Evidence for a role for cardiac myosin in regulating the contractile response

Evidence for a role for cardiac myosin in regulating the contractile response

ARCHIVES OF Evidence BIOCHEMISTRY AND BIOPHYSICS 171, 206-213 (1975) for a Role for Cardiac Myosin Response’ GARY in Regulating the Contrac...

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ARCHIVES

OF

Evidence

BIOCHEMISTRY

AND

BIOPHYSICS

171,

206-213

(1975)

for a Role for Cardiac Myosin Response’ GARY

in Regulating

the Contractile

BAILIN

Division of Cardiobgy, Department of Medicine, and Department of Physiology, Mount Sinai School of Medicine of The City University of New York, New York 10029 Received

April

24, 1975

Dinitrophenylated bovine cardiac myosin incorporates 1.3 mol of 1-fluoro-2,4-dinitrobenzene per 5 x 10’ g of protein. Concomitantly there was an activation of the Ca’+ATPase activity and an inhibition of the K+(EDTA)-ATPase activity. The dinitrophenyl group is located in the smallest active proteolytic fragment, subfragment 1. Virtually all of the labeling occurs in the region containing the heavy chains of cardiac myosin as judged by dissociation exneriments in sodium dodecyl sulfate. Dinitrophenylated myosin failed to form calcium-sensrtive actomyosin when tested in an ATPase assay system containing actin, tropomyosin, troponin and ethylene glycol-his@-aminoethyl ether)N,N’-tetraacetic acid. Thiolysis of the dinitrophenyl group from myosin with 2mercaptoethanol restored its ability to form a calcium-sensitive actomyosin. The Ca2+ and K+(EDTA)-ATPase activities were also restored to control values. These results indicate that cardiac myosin participates in the regulation of the interaction between the contractile proteins.

Troponin, tropomyosin and actin, the proteins of the thin filament of muscle, participate in a cooperative interaction (1) that is responsible for the regulation of contraction in cardiac muscle (2, 3). Less attention, however, has been paid to the possible involvement of myosin in this regulatory process. Scallop muscle (4) and smooth (chicken gizzard) muscle (5) lack the troponin complex, yet regulation of a myosin-linked type occurs in these muscles (4, 5). In other species such as insect flight (6) or rabbit skeletal muscle (7, 81, both troponin and myosin seem to be involved in the regulation of contraction. In the case of rabbit skeletal myosin, a regulatory role has been suggested for the NbsZ2 light chain (7-11). This light chain reportedly can bind Ca2+ (7) at physiological lev-

METHODS

1 This work was supported by grants from the Muscular Dystrophy Associations of America, Inc., and in part by an N.I.H. grant, No. AA 00316-01. 2 The abbreviations used are: Nbsz, 5,5’-dithiobis(2nitrobenzoic acid); N,ph-, 2,4-dinitrophenyl-; EGTA, ethylene glycol bis@aminoethyl ether)N,N’-tetraacetic acid; N*ph-F, l-fluoro-2,4dinitrobenzene.

Bovine cardiac myosin was prepared by a procedure previously described (14). Heavy meromyosin and light meromyosin were obtained by proteolytic digestion of cardiac myosin with trypsin at pH 6.2 for 50 min at room temperature (14). Subfragment 1 was prepared by digesting heavy meromyosin with chymotrypsin (14). A complex of cardiac actin, tropo206

Copyright All rights

0 1975 by Academic Press, Inc. of reproduction in any form reserved.

els (8) and the replacement of the EDTA light chains of scallop myosin with the Nbsz light chain restores the regulatory properties of the reconstituted scallop myosin (9). Furthermore, a homology may exist between the light chains and the Ca2+ receptor protein component of troponin, indicating a common genetic origin for these proteins (10, 11). Recent studies on cardiac myosin concerning the effects of Ca2+ and K+ on the ATPase activity (12) and the reactivity of -SH groups during exercise (13) suggest that there may be sites on myosin that govern its interaction with the actin-tropomyosin-troponin complex. This paper will present evidence in support of a regulatory role for cardiac myosin.

