THE JOURNAL OF BIOLOGICAL CHEMISTRY

Printed in U. S.A. Vol. 257, No. 2, Issue of January 25, pp. 1102-1105, 1982

(Received for publication, July 20, 1981, and in revised form, September 25, 1981)

Mathoor Sivaramakrishnan and Morris Burke From the Department of Biology, Case Western Reserve University, Cleveland, Ohio 44106

Vertebrate skeletal fast-twitch muscle myosin subfragment 1 is comprised of a heavy polypeptide chain of 95,000 daltons and one alkali light chain of either 21,000 daltons (Al) or 16,500 daltons (A2). In the present study, the heavy chain of subfragment 1 has been separated from the alkali light chain under non- denaturing conditions resembling those in vivo. The heavy chain exhibits the same ATPase activity as myosin subfragment 1, indicating that the heavy chain alone contains the catalytic site for ATP hydrolysis and that the alkali light chains are nonessential for activity. The free heavy chain associates readily at 4 “C or 37 “C with free A1 or A2 to form the subfragment 1 iso- zymes SFl(A1) or SFl(A2) respectively. Actin activates the MgATPase activity of the heavy chain in the same manner as occurs with the native isozyme, indicating that the heavy chain possesses the actin binding do- main.

It is now well established that skeletal muscle myosin is an oligomeric protein comprised of two heavy chains and two pairs of light chains known as the “regulatory” (DTNB)’ light chains and the essential light chains (1, 2). The smallest fragment containing the biological function, as defined by the ATPase activity and the ability to bind to actin, is the head region of the molecule known as subfragment 1 (3,4). Subfrag- ment 1 prepared by chymotryptic digestion of myosin is comprised of the NHz-terminal half of the heavy chain to- gether with an alkali light chain that exists as two distinct forms known as A1 and A2 (5, 6). It has been generally believed that the ATPase activity of the myosin molecule or subfragment 1 requires the specific interaction between the heavy chain and the alkali light chain in the head region. This conclusion is based on a number of dissociation-reassociation studies and observations that the isolated free subunits are essentially devoid of activity (7-10). More recent evidence suggests that the heavy chain subunit is the chief modulator of the ATPase activity. This is based on the photoaffiity labeling with an ATP analog (11) and ATPase kinetics of subfragment 1 and of myosin hybrids (12). The fact, that trapped nucleotide in the ATPase site (13) is not released by exchange of alkali light chain in the pPDM-modified heavy

* This work was supported by grants from the National Science Foundation (PCM-8007876) and the National Institutes of Health (2- RO1-N 515 319). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “aduertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

’ The abbreviations used are: DTNB is 5,5”dithiobis-2-nitrobenzoic acid; SFl(A1) and SFI(A2), the isozymes of subfragment 1 with the A1 and A2 alkali light chain, respectively; SDS, sodium dodecyl sulfate; HC, free heavy chain of subfragment 1; DEAE, diethylamino- ethyl; pPDM, p-N,N’-phenylenedimaleimide.

chain (14), suggests that the ATPase site may be solely restricted to the heavy chain. However, these studies do not establish that the heavy chain alone can hydrolyze MgATP. Only in the case of the aberrant myosin 1B of Acanthanoeba castellani has it been demonstrated that the isolated heavy chain possesses full ATPase activity (15).

Recent studies from our laboratory have suggested that at 37 “C in the presence of MgATP, the heavy chain-alkali light chain complex in subfragment 1 as well as myosin exists in a rapid equilibrium with their dissociated subunits (16, 17). If this conclusion is valid then it is clear that, by perturbing this equilibrium, it should be possible in principle to isolate the free heavy and alkali light chains. In the present study, we show, indeed, that the free heavy chain of subfragment 1 can be isolated under these conditions and, moreover, that this chain alone exhibits the same specific ATPase activity as the native subfragment 1 isozymes in the presence and absence of actin. Incubation of the free heavy chain with either free A1 or A2 in the presence of MgATP at either 4 “C or 37 “C results in the formation of SFl(A1) or SFl(A2), respectively, indicating that the alkali light chain association site is func- tional under these conditions. These results demonstrate un- equivocally that the heavy chain alone is the catalytic subunit of subfragment 1 and that actin activation of subfragment 1 MgATPase proceeds through the binding of the actin to the heavy chain.

