Peptide fragments of the heavy-chain region of phosphorylated and dinitrophenylated gizzard myosin.

Sulfopropyl (SP) Sephadex chromatography of trypsin-pepsin digests of dinitrophenylated gizzard myosin, pretreated with and without the myosin light-chain kinase calcium-calmodulin phosphorylating system, yielded similar elution patterns and nine peptide fractions were found. From a comparison with trypsin-pepsin digests of the heavy chains of dinitrophenylated myosin, pretreated with and without the phosphorylating system, it was established that peptide I, a major peptide fraction, was part of the 17-kDa dinitrophenylated light chain. Phosphorylation of myosin did not change the dinitrophenyl group content of peptide I but it did result in a significant increase in the dinitrophenylation of other peptides. The peptides contained only S-dinitrophenyl cysteine. Peptide III, previously considered to be part of the light-chain region (Bailin, G. and Lopez, F. (1982) J. Biol. Chem. 257, 264-270), was shown to originate in the heavy chains of myosin based on a comparison of the elution patterns of the digests of modified myosin and its heavy chains. Several neutral and basic peptides (peptides III to IX) originated in the heavy-chain region and they were different from those from the heavy chains of rabbit skeletal myosin. Phosphorylation of the 20-kDa light chain shifted the dinitrophenylation of the sulfhydryl groups from the 17-kDa light chain to the heavy chains of myosin, predominantly. These thiol groups do not resemble the fast-reacting -SH groups of rabbit skeletal myosin. The light chains are involved, in part, in making sites available on myosin that are necessary for actin-myosin interaction.

Crosslinking of sarcoplasmic reticulum ATPase protein with 1,5-difluoro 2,4-dinitrobenzene.

The reacton of 1,5-difluoro-2,4-dinitrobenzene with the ATPase protein of rabbit skeletal sarcoplasmic reticulum caused a marked loss of the Ca2+-dependent ATPase (ATP phosphohydrolase, EC 3.6.1.3) activity during an interval when 2 mol of the crosslinking reagent were incorporated/10(5) g of protein. The modified ATPase protein formed non-serial high molecular weight aggregates or oligomers during short (1--5 min) or long exposure (60 min) to the reagent at 25 degrees C or 4 degrees C. The same pattern was found when sarvoplasmic reticulum was treated similarly; only the ATPase protein formed oligomers (homopolymers). In all cases the ATPase protein monomer remained the predominant species present. During the appearance of the high molecular weight oligomers the Ca2+-ATPase activity was unaffected but Ca2+ uptake was inhibited. Major changes in the ATPase activity occurred when the monomeric ATPase protein was modified. Disubstituted dinitrophenylene derivatives of cysteine and tyrosine were found in modified ATPase protein and only a small amount of monosubstituted dinitrophenyl groups were identified. Thiolysis of the modified ATPase protein with 2-mercaptoethanol removed approx. 35% of the incorporated groups, but there was no restoration of the Ca2+-ATPase activity. Substrate MgATP2- protected the Ca2+-ATPase activity of the ATPase protein and sarcoplasmic reticulum but Ca2+ had no effect on the modificaton. Different conformational states of the ATPase protein could be ascertained from a comparison of the effects of Ca2+ and MgATP2- on the bifunctional reagent dinitrophenylation of the ATPOase protein with that of the monofunctional reagent 1-fluoro-2,4-dinitrobenzene (Bailin, G. (1980) Biochim. Biophys. Acta 623, 213-224). Intramolecular crosslinking of the ATPase protein predominated and oligomers which formed during the reaction were not essential for the maintenance of the ATPase activity.

Dinitrophenylation of rabbit skeletal sarcoplasmic reticulum ATPase protein.