DINITROPHENYLATION myosin and troponin was isolated from an acetonedried muscle powder at 37°C by the method of Katz (15). All the components were present, as judged by sodium dodecyl sulfate gel electrophoresis. The ATPase activity of myosin in the presence of Ca’+ and K+(EDTA) at high ionic strength (0.25 M KCl) was determined as described previously (16). For the assay of the actomyosin ATPase activity, the protein concentrations were: Myosin, 0.24 mgiml; the actin-tropomyosin-troponin complex, 0.08 mgiml. The assay was performed with or without 1 mM EGTA under the following conditions: 25 mM Tris buffer, pH 7.4, 5 mM MgClz, 0.01 mM CaC12, and 2 mM ATP in a final volume of 4 ml for 10 min at 25”. The reaction was initiated by the addition of ATP and it was stopped by the addition of 1 ml of 10% trichloroacetic acid (17). To study the effect of Ca*+ on the actomyosin ATPase activity in the presence of Mgz+, stock Ca-EGTA buffer solutions were prepared. The desired free Ca2+ concentration was obtained using a binding constant for Ca-EGTA equal to 4.4 x lo5 at pH 6.8 (18) and a stock CaCl, solution 1.5 x 10M4 M, which represents total calcium. The CaClz solution was titrated with a stock 0.3 M EGTA solution to obtain the free Ca2+ concentration at pH 6.8. For further details see the legend to Fig. 4. Dinitrophenylation reaction. Chemical modification of cardiac myosin (5 mg/ml) in 0.6 M KC1 with VHlN,ph-F was performed at 25”C, pH 7.5 (16). Myosin treated in the absence of [3H]N,ph-F served as a control. The reaction was stopped by precipitation of the modified myosin in 10 volumes of cold distilled water. The myosin was reprecipitated three times from 0.6 M KCl. Analysis of the amino acids dinitrophenylated has been described (16). Thiolysis of dinitrophenylated myosin. Dinitrophenylated myosin (5 mg/ml) in 0.6 M KC1 was treated with 2-mercaptoethanol at a final concentration of 0.05 M, pH 8.5, 25”C, for 40 min (16). The pH was maintained by the addition of 0.5 N NaOH. The reaction was stopped by precipitating the myosin in cold distilled water as described above. Cardiac myosin not treated with Ngh-F served as a control. Gel electrophoresis. Polyacrylamide-gel electrophoresis was performed with 10% gels in 7-cm tubes by using 0.10 M sodium phosphate buffer (pH 7.1) and 0.1% sodium dodecyl sulfate (19). The protein samples were dialyzed overnight at room temperature against 0.1 M sodium phosphate buffer (pH 7.1), 1% sodium dodecyl sulfate and 1 mM dithiothreitol. Electrophoresis, at a current of 8 mA/tube was terminated when the tracking dye moved to within 1 cm of the bottom of the tubes (about 5 h). The gels were fixed and stained in a solution containing 0.23% Coomassie blue, 45% methanol, 9.1% acetic acid and 11.5% trichloroacetic acid for 3-5 h. The gels were destained overnight with a solution of 45% methanol and 9.2% acetic acid. The distribution of the radioactivity in the various protein bands was determined

OF

CARDIAC

207

MYOSIN

by cutting the gels in l-mm slices followed by incubation with 0.5 ml of 30% hydrogen peroxide at 60°C for 18 h prior to counting (20). Protein determination. The biuret method of Gornall et al. was used (21). Materials. Radioactive N,ph-F labeled with ‘H was obtained from New England Nuclear Corp. A 0.1 M solution was standardized by the method of Murdock et al. (22), and it had a specific activity of 5.96 x lo6 cpm//lmol. NZph-F was obtained from Schwarz/Mann and was recrystallized before use (23). Trypsin, soybean trypsin inhibitor and chymotrypsin were obtained from Worthington Biochemical Corp. The assay of the proteolytic enzymes has been reported (14). RESULTS

