Modification of cardiac and smooth muscle myosins with 2,4,6-trinitrobenzenesulfonate. Evidence for differences in structure around the active sites of cardiac, smooth, and skeletal muscle myosin ATPase.

Myosins purified from cardiac (porcine heart) and smooth (chicken gizzard) muscles were modified with 2,4,6-trinitrobenzenesulfonate (TNBS) and the effects on the kinetic properties of myosin ATPase [EC 3.6.1.3] were studied. The following results were obtained. 1. About 0.5 mol of TNBS per mol of myosin head was incorporated rapidly, irrespective of the presence of PP1 (2mM), into both types of myosin studied. 2. The size of the initial burst of P1 liberation for both myosins was found to be 0.5--0.6 mol/mol head. 3. The rapid incorporation of TNBS into cardiac muscle myosin was accompanied by a rapid decrease in the size of the initial P1 burst, and it was completely lost after modification for 20 min. However, smooth muscle myosin retained its P1 burst. 4. The EDTA (K+)-ATPase activity of both myosins modified in the presence or absence of PP1 decreased sharply with incorporation of TNBS. 5. Superprecipitation and ATPase activity of reconstituted actomyosin from cardiac myosin and skeletal F-actin decreased only after 10 min of modification with TNBS in the absence of PP1. 6. The spectra of TNP bound to myosins from cardiac and smooth muscles were unchanged by the addition of PP1. The above findings are compared with those previously obtained for skeletal muscle myosin [Miyanishi, T., Inoue, A., & Tonomura, Y. (1979) J. Biochem. 85, 747--753], and the structural and functional differences among the myosins derived from skeletal, cardiac, and smooth muscles are discussed.     

Enzymatic activities and ATP-induced fluorescence enhancement of myosin from fast and slow skeletal and cardiac muscles.

The maximal ATP-induced enhancement of fluorescence and the dependence of this enhancement on ATP concentration were determined for myosins from fast and slow skeletal and cardiac muscle of the rabbit. With myosins from slow and cardiac muscle modifications in the preparative procedure and chromatography on DEAE-Sephadex were required to obtain preprations which were free of actin, which exhibited the maximal fluorescence enhancement and which bound two moles of ATP per mole of myosin. Since the fluorescence enhancement of cardiac and slow muscle myosins is labile at slightly alkaline pH, it was also necessary to minimize incubation at pH greater than 7 in order to attain the maximal enhancement. With fast muscle myosin the changes in preparative procedure, together with chromatography, led to a 50 to 100% increase in the steady-state rate of ATP hydrolysis and fluorescence enhancement, without changing the maximal binding of ATP. From a comparison of the rate of steady-state hydrolysis of ATP with the rate of decay of the enhanced fluorescence, it appears that for all three myosins, both ATP binding sites have the same enzymatic activity, the steady-state rate per site being slower for cardiac and slow muscle myosins than for fast muscle myosin.     

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.     

Formation and characterization of myosin hybrids containing essential light chains and heavy chains from different muscle myosins

Light chain exchange in 4.7 M NH4Cl was used to hybridize the essential light chain of cardiac myosin with the heavy chain of fast muscle myosin subfragment 1, S-1. The actin-activated ATPase properties of this hybrid were compared to those of the two fast S-1 isoenzymes, S-1(A1), fast muscle subfragment 1 which contains only the alkali-1 light chain, and S-1(A2), fast muscle myosin subfragment 1 which contains only the alkali-2 light chain. This hybrid S-1 behaved like S-1(A1)., At low ionic strength in the presence of actin, this hybrid had a maximal rate of ATP hydrolysis about the same as that of S-1(A1) and about one-half that of S-1(A2), while at higher ionic strengths the actin-activated ATPases of these three S-2 species were all similar. Light chain exchange in NH4Cl was also used to hybridize the essential light chains of fast muscle myosin with the heavy chains of cardiac myosin and to hybridize the essential light chains of cardiac myosin with the heavy chains of fast muscle myosin. In 60 and 100 mM KCl, the actin-activated ATPases of these two hybrid myosins were very different from those of the control myosins with the same essential light chains but were very similar to those of the control myosins with the same heavy chains, differing at most by one-third.     

