Dense precipitate of brain tubulin with skeletal muscle myosin

Purified tubulin from porcine brain formed a dense precipitate at 37 degrees C with muscle myosin filaments from rabbit skeletal muscle; this effect was greater than that with partially purified tubulin. ATP or GTP, which prevented the myosin filaments from precipitating, inhibited the formation of the dense precipitate, but did not dissociate the dense precipitate once formed. The dense precipitate was found by thin-section electron microscopy to be composed to side-by-side aggregates of myosin filaments whose projections might be decorated by tubulin. The decoration was also seen by negative-stain electron microscopy. The binding of tubulin to myosin filaments decreased the Mg2+- and Ca2+-GTPase activity of the myosin by about half, but did not affect either Mg2+- or Ca2+-ATPase activity. The binding ratio of tubulin to myosin in the presence of 5 mM MgCl2 was 2.2 mol/mol using purified tubulin and 1.8 mol/mol using partially purified tubulin. Five mM ATP and GTP in the presence of 5 mM MgCl2 decreased the tubulin binding by 1.6-2.0 and 1.1-1.3 mol/mol, respectively, when added before an encounter of tubulin with myosin filaments, but did not cause any decrease when added after such an encounter.     

Dissociation and reassociation of rabbit skeletal muscle myosin.

Whereas dissociation of rabbit skeletal muscle myosin light chains occurs at an increased temperature (25 degrees) and in the absence of divalent cations, reassociation of the myosin oligomer requires a low temperature (4 degrees C) and the presence of divalent cations, thus resulting in the original light to heavy chain stoichiometry. With a 5-10 per cent release of alkali light chains, LC1 and LC3, and a 50 per cent dissociation of the Ca2+ binding light chain, LC2, there is no significant decrease in myosin ATPase activity irrespective of the cation activator, however, there is an approximate 15-20 per cent decrease in actomyosin ATPase activity. With reassociation of the myosin oligomer, actomyosin ATPase activity is partially restored as well as the original number of Ca2+ binding sites.     

Autophosphorylation of skeletal muscle myosin light chain kinase.

Ca2+/calmodulin-dependent myosin light chain kinase phosphorylates the regulatory light chain of myosin. Rabbit skeletal muscle myosin light chain kinase also catalyzes a Ca2+/calmodulin-dependent autophosphorylation with a rapid rate of incorporation of 1 mol of 32P/mol of kinase and a slower rate of incorporation up to 1.52 mol of 32P/mol. Autophosphorylation was inhibited by a peptide substrate that has a low Km value for myosin light chain kinase. Autophosphorylation at both rates was concentration-independent, indicating an intramolecular mechanism. There were no significant changes in catalytic properties toward light chain and MgATP substrates or in calmodulin activation properties upon autophosphorylation. After digestion with V8 protease, phosphopeptides were purified and sequenced. Two phosphorylation sites were identified, Ser 160 and Ser 234, with the former associated with the rapid rate of phosphorylation. Both sites are located amino terminal of the catalytic domain. These results indicate that the extended "tail" region of the enzyme can fold into the active site of the kinase.     

Developmental modulation of myosin expression by thyroid hormone in avian skeletal muscle.

It is well established that a rise in circulating thyroid hormone during the second half of chick embryo development significantly influences muscle weight gain and bone growth. We studied thyroid influence on differentiation in slow anterior latissimus dorsi (ALD) and fast posterior latissimus dorsi (PLD) muscles of embryos rendered hypothyroid by hypophysectomy or administration of an anti-thyroid drug. The expression of native myosins and myosin light chains (MLCs) was studied by electrophoretic analysis, and the myosin heavy chain (MHC) was characterized by immunohistochemistry. The first effects of hypothyroid status were observed at day 21 of embryonic development (stage 46 according to Hamburger and Hamilton). Analysis of myosin isoform expression in PLD muscles of hypothyroid embryos showed persistence of slow migrating native myosins and slow MLCs as well as inhibition of neonatal fast MHC expression, indicating retarded differentiation of this muscle. In ALD muscle, hypothyroidism maintained fast embryonic MHC and induced noticeable amounts of fast MLCs, thus delaying slow muscle differentiation. Our results suggest that thyroid hormones play a role in modulating the appearance of neonatal fast MHC and the disappearance of isomyosins transiently present during embryogenesis. However, T3 supplemental treatment would seem to compensate in part for the effects of hypothyroidism induced by hypophysectomy, suggesting that thyroid hormone might interfere with other factors also accounting for the observed effects.     