Dinitrophenylation

of Cardiac of 13H]N2ph-F

Incorporation concentration

into

cardiac

Myosin at 0.10 mM myosin

was

marked by an activation of the Ca2+-ATPase activity and an inhibition of the K+(EDTA)-ATPase activity as a function of time (Fig. 1). These effects appeared

x 5c I-

/

,, ” I’ ,,’ x’ 1, \

,,,,,/- /

,c ; 1

r

FIG. 1. The effect of [3H]Nzph-F on the ATPase activity of cardiac myosin. Myosin (500 mg) in 100 ml of 0.6 M KCl, pH 7.5, was treated with [3H]N,ph to a final concentration of 0.10 mM at 25°C. At specified intervals, aliquots (2 ml) were removed for the determination of t3HlN,ph bound to myosin. At the same time, 0.2-ml aliquots were removed and assayed for ATPase activity. A control myosin was treated with ethanol. (O-O), Ca*+-ATPase activity (100% = 0.083 Fmol of Pi per minute per milligram of myosin); (O-O), K+(EDTA)-ATPase activity (100% = 0.133 I*mol of P, per minute per milligram of myosin); (X---X), incorporation of 13HlNzph into myosin. DNP = N,ph = dinitrophenol.

208

GARY

maximal when 1.3 mol of the reagent were incorporated per 5 x lo5 g of myosin. At higher concentrations of Nzph-F, i.e., 0.2 and 0.4 mM, there was a significant increase in the incorporation of Nzph-F but there was also a loss of the Ca2+-ATPase activity and a general denaturation of the myosin. At lower concentrations of the reagent, 0.05 mM or less, the effects were less pronounced. For example, after 30 min, when 0.53 mol of 13HlNzph-F was incorporated into myosin per 5 x lo5 g of protein, there was a slight activation of the Ca2+-ATPase activity and 57% of the K+(EDTA)-ATPase activity remained. The localization of the dinitrophenylation reaction became apparent when a comparison was made of the 13HlNZph bound to the various proteolytic fragments isolated from the tryptic digest of 13HlN,ph-cardiac myosin and the chymotryptic digest of the resulting heavy meromyosin (Table I). The bulk of the radioactivity is present in heavy meromyosin although some dinitrophenylation of light meromyosin has occurred. Subfragment 1 contains about onehalf of the Nzph-group that is present in heavy meromyosin (Table I). This was observed in a number of cases where the Nzph content was varied from 1.2-1.6 mol of r3H]Nzph-F incorporated per 5 x lo5 g of myosin. When the data of Table I are expressed in terms of moles of r3H]NZph bound per 100 mg of protein, there was a TABLE

I

INCORPORATION OF 3H-L~~~~~~ FLUORODINITROBENZENE INTO CARDIAC MYOSIN ITS PROTEOLYTIC FRAGMENTS~ Protein

Myosin Heavy meromyosin Light meromyosin Subfragment 1

Molecular

AND

weighP

[sH]N,ph bound per mole of protein

500,000 350,000 150,000 120,000

1.46 1.27 0.22 0.60

D Cardiac myosin (250 mg) in 50 ml of 0.6 M KC1 was treated with 13H]N,ph-F to a final concentration of 0.10 m&r for 30 min at 25”C, pH 7.5. The isolation of [SH]Nzph-myosin and its digestion with trypsin and chymotrypsin to form the various proteolytic fragments are described under Methods. * See Ref. (2).

BAILIN

HC

HC

IO

20 ZONE

30

NUMBER

FIG. 2. Distribution of radioactivity in 13H]Nzphmyosin. PH]N,ph-myosin, 150 pg (containing 1.2 mol of reagent per 5 x lo5 g of protein) was placed on each of 12 gels. After electrophoresis was accomplished the gels were cut and counted as described under Methods. HC, heavy chains; A, actin; L, and Lz, light chains of cardiac myosin. Insert, gel electrophoretogram of [3H]Nzph-myosin. The protein bands HC, L1 and Lz correspond to the radioactive zones HC, L, and Lz. The arrow represents the bottom of the gel.

