Comparative analyses of the kinetics and subunits of myosins from canine skeletal muscle and cardiac tissue.

Two types of canine cardiac myosins, myosin from the free wall of the right ventricle and the free wall of the left ventricle, were compared with canine skeletal muscle myosin from the gastrocnemius. The Vmax values for the ATPase reaction catalyzed by myosin were significantly different among the three types of tissues. For K+-activated myosin the Vmax values in micromoles of Pi per mg per min were: right ventricle, 0.57; left ventricle, 0.72; and gastrocnemius, 0.95. For Ca-2+ -activated myosin the Vmax values were: right ventricle, 0.32; left ventricle, 0.42; gastrocnemius, 0.50. All differences were significant (p smaller than 0.001). For all three types of tissues the Vmax values for NH4+ -activated myosin were the same (2.30). Light chains among all three types of tissues were immunologically identical, whereas the heavy chains of the two cardiac ventricles were immunologically identical with each other; however both were immunologically nonidentical with those of the gastrocnemius. The proportion of myosin light chains to heavy chains was different in the three types of tissue. Of the total protein present in each of the myosins, there was 18% in the light chains of right ventricle myosin, 10% in the light chains of left ventricle myosin, and 13% in the light chains of gastrocnemius. Both left ventricle myosin and myosin from gastrocnemius had significantly less C1d light chain, as compared to myosin from the right ventricle.     

Canine cardiac myosin with special referrence to pressure overload cardiac hypertrophy. I. Subunit composition.

In studies of myosin from left and right ventricles of normal hearts and hypertrophic hearts at 5 weeks and 13 weeks after aortic banding, polyacrylamide gel electrophoresis shows intermediate molecular weight components which derive from heavy chains fragmented in the presence of dodecyl sulfate. The proportion of degraded heavy chains is greater in myosin from hypertrophic hearts than normal hearts, with comparable degradation in left and right ventricle myosin. The observed fragmentation of myosin results from proteolysis due to contaminant proteases or a thermally activated, heat-stable nonenzymatic process, or both. The susceptibility of heavy chains to crude myofibrillar proteases differs in normal and hypertrophic cardiac myosin; however, the kinetics of tryptic digestion are identical for both myosins. With precautions to minimize proteolytic artifacts on dodecyl sulfate-polyacrylamide gel electrophoresis, preparations of myosin from left and right ventricles of normal and hypertrophic hearts exhibit comparable subunit composition, with approximately molar ratios of heavy chains, light chain L1, and light chain L2. Comparable stoichiometry for the light chain fraction is determined by high speed sedimentation equilibrium at pH 11 and direct fractionation of the different cardiac myosins. We do not confirm reports (e.g. Wikman-Coffelt, J., Fenner, C., Smith, A., and Mason, D. T. (1975) J. Biol. Chem. 250, 1257-1262) of different proportions of light chains in left and right ventricle myosin of normal and hypertrophic canine hearts. The light chains display microheterogeneity, with L1 generating two isoelectric variants and L2 generating two major and two minor variants, but identical mobilities and isoelectric values are obtained in the different myosin preparations.     

A new cardiac myosin characterized from the canine atria.

Canine atrial myosin light chains were electrophoretically distinct from myosins of canine ventricles on 5–20% polyacrylamide gradient slab gels (SDS), giving molecular weights of 26,000 and 21,000 as compared to 28,000 and 18,500 for ventricular myosin light chains. While atrial myosin heavy chains were immunologically identical with ventricular myosin heavy chains, in contrast, there was 8.0% relative cross-reactivity of atrial myosin light chains with left ventricular myosin light chains by radioimunoassay. According to charge separation on two-dimensional polyacrylamide urea gels, atrial myosin light chains were different from those of ventricular myosins. Variances in ATPase activities between atrial and ventricular myosins were strongly demonstrated. There was a lower K⁺ activated ATPase activity in atrial myosin, however the Ca²⁺ activated ATPase activity, at ATP saturation levels, was higher in atrial myosin as compared to ventricular myosins.     

Myosin and paramyosin of Caenorhabditis elegans: biochemical and structural properties of wild-type and mutant proteins.