Calcium sensitivity of vertebrate skeletal muscle myosin

The calcium sensitivity of vertebrate skeletal muscle myosin has been investigated. Adenosinetriphosphatase (ATPase) activity was assayed in a reconstituted system composed of either purified rabbit myosin plus actin or myosin plus actin, tropomyosin, and troponin. The calcium sensitivity of actomyosin Mg-ATPase activity was found to be directly affected by the ionic strength of the assay medium. Actomyosin assayed at approximately physiological ionic strength (120 mM KCl) demonstrated calcium sensitivity which varied between 6 and 52%, depending on the myosin preparation and the age of the myosin. Mg-ATPase activity was increased when calcium was present in the assay medium at physiological ionic strength. Conversely, actomyosin Mg-ATPase activity assayed at a lower ionic strength (15 mM KCl) was inhibited by addition of calcium. Addition of tropomyosin and troponin to the assay increased the calcium sensitivity of the system at the physiological ionic strength still further (up to 99% calcium sensitivity) and conferred calcium sensitivity on the system at the lower ionic strength (greater than 90% calcium sensitivity). A correlation also existed between myosin's calcium sensitivity and the phosphorylated state of light chain 2.     

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.     

The influence of ethylenediaminetetraacetate on white skeletal muscle myosin.

Myosin from rabbit white skeletal muscle was treated with 10 mM EDTA in 150 mM phosphate buffer. After precipitation of myosin by dialysis against a 14-fold volume of water, EDTA-treated myosin, myosin before treatment and the supernatant from the treatment of myosin with EDTA were examined on sodium dodecyl sulphate-polyacrylamide gels by electrophoresis. It has been found that the quantity of LC2 light chains diminished after treatment with EDTA, and the supernatant contained the LC2 light chains. Treatment of myosin with EDTA in the presence of Mg2+ does not change the stoichiometry of the LC2 light chain and the supernatant is free from LC2 light chains. The treatment of myosin with p-chloromercuri-benzoate leads to dissociation of the same amount of LC2 light chains. It is suggested that divalent cations and thiol groups are engaged in the attachment of LC2 light chain to the myosin molecule.     

Mammalian skeletal muscle myosin light chain kinases. A comparison by antiserum cross-reactivity

Purified myosin light chain kinases from skeletal muscle are reported to be significantly smaller (Mr = 75,000-90,000) than the kinases purified from smooth muscle (Mr = 130,000-155,000). It has been suggested that the smaller kinases from striated muscle are proteolytic fragments of a larger enzyme which is homologous, if not identical, to myosin light chain kinase from smooth muscle. Therefore, we have used an antiserum to rabbit skeletal muscle myosin light chain kinase and Western blot analysis to compare the subunit molecular weight of the kinase in skeletal muscle extracts of several mammalian species. In rabbit skeletal muscle, the antiserum only recognized a polypeptide of Mr = 87,000, with no indication that this polypeptide was a proteolyzed fragment of a larger protein. The apparent molecular weights observed in different animal species were 75,000 (mouse), 83,000 (guinea pig), 82,000 (rat), 87,000 (rabbit), 100,000 (dog), and 108,000 (steer). The molecular weight of myosin light chain kinase was constant within an animal species, regardless of skeletal muscle fiber type. The antiserum inhibited the catalytic activity of skeletal muscle myosin light chain kinase. Similar antibody dilution curves for inhibition of myosin light chain kinase activity in extracts were observed for all animal species (rabbit, rat, mouse, guinea pig, dog, cat, steer, and chicken) and different fibers (slow twitch oxidative, fast twitch oxidative glycolytic, and fast twitch glycolytic) tested. The antiserum did not inhibit the activity of rabbit smooth muscle myosin light chain kinase. These results suggest that there may be at least two classes of muscle myosin light chain kinase represented in skeletal and smooth muscles, respectively.     

Phosphorylation of synthetic peptides by skeletal muscle myosin light chain kinases

Substrate determinants for rabbit and chicken skeletal muscle myosin light chain kinases were examined with synthetic peptides. Both skeletal muscle myosin light chain kinases had similar phosphorylation kinetics with synthetic peptide substrates. Average kinetic constants for skeletal muscle myosin light chain heptadecapeptide, (formula; see text) where S(P) is phosphoserine, were Km, 2.3 microM and Vmax, 0.9 mumol/min/mg of enzyme. Km values were 122 and 162 microM for skeletal muscle peptides containing A-A for basic residues at positions 2-3 and 6-7, respectively. Average kinetic constants for smooth muscle myosin light chain peptide, (formula; see text), were Km, 1.4 microM and Vmax 27 mumol/min/mg of enzyme. Average Km values for the smooth muscle peptide, residues 11-23, were 10 microM which increased 6- and 11-fold with substitutions of alanine at residues 12 and 13, respectively. Vmax values decreased and Km values increased markedly by substitution of residue 16 with glutamate in the 11-23 smooth muscle tridecapeptide. Basic residues located 3 and 6-7 residues toward the NH2 terminus from phosphoserine in smooth muscle myosin light chain and 6-8 and 10-11 residues toward the NH2 terminus from phosphoserine in skeletal muscle myosin light chain appear to be important substrate determinants for skeletal muscle myosin light chain kinases. These properties are different from myosin light chain kinase from smooth muscle.     