MATERIALS AND METHODS

Distilled water was purified to reagent grade by a Millipore QTM system. All experiments were done at 4 “C unless otherwise specified. ATP was a product of the Sigma Chemical Co. and N-[3H]ethylmal- eimide (specific activity, 300 mCi/mmol) was the product of New England Nuclear. All other reagents were of analytical grade.

Zealand rabbits by the method of Godfrey and Harrington (18) and Myosin was prepared from the back muscles of male albino New

the alkali light chains by the procedure of Holt and Lowey (19). Subfragment 1 isozymes were prepared by chymotryptic digestion of myosin and subsequent separation on DEAE-cellulose as described by Weeds and Taylor (20). F-actin was isolated and purified by the procedure of Spudich and Watt (21). The concentrations of the protein were determined by absorbance at 280 nm employing E% nm of 5.5, 7.5, 2.0, and 11.0 for myosin, subfragment I, alkali light chain, and actin, respectively. Alternatively, the Bradford procedure (22) was used employing calibrations with known quantities of the respec- tive protein.

Free alkali light chain A2 (2 mg/ml) was modified with N-[3H] ethylmaleimide in 8 M urea, 0.05 M imidazole. HCl, pH 7.0, for 60 min at 25 “C. Subsequently, the excess N-[3H]ethylmaleimide and urea were removed from the protein by exhaustive dialysis versus 0.05 M imidazole.HC1, pH 7.0 at 4 “C. Prior to hybridization this 3H-labeled A2 was diluted 4-fold with unmodified A2 carrier. For hybridization of this [3H]A2 to the heavy chain of subfragment 1, SFl(A2) was incubated with a 4-fold molar excess of [3H]A2 in 4.7 M NH&l, 10 mM MgATP at 4 “C as described elsewhere (14). Following hybridization, the SFl([3H]A2) hybrid was separated from excess free [3H]A2 by ion exchange chromatography in 0.05 M imidazole-HC1, pH 7.0 at 4 “C (10). This SF1([3H]A2) hybrid was found to have a specific activity

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Isolation and ATPase Activity of Subfragment 1 Heavy Chain 1103

of 5.23 X lOI3 cpm/mol of subfragment 1 and essentially the same ATPase activities as that of the native SFl(A2). Isolation of the free heavy chain of subfragment 1 was accomplished by DEAE-cellulose Chromatography of SFl(A2) or SFl(['H]A2) as described below. A water-jacketed column (31 X 1.6 cm) of DEAE-cellulose was equili- brated in 0.093 M imidazole. HCI, 0.01 M ATP, 0.014 M MgC12,O.l mM dithiothreitol, pH 7.0, and maintained at 37 "C by means of a constant temperature water bath equipped with a circulating pump. 10 mg of SFl(A2) (1 mg/ml) in 0.05 M imidazole.HC1, 10 mM MgATP, 0.1 mM dithiothreitol, pH 7.0, was incubated a t 37 "C for 15 min and imme- diately applied to the ion exchange column. A flow rate of 60 ml/h was employed and 2.0-ml fractions were collected in test tubes im- mersed in ice water. Aliquots of 0.05 ml and 0.2 ml were removed from each fraction for determination of protein by the Bradford method (22) and estimation of [:'H]A2 by scintillation counting, respectively. Between 1.5 and 2.0 mg of free heavy chain could be isolated from 10 mg of SFl(A2) by this procedure. Free heavy chain fractions were concentrated using the Micro-Pro-Dicon apparatus (Bio-Molecular Dynamics, OR). Although the heavy chain was con- centrated the yields were low presumably due to a membrane binding effect.

