A new concept in the molecular process of muscle contraction: functional role of phosphorylated amino acids in myosin

In this report a summary is given of our experiments concerning the in vivo endogenous phosphate (P) content of myosin. It was found that besides the ester type phosphates of myosin there was a considerable amount of N-P type energy-rich phosphoryl groups bond to the basic amino acids of peptide chains. The endogenous P concentration of myosin depends on the source of the preparation. The concentration of P is much higher in myosin preparations of well-trained animals and human subjects compared to those found in the control muscles. As the P binding sites of fresh purified myosin are only partially saturated, the preparations can incorporate P up to a definite saturation only. The phosphorylating ability of myosins disappears after prolonged storage as a consequence of an alteration in structure of the myosin molecule. The P groups are moving inside the myosin molecule. It is supposed that the inorganic P release promoted by actin is connected with the thin filament movements towards the centre of the sarcomere, furthermore P replenishment, P linking and movement involve N3-trimethyl-lysine, 3-methylhistidine, P-Arg and two conformers of P-His. The two net negative charges of P group form electric monopoles of a minor battery (myosin head). They help to force generation at head rotation (90 degrees-45 degrees angle) and produce free energy changes that can be calculated from the number of N-P bonds.     

Purification and properties of myosin from the "hatching muscle" (m. complexus) of geese

The present work is concerned with the study of myosin fractions prepared from the hatching muscle (m. complexus) and a control muscle (m. pectoralis) of the developing goose embryo. The m. complexus attained its maximum mass at hatching and in the 4-day-old bird the mass of this muscle was only one fourth of that recorded at hatching. The m. complexus was hypertrophied already on the 21st day. At days 21, 27 and 28 of incubation and at posthatching days myosin preparations were made from both muscles. Partial purification of myosins from both sources yielded a high molecular weight fraction characteristic of the adult bird and one other protein fraction with molecular mass half of myosin. Both preparations exhibited the characteristic properties of myosin. The lower molecular weight fraction was also shown to develop filamentous aggregates as did the higher molecular-weight, gel filtrated myosin. The phosphate content of the half molecular mass myosin fraction prepared from the embryonic m. complexus at days prior to hatching was considerably higher than that of the high molecular weight fraction and the predominant component was P-Arg. Since the embryonic myosin was still not available in the m. complexus of the 4-day-old birds and the hypertrophied muscle underwent regression after hatching it appears that this myosin fraction is actively involved in breaking through the shell during the hatching period in geese.     

Preparation and characterization of mitochondrial myosins of rat and human liver

This paper confirmed the reality of the mitochondrial myosin (mt-myosin in human and rat liver. Simultaneously, cytoplasmic myosin (cp-myosin) was prepared from the large particle-free supernatant. The yield of purified mt- and cp-myosin from 1 kg fresh liver was altogether 5-600 mg (= 1-1.2 mumol). Half of the myosin originated from the mitochondrial fraction (composed of about 60 g of mitochondrial protein), while the remaining portion (cp-myosin) was derived from a translucent, but voluminous supernatant (containing about two-third of liver proteins). Comparing the molecular mass of mt- and cp-myosin to the skeletal muscle myosin (about 480 kDa)--on the basis of gel filtration profiles--they proved to have similar profiles. The characteristic properties of both preparations were similar to other myosins developing filamentous aggregations and showing ATP-induced superprecipitations, but they had lower ATPase activities thus being more similar to smooth muscle and cell myosins than to skeletal muscle myosin. The mitochondria and both myosins contained abundant covalently bound P and their endogeneous Si content was low, 2-3 mumol/g in fresh mitochondria and 5-7 mol Si per mol in the mt-myosin. The Si content was resolved into 2-3, while P into 5-6 fractions as revealed by ion exchange chromatographic technique. The mt-myosin could be saturated to a higher P level by autophosphorylation than the cytoplasmic myosin. The interaction of actin with myosin induced a release of significant amounts of P, depending on the ATP concentration. The Cu2+ treatment of mt-myosin caused also P release, and a limited amount of Cu remained bound in the preparations.     

Preparation and characterization of myosin from cultured cells of Escherichia coli (02:K1 30156 strain)

In this study the myosin preparations isolated from E. Coli cell cultures were analysed. The isolation of myosin from fresh cultures resulted in a substantially higher (approximately 10-fold) yield than from stored cells. The coli myosin, despite the two DEAE-cellulose treatments, contained more RNA and P-lipid than the myosin prepared from skeletal muscle. The RNA content can be removed gradually by acetone denaturation and lipid removal followed by subsequent washings. The fresh preparations contained protein-bound alkali-stable P; part of this was released by Cu ions. The coli myosin can be phosphorylated. The phosphate uptake depended on the concentrations of ATP and Mg2+ and on the time of incubation. The alkaline hydrolysate of the phosphorylated, washed lipid-free preparation was resolved into 8 P-containing peaks on ion-exchange chromatography. Of fractions P-Arg, Pi, P-Lys and 2 P-His conformer were identified by means of synthetic compounds, elution pattern and specific reactions. The remaining compounds could not be identified. The most abundant component was P-Arg suggesting that this compound might play an important role in the cytokinetic movements of E. coli.     

