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.     

Proteolytic susceptibility of both isolated and bound light chains from various myosins to myopathic hamster protease

Myopathic hamster protease was incubated with turkey gizzard, scallop adductor, and Loligo mantle retractor myosins in order to establish if the regulatory light chain could be selectively digested. In contrast to cardiac or skeletal muscle myosin in which almost all of the regulatory light chain is degraded, these light chains from smooth and invertebrate muscle myosins were remarkably resistant to proteolysis. In the case of scallop myosin, increasing the protease to myosin ratio resulted in comparable digestions of both the regulatory and essential light chains regardless of the presence of Mg2+. The isolated light chains on the other hand were readily digested into smaller fragments. In addition, it was observed that the myosin heavy chains were extremely sensitive and that it was possible to cleave them quantitatively to produce a new band moving with a mobility on SDS gels corresponding to an Mr of approximately 150,000. This was again at variance with cardiac or skeletal myosin where the breakdown of the heavy chains was shown to be minimal. In spite of the significant extent of heavy chain cleavage, gizzard myosin appears to maintain its tertiary structure as demonstrated by sedimentation velocity and equilibrium ultracentrifugation analysis. Moreover, upon examination by electron microscopy, both intact and cleaved gizzard myosin revealed the characteristic folded structure which had a sedimentation rate of about 10 S when dialyzed into a low salt, Mg X ATP-containing buffer. The effects and implications of such modifications on catalytic activities of gizzard, scallop, and Loligo myosins are discussed in detail.     

Inhibition of conformational change in smooth muscle myosin by a monoclonal antibody against the 17-kDa light chain

Monoclonal antibodies against gizzard smooth muscle myosin were generated and characterized. One of these antibodies, designated MM-2, recognized the 17-kDa light chain and modulated the ATPase activities and hydrodynamic properties of smooth muscle myosin. Rotary shadowing electron microscopy showed that MM-2 binds 51 (+/- 25) A from the head-rod junction. The depression of Ca2+- and Mg2+-ATPase activities of myosin and Ca2+-ATPase activity of heavy meromyosin at low KCl concentration were abolished by MM-2. Viscosity measurement indicated that MM-2 inhibits the transition of 6 S myosin to 10 S myosin. While the rate of the production of subfragment-1 by papain proteolysis of 6 S myosin was inhibited by MM-2, the rate of proteolysis of the heavy chain of 10 S myosin was enhanced by MM-2 and reached the same rate as that of 6 S myosin plus MM-2. These results suggest that MM-2 inhibits the formation of 10 S myosin by binding to the 17-kDa light chain which is localized at the head-neck region of the myosin molecule. MM-2 increased the Vmax of actin-activated Mg2+-ATPase activities of both dephosphorylated myosin and dephosphorylated heavy meromyosin about 10- and 20-fold, respectively. MM-2 also activated the actin-activated Mg2+-ATPase activity of phosphorylated myosin at a low MgCl2 concentration and thus abolished the Mg2+-dependence of acto phosphorylated myosin ATPase activity. These results suggest that MM-2 inhibits the formation of 10 S myosin, and this results in the activation of actin-activated Mg2+-ATPase activity even in the absence of phosphorylation.     

Dinitrophenylated thiols in tryptic fragments of the heavy chain from chicken gizzard myosin

Trypsin digestion of phosphorylated and 3H-labeled dinitrophenylated chicken gizzard myosin released major fragments of Mr 29,000, 50,000 and 66,000 in a ratio of close to one to one. They contained 58% of the label bound to thiols of the heavy chains; 28% of the label was bound to the light chains. The heavy chain fragments of Mr 29,000 and Mr 66,000 were dinitrophenylated when the enzyme activity was inhibited. The 3H-labeled dinitrophenylated myosin alone followed a somewhat different pattern in that the label was bound to the light chains predominantly. Thiolysis of the phosphorylated and dinitrophenylated myosin with 2-mercaptoethanol restored the K+ -ATPase (ATP phosphohydrolase, EC 3.6.1.32) activity and the dinitrophenyl group was removed from the N-terminal fragment of Mr 29,000 of the heavy chain, predominantly. In contrast, restoration of the enzymic activity occurred in thiolyzed dinitrophenylated myosin alone when the label was removed from the light chains rather than the tryptic fragments of the heavy chain. Phosphorylation induced conformational changes in gizzard myosin that altered the reactivity of the thiols in fragments of the globular heavy chain region.     