Polyacrylamide gel electrophoresis under nondenaturing condi- tions or in the presence of sodium dodecyl sulfate was done as previously described (16). Reassociation of free A I or A2 with the free heavy chain was examined by incubation of the heavy chain (0.25 mg/ml) with an &fold molar excess of light chain in 0.05 M imidazole, 10 mM MgATP, 0.1 mM dithiothreitol, pH 7.0, for 20 min a t 4 "C and 37 "C. The SFl(A2) isozyme was incubated with free AI under identical conditions. These samples were then immediately examined by gel electrophoresis a t 6 "C under nondenaturing conditions.

Ca2+, Mg", and K+/EDTA-activated ATPase activities were done by the procedures of Kielley and Bradley (23) and Kielley et al. (24). Actin activation studies were done by the pH-stat method employing a Radiometer TTT 60 titrator essentially as described by Eisenberg and Moos (25). In these studies, the SF1 or heavy chain a t 0.1 mg/ml in 50 mM KCI, 2 mM ATP, 3 mM MgCl2, 0.01 M Tris-histidine buffer was incubated with varying concentrations of actin (1.0 to 4.0 mg/ml) at pH 7.5 and 23 "C. These values were corrected by subtracting the amount of Pi liberated by these proteins in the absence of actin.

RESULTS

The elution profile obtained by subjecting subfragment 1 to ion exchange chromatography at 37 "C in the presence of MgATP is shown in Fig. 1. The elution profile shown here was obtained with the subfragment 1 hybrid, SFl(['H]AB), but identical profiles were obtained with the native subfrag- ment 1 species SFl(A2). The use of the hybrid with "-labeled light chain offered the advantage that the presence of the light chain, whether free or associated to the heavy chain, in the eluent could be readily monitored by scintillation count-

ing. Whereas this subfragment 1 species elutes as a single peak from both DEAE-cellulose (10) and agarose-ATP (26) at 4 "C, it is clear that ion exchange chromatography a t 37 "C in the presence of MgATP results in the separation of distinct peaks. It is unlikely that the peaks seen here result from proteolysis of the subfragment 1, since incubation of SFl(A2) at 37 "C with 10 mM MgATP showed no evidence of proteolysis on subsequent electrophoretic analyses in the presence or ab- sence of SDS (16).

On the basis of the radioactivity elution profie, it is evident that the first peak contains no "H label, suggesting that it is free of alkali light chain and, therefore, it represents free heavy chain. From the level of radioactivity observed the maximum contamination of this peak by A2 is less than 0.01 mol/mol of heavy chain. The second peak does contain a small amount of radioactivity, corresponding to about 0.15 mol of [:'H]A2/mol of heavy chain, indicating that it is pre- dominantly comprised of free heavy chain. The fwst two peaks were next examined by gel electrophoresis under nondenatur- ing conditions and the results are presented in Fig. 2. It is clear that, within the resolution of this method, the presump- tive heavy chain (peak 1) is comprised predominantly of a single component having an electrophoretic mobility distinct from either the two subfragment 1 isozymes SFl(A1) and SFl(A2) and from the free alkali light chains A1 and A2 (Fig. 2B). A minor band with a mobility slightly higher than the major component can also be discerned. Since the heavy chain population of both SFl(A2) and SFl(A1) is known to be heterogeneous (27), it is tempting to speculate that these two components may represent isozymic forms of the heavy chains. The second peak showed a major component which moved with a mobility close to that of native SFl(A1) but, since only SFl(A2) was employed in this study, this fraction must be a different species (Fig. 2 A ) .

The composition of peak 1 was next examined by gel elec- trophoresis in the presence of SDS and the resulting electro- phoretogram is shown in Fig. 3A. It is clear that this fraction is comprised of a single polypeptide chain with the same electrophoretic mobility as the heavy chains of SFl(A1) and SFl(A2) (Fig. 3, B to E ) . This SDS-gel electrophoretogram of a highly concentrated peak 1 sample shows negligible amounts of light chain components. Although the radioactivity profile of SFl([:'H]A2) on DEAE-cellulose shown in Fig. 1 shows no

1 Peak 27 1

.. I I

0 20 40 60 80 100 120

FRACTION NUMBER

FIG. 1. DEAE-cellulose chromatography of SF1([sH]A2) at 37 "C in the presence of 10 m~ MgATP. -, absorbance a t 595 nm; . . . . , cpm. Steps of 0.12 M KC1 and 1.0 M KC1 were introduced as indicated by the arrows. Further details are given under "Materials and Methods."

