased (Fig. 3h). Thus, it is clear that the 55 000 mol. wt. species is soluble in 0.1 M KC1, pH 6, but not at pH 4.5; hence when the digest is dialyzed at pH 6 this component appears in the soluble fraction (i.e. note the absence of this band in Fig. 3g). But if the digest is dialyzed at pH 4.5 this component appears in the insoluble fraction {Fig. 3h). The 83 000 mol. wt. constituent is partially soluble at pH 6; hence it appears in both the soluble phase (Fig. 3b) and insoluble phase (Fig. 3g) when the digestion products are dialyzed at this pH but only the insoluble phase (Fig. 3h) when the digest is dialyzed at pH 4.5. Both component~ are ethanol resistant and therefore are presumed to have a rod-like structure. More limited digestion gives rise to an additional ethanolresistant component with mass 111 000 and the band corresponding to the component with chain weight of 96 000 is relatively more intense in the gel patterns. These results indicate that a simple mono-disperse light meromyosinlike species is not produced by tryptic digestion of lobster myosin and that the molecule has more sites susceptible to tryptic proteolysis than does rabbit myosin.
Tryptic digestion of papain long rods from lobster and rabbit myosin In view of the complexity of the lobster heavy meromyosin and light meromyosin-like fractions, and the presence in the soluble fraction of the myosin digests, of fragments from the globular part of the molecule, efforts to analyze further the proteolysis of the rod portion of the lobster myosin molecule were directed to tryptic digestion of long rods produced by papain digestion. The results of tryptic digestion of lobster myosin papain long rods for intervals ranging from 0 to 120 min are shown in Fig. 4a. Nine bands corresponding to fragments with chain molecular weights of 127 000, 113 000, 102 000, 95 000, 83 000, 68 000, 55 000, 45 000, and 38 000 are apparent, along with additional weak, diffuse bands at 29 000. Bands at 21 000 (due to trypsin inhibitor) and 15 000--17 000 mol. wt. (which appears after about 15 min digestion and is attributed to smaller, heterogeneous degradation products) are also apparent. The band pattern illustrated was reproduced with several different papain long rod samples. The order of appearance of the rod fragments was approximately as expected for sequential degradation of the rod except that the 38 000 fragment appeared before the 45 000 fragment. Except for the 55 000 mol. wt. component (which accounted for approx. 38% of the total sample in digests from 45 to 90 min) none of the various fragments seemed to accumulate preferentially during the course of digestion. Simultaneous gel electrophoresis of the pH 4.5, low ionic strength insoluble fraction from tryptic digestion of lobster myosin (Fig. 3h) with the papain long rod digests showed that the bands in the light meromyosin-like fraction corresponded to the long rod fragments with molecular weights of 95 000, 83 000, 68 000, and 55 000. Concurrent tryptic digestion and gel electrophoresis of both lobster and rabbit papain long rods revealed differences both in the number and molecular weights of the components produced from the two long rod samples (compare Figs. 4a and 4b). Six principal bands (corresponding to chain weights of
150
!
8o
Fig. 4. S D S gel e l e c t r o p h o r e s i s o f t r y p t i c d i g e s t s o f papain long r o d s f r o m lobster and r a b b i t m y o s i n . D i g e s t i o n c o n d i t i o n s as d e s c r i b e d in p r o c e d u r e s , t i m e s as s h o w n o n gels (in r a i n ) , a, t r y p t i c d i g e s t i o n o f l o b s t e r m y o s i n p a p a i n l o n g r o d ; b , tryptic digestion o f r a b b i t m y o s i n p a p a i n l o n g r o d .