Myosin and paramyosin have been purified from the nematode, Caenorhabditis elegans. The properties of the myosin in general resemble those of other myosins. The native molecule is a dimer of heavy (210,000 dalton) polypeptide chains and contains 18,000 and 16,000 dalton light chains. When rapidly precipitated from solution, it forms small, bipolar aggregates, about 150 nm long, consistent with the expected molecular structure of a rigid rod with a globular head region at one end. Its ATPase activity is stimulated by Ca2+ and EDTA. The myosin binds to F actin in a polar and ATP-sensitive manner, and the Mg2+-ATPase is activated by either F actin or nematode thin filaments. Dialysis of myosin to low ionic strength produces very long filaments. When a myosin-paramyosin mixture is dialyzed under the same condtions, co-filaments form which consist of a myosin cortex, surrounding a paramyosin core. Some properties of myosin from the mutants E675 and E190, which have functionally and structurally altered body wall muscles, are compared with those of wild-type myosin. These myosins of these results are discussed in terms of the myosin heavy chain composition.     

Changes in composition of cardiac myosin light chains in dilated cardiomyopathy: effect on functional properties

A reduction (by 16-24%) in the amount of myosin regulatory light chains (LC2) in all heart sections of patients with dilated cardiomyopathy was found. The appearance of atrial essential light chains in ventricular myosin (up to 23%) not typical for this heart section in norm was also revealed. The decrease in LC2 content leads to a considerable inhibition of actin-activated ATPase activity and a loss of Ca2+ sensitivity of reconstructed filaments of myosin isolated from atria and ventricles of patients with dilated cardiomyopathy. The hybridization of myosin molecules from heavy chains of pathological human left ventricular myosin and light chains of pig left ventricular myosin leads to an increase in actin-activated ATPase activity of myosin and its Ca2+ sensitivity to the control level. The data suggest strongly the contribution of LC2-deficit to the distortion of functional properties of myosin in dilated cardiomyopathy. In contrast, the appearance of atrial LC1 in ventricle in dilated cardiomyopathy is a factor improving these properties.     

Structural differences between atrial and ventricular myosins from normal human hearts

Comparisons were made between myosins isolated from the right and left ventricles and the atria of normal human hearts. Parameters examined included electrophoretic mobilities of native molecules, K+ and Ca2+ dependent enzymatic activities, light chain composition, peptide patterns from partial proteolytic digests of entire heavy chains or rods, and maps of complete digests of specific 21 and 25 kilodalton heavy chain fragments. Human ventricular and atrial myosins differ in all parameters except in the charge of molecules. Structural differences between cardiac myosins derived from the two sources were apparent in both the head and tail portions of the heavy chains. With respect to the above parameters no differences were found between myosins from left and right human ventricles.     

Chimeric myosin regulatory light chains identify the subdomain responsible for regulatory function.

Regulatory light chains, located on the 'motor' head domains of myosin, belong to the family of Ca2+ binding proteins that consist of four 'EF-hand' subdomains. Vertebrate regulatory light chains can be divided into two functional classes: (i) in smooth/non-muscle myosins, phosphorylation of the light chains by a calcium/calmodulin-dependent kinase regulates both interaction of the myosin head with actin and assembly of the myosin into filaments, (ii) the light chains of skeletal muscle myosins are similarly phosphorylated, but they play no apparent role in regulation. To discover the basis for the difference in regulatory properties of these two classes of light chains, we have synthesized in Escherichia coli, chimeric mutants composed of subdomains derived from the regulatory light chains of chicken skeletal and smooth muscle myosins. The regulatory capability of these mutants was analysed by their ability to regulate molluscan myosin. Using this test system, we identified the third subdomain of the regulatory light chain as being responsible for controlling not only the actin-myosin interaction, but also myosin filament assembly.     

Myosin from human erythrocytes

We have purified myosin from human erythrocytes using methods similar to that for other cytoplasmic myosins with a yield of about 500 micrograms/100 ml of packed cells. It consists of a 200-kDa heavy chain and light chains of 26- and 19.5 kDa and therefore differs from the isozyme in platelets which has light chains of 20- and 15 kDa. At low ionic strength, the myosin forms short bipolar filaments like those of platelet myosin. Eight of eight monoclonal antibodies to platelet myosin also bind to erythrocyte myosin. Like most myosins, it has a high ATPase activity in the presence of Ca2+ or EDTA, but is inhibited by Mg2+. Myosin light-chain kinase transfers 1 phosphate from ATP to the 20-kDa light chain, and this stimulates the actin-activated ATPase. Thus, myosin may play a role in shape changes in the erythrocytes.     