Localization and topography of antigenic domains within the heavy chain of smooth muscle myosin

We have produced and characterized monoclonal antibodies that label antigenic determinants distributed among three distinct, nonoverlapping peptide domains of the 200-kD heavy chain of avian smooth muscle myosin. Mice were immunized with a partially phosphorylated chymotryptic digest of adult turkey gizzard myosin. Hybridoma antibody specificities were determined by solid-phase indirect radioimmunoassay and immunoreplica techniques. Electron microscopy of rotary-shadowed samples was used to directly visualize the topography of individual [antibody.antigen] complexes. Antibody TGM-1 bound to a 50-kD peptide of subfragment-1 (S-1) previously found to be associated with actin binding and was localized by immunoelectron microscopy to the distal aspect of the myosin head. However, there was no antibody-dependent inhibition of the actin-activated heavy meromyosin ATPase, nor was antibody TGM-1 binding to actin-S-1 complexes inhibited. Antibody TGM-2 detected an epitope of the subfragment-2 (S-2) domain of heavy meromyosin but not the S-2 domain of intact myosin or rod, consistent with recognition of a site exposed by chymotryptic cleavage of the S-2:light meromyosin junction. Localization of TGM-2 to the carboxy-terminus of S-2 was substantiated by immunoelectron microscopy. Antibody TGM-3 recognized an epitope found in the light meromyosin portion of myosin. All three antibodies were specific for avian smooth muscle myosin. Of particular interest is that antibody TGM-1, unlike TGM-3, bound poorly to homogenates of 19-d embryonic smooth muscles. This indicates the expression of different myosin heavy chain epitopes during smooth muscle development.     

N-terminal region of gizzard myosin heavy chain is critical for the ATPase activity

Two different HMM species of gizzard myosin were prepared under conditions such that the phosphorylation of light chain was fully maintained. They were different in the N-terminal structure of the heavy chain but not in the light chain composition. A significant decrease in the Mg2+-ATPase activity was observed in one class of HMM which was proteolytically cleaved intramolecularly at site 1, 5 K daltons from the masked N terminus. Another class of HMM without the cleavage at site 1 showed ATPase activity similar to that of myosin. The decrease in ATPase activity was not caused by denaturation since similar amounts of initial burst of Pi liberation were observed with both HMMs and myosin. Kinetic and substructure analyses of HMM revealed that the activity change depended solely on the cleavage at site 1. The N-terminal region of gizzard myosin heavy chain may thus have an important role in maintaining the active site structure.     

Proteolysis of smooth muscle myosin by Staphylococcus aureus protease: preparation of heavy meromyosin and subfragment 1 with intact 20 000-dalton light chains

The proteolysis of gizzard myosin by Staphylococcus aureus protease produces both heavy meromyosin and subfragment 1 in which the 20 000-dalton light chains are intact, and conditions are suggested for the preparation of each. Cleavage of the myosin heavy chain to produce subfragment 1 is dependent on the myosin conformation. Proteolysis of myosin in the 10S conformation yields predominantly heavy meromyosin, and myosin in the 6S conformation yields mostly subfragment 1 and some heavy meromyosin. Two sites are influenced by myosin conformation, and these are located at approximately 68 000 and 94 000 daltons from the N-terminus of the myosin heavy chain. The latter site is thought to be located at the subfragment 1-subfragment 2 junction, and cleavage at this site results in the production of subfragment 1. The time courses of phosphorylation of both heavy meromyosin and subfragment 1 can be fit by a single exponential. The actin-activated Mg2+-ATPase activity of heavy meromyosin is markedly activated by phosphorylation of the 20 000-dalton light chains. From the actin dependence of Mg2+-ATPase activity the following values are obtained: for phosphorylated heavy meromyosin, Vmax approximately 5.6 s-1 and Ka (the apparent dissociation constant for actin) approximately 2 mg/mL; for dephosphorylated heavy meromyosin, Vmax approximately 0.2 s-1 and Ka approximately 7 mg/mL. The actin-activated ATPase activity of subfragment 1 is not influenced by phosphorylation, and Vmax and Ka for both the phosphorylated and dephosphorylated forms are 0.4 s-1 and 5 mg/mL, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)     

The effect of actin and phosphorylation on the tryptic cleavage pattern of Acanthamoeba myosin IA