Phosphorylation of skeletal and cardiac muscle C-proteins by the catalytic subunit of cAMP-dependent protein kinase

Catecholamines are known to influence the contractility of cardiac and skeletal muscles, presumably via cAMP-dependent phosphorylation of specific proteins. We have investigated the in vitro phosphorylation of myofibrillar proteins by the catalytic subunit of cAMP-dependent protein kinase of fast- and slow-twitch skeletal muscles and cardiac muscle with a view to gaining a better understanding of the biochemical basis of catecholamine effects on striated muscles. Incubation of canine red skeletal myofibrils with the isolated catalytic subunit of cAMP-dependent protein kinase and Mg-[gamma-32P]ATP led to the rapid incorporation of [32P]phosphate into five major protein substrates of subunit molecular weights (MWs) 143,000, 60,000, 42,000, 33,000, and 11,000. The 143,000 MW substrate was identified as C-protein; the 42,000 MW substrate is probably actin; the 33,000 MW substrate was shown not to be a subunit of tropomyosin and, like the 60,000 and 11,000 MW substrates, is an unidentified myofibrillar protein. Isolated canine red skeletal muscle C-protein as phosphorylated to the extent of approximately 0.5 mol Pi/mol C-protein. Rabbit white skeletal muscle and bovine cardiac muscle C-proteins were also phosphorylated by the catalytic subunit of cAMP-dependent protein kinase, both in myofibrils and in the isolated state. Cardiac C-protein was phosphorylated to the extent of 5-6 mol Pi/mol C-protein, whereas rabbit white skeletal muscle C-protein was phosphorylated at the level of approximately 0.5 mol Pi/mol C-protein. As demonstrated earlier by others, C-protein of skeletal and cardiac muscles inhibited the actin-activated myosin Mg2+-ATPase activity at low ionic strength in a system reconstituted from the purified skeletal muscle contractile proteins (actin and myosin).(ABSTRACT TRUNCATED AT 250 WORDS)     

The biosynthesis of plasmodial myosin during starvation of Physarum polycephalum

The actomyosin protein complex of Physarum polycephalum was prepared from vegetative and starved plasmodia. The yield of actomyosin per unit wet wt. was the same from both types of plasmodia. Myosin was resolved from the complex by gel filtration and purified by ion-exchange chromatography. The Ca(2+)-stimulated adenosine triphosphatase activities of myosin preparations from vegetative and starved plasmodia were not appreciably different. Synthesis of myosin de novo was shown to occur during the starvation phase of the life-cycle by the isolation of labelled myosin preparations from plasmodia starved in the presence of [2-(14)C]glycine. Fractionation of polyacrylamide gels after gel filtration of labelled myosin confirmed the presence of label in the adenosine triphosphatase-active myosin band. It is concluded that during starvation myosin synthesis continues although there is a net loss of approx. 50% of the total protein. Sodium dodecyl sulphate-polyacrylamide-gel electrophoresis of Physarum myosin showed the presence of low-molecular-weight components of the molecule, similar to those of muscle myosins. The content and composition of the free amino acid pool of Physarum was measured at various time-intervals during the vegetative and starvation phases of the life-cycle.     

Evidence for myosin heterogeneity inDrosophila melanogaster

Summary Electrophoresis of myosin extracts from larvae and adult tissues ofDrosophila melanogaster under non-dissociating conditions indicate that two of the bands seen are myosins. They stain for Ca²⁺ ATPase activity and when cut and re-run under dissociating conditions are found to contain a myosin heavy chain that co-migrates with rabbit skeletal muscle myosin heavy chain. One of the forms of myosin seen is found primarily in extracts from the leg. The other is common to the adult fibrillar flight muscles and the larval body wall muscles.The electrophoretic evidence for two myosin types is strengthened by the histochemical demonstration of two myofibrillar ATPases on the basis of their lability to acid or alkali preincubation. The myofibrillar ATPase in the leg and the Tergal Depressor of the Trochanter (TDT) are shown to be relatively acid labile and alkali stable. The larval body wall muscles and the adult fibrillar flight muscles have an ATPase which is acid stable and alkali labile. This distribution of the two myofibrillar ATPase coincides with that predicted by electrophoresis of extracts from whole tissue and also locates the two myosins to specific muscle types.     