FIG. 2. Gel electrophoresis under nondenaturing conditions of peaks 1 and 2 of Fig. 1. A, peak 2; B, peak 1; C and D, SFl(A2); E , SFl(A1).

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1104 Isolation and ATPase Activity of Subfragment 1 Heavy Chain

'H label due to A2 in peak 1, we have on occasion observed traces of material ( ~ 5 % ) running with the same mobility as A2 on overloaded SDS-gels of this peak.

Additional evidence that peak 1 is indeed the free heavy chain subunit of subfragment 1 is provided by the reassocia- tion studies with free A1 or A2. In this study, peak 1 was incubated alone and with A1 or A2 a t 4 "C and 37 "C in the presence of MgATP for 20 min and then immediately sub- jected to gel electrophoresis under nondenaturing conditions. The electrophoretograms obtained are shown in Fig. 4. It is evident that incubation of peak 1 in the absence of alkali light chain did not alter its electrophoretic mobility. On the other hand, incubation of peak 1 with A1 or A2 resulted in the disappearance of the peak 1 species and the concomitant appearance of bands migrating with electrophoretic mobilities corresponding to SFl(A1) or SFl(A2), respectively (Fig. 4, E to H). The fact that, even at 4 "C, peak 1 is capable of combining with free alkali light chain to generate the corre- sponding subfragment 1 isozyme confirms that peak 1 is the free heavy chain. It should be pointed out that at the low ionic strengths employed in these reassociation studies, the subunit

ea.

FIG. 3. SDS-gel electrophoresis of Peak 1, SFl(A1). SFl(A2). free A1 and A2. A, Peak 1; R and C, SFl(A1); C and 19. SFl(A2); E , Al; F, A2. The diffuse band under the heavy chain bands is an artifact of staining.

TABLE I ATPase activities of isolated heavy chain and other SF1 species

k ( s - ' ) (37 "C) k ( s - ' ) (23 "C)

PM SF1 (Al) 8.2 19.3 0.043 16.5 74 SF1 (A2) 9.8 19.8 0.043 29.0 198 Heavv chain 9.2 18.6 0.055 29.0 164

0.2 - J I I 1 J

0.0 1 0.02 0.03 l lAct in (yM")

FIG. 5. Actin-activated ATPases. Assays were done as described under "Materials and Methods". 0, SFl(A2); 0, free heavy chain. The plots represent linear regression fits to the experimental data.

interactions in subfragment 1 are very strong, as evidenced by the fact that no hybrid is formed when SFl(A2) is incubated with free A1 under the same conditions at 4 "C (Fig. 40). At 37 "C, it appears that some SFl(A1) hybrid is formed in the SFl(AB)-free A1 system (Fig. 4 0 .

It was of interest to examine whether the isolated heavy chain possessed any ATPase activity. After exhaustive dialysis against 0.05 M imidazole-HC1, 0.1 mM dithiothreitol, 2 lll~ EDTA, pH 7.0, to remove MgATP, the free heavy chain was then assayed for ATPase activity in the presence and absence of actin. These results are shown in Table I, from which it is clear that the isolated heavy chain exhibits full ATPase activ- ity comparable to that of the native subfragment 1. The data obtained with actin are shown in Fig. 5 and Table I. It is clear

FIG. 4. Gel electrophoresis under nondenaturing conditions of Peak 1 incubated in the presence and absence of A1 and A2. A, SFl(A2); E , SFl(A1); C and D. SFl(A2)-free A1 mixture; E , and F, Peak 1-A2 mixture; G and H, Peak 1-A1 mixture; I and J, Peak 1 alone. C, E , G, and I are 37 "C incubations. D, F, H, and J , are 4 "C incubations. Other details are given under "Materials and Methods."