The Mg2+-ATPase activity of Acanthamoeba myosin IA is activated by F-actin only when the myosin heavy chain is phosphorylated at a single residue. In order to gain insight into the conformational changes that may be responsible for the effects of F-actin and phosphorylation on myosin I ATPase, we have studied their effects on the proteolysis of the myosin IA heavy chain by trypsin. Trypsin initially cleaves the unphosphorylated, 140-kDa heavy chain of Acanthamoeba myosin IA at sites 38 and 112 kDa from its NH2 terminus and secondarily at sites 64 and 91 kDa from the NH2 terminus. F-actin has no effect on tryptic cleavage at the 91- and 112-kDa sites, but does protect the 38-kDa site and the 64-kDa site. Phosphorylation (which occurs very near the 38-kDa site) has no detectable effect on the tryptic cleavage pattern in the absence of F-actin or on F-actin protection of the 64-kDa site, but significantly enhances F-actin protection of the 38-kDa site. Protection of the 64-kDa site is probably due to direct steric blocking because F-actin binds to this region of the heavy chain. The protection of the 38-kDa site by F-actin may be the result of conformational changes in this region of the heavy chain induced by F-actin binding near the 64-kDa site and by phosphorylation. The conformational changes in the heavy chain of myosin IA that are detected by alterations in its susceptibility to proteolysis are likely to be related to the conformational changes that are involved in the phosphorylation-regulated actin-activated Mg2+-ATPase activities of Acanthamoeba myosins IA and IB.     

Trinitrophenylation of smooth muscle myosin

The reaction of trinitrobenzenesulfonate with gizzard myosin was studied. The initial phase of the reaction involved two residues and at this level of modification the following was observed: the Mg2+-ATPase of myosin, the actin-activated ATPase of phosphorylated myosin and the phosphorylation kinetics of myosin were not affected. However, trinitrophenylation did induce an activation of the actin-activated ATPase of dephosphorylated myosin and in this respect mimicked the effect of light chain phosphorylation. The Mg2+-dependence of actin-activated ATPase also is altered on trinitrophenylation. These alterations of enzymatic properties could be at least partly explained by the finding that trinitrophenylation favored the 6S conformation of myosin.     

Effect of monoclonal antibodies on the properties of smooth muscle myosin

Monoclonal antibodies were generated against turkey gizzard myosin, and their effects on some of the properties of myosin were assayed. Ca2+- and Mg2+-ATPase activities of myosin were enhanced by the anti-subfragment 2 antibodies at low ionic strength (i.e., with 10S myosin). Tryptic fragments of an anti-S2 IgM also activated these activities. Antibodies directed against subfragment 1 and light meromyosin had no effect. The Mg2+-ATPase activity of heavy meromyosin also was activated by an anti-S2 antibody. Actin-activated ATPase activity of phosphorylated myosin was enhanced by the anti-S2 IgM fragments at low MgCl2 concentrations. This increase was reflected by a 5-fold increase in Vmax and a slight decrease in the apparent dissociation constant for actin. The actin-activated ATPase of dephosphorylated myosin was not affected by intact anti-S2 antibody or its fragments. The rates of phosphorylation and dephosphorylation of the 20,000-dalton light chains were increased by interaction of myosin with anti-S2 antibody. Limited proteolysis of myosin was used as a conformational probe. Interaction of anti-S2 antibody with 10S myosin increased the extent of cleavage at the S1-S2 junction. Proteolysis of 6S myosin was rapid and was not influenced by anti-S2 antibody. Our interpretation of these results is that interaction of the anti-S2 antibodies with myosin alters the conformation in the S2 region and this in turn modifies some of the properties of myosin. This is consistent with the hypothesis that the S2 region of smooth muscle myosin is a determinant of its biological properties.     