127 000, 76 000, 62 000, 56 000, 45 000, and 40 000) are apparent in the gel electrophoresis pattern of extended digests of rabbit papain long rods. In addition a trace band is apparent at 29 000, as well as trypsin inhibitor at 21 000. Digestion of rabbit papain long rods did not proceed quite as had been anticipated on the basis of previous work by others: apart from the minor contamination of the initial papain long rod sample with light meromyosin it is apparent that thte initial stages of digestion were accompanied by the appearance of a component with chain weight of 59 000 rather than 30 000--40 000 as anticipated on the basis of the rods being cleaved directly to light meromyosin and subfragment-2. As digestion proceeded this component gradually disappeared with the concurrent appearance of bands at 62 000, 56 000, 45 000, and 40 000 mol. wt. In contrast to digestion of lobster papain long rods, none of the components in the rabbit papain long rod digest seemed to accumulate preferentially. Tentative identification of the various components in the gel patterns of the rabbit long rod digests was facilitated by comparison
151 with rod fragments prepared from papain digestion of rabbit tryptic heavy meromyosin and tryptic digestion of rabbit tryptic light meromyosin. Brief digestion of rabbit tryptic heavy meromyosin (6 min, 100/1 substate per enzyme, pH 7.8, 0.5 M KC1) followed by passage of the digest mixture through gel filtration columns yielded two fractions. The faster moving fraction was ethanol resistant, appeared as either two bands when electrophoresed on nondissociating gels, or two doublet bands when run on SDS gels (mol. wt. 59 000 and 44 000, Fig. 5a) and ran as a single component in the centrifuge with a sedimentation coefficient of 2.64 S. High speed equilibrium centrifugation showed this fraction to be heterogeneous with a cell average molecular weight of 107 000. More extended papain digestion of rabbit tryptic heavy meromyosin (15 min, 15/1 substrate per enzyme) followed by the same procedure yielded subfragment-2 (ethanol resistant, high speed equilibrium mol. wt = 76 000, SDS chain weight 38 000, Fig. 5b). Subfragment-1 was also produced in this digestion but was not of interest here. Comparison of these results with the tryptic proteolysis of rabbit papain long rods suggests that the initial products of digestion were light meromyosin (76 000) and a rod-like precursor of subfragment-2 with chain weight 59 000. Upon further digestion, this component was degraded to subfragment-2 which appears as a band at 40 000 in the extended digest (Fig. 4b, 120 min digest gel). Controlled tryptic digestion of light meromyosin fraction 1 followed by gel filtration (Sephadex G-200) was used to produce the LF-1, LF-2 and LF-3 fragments with molecular weights of 112 000, 86 000, and 57 000 determined from high speed equilibrium ultracentrifugation and chain weights of 59 000, 44 000, and 30 000 from SDS gels (Figs. 5c--5e). Thus bands in the extended tryptic digestion of rabbit papain longs rods (Fig. 4b, 120 min digest gel) corre-
59~
i
44~
• ~
Hi
--59
~"~ ~IHDP --
a MxlO. 3 ~
~.b ~L..__~-J~
d
--44
-111' - 3 0
e
m.~.,..~.-~-:=,,~
Fig. 5. SDS gel e l e c t r o p h o r e s i s of f r a g m e n t s isolated f r o m p a p a i n d i g e s t i o n o f r a b b i t tryptic heavy m e r o m y o s i n (a a n d b) a n d txyptic digestion o f r a b b i t t r y p t i c light m e r o m y o s i n (c--e). a, subfragment-2 precursor (59 0 0 0 a n d 4 4 0 0 0 ) ; b , s u b f r a g m e n t - 2 (38 0 0 0 ) ; c, LF-1 (59 0 0 0 ) ; d, LF-2 (44 0 0 0 ) ; e, L F - 3 (30 000).
152
sponding to molecular weights of 56 000, 45 000, and 29 000 were identified as LF-1, LF-2, and LF-3, respectively. The remaining c o m p o n e n t at 62 000 has not been specifically identified but is assumed to be partially degraded light meromyosin. Discussion The polydispersity evident in the long rod preparations made by a-chymotryptic or papain proteolysis of lobster myosin suggests that there are several sites susceptible to enzymic cleavage in the region of the molecule where the globular head units join the rod. The molecular weight differences between the largest and smallest components in these fractions indicate that the cleavage sites could be as much as 4 0 0 / ~ apart along the myosin rod (if the relative proportions of the components are taken into consideration it appears that the principal cleavage sites occur with a maximum spread of about 300 A). In contrast, rabbit myosin long rod preparations are monodisperse with extended digestion yielding light meromyosin in addition to the long rods. These results provide additional support for the conclusion drawn from previous kinetic studies on tryptic hydrolysis of lobster myosin [17]; namely, that the lobster myosin molecule is much more susceptible to enzymic cleavage in the subfragment-2 region than is rabbit myosin. Although the long rods produced