Chick brain actin and myosin. Isolation and characterization

Brain actin extracted from an acetone powder of chick brains was purified by a cycle of polymerization-depolymerization followed by molecular sieve chromatography. The brain actin had a subunit molecular weight of 42,000 daltons as determined by co-electrophoresis with muscle actin. It underwent salt-dependent g to f transformation to form double helical actin filaments which could be "decorated" by muscle myosin subfragment 1. A critical concentration for polymerization of 1.3 microM was determined by measuring either the change in viscosity or absorbance at 232 nm. Brain actin was also capable of stimulating the ATPase activity of muscle myosin. Brain myosin was isolated from whole chick brain by a procedure involving high salt extraction, ammonium sulfate fractionation and molecular sieve chromatography. The purified myosin was composed of a 200,000-dalton heavy chain and three lower molecular weight light chains. In 0.6 M KCl the brain myosin had ATPase activity which was inhibited by Mg++, stimulated by Ca++, and maximally activated by EDTA. When dialyzed against 0.1 M KCl, the brain myosin self-assembled into short bipolar filaments. The bipolar filaments associated with each other to form long concatamers, and this association was enhanced by high concentrations of Mg++ ion. The brain myosin did not interact with chicken skeletal muscle myosin to form hybrid filaments. Furthermore, antibody recognition studies demonstrated that myosins from chicken brain, skeletal muscle, and smooth muscle were unique.     

Isolation and characterization of myosin from amoebae of Physarum polycephalum

Myosin was isolated from amoebae of Physarum polycephalum and compared with myosin from plasmodia, another motile stage in the Physarum life cycle. Amoebal myosin contained heavy chains (Mr approximately 220,000), phosphorylatable light chains (Mr 18,000), and Ca2+-binding light chains (Mr 14,000) and possessed a two-headed long-tailed shape in electron micrographs after rotary shadow casting. In the presence of high salt concentrations, myosin ATPase activity increased in the following order: Mg-ATPase activity less than K-EDTA-ATPase activity less than Ca-ATPase activity. In the presence of low salt concentrations, Mg-ATPase activity was activated approximately 9-fold by skeletal muscle actin. This actin-activated ATPase activity was inhibited by micromolar levels of Ca2+. Amoebal myosin was indistinguishable from plasmodial myosin in ATPase activities and molecular shape. However, the heavy chain and phosphorylatable light chains of amoebal myosin could be distinguished from those of plasmodial myosin in sodium dodecyl sulfate-polyacrylamide gel electrophoresis, peptide mapping, and immunological studies, suggesting that these are different gene products. Ca2+-binding light chains of amoebal and plasmodial myosins were found to be identical using similar criteria, supporting our hypothesis that the Ca2+-binding light chain plays a key role in the inhibition of actin-activated ATPase activity in Physarum myosins by micromolar levels of Ca2+.     

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.     

Characteristics of ATPase activity and the subunit composition of myosin in the conduction system of bovine heart

The ATPase activity, light chains and isoenzymes of myosin from specialized myocardial tissue (the A-V node, bundle of His, and right and left bundle branches) of bovine heart were compared with those of atrial and ventricular myosins. The order of Ca2+-activated ATPase activity was atrial greater than specialized myocardial tissue greater than ventricular myosin. SDS-polyacrylamide gel electrophoresis showed that myosin from the specialized myocardial tissue contained the light chains of both atrial and ventricular myosins. On the other hand, the specialized myocardial tissue contained one V3 isomyosin and showed no difference from ventricular myocardial tissue on pyrophosphate gel.     

Phylogenetic studies of cardiac myosins from amphibia to mammals

Comparison between pig atrial and ventricular myosins was performed on the light chains (using SDS-PAGE) and on the heavy chains (using Ca2+-ATPase measurements and NTCBA peptide mapping). Light chain composition of pig cardiac myosins was compared to three other species ones (frog, chicken and human). Up to birds, atrial and ventricular myosin light chain composition was identical whereas in mammals atrial and ventricular myosin light chain composition was different; likewise the heavy chains. Six cardiac myosin isoenzymes have been thus characterized. No correlation can be established between cardiac myosin light chain pattern and species evolution.     