The ATPase (ATP phosphohydrolase (EC 3.6.1.3)) protein of rabbit skeletal sarcoplasmic reticulum rapidly incorporated three mol of 1-fluoro-2,4-dinitrobenzene per 10(5) g of protein with little change in the Ca2+-dependent ATPase activity. When 2 additional mol of the reagent were bound the Ca2+-dependent ATPase activity was inhibited. The dinitrophenyl group was located mainly in the ATPase protein and a small amount of the label was found in the proteolipid component of the ATPase preparation as judged by dissociation experiments in sodium dodecyl sulfate. Cysteine and tyrosine residues were dinitrophenylated in the modified ATPase protein. Thiolysis of the dinitrophenylated ATPase protein with 2-mercaptoethanol under various conditions did not restore the Ca2+-dependent ATPase activity. Solubilization of the ATPase protein with sodium deoxycholate increased the reactivity of the reagent and the Ca2+-dependent ATPase activity was inhibited to a greater extent. Dinitrophenylation of the ATPase protein was Ca2+-dependent; in the presence of high Ca2+ the incorporation increased by 50% and a large decrease in the Ca2+-ATPase activity was noted. The half-maximal changes for the incorporation of the reagent and the inhibition of the Ca2+-ATPase activity occurred at 3--4 microgram Ca2+-concentration, consistent with the binding of Ca2+ to high affinity sites on the ATPase protein. These results indicate that the ATPase protein as a Ca2+-free and a Ca2+-bound conformation. The reagent, 1-fluoro-2,4-dinitrobenzene reacts differentially and thus characterizes these two conformations.

Reaction of 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole with the (Ca2+ + Mg2+)- ATPase protein of sarcoplasmic reticulum at low temperature.

Modification of the (Ca2+ + Mg2+)-ATPase protein of rabbit skeletal sarcoplasmic reticulum (SR) with 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole, NBD-Cl, at 4 degrees C for 5 min caused a 63% loss of the Ca(2+)-dependent ATPase activity when 1 mol of the adenine analog was incorporated per 10(5) g of protein. At 25 degrees C, above the lipid phase transition, the extent of labeling was 3-fold higher although the Ca(2+)-ATPase activity was inhibited to the same extent. MgATP protected the ATPase activity at 4 degrees C and 25 degrees C but there was little change in the extent of labeling at 4 degrees C suggesting that changes in the fluidity of the lipid moiety made different sites on the ATPase protein accessible to the reagent. At 4 degrees C, addition of sodium deoxycholate enhanced the inactivation (6% ATPase activity remained) but the labeling of the SR-ATPase protein did not increase significantly. Incubation with MgATP prior to solubilization with deoxycholate resulted in the protection of the Ca(2+)-ATPase activity and only a small decrease in the labeling occurred. At 25 degrees C, a similar pattern was found with deoxycholate but the loss of ATPase activity was less dramatic and the extent of labeling by NBD-Cl was greater than that at 4 degrees C. MgATP induced changes in the conformation of the ATPase protein protecting essential cysteine residues while shifting the reaction of NBD-Cl with the ATPase protein to non-essential sites in the absence or presence of deoxycholate. An analysis of tryptic digests of the NBD-ATPase protein showed that MgATP shifted the labeling from the A2 subfragment to the A1 subfragment in the absence of deoxycholate and from the A1 subfragment to the A2 subfragment in the presence of deoxycholate. The reagent, NBD-Cl, can distinguish between different temperature dependent conformational states of the ATPase protein.

Dinitrophenylation of rabbit skeletal actomyosin.

Dinitrophenylated reconstituted or natural actomyosin effected changes in the Ca2+ sensitivity which were dependent upon the ionic strength of the reaction medium. Dinitrophenylation of reconstituted actomyosin in 0.6 M KCl led to the incorporation of 2-6 mol of the reagent per 5-10(5) g of protein and it possessed considerable Ca2+ sensitivity. Dinitrophenylated natural actomyosin under the same conditions lost most of its Ca2+ sensitivity when 1.3-5.4 mol of the dinitrophenyl group were bound. The myosin from these modified actomyosins did not lose Ca2+ sensitivity and the myosin was labeled only with 0.4-1.7 mol of the dinitrophenyl group. Dinitrophenylation of both kinds of actomyosin in 0.06 M KCl abolished the Ca2+ sensitivity; the myosin from the modified actomyosins also lost Ca2+ sensitivity. Myosin alone was more susceptible to a loss of Ca2+ sensitivity than myosin in actomyosin. Actin protected the ability of myosin to sense Ca2+ regulated actin in modified actomyosin at 0.6 M KCl but not at 0.06 M KCl. Actomyosin dinitrophenylated in the presence of ATP lost Ca2+ sensitivity. However, the myosin from this actomyosin possessed Ca2+ sensitivity. Thiolysis of the dinitrophenylated actomyosin by 2-mercaptoethanol at low ionic strength did not restore the Ca2+ sensitivity of this actomyosin or its myosin although there was a significant loss of the dinitrophenyl group.