significant increase in the specific radioactivity of the proteolytic fragments from 13H]Nzph-myosin. For instance, myosin, heavy meromyosin and subfragment 1 contained 0.292, 0.363, and 0.498 pmol of r3H]Nzph bound per 100 mg of protein, respectively. It is clear that only nonlabeled protein was removed during proteolysis of heavy meromyosin. The N,ph-group is conserved in subfragment 1. Dissociation of [3HlNzph-cardiac myosin in sodium dodecyl sulfate followed by gel electrophoresis resolved the protein into its heavy and light chain components (insert of Fig. 2). The heavy chains are present (band HC of gel, insert of Fig. 2) and the myosin contained a small amount of actin (band A of gel, insert of Fig. 2). The light chain components, L, and L2 of molecular weight 27,000 and 18,000, respectively, are characteristic of a cardiac myosin (24). Apparently, none of the light chains was lost during dinitrophenylation and the incorporation of 13HlN,ph-F was limited to the heavy chain region (Fig. 2). The heavy chain region contained 93% of the total radioactivity (zone HC and band

DINITROPHENYLATION

HC of gel, insert of Fig. 2). The two radioactive zones corresponding to band HC of the gel indicates that the heavy chains are heterogeneous and contaminating proteins of the M-line may be present (25, 26). The doublet band between band HC and A of the gel is probably C-protein (27) and some lower molecular weight M-line proteins (25, 26), but the doublet did not contain any radioactivity. Moreover, the light chain components L, and LZ contained only 5 and 2%, respectively, of the total radioactivity recovered. There was no incorporation of N,ph-F into the small amount of actin present. The recovery of radioactivity from the gel was 90%. To determine the specific amino acid residues dinitrophenylated, the [3H]Nzphmyosin was hydrolyzed in 6 N HCl and chromatographed on paper. Figure 3 shows a typical chromatogram of an 13HlNzph-myosin hydrolysate. Cysteine appears to be the major amino acid dinitrophenylated in cardiac myosin. More than 65% of the radioactivity recovered was found as S-N,ph-cysteine whereas the radioactive content of l -N,ph-lysine and ONzph-tyrosine was 13 and 11% respectively. In addition to changes in the Ca2+ and K+(EDTA)-ATPase activities there were major changes in the ability of the N,phmyosin to form Ca2+-sensitive actomyosin (Table II, upper part). There was an activation of the ATPase activity of an actomyosin made with Nzph-myosin when compared to a control (see Without EGTA samples in Table II, upper part). The enzymic activity of a reconstituted actomyosin made with a control cardiac myosin and the actin-tropomyosin-troponin complex was inhibited in the presence of EGTA. The actomyosin ATPase activity in the presence of EGTA was only 21% of an actomyosin assayed in the absence of EGTA (see With and Without EGTA samples in Table II, upper part). This is usually the case when an actomyosin is Ca2+ sensitive (1, 17). In contrast, the enzymic activity of a reconstituted actomyosin made with N2ph-cardiac myosin (which had incorporated 1.3 mol of [3H]N,ph-F per 5 x 105 g of protein) and the actin complex was not

OF

CARDIAC

209

MYOSIN

S-rlNP-CYS

2-

1-

Lone

number

FIG. 3. Paper chromatography of a t3H]N2ph-carmyosin hydrolysate treated with and without 2mercaptoethanol. The 13H]N2ph-myosin was treated with 2-mercaptoethanol as described under Methods and in Table II. The samples were hydrolyzed in uucuo with 6 N HCl for 16 h at 105°C and subsequently chromatographed on Whatman No. 3MM paper (44 x 45 cm). The hydrolysate (0.1 ml) from 2.5 mg of modified cardiac myosin was applied to the paper and ascending chromatography was carried out for 40 h in l-butanoliacetic acid/water (4:1:5, v/v/v; upper layer). The paper was cut into 1.2 x 5cm strips and counted for radioactivity. Solid line, 13HlNZph-myosin; dashed line, 13H]Nzph-myosin with 2-mercaptoethanol. DNP=N,ph=dinitrophenyl.

disc

inhibited in the presence of EGTA. The ATPase activity in the presence of EGTA was 86% of the actomyosin assayed in the absence of EGTA (see With and Without EGTA samples in Table II, upper part). The dinitrophenylation of cardiac myosin desensitized an actomyosin made with this modified myosin to calcium. A comparison of the influence of Ca2+ on the ATPase activity of a reconstituted actomyosin made from N,ph-myosin and the actin complex with an actomyosin made from a control myosin revealed a similar pattern of desensitization. In the presence of 5 mM Mg2+ and various concentrations of free Ca2+, activation of the actomyosin (containing a control cardiac myosin), ATPase activity was found to be half maxi-