Cytoplasmic myosin from Drosophila melanogaster

Myosin is identified and purified from three different established Drosophila melanogaster cell lines (Schneider's lines 2 and 3 and Kc). Purification entails lysis in a low salt, sucrose buffer that contains ATP, chromatography on DEAE-cellulose, precipitation with actin in the absence of ATP, gel filtration in a discontinuous KI-KCl buffer system, and hydroxylapatite chromatography. Yield of pure cytoplasmic myosin is 5-10%. This protein is identified as myosin by its cross-reactivity with two monoclonal antibodies against human platelet myosin, the molecular weight of its heavy chain, its two light chains, its behavior on gel filtration, its ATP-dependent affinity for actin, its characteristic ATPase activity, its molecular morphology as demonstrated by platinum shadowing, and its ability to form bipolar filaments. The molecular weight of the cytoplasmic myosin's light chains and peptide mapping and immunochemical analysis of its heavy chains demonstrate that this myosin, purified from Drosophila cell lines, is distinct from Drosophila muscle myosin. Two-dimensional thin layer maps of complete proteolytic digests of iodinated muscle and cytoplasmic myosin heavy chains demonstrate that, while the two myosins have some tryptic and alpha-chymotryptic peptides in common, most peptides migrate with unique mobility. One-dimensional peptide maps of SDS PAGE purified myosin heavy chain confirm these structural data. Polyclonal antiserum raised and reacted against Drosophila myosin isolated from cell lines cross-reacts only weakly with Drosophila muscle myosin isolated from the thoraces of adult Drosophila. Polyclonal antiserum raised against Drosophila muscle myosin behaves in a reciprocal fashion. Taken together our data suggest that the myosin purified from Drosophila cell lines is a bona fide cytoplasmic myosin and is very likely the product of a different myosin gene than the muscle myosin heavy chain gene that has been previously identified and characterized.     

Structure-function studies on Acanthamoeba myosins IA, IB, and II

Myosins IA and IB are globular proteins with only a single, short (for myosins) heavy chain (140,000 and 125,000 daltons for IA and IB, respectively) and are unable to form bipolar filaments. The amino acid sequence of IB heavy chain shows 55% similarity to muscle myosins in the N-terminal 670 residues, which contain the active sites, and a unique 500-residue C-terminus highly enriched in proline, glycine, and alanine. The C-terminal region contains a second actin-binding site which allows myosins IA and IB to cross-link actin filaments and support contractile activity. Myosins IA and IB are regulated solely by phosphorylation of one serine on the heavy chain positioned between the catalytic site and the actin-binding site that activates ATPase. Myosin II is a more conventional myosin in composition (two heavy chains and two pairs of light chains), heavy chain sequence (globular head 45% identical to muscle myosins and a coiled-coil helical tail), and structure (bipolar filaments). The tail of myosin II is much shorter than that of other conventional myosins, and it contains a 25 amino acid sequence in which helical structure is predicted to be weak or absent. The position of this sequence corresponds to the position of a bend in the monomer. Myosin II heavy chains also have a 29-residue nonhelical tailpiece which contains three regulatory, phosphorylatable serines. Phosphorylation at the tip of the tail regulates ATPase activity in the globular head apparently through an effect on filament structure.     

A comparative study of reactive--SH groups of cardiac and skeletal muscle myosins.

Cardiac and skeletal muscle myosins have been treated by N-ethylmaleimide in presence or absence of Mg-ADP. The variations of Ca2+ and K+-ATPase activities and the incorporation of N-[14C]ethylmaleimide into the whole myosin molecule and into its separated subunits (heavy and light chains) have been measured with N-ethylmaleimide treatment for different lengths of time. The results reported here show the following: 1. The Ca2+-ATPase activity of cardiac myosin is activated by N-ethylmaleimide treatment to a lesser extent than that of skeletal myosin. 2. The K+-ATPase activity of both myosins is inhibited in the same quantitative way. 3. The cardiac light chain L1 contains one highly reactive thiol group which is absent from the skeletal light chains. 4. The labelling of three SH-groups localized in the heavy subunits of both myosins induced the same degree of inactivation. 5. The difference observed between the degree of inhibition of the Ca2+-ATPase activity for the two types of myosin with longer treatments appears to be due to differences in the reactivity of the fourth--SH group labelled on the heavy chains.     

Purification and characterization of a third isoform of myosin I from Acanthamoeba castellanii

A third isoform of myosin I has been isolated from Acanthamoeba and designated myosin IC. Peptide maps and immunoassays indicate that myosin IC is not a modified form of myosin IA, IB, or II. However, myosin IC has most of the distinctive properties of a myosin I. It is a globular protein of native Mr approximately 162,000, apparently composed of a single 130-kDa heavy chain and a pair of 14-kDa light chains. It is soluble in MgATP at low ionic strength, conditions favoring filament assembly by myosin II. Myosin IC has high Ca2+- and (K+,EDTA)-ATPase activities. Its low Mg2+-ATPase activity is stimulated to a maximum rate of 20 s-1 by the addition of F-actin if its heavy chain has been phosphorylated by myosin I heavy chain kinase. The dependence of the Mg2+-ATPase activity of myosin IC on F-actin concentration is triphasic; and, at fixed concentrations of F-action, this activity increases cooperatively as the concentration of myosin IC is increased. These unusual kinetics were first demonstrated for myosins IA and IB and shown to be due to the presence of two actin-binding sites on each heavy chain which enable those myosins I to cross-link actin filaments. Myosin IC is also capable of cross-linking F-actin, which, together with the kinetics of its actin-activated Mg2+-ATPase activity, suggests that it, like myosins IA and IB, possesses two independent actin-binding domains.     