from lobster myosin are polydisperse, the corresponding subfragment-1 preparations have only one principal heavy chain c o m p o n e n t in contrast to the two or three major heavy chain components observed in SDS gel electrophoresis of rabbit myosin subfragment-1. The enzymatic and molecular weight (ultracentrifuge) characteristics of the low ionic strength soluble fragments will be discussed elsewhere. Although the low ionic strength soluble and insoluble fractions obtained after dialysis of tryptic digests of lobster myosin may have one or more c o m m o n components (i.e. the 83 000 and 55 000 mol. wt. components) and thus not be completely analogous to rabbit meromyosins, it is nevertheless instructive to compare the fractions obtained from lobster myosin with the rabbit meromyosins as well as the fragments produced by tryptic digestion of long rods from the two species of myosin. The major ethanol-sensitive component (presumably derived from subfragment-1) of lobster heavy meromyosin has a molecular weight of 71 000 as compared with the 76 000 component in rabbit heavy meromyosin. Comparison of the results obtained here with those of Balint et al. [32] suggests that the ethanol-resistant components of 83 000 and 55 000 mol. wt. might be related to the 84 000 c o m p o n e n t in rabbit heavy meromyosin which on further proteolysis yields a 64 000 fragment that in turn is split to produce ethanol-resistant fragments of 37 000 and 21 00. If the 83 000 mol. wt. c o m p o n e n t is essentially a-helical as implied by its resistance to ethanol, it would certainly be the maximum size rod fragment that could be accomodated in heavy meromyosin. Weeds and Pope [29] have recently suggested that subfragment-2 may be as large as 73 000 when produced from rabbit myosin by limited a-chymotryptic digestion and observed on dissociating gels. Alternatively, the limited low ionic strength solubility of the 83 000 c o m p o n e n t suggests that it may relate more directly to
153 light meromyosin. That this component does come from the myosin rod and does not include part of the globular head is indicated by its appearance in tryptic digests of lobster papain long rods. The low ionic strength precipitable components {ethanol resistant) from tryptic digests of lobster myosin are rod fragments that can be compared directly with those observed by SDS gel electrophoresis of tryptic digests of the papain long rods. The lobster light meromyosin fraction always contains several major components in contrast to rabbit light meromyosin which has just one principal constituent (76 000 chain weight) although trace bands for minor components are frequently observed also. From direct comparison of SDS gel electrophoretic patterns of tryptic digests of lobster and rabbit papain long rods, it is evident that under comparable digestion conditions lobster long rods yield more fragments than do the rabbit long rods. The large component chain weights {>70 000) and low ionic strength insolubility near neutral pH of these fragments, indicates that five cleavage sites occur in the subfragment-2 region of the lobster myosin rod; whereas, for rabbit myosin there is only one cleavage locus (producing long rods) in the corresponding region. Weeds and Pope [29] have suggested that light meromyosin and subfragment-2 produced by a-chymotryptic digestion of rabbit myosin are multicomponent species having fragments with chain weights of 77 000, 69 000, 66 000, and 61 000 for light meromyosin and 73 000, 59 000, and 46 000 for subfragment-2. Rabbit light meromyosin produced here by tryptic digestion had one major component (76 000) with trace bands at 68 000, 65 000, and 60 000, while the subfragment produced by limited papain digestion of rabbit heavy meromyosin had components of 59 000 and 44 000 similar to the subfragment-2 of Weeds and Pope. A 73 000 mol. wt. component for subfragment-2 was not detected in these preparations. The molecular weight of the subfragment-2 (76 000 from equilibrium centrifugation and SDS gels) isolated after more extended papain digestion of heavy meromyosin is in good agreement with the results of Biro et al. [4] but somewhat larger than the value of 60 000 frequently reported [5,6]. The molecular weights of the three rabbit light meromyosin fragments isolated here are in good agreement with those reported by Biro et al. [4] and correspond well with the bands observed in SDS gel electrophoresis of tryptic digests of the rabbit myosin long rods {Fig. 4b). In tryptic digests of lobster long rods two bands were clearly observed (45 000 and 38 000 mol. wt.} that would indicate cleavage of the light meromyosin portion of the molecule. A third band {29 000) was faintly observed and increased in intensity somewhat with further digestion. Thus, both rabbit and lobster myosin have three principal tryptic sensitive sites in the light meromyosin region but whereas rabbit myosin has only one sensitive locus in the subfragment-2 region lobster myosin has five. Although the chain weights of two of the three light meromyosin-like lobster subfragments (45 000, 38 000, and 29 000) were the same as those for light fragments from rabbit light meromyosin proteolysis (56 000, 45 000, and 29 000), further information concerning their composition and position in the myosin rod will be required to ascertain if they do indeed correspond to each other in the rod structure. If susceptibility to proteolysis is interpreted as a measure of molecular