210

GARY TABLE

II

ATPASE ACTIVITY OF ACTOMYOSIN RECONSTITUTED FROM 3H-L~~~~~~ DINITROPHENYL~~ARDIAC MYOSIN TREATED IN THE PRESENCE AND ABSENCE OF ZMERCAPTOETHANOL~ Sample

[WN,ph bound per 5 x 105g of myosin (mol)

Control N,ph-myosin

1.3

Control + mercaptoethanol N,ph-myosin + mercaptoethanol

0.62

ATPase activity (~mol of P, mu-’ mg-‘) Without EGTA

With EGTA

0.061 0.077

0.013 0.066

0.052

0.016

0.063

0.022

a Dinitrophenylated myosin (200 mg) in 40 ml of 0.6 M KC1 was treated with 2-mercaptoethanol at a final concentration of 0.05 M for 40 min at pH 6.5, 25°C. The [8H1N,phmyosin was isolated as described under Methods. Subsequently, 10 mg of protein were removed for determination of 13HlNzph-F bound to myosin. An actomyosin was reconstituted from this modified myosin and it was assayed for ATPase activity in the presence and absence of EGTA. For details, see Methods.

ma1 at 5 X lo+ M Ca2+ concentration (Fig. 4). This is an agreement with the data of Katz et al. (28). However, the ATPase activity of an actomyosin reconstituted from N,ph-cardiac myosin and the actin complex did not change at various Ca2+ concentrations (Fig. 4). The N,ph-myosin has failed to form Ca2+-sensitive actomyosin. Thiolysis Myosin

of Dinitrophenylated

BAILIN

stored. Furthermore, there was a loss of 0.7 mol of the N,ph-group per 5 x lo5 g of protein when N,ph-cardiac myosin was treated with 2-mercaptoethanol. Analysis of the N2ph-amino acids thiolyzed showed that more than half of the N2ph-group was cleaved from modified cysteine residues and there was some removal of the label from tyrosine and lysine residues (Fig. 3). Thiolytic cleavage of the Nzph-group from dinitrophenylated myosin by 2-mercaptoethanol also restored the Ca2+ and K+(EDTA)-ATPase activities (Table III). In this case, the removal of nearly 0.6 mol of the N2ph-group per 5 x lo5 g of protein resulted in the restoration of 62% of the K+(EDTA)-ATPase activity and the Ca2+ATPase activity (which was activated to 165%) was restored to control values (Table III). The restoration of the enzymic activities could also be due to the reduction of disulfide bonds by X-mercaptoethanol that could have formed in the modified myosin during the dinitrophenylation reaction. To test this possibility the N,phmyosin was carboxymethylated with iodoacetate and analyzed for its S-carboxy-

Cardiac

Treatment of [3H]N2ph-myosin with 0.05 2-mercaptoethanol at pH 8.5 for 40 min restored its ability to form a calcium-sensitive actomyosin (Table II, lower part). In the presence of EGTA, the ATPase activity of a reconstituted actomyosin made with thiolyzed [3H]N2ph-cardiac myosin and the actin complex was only 35% of the actomyosin assayed in the absence of EGTA. By comparison, a reconstituted actomyosin made with a control cardiac myosin had only 31% ATPase activity in the presence of EGTA (see Without and With EGTA samples in Table II, lower part). In other words, the Ca2+-sensitive response, measured by the extent of inhibition of the actomyosin ATPase activity with EGTA, is nearly the same in both has been recases. The Ca2+ sensitivity M

0 10-a

10-7

10-6 Cc?+

10-5

Wi

(Ml

FIG. 4. Effect of Ca2+ on the ATPase activity of The cardiac reconstituted cardiac actomyosin. myosin concentration was 0.24 mg/ml and the actin complex was 0.08 mg/ml. The assay was carried out at 25°C in 0.04 M KCl, 5 mM MgCl,, 25 mM histidine, 2 mM ATP at pH 6.8. Free Ca2+ concentrations were determined as described under Methods. Solid line, control myosin; dashed line, N,ph-myosin.