Myosins and pathology: genetics and biology

This article summarizes current knowledge on the genetics and possible molecular mechanisms of Human pathologies resulted from mutations within the genes encoding several myosin isoforms. Mutations within the genes encoding some myosin isoforms have been found to be responsible for blindness (myosins III and VIIA), deafness (myosins I, IIA, IIIA, VI, VIIA and XV) and familial hypertrophic cardiomyopathy (beta cardiac myosin heavy chain and both the regulatory and essential light chains). Myosin III localizes predominantly to photoreceptor cells and is proved to be engaged in the vision process in Drosophila. In the inner ear, myosin I is postulated to play a role as an adaptive motor in the tip links of stereocilia of hair cells, myosin IIA seems to be responsible for stabilizing the contacts between adjacent inner ear hair cells, myosin VI plays a role as an intracellular motor transporting membrane structures within the hair cells while myosin VIIA most probably participates in forming links between neighbouring stereocilia and myosin XV probably stabilizes the stereocilia structure. About 30% of patients with familial hypertrophic cardiomyopathy have mutations within the genes encoding the beta cardiac myosin heavy chain and both light chains that are grouped within the regions of myosin head crucial for its functions. The alterations lead to the destabilization of sarcomeres and to a decrease of the myosin ATPase activity and its ability to move actin filaments.     

Myosin from abdominal flexor muscle in a crayfish, Procambarus clarki Girard.

1. Crayfish (Procambarus clarki) myosin was obtained from abdominal flexor muscle. The Ca2+-ATPase activity of crayfish myosin was much lower than that of rabbit skeletal myosin. However, F-actin-activated Mg2+-ATPase of crayfish and its superprecipitation closely resembled those of rabbit skeletal myosin. This fact suggests that the ability of crayfish myosin to combine with F-actin is essentially the same as that of skeletal myosin, although the chemical structures of both the myosin molecules when involved in their Ca2+-ATPast activity must be different from each other. 2. Crayfish and rabbit skeletal myosins were subjected to SDS-polyacrylamide gel electrophoresis. Crayfish myosin was found to have one heavy chain and two distinct light chain components (CF-gl and CF-g2), which have molecular weights of 18,000 and 16,000, respectively. These light chains correspond in molecular weight to the light chains (SK-g2 and SK-g3) in rabbit skeletal myosin. 3. CF-g1 could be liberated from the crayfish myosin molecule reacting with 5,5'-dithio-bis (2-nitrobenzoic acid), (Nbs2), without recovery of ATPase activity by the addition of DTT. These properties are equivalent to those of SK-g2 in rabbit skeletal myosin, although Nbs2-treated crayfish myosin did not recover its ATPase activity at all.     

Regulation of myosin filament assembly by light-chain phosphorylation

Myosins isolated from vertebrate smooth muscles and non-muscle cells such as lymphocytes and platelets contain regulatory light chains (Mr = 20000), which are phosphorylated by a Ca2+-calmodulin-dependent kinase and dephosphorylated by a Ca2+-insensitive phosphatase. Phosphorylation of the regulatory light chains of these myosins in vitro regulates not only their interactions with actin but also their assembly into filaments. Under approximately physiological conditions (0.15 M NaCl, pH 7.0) stoichiometric levels of Mg-ATP disassemble these non-phosphorylated myosin filaments into species with sedimentation coefficients (So20,w) of approximately 11S. Hydrodynamic and electron microscope observations have indicated that this 11S species is a monomer with a folded conformation (Trybus et al., Proc. natn. Acad. Sci. U.S.A. 79, 6151 (1982)). Rotary shadowing reveals that the tails of disassembled gizzard and thymus myosins are folded twice at two hinge points to form a folded three-segment structure. Phosphorylation of the regulatory light chains of these myosins causes these folded 11S molecules to unfold into the conventional extended monomeric form (6S), which is able to assemble into filaments. Thus in vitro these myosin filaments can be assembled or disassembled by phosphorylation or dephosphorylation of their light chains. Whether these results have any relevance to the situation within living non-muscle and smooth muscle cells remains to be established.