154
stability [5], these results suggest that the subfragment-2 region of lobster myosin is less stable than the corresponding region in rabbit myosin. Similarly, the results suggest significant differences in residue composition for the heavy chains of the two myosins in these regions. Amino acid composition data for rabbit myosin show that the number of lysine and arginine residues (per l 0 s g protein, [6] ) is approximately the same in the light meromyosin and subfragment-2 regions of the molecule. However, preliminary amino acid analysis results for lobster myosin indicate an excess of lysine residues i n t h e subfragment-2 region of the molecule relative to the light meromyosin region. These data are consistent with the relative susceptibilities of the molecular rods to tryptic proteolysis but leave open the question of their sensitivity to papain or chymotryptic cleavage. Isolation and analysis of the various fragments produced in the digestion process can be expected to yield further insight into the relative substructure of both the various regions of the lobster myosin molecule and differences between lobster and rabbit myosin that have a bearing on their assembly into thick filaments and functional role in muscle contraction. Acknowledgements The author is indebted to C. Brexel and E. Edwards for technical assistance during part of this work and to H. Manuel for the ultracentrifuge molecular weight determinations for the ~-chymotrypsin and papain lobster long rods. Extensive helpful discussions with R. Siemankowski concerning various aspects of this work are also gratefully acknowledged. References 1 2 3 4 5 6 7 8 9 10 11 12 3 14 15 16 17 18 19 20 21 22 23 24
Szent-GyorgyL A.G. (1953) A r c h . B i o c h e m . B i o p h y s . 42. 305--320 Mihalyi, E. and Harrington, W.F. (1959) Biochirn. B i o p h y s . A c t a 36, 447--466 Young, D.M., Himmelfarb. S. and HHarrington, W.F. (1965) J. Biol. C h e m . 240, 2428--2436 Bixo, N.A.0 Szflagyi, L. and Ballnt, M. (1972) Cold Spring I-Iarb. Syrup. Quant. Biol. 37, 55--63 Burke, M., Himmelfarb. S. and Harrington, W.F. (1973) Biochemistry 12, 701--710 Lowey , S. (1971) in Subunits in Biological S y s t e m s ( T i m a s h e f f , S . N . and F a s m a n . C.D., eds.), pp. 201--259, Marcel Dekker, New Y o r k Pepe. F.A. (1971) in Progress in B i o p h y s i c s and Molecular B i o l o g y (Butler, J . A . V . and N o b l e , D., eds.), pp. 75--96, P e r g a m o n Press, N e w York Harrington, W.F. (1971) Proc. Natl. A c a d . Sci. U.S. 68, 685--689 Huxley, H.E. (1969) Science 164, 1356--1366 H u x l e y , H.E. ( 1 9 6 3 ) J. Mol. Biol. 7 , 2 8 1 - - 3 0 8 Kaminer. B. and Bell, A.L. (1966) J. Mol. Biol. 20, 391--401 Harrison, R.G., Lowey, S. and C o h e n , C. (1971) J. Mol. Biol. 59, 531--535 Kaminer, B. (1969) J. Mol. Biol. 39, 257--264 Nakamura, A., Sreter0 F. and Gergely, J. (1971) J. Cell Biol. 49. 883--898 Hayes, D., Huang, M. and Zobel, C.R. (1971) J. Ultrastruct. Res. 3 7 . 1 7 - - 3 0 Siemankowski. R.F. and Zobel, C.R. (1976) B i o c h i m . B i o p h y s . A c t a 420, 406--416 Siemankowski. R.F. and Zobel, C.R. (1976) J. M e c h a n o c h e m . Cell Motility 3 , 1 7 1 - - 1 8 4 Szent-Gyorgyi, A. (1951) Chemistry of Muscular C o n t r a c t i o n . 2nd e d n . , A c a d e m i c Press, N e w Y ork Weeds. A.G. and Taylor, R.S. (1975) Nature 257, 54--56 Szent-Gyorgyi, A.G., Cohen, C. a n d Philpott, D.E. (1960) J. Mol. Biol. 2, 133--142 Weber, K. and Osborn, M. (1969) J. Biol. C h e m . 244, 44 06--4412 Gazith, J., Himmelfarh, S. and Harrington, W.F. (1970) J. Biol. C h e m . 245, 15--22 L o w e y , S., Slayter, H.S., Weeds, A . G . and Baker, H. (1969) J. Mol. Biol. 42, 1--29 Szuchet. S. and Zohel. C.R. (1974) Biochemistry 13, 1482--1491
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25 26 27 28 29 30 31 32
Mattice, W.L., Riser, J.M. and Clark, D.S. (1976) Biochemistry 15. 4 2 6 4 - - 4 2 7 2 Halsey, J.F. and Harrington, W.F. (1973) Biochemistry 12, 693--701 Cowgill, R.W. (1975) Biochemistry 14, 503--509 Yphantis, D.A. (1964) Biochemistry 3. 297--317 Weeds, A.G. and Pope, B. (1977) J. Mol. Biol. 1 1 1 , 1 2 9 - - 1 5 7 Balint, M., Sreter, F.A. and Gergely, J. (1975) Arch. Biochem. Biophys. 1 6 8 , 5 5 7 - - 5 6 6 Margossian, S.S. and Lowey, S. (1973) J. Mol. Biol. 74. 301--311 Balint, M., Sreter, F.A.0 Wolf, I., Nagy, B. and Gergely, J. (1975) J. Biol. Chem. 250, 6168--6177