Properties of porcine platelet myosin. I. Similarity between vertebrate smooth muscle and nonmuscle myosins in their binding properties with F-actin

The mechanism of the ATPase [EC 3.6.1.3] reaction of porcine platelet myosin and the binding properties of platelet myosin with rabbit skeletal muscle F-actin were investigated. The kinetic properties of the platelet myosin ATPase reaction, that is, the rate, the extent of fluorescence enhancement of myosin, the size of the initial P1 burst of myosin, and the amount of nucleotides bound to myosin during the ATPase reaction, were very similar to those found for other myosins. Strong binding of platelet myosin with rabbit skeletal muscle F-actin, as found for smooth muscle myosin, was suggested by the following results. The rate of the ATP-induced dissociation of hybrid actomyosin, reconstituted from platelet myosin and skeletal muscle F-actin, was very slow. The amount of ATP necessary for complete dissociation of hybrid actomyosin was 2 mol/mol of myosin, although skeletal muscle actomyosin is known to dissociate completely upon addition of 1 mol ATP per mol of myosin. Unlike skeletal muscle myosin, the EDTA(K+)-ATPase activity of platelet myosin was inhibited by skeletal muscle F-actin. These observations indicate that ATP hydrolysis by vertebrate nonmuscle myosin follows the same mechanism as with other myosins and that the binding properties of nonmuscle myosin with F-actin are similar to those of smooth muscle myosin but not to those of skeletal muscle myosin.     

Comparative analysis of chicken atrial and ventricular myosins.

1. Structural and enzymic properties of myosins from atrial and ventricular cardiac muscle of the chicken were investigated and compared with myosins from the fast skeletal pectoralis and the slow skeletal anterior latissimus dorsi muscle. 2. The Ca2+-ATPase activity, both in function of pH and [K+], of atrial myosin closely resembled that of the fast pectoralis myosin, whereas the enzymic properties of ventricular myosin were similar to those of slow skeletal myosin. 3. By sodium dodecyl sulphate polyacrylamide gel electrophoresis on gradient gel and two-dimensional electrophoresis, involving isoelectric focusing in the first dimension and SDS gel electrophoresis in the second dimension, no difference could be demonstrated in the light-chain pattern of atrial and ventricular myosin. Complete identity was also found between anterior latissimus dorsi and cardiac light chains. 4. Electrophoretic analysis of soluble peptides released by tryptic digestion of myosin and electron microscopic study of light meromyosin paracrystals showed significant differences between the heavy chains of atrial and ventricular myosins, as well as between the heavy chains of cardiac and skeletal myosins. 5. The results confirm previous immunochemical findings and provide direct biochemical evidence for the existence of a new, unique type of myosin in the chicken atrial tissue.     

Thyroxine-induced cardiomegaly: assessment of nucleic acid, protein content and myosin ATPase of rat heart.

1 mg/kg L-thyroxine was administered to rats for 14 days to evaluate the potential of the hyperthyroid state to induce heart hypertrophy and its effect on myosin adenosine-triphosphatase (ATPase) activity. Evidence of hyperthyroidism such as weight loss, elevation of rectal temperature, increased heart rate and oxygen consumption, was observed in all treated rats. Cardiac enlargement was determined by comparison of wet and dry ventricle weights, myocardial RNA, DNA and protein content. Wet and dry ventricle weights and the level of cardiac RNA and protein were augmented by thyroxine treatment. ATPase activity of cardiac myosin was stimulated as the Ca2+ concentration in the incubation medium increased. No difference was found in Ca2+-activation, salt sensitivity or ATPase activity of unreacted and sulphydrylmodified cardiac myosins from euthyroid or hyperthyroid groups. The results showed that in hyperthyroid rats, in contrast to some other species, the biochemical mechanism responsible for the enhancement of cardiac contractility is not an increased myosin ATPase.     

Immunological, electrophoretic and kinetic properties of cardiac myosins from various species.

1. In a homologous radioimmunoassay for canine ventricular myosin light chains, the following percentages of cross-reactivities were obtained using the dog as a reference: human, 28%; sheep, 21%; cat, 8%; guinea-pig, 7%; rabbit, 5%; and rat, 4%. 2. In a homologous double diffusion immunoassay using specific gamma G to canine cardiac myosin heavy chains, dog cardiac myosin showed immunological identity with human and sheep cardiac myosin but partial identity with myosins of other species. 3. On a 5-20% polyacrylamide gradient, light chain C1 was electrophoretically distinct in some species; light chain C2 was electrophoretically identical in all species. 4. The K+-activated myosin ATPase of small animals was higher than that of larger animals at an alkaline pH; the same was true for Ca2+-activated myosin when assayed at pH 6.3.     