Myosin and actomyosin from human skeletal muscle.

Human skeletal natural actomyosin contained actin, tropomyosin, troponin and myosin components as judged by polyacrylamide gel electrophoresis in sodium dodecyl sulfate. Purified human myosin contained at least three light chains having molecular weights (+/-2000) of 25 000, 18 000 and 15 000. Inhibitory and calcium binding components of troponin were identified in an actin-tropomyosin-troponin complex extracted from acetone-dried muscle powder at 37 degrees C. Activation of the Mg-ATPase activity of Ca2+-sensitive human natural or reconstituted actomyosin was half maximal at approximately 3.4 muM Ca2+ concentration (CaEGTA binding constant equals 4.4 - 10(5) at pH 6.8). Subfragment 1, isolated from the human heavy meromyosin by digestion with papain, appeared as a single peak after DEAE-cellulose chromatography. In the pH 6-9 range, the Ca2+-ATPase activity of the subfragment 1 was 1.8- and 4-fold higher that the original heavy meromyosin and myosin, respectively. The ATPase activities of human myosin and its fragments were 6-10 fold lower than those of corresponding proteins from rabbit fast skeletal muscle. Human myosin lost approximately 60% of the Ca2+-ATPase activity at pH 9 without a concomitant change in the number of distribution of its light chains. These findings indicate that human skeletal muscle myosin resembles other slow and fast mammalian muscles. Regulation of human skeletal actomyosin by Ca2+ is similar to that of rabbit fast or slow muscle.

Phosphorylation of an actin.tropomyosin.troponin complex from human skeletal muscle.

A human skeletal actin.tropomyosin.troponin complex was phosphorylated in the presence of [gamma-32 P]ATP, Mg2+, adenosine 3':5'-monophosphate (cyclic AMP) and cyclic AMP-dependent protein kinase (protein kinase). Phosphorylation was not observed when the actin complex was incubated in the absence of protein kinase or 1 microM cyclic AMP. In the presence of 10(-7) M Ca2+ and protein kinase 0.1 mole of [32P]phosphate per 196 000 g of protein was incorporated. This was two-fold higher than the [32P]phosphate content of a rabbit skeletal actin complex but two-fold lower than that of a bovine cardiac actin complex. At high Ca2+, 5.10(-5) M, little change in the phosphorylation of a human skeletal actin complex occurred. Phosphoserine and phosphothreonine were identified in the [32P]phosphorylated actin complex. Polyacrylamide gel electrophoresis in sodium dodecyl sulfate showed that 60% of the label was associated with the tropomyosin binding component of troponin. The inhibitory component of troponin contained 16% of the bound [32P]phosphate. Increasing the Ca2+ concentration did not significantly decrease the [32P]phosphate content of the phosphorylated proteins in the actin complex. No change in the distribution of phosphoserine or phosphothreonine was observed. Half maximal calcium activation of the ATPase activity of reconstitute human skeletal actomyosin made with the [32P] phosphorylated human skeletal actin complex was the same as a reconstituted actomyosin made with an actin complex incubated in the absence of protein kinase at low or high Ca2+.

Dinitrophenylation of phosphorylated gizzard myosin. Labelling of the subfragment 1 region.

Chymotrypsin and papain digestion of 3H-labelled dinitrophenylated chicken gizzard myosin, pretreated with the myosin light chain catalyzed phosphorylating system, released a subfragment 1 devoid of its light chains but containing all of the label associated with the thiols of the heavy chain region. This was also the case when myosin was incubated in the presence of the phosphorylating system but without ATP. Dinitrophenylation of a reconstituted actomyosin made from phosphorylated myosin occurred mainly on -SH groups of the heavy chains, in contrast to the predominant modification of the light chains of myosin from a control actomyosin that was treated similarly. Conformational changes in myosin may govern, in part, actin-myosin interaction through the phosphorylation of the light chain of Mr 20.000.