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Isolation and ATPase Activity of Subfragment 1 Heavy Chain 1105

that the V,,, values for the free heavy chain and the subfrag- ment 1 isoenzyme, from which it was derived, are essentially the same. The differences in the K,,, values may not be significant, since we have observed that, in some preparations of heavy chain, both the V,,, and Kapp values are the same as those of SFl(A2).

DISCUSSION

The strategy for isolating the heavy chain with full ATPase activity was based on the following observations: (i) At 37 "C in the presence of MgATP, subfragment 1 exists in a rapid, reversible equilibrium with its dissociated subunits with no evidence for inactivation (16, 17). (ii) The anionic mobilities of the subfragment 1 isozymes and the free alkali light chains are in the order A2 > A1 > HC-A2 > HC-A1 and presumably in an anionic exchanger, such as DEAE-cellulose, a similar order of binding would be observed with the free heavy chain expected to have the weakest binding. Assuming a similar relative order for the binding of these species to DEAE- cellulose at 37 "C in the presence of MgATP, it would be anticipated that A2 would bind very strongly and the free heavy chain weakly, while the undissociated isozyme (HC-A2) would have intermediate binding capacity. Since, under these conditions, subfragment 1 (HC-AB) is an equilibrium system of these three species, it is clear that this equilibrium will be shifted toward more dissociation by passage through DEAE- cellulose and should result in a separation of these species. An additional advantage of employing DEAE-cellulose is that the anticipated weak binding of the free heavy chain would ensure its rapid elution and prevent excessive exposure of this subunit to 37 "C. The results presented in Fig. 1 show that HC-A2 can indeed be separated into different species differing in their affinities for DEAE and that peak 1 represents the free heavy chain. The subfragment 1 isozyme SFl(A2) was chosen for this study since previous results have shown that the subunit interactions of this isozyme are weaker than those of SFl(A1) (17).

As in the case of myosin of Acanthamoeba castellani (15), the heavy chain of skeletal myosin subfragment 1 binds quan- titatively to agarose-ATP.* We have also observed that the free heavy chain does not bind to DEAE-cellulose at 4 "C under normal conditions (20) where the subfragment 1 iso- zymes do bind. This can be exploited as a further purification procedure if necessary to remove any residual SF1 from the heavy chain fraction.

Perhaps the most significant finding in the present study is the observation that the free heavy chain in the presence and absence of actin exhibits specific ATPase activities compara- ble to that of the native subfragment 1 (Table I and Fig. 5 ) . The observation that the actin activation of free heavy chain is the same as that of the isoenzyme SFl(A2) from which it was isolated rather than that of SFl(A1) suggests that the extent of heavy chain heterogeneity in the isoenzymes may not be identical. Although this must await further study, it raises the possibility that different actin-activated ATPase activities will be observed for the different forms of heavy chains. This would be in agreement with the conclusion of Wagner (12) that the heavy chain component of myosin is the chief modulator of this activity. These studies demonstrate that the alkali light chain subunits are not essential for the

* M. Burke and M. Sivaramakrishnan, unpublished observations.

ATPase function and raises once more the question of what is their role in the biological and physiological functions of myosin. Under conditions resembling the resting state of muscle, subfragment 1 apparently exists in a dynamic equilib- rium with its dissociated subunits (16). This observation to- gether with recent reports that the free alkali light chains show weak binding affinity for ATP (26,28) suggest that they may play a regulatory role. Alternatively, it is conceivable that the alkali light chains together with the DTNB chains may be involved in a thick filament Ca2' regulation or mod- ulation of the vertebrate actomyosin interaction in a manner similar to that suggested for the invertebrate scallop myosin (29).

Addendum-Since this paper was submitted, an independent re- port by Wagner and Giniger (30) has appeared on the isolation of free heavy chain by another procedure and showing that the heavy chain alone possesses ATPase activity in the presence and absence of actin.

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M Sivaramakrishnan and M Burkeenzymatic activity.

The free heavy chain of vertebrate skeletal myosin subfragment 1 shows full

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