The effect of cleavage at site 1 of gizzard HMM in the interaction with skeletal muscle actin

Two different classes of gizzard heavy meromyosins (HMMs) were prepared from phosphorylated myosin by chymotryptic digestion in the presence and absence of ATP and were compared with respect to their actin-activated Mg2+-ATPase reactions. One class of HMM, named HMM(+), had a cleavage at site 1 in the N terminal portion of the heavy chain and the other class of HMM, named HMM(-), had no cleavage at this site. Maximum turnover rate (Vmax) of the skeletal acto-gizzard HMM Mg2+-ATPase reaction was obviously different between HMM(+) and HMM(-). The Vmax value of HMM(+) was 2.5-fold larger than that of HMM(-). On the other hand, the apparent association constants (Ka) of skeletal muscle actin for both HMMs which were deduced from double reciprocal plots (v-1 versus [actin]-1) seemed to be identical. The difference in Vmax value was attributed to the cleavage at site 1 since a following chymotryptic cleavage of HMM(-) at site 1 caused a 2.5-fold increase in the Vmax value. That site 1 in the N terminal portion of the gizzard myosin heavy chain was the key locus for the actin-myosin interaction was shown in addition to our previous finding of the effects of cleavage at site 1 on the ATPase activity and nucleotide binding ability of gizzard HMM (Okamoto, Y. & Sekine, T. (1981) J. Biochem. 90, 833-843; 843-949).     

In situ phosphorylation of human platelet myosin heavy and light chains by protein kinase C

Treatment of human platelets with 162 nM 12-O-tetradecanoylphorbol-13-acetate (TPA) resulted in phosphorylation of a number of peptides, including myosin heavy chain and the 20-kDa myosin light chain. The site phosphorylated on the myosin heavy chain was localized by two-dimensional peptide mapping to a serine residue(s) in a single major tryptic phosphopeptide. This phosphopeptide co-migrated with a tryptic peptide that was produced following in vitro phosphorylation of platelet myosin heavy chain using protein kinase C. The sites phosphorylated in the 20-kDa myosin light chain in intact cells were analyzed by two-dimensional mapping of tryptic peptides and found to correspond to Ser1 and Ser2 in the turkey gizzard myosin light chain. In vitro phosphorylation of purified human platelet myosin by protein kinase C showed that in addition to Ser1 and Ser2, a third site corresponding to Thr9 in turkey gizzard myosin light chain is also phosphorylated. The phosphorylatable myosin light chains from human platelets were found to consist of two major isoforms present in approximately equal amounts, but differing in their molecular weights and isoelectric points. A third, minor isoform was also visualized by two-dimensional gel electrophoresis. Following treatment with TPA, both the mono- and diphosphorylated forms of each isoform could be visualized, and the sites of phosphorylation were identified. The phosphate content rose from negligible amounts found prior to treatment with TPA to 1.2 mol of phosphate/mol of myosin light chain and 0.7 mol of phosphate/mol of myosin heavy chain following treatment. These results suggest that TPA mediates phosphorylation of both myosin light and heavy chains in intact platelets by activation of protein kinase C.     

Effects of tryptic digestion on the enzymatic activites of chicken gizzard myosin.

Tryptic digestion of gizzard myosin resulted in the degradation of the 20K light chain (G1) to its 17K fragment, which could not be phosphorylated. The rapid loss of Ca2+-dependent activation of actomyosin ATPase activity accompanied the degradation of G1. Increase in the Ca2+-ATPase activity and decrease in the EDTA-ATPase activity of myosin accompanied the degradation of myosin heavy chain, but not the cleavage of G1.     

Changes in myosin isozymes during development of chicken gizzard muscle

The distribution of myosin isozymes in embryonic and adult chicken gizzard muscle were examined by electrophoresis in a non-denaturing gel system (pyrophosphate acrylamide gel electrophoresis), and both light and heavy chains of embryonic and adult myosin isozymes were compared. In pyrophosphate acrylamide gel electrophoresis, there were three isozyme components in embryonic gizzard myosin, but only one isozyme in adult gizzard myosin. The mobility of the fastest migrating embryonic isozyme was similar to that of the adult isozyme. The three embryonic isozymes differ from each other in the light chain distribution. Two of them contain an embryo-specific myosin light chain, which is characterized by its molecular weight and isoelectric point, whereas the other embryonic myosin isozyme contained the same light chains as the adult myosin. The pattern of peptide fragments of embryonic heavy chain produced by digestion with alpha-chymotrypsin in the presence of SDS was not distinguishable from that of adult myosin heavy chain. Thus there are myosin isozymes specific to embryonic gizzard muscle which exhibit embryo-specific light chain compositions, but are similar to adult gizzard myosin in their heavy chain structure.     