Calmodulin and myosin light-chain kinase of rabbit fast skeletal muscle.

1. It is confirmed that myosin light-chain kinase is a protein of mol.wt. about 80,000 that is inactive in the absence of calmodulin. 2. In the presence of 1 mol of calmodulin/mol of kinase 80-90% of the maximal activity is obtained. 3. Crude preparations of the whole light-chain fraction of rabbit fast-skeletal-muscle myosin contain enough calmodulin to activate the enzyme. A method for the preparation of calmodulin-free P light chain is described. 4. A procedure is described for the isolation of calmodulin from rabbit fast skeletal muscle. 5. Rabbit fast-skeletal-muscle calmodulin is indistinguishable from bovine brain calmodulin in its ability to activate myosin light-chain kinase. The other properties of these two proteins are also very similar. 6. Rabbit fast-skeletal-muscle troponin C was about 10% as effective as calmodulin as activator for myosin light-chain kinase. 7. By chromatography on a Sepharose-calmodulin affinity column evidence was obtained for the formation of a Ca2+-dependent complex between calmodulin and myosin light-chain kinase. 8. Troponin I from rabbit fast skeletal muscle and histone IIAS were phosphorylated by fully activated myosin light-chain kinase at about 1% of the rate of the P light chain.     

Autophosphorylation of untrained and well-trained skeletal muscle myosin

Earlier the autophosphorylation of myosin and the labile phosphate (P) content of rabbit skeletal muscle was reported [6, 7, 9]. The present paper describes that the endogeneous preformed P level in fresh preparation of exercised muscle is higher than that of untrained control one. It was revealed that the presence of a significant amount of mitochondrial myosin (with much higher P content) in the well-trained human muscle preparations falsified the appreciation of myofibrillar myosin. Therefore, a reliable myofibrillar preparation with correct P content from exercised subjects was obtained only after the separation of mitochondrial fraction. The P content of fresh preparations can be increased by phosphorylation even in the exercised muscle myosins up to the most higher level in human samples. The phosphoryl group incorporation from [gamma-32P]ATP into the rabbit and hare myosins was checked by radioactive tracer technique, and confirmed by total P content determination performed parallel with molybdate test. It was stated that under present circumstances the labelled 32P incorporation was lower even at an optimal substrate concentration than that of P value obtained directly with molybdate method; because the total P content of preparations had not exchanged during 2 min incubation. So it has been concluded from [gamma-23P] phosphoryl group assayments that much higher amount of P was incorporated into P-Arg, N pi-P-His and fraction 2 as compared with unappreciated labelled P level of the inorganic P (P-Ser, P-Thr), P-Lys, N tau-P-His and minor fractions. From these observations it has been considered that the P-Arg, N pi-P-His and fraction 2 take part in the contraction mechanism and in the course of physical training.     

A model for the active site of skeletal muscle myosin.

A model for a main element of the active site of skeletal muscle myosin is presented that relates directly to the 92 amino acid fragment (p₁₀) of myosin recently described by Elzinga &; Collins (1977). In this model, the substrate, an eight-membered cyclic complex of MgATP, fits tightly into a 16 amino acid segment of p₁₀ and interacts with seven of its amino acids. A main feature of the model is the important role played by the one molecule of N^τ-methylhistidine² that is present in each myosin heavy chain. At the site, it is postulated that this rare amino acid functions as a donor ligand to Mg²⁺. Once N^τ-methylhistidine is put in place next to the metal, the other amino acids that appear to form a pocket come easily into position around the MgATP. These amino acids with their postulated functions are: tyrosine 72, which through a Mg-bound water, or perhaps directly, is attached to the Mg; histidine 76, which donates a proton to the P_γ of ATP; lysine 78, which binds electrostatically to P_β of ATP; phenylalanines 80 and 81, which flank the purine ring of ATP; and aspartate 66, which forms a hydrogen bond to the 6-amino group of adenine. The Mg-coordination role ascribed to N^τ-methylhistidine 69 in skeletal muscle myosin could be taken by histidine 69 in cardiac myosin and in other muscle myosins that do not contain the methylated amino acid.The choice of p₁₀ to contain a main element of the active site is based on: (a) the presence in p₁₀ of the essential sulfhydryl groups, SH1 and SH2, whose modification affects the ATPase activity of myosin; (b) the presence in ρ₁₀ of N^τ-methylhistidine, an unusual amino acid whose methylation in skeletal muscle we take as an indicator for a special function at the active site; (c) the position of p₁₀ in the primary structure near the junction between subfragment 1 and subfragment 2 (the hinge region) where, we postulate, enzymatic events at the active site are coupled to movements of the hinge that occur during contraction; (d) indications that the DTNB light chain, probably involved in regulation, is also near the hinge; (e) the effects of MgATP at the active site on the chemical reactivity of three SH groups (SH1, SH2 and SH3) located near the hinge; and (f) the effect of hinge cleavage on the oxygen exchange reaction catalyzed at the active site. The correlation of all these observations forms the basis for our placement of part of the active site on p₁₀ near the subfragment 1-subfragment 2 hinge.     