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.     

Studies on the amino groups of myosin ATPase. Trinitrophenylation of reactive lysyl residue in ventricular and atrial myosins

Myosin and heavy meromyosin from ventricular, atrial, and skeletal muscle were purified and trinitrophenylated by 2,4,6-trinitrobenzene sulfonate. The trinitrophenylation reaction followed a complex kinetics consisting of a fast and slow reaction in all preparations studied. Reactive lysine residues were trinitrophenylated during the fast reaction with a concomitant decrease in K⁺ (EDTA)-activated ATPase and an increase in Mg²⁺-stimulated ATPase activities of myosin. The extent of increase in Mg²⁺-mediated ATPase was the highest with skeletal and the lowest with atrial myosin. The trinitrophenylation of the less reactive lysyl residues continued during the slow reaction. The rate constants of the reactions and the number of reactive lysine residues were evaluated by computer analyses of the trinitrophenylation curves. Two reactive lysine residues were found in skeletal and ventricular myosins while their number in atrial myosin was somewhat lower. The rate of trinitrophenylation in skeletal muscle myosin or heavy meromyosin was always higher than in the two cardiac myosin isozymes. Addition of KCl increased the trinitrophenylation of both highly reactive and slowly reactive lysyl residues in all of the three heavy meromyosins, however, the effect was more profound with cardiac heavy meromyosins. Addition of MgADP induced spectral changes in trinitrophenylated skeletal but not in cardiac myosins. Similar changes occurred in skeletal and to a lesser degree in ventricular heavy meromyosin, but no definite spectral changes were observed in atrial heavy meromyosin. The findings suggest that structural differences exist around the reactive lysyl residue in the head portion of the three myosins.     

Phosphorylation of the light-chain components of myosin from cardiac and red skeletal muscles.

1. The light-chain components of myosin from cardiac muscle (19000 and 27000 daltons) and of rabbit soleus and crureus muscles (19000, 27000 and 29000 daltons) were characterized. 2. The 19000-dalton components in carciac- and red-skeletal-muscle myosins were spontaneously modified to a component of slightly higher net negative charge. 3. The 19000-dalton component in cardiac and red skeletal muscles and their modified forms were phosphorylated by myosin light-chain kinase. 4. Evidence was obtained for the presence of myosin light-chain kinase in cardiac and red skeletal muscles. 5. Myosin light-chain kinase catalysed the phosphorylation of the whole light-chain fraction from white and red skeletal muscle at similar rates. The light-chain fraction of cardiac-muscle myosin was phosphorylated at a significantly lower rate. 6. The light-chain components of cardiac-muscle myosin and their phosphorylated froms were separated by ion-exchange chromatography and their amino acid compositions determined.     

Structural properties of myosin from the particulate fraction of human blood platelets

Fractionation of human blood platelets has revealed that myosin, a contractile and mechanochemical protein, is present in both the soluble and particulate fraction. The aim of this study was to elucidate whether platelets contain more than one myosin isoform, especially in view of the fact that in other cellular systems (cardiac muscle, amoeba) several myosin isoenzymes were found. The particulate fraction was solubilized by Triton X-100, and the myosin was purified by the same procedure used for the cytoplasmic myosin. The final preparation contained, in addition to myosin, a 130-kDa polypeptide, which was observed also in myosin preparations obtained from the soluble fraction. The electrophoretic mobilities of the two myosins were identical under both dissociating and nondissociating conditions. Comparison of the molecular structure of the heavy chain of the two myosins by limited proteolysis with Staphylococcus aureus V8 protease showed that the proteolytic fragments of the two myosins were rather similar, with only minor alterations in the quantitative distribution of the products. Two-dimensional peptide mapping of the iodinated tryptic peptides of the myosin heavy chains indicated that at least one peptide is missing in the map of the particulate myosin, as compared to its soluble counterpart. According to the two-dimensional peptide map, the 130-kDa polypeptide seems to be a proteolytic fragment of the myosin heavy chain and most probably the rod portion of the molecule. The observed minor variations in the structure of myosins isolated from the soluble and the fractions of human platelets may reflect differences in their respective physiological functions.     