Presence of covalently bound energy-rich phosphates in human tracheal smooth muscle myosin

In a preliminary report, the tracheal NaCl-myosin prepared from an old and a young subject was discussed. In the present paper, the total bound phosphate (P) content and its distribution is described in two parallel preparations of human muscle myosin. It was shown that a considerable amount of covalently bound P was present not only in NaCl, but in the fresh preparations of tracheal KCl-myosin. Analysing this phosphate fraction in the alkaline hydrolysate of RNA- and lipid-free preparations of myosin it was confirmed that phosphate was linked to the basic amino acid residues and their hydrolytic derivatives. As the phosphoryl binding sites are partly saturated, the phosphate concentration can be enhanced nearly three-fold compared to the fresh preparation. Phosphate incorporation is an autophosphorylation process depending on the ATP and Mg2+ concentration. Studying the actomyosin fraction in the presence of ATP it was found that its phosphate content can also be increased to a certain degree. It is supposed that the changes in phosphate content of myosin are associated with the formation of crossbridges between the actin and myosin filaments in the processes of muscle contraction and relaxation. The process can be influenced directly and indirectly by some natural factors and drugs resulting in the concentration or relaxation of bronchial muscles.     

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.     

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.     

Preparation and P content of non-pregnant and pregnant human uterine myosin

Myosin content and phosphorus (P) concentration of myosin preparations were measured in non-pregnant and pregnant human myometrial tissue specimens. It was found that the amount of myosin gained from 1 g of minced myometrial tissue is 0.5 mg in the early follicular phase of the menstrual cycle, 0.6-0.7 mg in the late luteal phase, and 6-7 mg during pregnancy. Considering the different functional stages of the myosin sources and the performance characteristics of the methods, the estimated myosin content of non-pregnant myometrium is 1.0-1.5 mg, while 10-15 mg in pregnant myometrial tissue. A considerable amount of P is bound to the preparations. It is the smallest in the post-menstrual period and increases towards the end of the cycle. The largest amount of P is gained from fresh pregnant uterine samples. Analysis of the alkaline hydrolysate showed that the phosphate group was bound to amino acids, in the largest amount to arginine, less to histidine and the smallest amount to lysine and serine. As a function of the duration of storage, especially the P-Arg concentration was decreasing. The prolonged hydrolysis time decreases again the concentration of P-Arg with a consecutive increase of No. 1 and 2 P-containing peaks in the chromatographic profile of alkaline hydrolysate.     

Fibrinoligase-catalyzed cross-linking of myosin from platelet and skeletal muscle.

Fibrinoligase (thrombin- and calcium-activated Factor XIII) from human plasma catalyzes the incorporation of dansylcadaverine and [¹⁴C]putrescine into myosin, prepared from either human platelets or rabbit skeletal muscle. At least 9 mol of amine is incorporated per mole of myosin of either type when the enzyme is used under saturating conditions. Both heavy and light chains of the platelet and muscle myosins incorporate dansylcadaverine and [ ¹⁴C]putrescine. However, in quantitative terms, the incorporation into the light chains of either type is much less than into the heavy chains. Profound fluorescent changes occurred when dansylcadaverine was bound to myosin. Highly cross-linked platelet and muscle myosin polymers form in the absence of added amines, indicating the presence of both acceptor and donor sites. ATPase activity was not altered by cross-linking of 50–60% of myosin. The nature of the cross-link in myosin was found to be a γ-glutamyl-?-lysine bond, with an average of 19 mol of dipeptide per mole of platelet myosin.     

Age-related changes in the incorporation of [14-C] leucine into myofibrillar and sarcoplasmic proteins of red and white muscles of chicks.

Studies on the incorporation of DL-[1- 14-C] leucine into myosin, total myofibrillar protein and total sarcoplasmic protein have shown age-dependent alterations in the rate of synthesis of these protiens in red and white skeletal muscles of chicks. During the early phase of ex ovo development white muscle synthesizes significantly higher amounts of myofibrillar proteins, especially myosin, in comparison with red muscle. The rate of sarcoplasmic protein synthesis in red and white muscles one day after hatching is almost identical. The red muscle shows a markedly higher rate of sarcoplasmic protein synthesis from 10 days after hatching. The incorporation of amino acid into various protein fractions of both the muscle types decreases with advancing age. In adult chicks red muscle displays a higher ability to synthesize sarcoplasmic and myofibrillar proteins.     