DINITROPHENYLATION TABLE COMPARISON

OF

III AND ATPASE DINITROPHENYL-MYOSIN

OF RADIOACTIVITY

ACTIVITY OF 3H-L~~~~~~ TREATED IN THE PRESENCE

AND

ABSENCE

OF 2-

MERCAPTOETHANOL”

Sample

Control + mercaptoethsn01 N,ph-myosin + mercaptoethanol

activity

Ca*+ (%I

1.20

100 165

100 16

100

100

92

70

Control N,ph-myosin

ATPase

WlNzph bound per 5 x 105 g of my&n (mol)

0.65

K+(EDTA) (%I

a Dinitrophenylated myosin wss treated as described in the footnote to Table II. The ATPase activity is expressed as Dercentaee of control. CaZ+-ATPase 100% value is 0.07 urn01 bf P, perminute per milligram of myosin. K+(EDTA)-kTPase 100% value is 0.15 qw31 of Pi per minute per milligram of myosin

methylcysteine content (16 and references therein). In a typical experiment, 35.6 and 34.0 mol of S-carboxymethylcysteine per 5 x lo5 g of protein were found for N,phmyosin treated with and without 2-mercaptoethanol, respectively. A control cardiac myosin value was 37.1 mol of S-carboxymethylcysteine. Taking into consideration the incorporation of N,ph-F (Tables I-III), the recovery of free -SH groups is nearly the same for both kinds of myosin. Therefore, the loss of the Nzph-group is responsible for the restoration of the ATPase activities. It is noteworthy that treatment of N,ph-cardiac myosin with 2-mercaptoethano1 or dithiothreitol did not result in the loss of any light chain components. DISCUSSION

Dinitrophenylation of cardiac myosin results in the activation of the Ca2+-ATPase activity and an inhibition of the K+(EDTA)-ATPase activity when only 1.3 mol of r3H]N,ph-F were incorporated per 5 x lo5 g of protein (Fig. 1). The reaction of cardiac myosin with different sulfhydryl reagents produces comparable changes in these activities (29-31). The same effects were also observed when rabbit skeletal myosin was treated with N,ph-F (23,32) or other reagents (33-37). These changes in ATPase activity seem to occur when cysteine residues of cardiac myosin are dini-

CARDIAC

MYOSIN

211

trophenylated (Fig. 3). It should be mentioned, however, that lysine and tyrosine residues were also modified. Similar results were obtained for N,ph-rabbit skeletal myosin (16, 23). The classical pattern of rapid changes in the ATPase activity of myosin has been interpreted in terms of the highly reactive -SH groups of the protein (16,32-37). Reisler et al. (32,38) have proposed that modification of sites containing these -SH groups prevents the formation of an inhibitory complex consisting of the -SH groups and Mg-ATP. The dinitrophenylation of rabbit skeletal myosin (23, 32) or cardiac myosin at reactive sulthydryl sites probably disrupts the complex effecting an increase in the Ca2+-ATPase activity. Accordingly, the reagents, N,ph-F or N-ethylmaleimide in vitro may be mimicking the way in which actin interacts with such a complex, and what follows is an activation of the ATPase activity (32, 38). The loss of K+(EDTA)-ATPase activity is more difficult to explain. In all probability both fast and slow reacting -SH groups are necessary (36). The dinitrophenyl group was located mainly in the globular head region of cardiac myosin which contains subfragment 1 (Table I). Only a small amount of the N2ph-group was found in light meromyosin, and subfragment 1 contained half of the label found in heavy meromyosin. From specific radioactivity calculations of the data in Table I (see Results), it is clear that all of the N,ph labeling is retained in subfragment 1. This would indicate that only one of the two subfragment 1 molecules was labeled. On the other hand, both of the subfragment 1 molecules could be equally labeled with N,ph-F. It is difficult to distinguish between these two possibilities at present. From dissociation experiments in sodium dodecyl sulfate it was found that the N2ph-group was localized in the heavy chain region, as only 7% of the label was present in light chains L, and L, (Fig. 2). This was the case for N,ph-rabbit skeletal myosin (23). It should be mentioned that the heterogeneous distribution of the radioactivity in the heavy chain region (Fig. 2) indicates that other compo-