Comparison of ATPase activity of cardiac myosins from different species

Ca2+--ATPase activity of myosins prepared from hearts with different shortening speeds was measured in order to determine whether an alteration in hydrolitic activity or in the affinity of myosin for its substrate, or both, may be responsible for the species differences in Ca2+--ATPase. The KM values of ATP for cardiac myosins from rat, guinea-pig and rabbit did not differ significantly, whereas the Vmax decreased in the following order: rat greater than guinea pig greater than rabbit. These facts lead us to assume that, in spite of a great similarity of the active centres of the these myosins, the catalytic sites may not be identical.     

Tissue-specific biosynthesis of epsilon-N-monomethyllysine and epsilon-N-trimethyllysine in skeletal and cardiac muscle myosin: a model for the cell-free study of post-translational amino acid modifications in proteins.

As a continuation of the study on post-ribosomal amino acid modifications in myosin, the regulation of tissue-specific biosynthesis of ϵ-N-monomethyllysine and ϵ-N-trimethyllysine was investigated. While ϵ-N-trimethyllysine is a component of both skeletal and cardiac muscle myosins, in certain species the monomethylated amino acids occur only in myosin from skeletal muscle. The methylation of skeletal and cardiac muscle myosin with cardiac or skeletal muscle enzymes was expected to elucidate whether the tissue-specific occurrence of the ϵ-N-monomethyllysine is related to the structure of skeletal and cardiac myosin or to the existence of the methylating enzyme in the skeletal and cardiac muscle cells. The experimental approach is based on cell-free methylation of lysines at 24 °C, at which temperature the myosin chains remain polysome-bound. The methylated myosin was digested with trypsin and the radioactive methyl group-containing peptides were fractionated with ion-exchange chromatography. The peptide peaks with radioactivity were subjected to amino acid analyses and the radioactive methylated lysine derivatives were identified. ϵ-N-trimethyllysine was found in hydrolysates of all the methylated myosins, and ϵN-monomethyllysine was also present in both skeletal and cardiac muscle myosin if they were incubated with skeletal muscle supernatant. Thus the experimental results agree with our earlier suggestion (Huszar &; Elzinga, 1972) that the lack of a certain methylated amino acid in cardiac muscle myosin is due to the absence of the methylating enzyme rather than to differences in the structure of cardiac versus skeletal myosin. The experimental design developed for this work should be useful to study post-translational modifications in proteins, as well as to investigate muscle and other diseases in which the post-translational processing of proteins contributes to the dys function.     

Comparison of biochemical and immunochemical properties of myosin II in taeniid parasites

Type II myosins are highly conserved proteins, though differences have been observed among organisms, mainly in the filamentous region. Myosin isoforms have been identified in Taenia solium, a helminth parasite of public health importance in many developing countries. These isoforms are probably associated with the physiological requirements of each developmental stage of the parasite. In this paper we extend the characterization of myosin to several other Taenia species. Type II myosins were purified from the larvae (cysticerci) of Taenia solium, T. taeniaeformis and T. crassiceps and the adult stages of T. solium, T. taeniaeformis and T. saginata. Rabbit polyclonal antibodies against some of these myosins were specific at high dilutions but cross-reacted at low dilutions. ATPase activity was evaluated and kinetic values were calculated for each myosin. Homologous actin-myosin interactions increased both the affinity of myosin for ATP and the hydrolysis rate. The results indicate immunological and biochemical differences among taeniid myosins. This variability suggests that different isoforms are found not only in different taeniid species but also at different developmental stages. Further characterization of myosin isoforms should include determination of their amino acid composition.     

The in vitro motility assay to study the calcium-mechanical relationship in skeletal and cardiac muscles

In a series of experiments on regulated contractile systems (i.e., in vitro mobile systems with reconstructed thin filaments), the velocities of the movement of a thin filament on the surface covered by either rabbit skeletal or rat cardiac myosin at various concentrations of calcium ions in solution (in the pCa range from 4 to 8) were assessed. The corresponding "pCa-velocity" relationships were plotted, which proved to be of the sigmoid form. It was found that, at a saturating calcium concentration (pCa 4), the velocity of regulated thin filaments was 65% higher than for unregulated ones in the case of skeletal myosin and 87% higher than for unregulated thin filaments in the case of cardiac myosin. It was also found that the Hill coefficient was 1.95 and 2.5 for skeletal and cardiac myosins, respectively. The difference in the Hill coefficients for skeletal and cardiac myosins is discussed in terms of the difference in contribution of cooperativity mechanisms of contractile and regulatory proteins in the regulation of contraction in these types of muscles.     