Dinitrophenylation of chicken gizzard myosin: reactivity of the 17 000-dalton light chain.

Chicken gizzard myosin rapidly incorporated 3 mol of 1-fluoro-2,4-dinitrobenzene per 4.7 x 10(5) g of protein with little change in the ATPase (ATP phosphohydrolase, EC 3.6.1.3) activity. During an interval when 2 additional mol of the reagent were bound the K+-ATPase activity in the presence of EDTA was inhibited and the Ca2+-ATPase activity was altered to a lesser extent. Cysteine residues were modified in the dinitrophenylated gizzard myosin. The dinitrophenyl group was located mainly in the active proteolytic fragment, subfragment 1. Dinitrophenylation of the heavy and light chains was observed but major changes in the ATPase activity occurred when the 17 000-dalton light chain and some heavy chains were modified as judged by dissociation experiments in sodium dodecyl sulfate. Thiolysis of the dinitrophenylated gizzard myosin with 2-mercaptoethanol restored the ATPase activity and approx. 2 mol of the dinitrophenyl group were removed. The restoration of the enzymic activity, however, occurred when 1 mol of the label was thiolytically cleaved from cysteine residues of the 17 000-dalton light chain. Substrate Mg-ATP(2-) or MgADP did not protect the ATPase activity of modified gizzard myosin. In the presence of nucleotide there was an increase in the incorporation of the reagent, and a change in its distribution into the light and heavy chains. Calcium had no effect on the dinitrophenylation of this myosin. these results indicate that the reagent, 1-fluoro-2,4-dinitrobenzene, could detect chemical differences in smooth muscle myosin when compared to the reactivity of other myosins. Thiol groups of the 17 000-light chain (and some heavy chains) are probably located peripheral to the active site region of gizzard myosin and they are involved in maintaining the enzymic activity of this protein.

Modification of the (Ca2+ + Mg2+)-ATPase protein of sarcoplasmic reticulum with 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole.

The (Ca2+ + Mg2+)-ATPase (ATP phosphohydrolase (Ca2+-transporting), EC 3.6.1.38) protein of rabbit skeletal sarcoplasmic reticulum (SR) rapidly incorporated 2 mol of 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole (NBD-Cl) per 10(5) g of protein with little change in the Ca2+-dependent ATPase activity. When 2 additional mol of the reagent were bound the Ca2+-ATPase, activity was inhibited. The same pattern was found for modified intact SR and the Ca2+ uptake ability was inhibited. MgATP, CaATP and MgADP protected the Ca2+-ATPase activity concurrent with a decrease of about 1 mol of the NBD group per 10(5) g protein, but the Ca2+ uptake ability was not protected. Calcium alone had no effect on the modification. The modified ATPase protein or SR formed non-serial oligomers or aggregates, but the ATPase protein remained the predominant species present. In the presence of MgATP, oligomer formation was reduced partially but the major changes in the Ca2+-ATPase activity were due to the modification of the ATPase monomer. Thiolysis of the NBD-ATPase protein with dithiothreitol did not restore the Ca2+-ATPase activity, although more than 1 mol of the NBD group was removed from cysteine residues. Cysteine residues were modified in the NBD-ATPase protein or SR when the enzyme activity was inhibited. Trypsin digestion of NBD-SR or its ATPase protein released the A, B, A1, and A2 fragments. The A fragment and its subfragment A2 contained most of the label. Substrate MgATP protection studies showed that the A1 and A2 fragments were involved in maintaining the Ca2+-ATPase activity. Reagent-induced conformational changes of these fragments rather than direct active site group labeling accounted for the loss of ATPase activity.

Cooperative interactions between the contractile proteins of cardiac and skeletal muscle.