212

GARY

nents, such as M-line protein and C-protein, may be present (25-27). N,ph-cardiac myosin failed to form a Caz+-sensitive actomyosin when 1.3 mol of r3H]Nzph-F were incorporated per 5 x lo5 g of protein (Table II). The ATPase activity of reconstituted actomyosin made with an untreated cardiac myosin was inhibited (79%) when assayed in the presence of EDTA. The actomyosin is CaZ+ sensitive (1, 17). However, the ATPase activity of a reconstituted actomyosin made from N,phmyosin assayed in the same way was only 14% inhibited. In other words, the Ca2+ sensitivity was lost. The findings are consistent with those obtained for rabbit skeletal myosin treated with N,ph-F (39) or other reagents (40-44). Moreover, the ATPase activity of this reconstituted actomyosin did not change as a function of Ca2+ concentration (Fig. 4). Thus, dinitrophenylation may block sites on myosin that are directly or indirectly involved in governing actin-myosin interaction. Thiolytic cleavage of Nzph-cardiac myosin by 2-mercaptoethanol results in the restoration of the ability of the modified myosin to form Ca’+-sensitive actomyosin (Table II, lower part). The inhibition values for the ATPase activity (assayed in the presence and absence of EGTA) of reconstituted actomyosins made from untreated cardiac myosin or N,phcardiac myosin were nearly the same: 69 and 65%, respectively. The Ca2+-sensitive response was restored, and 52% of the Nzph-group was removed from the thiolyzed Nzph-myosin. Furthermore, the Cazf and K+(EDTA)-ATPase activities were restored to control values when 46% of the Nzph-group was removed (Table III). Thiolysis of the Nzph-group from modified cysteine residues occurs during the restoration of the enzymic activity, but a loss also occurs from +Nzph-lysine and 0-N*ph-tyrosine (Fig. 3). These observations are quite similar to those of Nzph-rabbit skeletal myosin treated with 2-mercaptoethanol (16). Possibly, the site(s) which participate in the Ca2+ and K+(EDTA)-ATPase activities are also involved in the ability to form Ca2+-sensitive actomyosin (39). According to Seidel (45), however, the loss of Ca2+

BAILIN

sensitivity does not involve direct blocking of the -SH groups in rabbit skeletal myosin. Thus, the effects observed could be due to conformational changes induced by the reagent (45). The removal of the N2ph-group occurred at the subfragment 1 level and none of the light chains were lost before or after thiolytic cleavage of DNP-myosin (Fig. 2, gel insert). Similarly, Nbsz cardiac myosin did not lose any significant amount of light chain component (24, 46). This is somewhat surprising in view of recent studies which show that rabbit skeletal myosin treated with p-chloromercuribenzoate (47) or Nbsz (7, 46, 48) followed by addition of dithiothreitol, and scallop myosin (4) treated with EDTA result in the removal of light chain components (46-48) and a loss of the Ca2+-sensitive response (4, 7). Possibly the heavy chains of cardiac myosin are involved in regulating actinmyosin interaction. The dinitrophenylation of cysteine or tyrosine residues (16) at or near strategically located sites could make them unavailable for interaction with sites on the light chains and on the actin-troponin complex. As a result the Nzph-myosin loses its ability to form Ca’+sensitive actomyosin, and this is reflected in the actomyosin ATPase activity which was not inhibited in the presence of 1 mM EGTA (Table III). A regulatory role for cardiac myosin has been suggested (12) and -SH groups may be involved (13). Indeed, myosin-linked Ca2+ regulation has been indicated in muscles of various species (4-8). For example, scallop (4) and smooth muscles (5,49) do not contain troponin and contraction is regulated by myosin. Furthermore, the light chains of scallop (4) and rabbit skeletal myosin (7, 8) have been implicated in the regulatory process. In conclusion, cardiac myosin contains sites that may play a role in modulating interactions with the actin-tropomyosintroponin complex. ACKNOWLEDGMENTS The expert appreciated.

assistance

of Mu-Ju

Shen

is greatly

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