Immunochemical evidence for the species-specificity of mammalian cardiac myosin and heavy meromyosin.

Structural differences between various myosins were investigated by means of antibodies to heavy meromyosin, a tryptic subfragment of myosin. Heavy meromyosin was purified from rabbit white skeletal and from pig and human cardiac muscles by gel filtration, and antisera were produced in guinea pigs. Analyses, carried out with the quantitative micro-complement fixation technique, indicated that the antibodies were specific to heavy meromyosin and myosin and not to other contractile proteins. For each muscle type, the corresponding intact myosin reacted, and the degree of dixation was always lower than with heavy meromyosin (50 and 70% fixation respectively). This vertical shift was the same for the three muscle types, indicating that the heavy meromyosin represent corresponding fragments of the myosin molecule from one muscle to the other. Antisera to pig or human cardiac heavy meromyosin clearly distinguished antigens (heavy meromyosins, myosins, or crude extracts) from the ventricles of various heterologous species. Relative to pig, the immunological distances were 50 for the rabbit, 73 for the rat and greater than 100 for human and mice. Relative to human, these values were 20 for the rat, 60 for the rabbit, 72 for the pig. These data provide direct evidence that mammalian cardiac myosin is species-specific.     

The magnesium-ion-dependent adenosine triphosphatase of bovine cardiac Myosin and its subfragment-1.

The kinetics of the Mg2+-dependent ATPase (adenosine triphosphatase) activity of bovine cardiac myosin and its papain subfragment-1 were studied by using steady-state and pre-steady-state techniques, and results were compared with published values for the corresponding processes in the ATPase mechanism of rabbit skeletal-muscle myosin subfragment-1. The catalytic-centreactivity for cardiac subfragment-1 is 0.019s-1, which is less than one-third of that determined for the rabbit protein. The ATP-induced isomerization process, measured from enhancement of protein fluorescence on substrate binding, is similarly decreased in rate, as is also the isomerization process associated with ADP release. However, the equilibrium constant for ATP cleavage, measured by quenched-flow by using [gamma-32P]ATP, shows little difference in the two species. Other experiments were carried out to investigate the rate of association of actin with subfragment-1 by light-scattering changes and also the rate of dissociation of the complex by ATP. The dissociation rate increases with increasing substrate concentration, to a maximum at high ATP concentrations, with a rate constant of about 2000s-1. It appears that isomerization processes which may involve conformational changes have substantially lower rate constants for the cardiac proteins, whereas equilibrium constants for substrate binding and cleavage are not significantly different. These differences may be related to the functional properties of these myosins in their different muscle types. Kinetic heterogeneity has been detected in both steady-state and transient processes, and this is discussed in relation to the apparent chemical homogeneity of cardiac myosin.     

The ATPase reaction in the steady state and in the initial burst catalyzed by chicken gizzard myosin in 0.6 M KCl1.

On studying the steady-state activity in 0.6 M KCl, it was found that Mg-ATPase of chicken gizzard myosin was identical with that of rabbit skeletal myosin in the pH-activity profile, Michaelis-Menten constant, and maximum velocity. As regards the "initial burst" of ATP splitting in the presence of Mg (0.6 M KCl), it was found that gizzard and skeletal myosins were identical both in the size of the initial burst and in the velocity-ATP concentration relationship. The only difference we observed was that the Ca- and EDTA-ATPase activities of gizzard myosin were, as reported by other investigators, approximately one-half to one-third of those of skeletal myosin, although the pH-activity profiles for the ATPase of gizzard myosin was essentially the same as that of skeletal myosin.     

The calcium binding properties of phosphorylated and unphosphorylated cardiac and skeletal myosins.

Porcine left ventricular cardiac myosin and rabbit white skeletal myosin were phosphorylated by rabbit skeletal myosin light chain kinase and their Ca2+ binding properties were examined by equilibrium dialysis techniques. No significant effect of phosphorylation on the Ca2+ binding properties of these myosins was observed. Both types of striated muscle myosins bound approximately 2 mol of Ca2+/mol of myosin with similar affinities of 3 x 10(7) M-1. In the presence of 3 x 10(-4) M Mg2+ the myosins bound Ca2+ with a reduced affinity of 3 to 4 x 10(5) M-1. Assuming competition between Mg2+ and Ca2+ for the binding sites on myosin, the changes in Ca2+ binding can be accounted for by a Mg2+ affinity of 2.5 to 3.0 x 10(5) M-1.