Liver cell myosin: isolation and properties

We have purified myosin from isolated rabbit liver cells that had been previously shown to be well separated from blood vessels and connective tissue (Okamoto, Y. et al. (1983) J. Biochem. 94, 645-653). It comprises a 200-kDa heavy chain and light chains of 24-kDa, 22-kDa, and 17-kDa. In the light chain composition and in the mobility in PPi-PAGE, liver cell myosin differs from the myosin in liver blood vessels. The light chains of liver cell myosin were phosphorylated by myosin light-chain kinase from chicken gizzard and the Mg2+-ATPase activity of phosphorylated myosin was activated 10-fold by F-actin.     

Interaction of the heavy chain of gizzard myosin heads with skeletal F-actin

To probe the molecular properties of the actin recognition site on the smooth muscle myosin heavy chain, the rigor complexes between skeletal F-actin and chicken gizzard myosin subfragments 1 (S1) were investigated by limited proteolysis and by chemical cross-linking with 1-ethyl-3-[3-(dimethyl-amino)propyl]carbodiimide. Earlier, these approaches were used to analyze the actin site on the skeletal muscle myosin heads [Mornet, D., Bertrand, R., Pantel, P., Audemard, E., & Kassab, R. (1981) Biochemistry 20, 2110-2120; Labbé, J.P., Mornet, D., Roseau, G., & Kassab, R. (1982) Biochemistry 21, 6897-6902]. In contrast to the case of the skeletal S1, the cleavage with trypsin or papain of the sensitive COOH-terminal 50K-26K junction of the head heavy chain had no effect on the actin-stimulated Mg2+-ATPase activity of the smooth S1. Moreover, actin binding had no significant influence on the proteolysis at this site whereas it abolished the scission of the skeletal S1 heavy chain. The COOH-terminal 26K segment of the smooth papain S1 heavy chain was converted by trypsin into a 25K peptide derivative, but it remained intact in the actin-S1 complex. A single actin monomer was cross-linked with the carbodiimide reagent to the intact 97K heavy chain of the smooth papain S1. Experiments performed on the complexes between F-actin and the fragmented S1 indicated that the site of cross-linking resides within the COOH-terminal 25K fragment of the S1 heavy chain. Thus, for both the striated and smooth muscle myosins, this region appears to be in contact with F-actin.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effects of proteolysis on the adenosinetriphosphatase activities of thymus myosin

Limited proteolysis was used to identify regions on the heavy chains of calf thymus myosin which may be involved in ATP and actin binding. Assignments of the various proteolytic fragments to different parts of the myosin heavy chain were based on solubility, gel filtration, electron microscopy, and binding of 32P-labeled regulatory light chains. Chymotrypsin rapidly cleaved within the head of thymus myosin to give a 70,000-dalton N-terminal fragment and a 140,000-dalton C-terminal fragment. These two fragments did not dissociate under nondenaturing conditions. Cleavage within the myosin tail to give heavy meromyosin occurred more slowly. Cleavage at the site 70,000 daltons from the N-terminus of the heavy chain caused about a 30-fold decrease in the actin concentration required to achieve half-maximal stimulation of the magnesium-adenosinetriphosphatase (Mg-ATPase) activity of unphosphorylated thymus myosin. The actin-activated ATPase activity of this digested myosin was only slightly affected by light chain phosphorylation. Actin inhibited the cleavage at this site by chymotrypsin. In the presence of ATP, chymotrypsin rapidly cleaved the thymus myosin heavy chain at an additional site about 4000 daltons from the N-terminus. Cleavage at this site caused a 2-fold increase in the ethylenediaminetetraacetic acid-ATPase activity and 3-fold decreases in the Ca2+- and Mg-ATPase activities of thymus myosin. Thus, cleavage at the N-terminus of thymus myosin was affected by ATP, and this cleavage altered ATPase activity. Papain cleaved the thymus myosin heavy chain about 94,000 daltons from the N-terminus to give subfragment 1.(ABSTRACT TRUNCATED AT 250 WORDS)     

Functional consequences of the proteolytic removal of regulatory serines from the nonhelical tailpiece of Acanthamoeba myosin II