Phosphorylation of rabbit skeletal muscle myosin in situ

Myosin light chain (P light chain) is phosphorylated by Ca2+ X calmodulin-dependent myosin light chain kinase. Based on studies with rat skeletal muscles, it has been shown that P light chain phosphorylation correlated to the extent of potentiation of isometric twitch tension. It is not clear whether this correlation exists in rabbit skeletal muscle, which has been the primary source of contractile proteins for biochemical studies. Therefore, phosphorylation of myosin P light chain in rabbit slow-twitch soleus and fast-twitch plantaris muscles in situ was examined. Electrical stimulation (5 Hz, 20 seconds) of plantaris muscle produced an increase in the phosphate content of P light chain from 0.17 to 0.45 mol phosphate/mol P light chain. This increase in phosphate content was accompanied by a 58% increase in maximal isometric twitch tension. Tetanic stimulation (100 Hz, 15 seconds) of rabbit soleus muscle resulted in only a small increase in P light chain phosphate content from 0.02 to 0.10 mol phosphate/mol P light chain, and posttetanic twitch tension did not increase significantly. The correlation between potentiated isometric twitch tension and P light chain phosphorylation in rabbit fast-twitch muscle is similar to that observed in rat skeletal muscle. These results were consistent with the hypothesis that phosphorylation of rabbit skeletal muscle myosin, which results in an increase in actin-activated ATPase activity, may be related to isometric twitch potentiation.     

Dissociation and reassociation of rabbit skeletal muscle myosin

Whereas dissociation of rabbit skeletal muscle myosin light chains occurs at an increased temperature (25°) and in the obsence of divalent cations, reassociation of the myosin oligomer requires a low temperature (4°C) and the presence of divalent cations, thus resulting in the original light to heavy chain stoichiometry. With a 5–10 per cent release of alkali light chains, LC₁ and LC₃, and a 50 per cent dissociation of the Ca²⁺ binding light chain, LC₂, there is no significant decrease in myosin ATPase activity irrespective of the cation activator, however, there is an approximate 15–20 per cent decrease in actomyosin ATPase activity. With reassociation of the myosin oligomer, actomyosin ATPase activity is partially restored as well as the original number of Ca²⁺ binding sites.     

The mechanism of the skeletal muscle myosin ATPase. I. Identity of the myosin active sites.

In the present study, the question of whether the two myosin active sites are identical with respect to ATP binding and hydrolysis was reinvestigated. The stoichiometry of ATP binding to myosin, heavy meromyosin, and subfragment-1 was determined by measuring the fluorescence enhancement caused by the binding of MgATP. The amount of irreversible ATP binding and the magnitude of the initial ATP hydrolysis (initial Pi burst) was determined by measuring [gamma-32P]ATP hydrolysis with and without a cold ATP chase in a three-syringe quenched flow apparatus. The results show that, under a wide variety of experimental conditions: 1) the stoichiometry of ATP binding ranges from 0.8 to 1 mol of ATP/myosin active site for myosin, heavy meromyosin, and subfragment-1, 2) 80 to 100% of this ATP binding is irreversible, 3) 70 to 90% of the irreversibly bound ATP is hydrolyzed in the initial Pi burst, 4) the first order rate constant for the rate-limiting step in ATP hydrolysis by heavy meromyosin is equal to the steady state heavy meromyosin ATPase rate only if the latter is calculated on the basis of two active sites per heavy meromyosin molecule. It is concluded that the two active sites of myosin are identical with respect to ATP binding and hydrolysis.     

Binding of myosin cross-bridges to thin filaments of rabbit skeletal muscle.

Cross-linking of myosin subfragment 1 (S1) with a molar excess of actin in vitro reveals the presence of an actin-S1-actin complex. It is absolutely essential that actin be present in molar excess over S1 so that the decoration of F-actin with S1 be incomplete. However, the excess of actin may not be available in the overlap zone of sarcomeres of skeletal muscle. We therefore found it necessary to test for the presence of the actin-S1-actin complex in vivo. Myofibrils from rabbit skeletal muscle were reacted with zero-length cross-linker, the products were resolved by polyacrylamide gel electrophoresis and analyzed by Western blots using antibodies against actin and against heavy and light chains of myosin. The cross-linking produced the evidence of formation of actin-S1-actin complex.