Actomyosin content of Physarum plasmodia and detection of immunological cross-reactions with myosins from related species

The content of myosin in plasmodia of the myxomycete Physarum polycephalum was measured by an immunological technique, quantitative microcomplement (C') fixation. Migrating plasmodia (starved after growth on rolled oats) contained 0.60 +/- 0.08 (SD) mg myosin per g fresh plasmodia. Myosin comprised 0.77% +/- 0.05 (SD) of the total plasmodial protein. When total plasmodial proteins were separated by electrophoresis on SDS-polyacrylamide gels, a large amount of protein appeared in a band comigrating with muscle actin. Densitometry performed after Coomassie blue staining indicated that as much as 15- 25% of the total protein in the plasmodium could be actin. This gives an actin/myosin ratio by weight in the myxomycete plasmodium as high as 19-33, a very "actin-rich" actomyosin compared with rabbit skeletal muscle actomyosin with an actin/myosin ratio of 0.6. Starvation stimulates rapid migration and is correlated with a higher percent of both myosin and actin in the total protein of the plasmodium compared with normally growing cultures. Immunological cross-reaction of myosins from a variety of species was measured by C' fixation using an antiserum produced against purified native myosin from P. polycephalum. Although myxomycete and vertebrate striated muscle myosins have very similar morphological and biochemical properties, and apparently possess similar binding properties to F-actin, only myosins from myxomycetes in the order Physarales, rather closely related to P. polycephalum, gave detectable cross-reactions. This finding suggests that many amino acid sequences in myosin have been variable during evolution.     

Presence of phospho-tyrosine in alkaline hydrolysate of pig skeletal muscle myosin

A small part of the P-containing fraction of myosin hydrolysate which could be eluted with basic solvent (triethylamine) buffer was accumulated on chromatographic column. On the basis of the elution profile, P- and tyrosine content determination, moreover the thin layer chromatographic separation and specific Tyr reaction, this fraction is suggested to be phosphotyrosine. The concentration of P-Tyr is about 2 mol in pig muscle myosin calculated for 500 kDa protein. As the higher P-containing myosins also have an approx. 2 mol P-Tyr, it is thought that this concentration had been present in myosins already before alkaline hydrolysis.     

Protein kinase activity of purified rat liver glucocorticoid receptor

Molybdate-stabilized nonactivated rat liver glucocorticoid receptor (GR) was purified to near homogeneity using a biospecific affinity adsorbent, Bio Gel A 0.5 m and DEAE-Sephacel. The purified GR sedimented in the 9-10S region in 5-20% sucrose gradients containing 0.10M KCl and 20mM Na2MoO4. SDS-polyacrylamide gel electrophoresis revealed a major single band with an apparent molecular weight of 90,000 +/- 2,000. Affinity labeling of GR with [3H]-dexamethasone mesylate showed association of the radioactivity with a peptide of 90,000 molecular weight. Purified receptor preparation was dialyzed to remove molybdate and was incubated with different protein substrates in the presence of 50 microM [gamma-32P]-ATP and divalent cations. Radioactive phosphate from [gamma-32P]-ATP was seen to be incorporated into calf thymus histones, turkey gizzard myosin light chain kinase and rabbit skeletal muscle kinase in the presence of Mg2+ and Ca2+ ions. Addition of steroid ligand exogenously to the reaction mixture appeared to increase the extent of protein phosphorylation. No autophosphorylation of GR was evident under the above conditions. The data suggest that purified rat liver GR displays protein kinase activity.     

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.     

Polymorphism of myosin light chains. An electrophoretic and immunological study of rabbit skeletal-muscle myosins.

Antibodies specific for rabbit fast-twitch-muscle myosin LCIF light chain were purified by affinity chromatography and characterized by both non-competitive and competitive enzyme-linked immunosorbent assay (ELISA) and a gel-electrophoresis-derived assay (GEDELISA). The antibodies did not cross-react with myosin heavy chains, and were weakly cross-reactive with the LC2F [5,5'-dithio-(2-nitrobenzoic acid)-dissociated] light chain and with all classes of dissociated light chains (LC1Sa, LC1Sb and LC2S), as well as with the whole myosin, from hind-limb slow-twitch muscle. The immunoreactivity of myosins with a truly mixed light-chain pattern (e.g. vastus lateralis and gastrocnemius) correlated with percentage content of fast-twitch-muscle-type light chains. A more extensive immunoreactivity was observed with diaphragm and masseter myosins, which were also characterized, respectively, by a relative or absolute deficiency of LC1Sa light chain. Furthermore, it was found that the LC1Sb light chain of masseter myosin is antigenically different from its slow-twitch-muscle myosin analogue, and is immunologically related to the LC1F light chain. Rabbit masseter muscle from its metabolic and physiological properties and the content, activity and immunological properties of sarcoplasmic-reticulum adenosine triphosphatase, is classified as a red, predominantly fast-twitch, muscle. Therefore our results suggest that the two antigenically different iso-forms of LC1Sb light chain are associated with the myosins of fast-twitch red and slow-twitch red fibres respectively.     

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.     

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.