The actin-activated Mg2(+)-ATPase activity of myosin II from Acanthamoeba castellanii is regulated by phosphorylation of 3 serines in its 29-residue, nonhelical, COOH-terminal tailpiece, i.e., serines-1489, -1494, and -1499 or, in reverse order, residues 11, 16, and 21 from the COOH terminus. To investigate the essential requirements for regulation, myosin II filaments in the presence of F-actin were digested by arginine-specific submaxillary gland protease. Two-dimensional peptide mapping of purified, cleaved myosin II showed that the two most terminal phosphorylation sites, serines-1494 and -1499, had been removed. Cleaved dephosphorylated myosin II retained full actin-activated Mg2(+)-ATPase activity (with no change in Vmax or Kapp) and the ability to form filaments similar to those of the native enzyme. However, higher Mg2+ concentrations were required for both filament formation and maximal ATPase activity. The one remaining regulatory serine in the cleaved myosin II was phosphorylatable by myosin II heavy-chain kinase, and phosphorylation inactivated the actin-activated Mg2(+)-ATPase activity, as in the case of the native myosin II. Also as in the case of the native myosin II, phosphorylated cleaved myosin II inhibited the actin-activated Mg2(+)-ATPase activity of dephosphorylated cleaved myosin II when the two were copolymerized. These results suggest that at least 18 of the 29 residues in the nonhelical tailpiece of the heavy chain are not required for either actin-activated Mg2(+)-ATPase activity or filament formation and that phosphorylation of Ser-1489 is sufficient to regulate the actin-activated Mg2(+)-ATPase activity of myosin II.     

Amino acid sequence of a segment of the Acanthamoeba myosin II heavy chain containing all three regulatory phosphorylation sites

The actin-activated Mg2+-ATPase activity of myosin II from the soil amoeba Acanthamoeba castellanii is regulated by phosphorylation of 3 serine residues on the myosin II heavy chain. Partial chymotryptic digestion of 32P-labeled myosin II cleaves from the tail end of the myosin II heavy chain a small peptide which contains all three phosphorylation sites. During purification the phosphorylated peptide is resolved into several different species as a result of heterogeneity both in phosphate content and in size (probably due to chymotryptic cleavage at the carboxyl terminus). However, all forms of the peptide have an identical amino terminus. The sequence of the first 58 residues of the peptide is: N-S-A-L-E-S-D-K-Q-I10-L-E-D-E-I-G-D-L-H- E20-K-N-K-Q-L-Q-A-K-I-A30-Q-L-Q-D-E-I-D-G-T- P40-S-S-R-G-G-S-T-R-G-A50-S-A-R-G-A-S-V-R. The phosphorylated serines are at positions 46, 51, and 56. The first 36 residues of the sequence display a repeating 3-4-3-4 pattern of hydrophobic residues suggesting that this section of the peptide forms an alpha-helical coiled-coil structure. A -Gly-Thr-Pro sequence at residues 38-40 disrupts the alpha-helix and, at the same point, the repeating pattern of non-polar residues is lost. It is likely that the residues extending from Gly-38 to the end of the myosin II tail, which include the 3 phosphorylatable serines, form a randomly coiled or small globular structure. This is the first report of the sequence around the regulatory phosphorylation sites on any myosin heavy chain.     

Purification and identification of myosin heavy chain kinase from bovine brain

A high salt extract of bovine brain was found to contain a protein kinase which catalyzed the phosphorylation of heavy chain of brain myosin. The protein kinase, designated as myosin heavy chain kinase, has been purified by column chromatography on phosphocellulose, Sephacryl S-300, and hydroxylapatite. During the purification, the myosin heavy chain kinase was found to co-purify with casein kinase II. Furthermore, upon polyacrylamide gel electrophoresis of the purified enzyme under non-denaturing conditions, both the heavy chain kinase and casein kinase activities were found to comigrate. The purified enzyme phosphorylated casein, phosvitin, troponin T, and isolated 20,000-dalton light chain of gizzard myosin, but not histone or protamine. The kinase did not require Ca2+-calmodulin, or cyclic AMP for activity. Heparin, which is known to be a specific inhibitor of casein kinase II, inhibited the heavy chain kinase activity. These results indicate that the myosin heavy chain kinase is identical to casein kinase II. The myosin heavy chain kinase catalyzed the phosphorylation of the heavy chains in intact brain myosin. The heavy chains in intact gizzard myosin were also phosphorylated, but to a much lesser extent. The heavy chains of skeletal muscle and cardiac muscle myosins were not phosphorylated to an appreciable extent. Although the light chains isolated from brain and gizzard myosins were efficiently phosphorylated by the same enzyme, the rates of phosphorylation of these light chains in the intact myosins were very small. From these results it is suggested that casein kinase II plays a role as a myosin heavy chain kinase for brain myosin rather than as a myosin light chain kinase.