The calcium activation of the ATPase (ATP phosphohydrolase, EC 3.6.1.3) activity of cardiac actomyosin reconstituted from bovine cardiac myosin and a complex of actin-tropomyosin-troponin extracted from bovine cardiac muscle at 37 degrees C was studied and compared with similar proteins from rabbit fast skeletal muscle. The proteins of the actin complex were identified by polyacrylamide gel electrophoresis in sodium dodecyl sulfate. Half-maximal activation of the cardiac actomyosin was seen at a calcium concentration of 1.2 +/- 0.002 (S.E. of mean) muM. A hybridized reconstituted actomyosin made with cardiac myosin and the actin-tropomyosin-troponin complex extracted from rabbit skeletal muscle was also activated by calcium but the half-maximal value was shifted to 0.65 +/- 0.02 (S.E. of mean) muM Ca2+. Homologous rabbit skeletal actomyosin showed half-maximal activation at 0.90 +/- 0.01 (S.E. of mean) muM Ca2+ and the value for a hybridized actomyosin made with rabbit skeletal myosin and the actin-complex from cardiac muscle was found at 1.4 +/- 0.03 (S.E. of mean) muM Ca2+ concentration. Kinetic analysis of the Ca2+ activated ATPase activity of reconstituted bovine cardiac actomyosin indicated some degree of cooperativity with respect to calcium. Double reciprocal plots of reconstituted actomyosins made with bovine cardiac actin complex were curvilinear and significantly different than those of reconstituted actomyosins made with the rabbit fast skeletal actin complex. The Ca2+-dependent cooperativity was of a mixed type as determined from Hill plots for homologous reconstituted bovine cardiac and rabbit fast skeletal actomyosin. The results show that cooperative interactions in reconstituted actomyosins were greater when the actin-tropomyosin-troponin complex was derived from cardiac than skeletal muscle.

Fluorescence properties of the Ca2+,Mg2(+)-ATPase protein of sarcoplasmic reticulum labeled with 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole.

The fluorescence intensity of the Ca2+,Mg2(+)-ATPase protein of rabbit skeletal sarcoplasmic reticulum that incorporated about 2 mol of 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole (NBD-Cl) was enhanced at high MgATP concentrations with or without 50 microM calcium. The observed enhancement indicates that the fluorophore, NBD-Cl, can detect conformational changes in the ATPase protein.

Effect of phosphorylation and dinitrophenylation on chicken gizzard myosin.

Treatment of phosphorylated chicken gizzard myosin which had incorporated 1.5 mol of phosphate per 4.7 x 10(5) g of protein with 1-fluoro-2,4-dinitrobenzene resulted in the modification of the heavy and light chains when 5.8 mol of the reagent were bound to myosin. Concurrently, the K+-ATPase activity was inhibited and the modified myosin possessed actin activated-ATPase activity. Thiolysis of nearly 2 mol of the dinitrophenyl group mainly from the heavy chains (and some light chains) of the modified myosin with 2-mercaptoethanol restored the K+-ATPase activity. Digestion of phosphorylated gizzard myosin with chymotrypsin or papain occurred to a lesser extent than a control myosin. Chymotryptic fragments of phosphorylated and dinitrophenylated myosin were formed at a faster rate than those of dinitrophenylated myosin alone suggesting that phosphorylation of the light chain of Mr 20,000 altered the susceptibility of the heavy chains of myosin to proteolysis. Phosphorylation of dinitrophenylated gizzard myosin which had incorporated 5.5 mol of 1-fluoro-2,4-dinitrobenzene per 4.7 x 10(5) g of protein was the same as that of a control myosin; this was also the case for the thiolyzed dinitrophenylated myosin. In the absence of calcium, phosphorylation of control and dinitrophenylated myosins decreased by 73% suggesting that the phosphorylation reaction was calcium dependent. Phosphorylation and dinitrophenylation induced conformational changes in the light chains of gizzard myosin that may be involved in maintaining the structure of the heavy chain region.

Modification of gizzard myosin and reconstituted actomyosin with 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole at low and high ionic strength.

Treatment of reconstituted gizzard actomyosin at 0.15 M or 0.6 M KCl with the fluorescent adenine analog 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole, NBD-Cl, resulted in a significant decrease in the labeling of the myosin from actomyosin compared to that of myosin alone. Actin protected partially the K(+)-ATPase activity of myosin from modified actomyosin. The reagent was able to detect changes in the conformation of myosin as the distribution of the label in the heavy and light chains of myosin and actin was different at 0.15 M and 0.6 M KCl. The 6S and 10S transition, unique to smooth muscle myosin, can be monitored with the aid of this reagent.