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.     

Peroxidative crosslinking of myosins.

The effect of myoglobin, free hemin and H2O2 on myosins from heart and skeletal muscle was studied. SDS-gel electrophoresis revealed that each agent caused intermolecular thiol crosslinking of both myosins dissociable by excess of beta-mercaptoethanol. In the simultaneous presence of H2O2 and myoglobin or H2O2 and free hemin, myosin formed covalent aggregates undissociable by beta-mercaptoethanol and therefore assessed to formation of non S-S inter molecular covalent bonds. The latter aggregates are suggested to result from pairing of myosin radicals formed by the H2O2 induced ferryl iron state in myoglobin, free hemin or hemo-myosin.     

Autoantibodies specific for the cardiac myosin isoform are found in mice susceptible to Coxsackievirus B3-induced myocarditis

Several mouse strains are susceptible to immunopathic myocarditis after infection with Coxsackievirus B3 (CB3). This disease is associated with autoantibodies that are directed against myosin. In this study we characterized sera from CB3-infected mice for their reactivity with three different myosin isoforms (heart, skeletal muscle, and brain myosins) and for autoantibody isotype by using an ELISA. Competitive inhibition assays and absorption studies with various myosins demonstrated the presence of two autoantibody populations in sera of susceptible A.CA and A.SW mice. The first was specific for cardiac myosin and was mainly IgG. The second antibody population cross-reacted with heart, skeletal muscle, and brain myosin and was mainly IgM. B10.PL/SgSf and B10.A/SgSf mice, which do not develop immunopathic myocarditis, produced only the IgM autoantibody population cross-reactive with all three myosin isoforms. Because the heart-specific myosin autoantibodies were found exclusively in the mouse strains that developed immunopathic myocarditis, they can be considered a serologic marker for autoimmune heart disease.     

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.     

Ca2+-activated ATPase reaction of myosin from human cardiac, skeletal and smooth muscle cells

A comparative study of Ca2+ activated ATPase reaction of myosin isolated from cardiac, skeletal and smooth human muscles shows that on the molecules of all kinds of myosins there exist two types of centres binding the substrate CaATP with high and low affinity (Km approximately 1.6 x 10(-6) M and Km approximately 200 x 10(6) M respectively). Hydrolysis of ATP by myosin is noncompetitively activated by three types of Ca2+ binding sites on its molecule, having different affinity. The association constants (KaCa) for the smooth muscles are several times higher than those for skeletal and cardiac muscles.     

Androgen and estrogen binding in rat skeletal and perineal muscles.

Specific in vitro binding of [3H]testosterone (T), 5ALPHA[3H]dihydrotestosterone (DHT), and [3H[estradiol (E2) was demonstrated in the 30 000 X g supernatant (cytosol) of thigh muscles (TM) and of the levator ani - bulbocavernosus muscle complex (LA-BC) by gel filtration through Sephadex G-25 columns. In TM cytosol, T and E2 [are bound with high affinity (Ka = 1.1 X 10(9) M-1, and 2.3 X 10(9) M-1 respectively) whereas DHT binding is of lower affinity (Ka = 5.0 X 10(7) M-1).] In LA-BC cytosol, T, E2, and DHT are bound with high affinity (Ka = 1.9 X 10(9) M-1, 0.3 X 10(9) M-1, and 0.5 X 10(9) M-1, respectively). Competition experiments suggest that the binding of the three hormones (T, E2, and DHT) is due to different proteins. In addition to TM and LA-BC, T and E2 binding was found in other muscles of male and female rats, including gastrocnemius, the pectoralis, diaphragm, and heart.     

Heavy meromyosin binds actin with negative cooperativity.

The association of fluorescently labeled heavy meromyosin (HMM) and F-actin was measured by time-resolved fluorescence depolarization. The effects of varying the protein concentrations, temperature, KCl concentration, and pH were determined. Measurements of HMM mobility supported a model of no interaction between the two heads in the absence of actin. Measurements of actin binding, when compared with results for myosin subfragment I, indicated that the two heads of HMM do not bind independently in the rigor complex. This could result from actin-transmitted negative cooperativity or from steric inhibition due to the structure of HMM. For HMM and actin in 0.15 7 kcl at 25 degrees C: Ka = 3.9 X 10(7) M-1, deltaHco' = 36 +/- 2 J M-1, deltaSco' = 0.26 +/- 0.02 kJ M-1 K-1; the slope of ln Ka vs. [KCl]1/2 = -3.88 and the pH of maximum association was 6.9.     

Order-disorder structural transitions in synthetic filaments of fast and slow skeletal muscle myosins under relaxing and activating conditions

In the previous study (Podlubnaya et al., 1999, J. Struc. Biol. 127, 1-15) Ca2+-induced reversible structural transitions in synthetic filaments of pure fast skeletal and cardiac muscle myosins were observed under rigor conditions (-Ca2+/+Ca2+). In the present work these studies have been extended to new more order-producing conditions (presence of ATP in the absence of Ca2+) aimed at arresting the relaxed structure in synthetic filaments of both fast and slow skeletal muscle myosin. Filaments were formed from column-purified myosins (rabbit fast skeletal muscle and rabbit slow skeletal semimebranosusproprius muscle). In the presence of 0.1 mM free Ca2+, 3 mM Mg2+ and 2 mM ATP (activating conditions) these filaments had a spread structure with a random arrangement of myosin heads and subfragments 2 protruding from the filament backbone. Such a structure is indistinguishable from the filament structures observed previously for fast skeletal, cardiac (see reference cited above) and smooth (Podlubnaya et al., 1999, J. Muscle Res. Cell Motil. 20, 547-554) muscle myosins in the presence of 0.1 mM free Ca2+. In the absence of Ca2+ and in the presence of ATP (relaxing conditions) the filaments of both studied myosins revealed a compact ordered structure. The fast skeletal muscle myosin filaments exhibited an axial periodicity of about 14.5 nm and which was much more pronounced than under rigor conditions in the absence of Ca2+ (see the first reference cited). The slow skeletal muscle myosin filaments differ slightly in their appearance from those of fast muscle as they exhibit mainly an axial repeat of about 43 nm while the 14.5 nm repeat is visible only in some regions. This may be a result of a slightly different structural properties of slow skeletal muscle myosin. We conclude that, like other filaments of vertebrate myosins, slow skeletal muscle myosin filaments also undergo the Ca2+-induced structural order-disorder transitions. It is very likely that all vertebrate muscle myosins possess such a property.     

Comparative studies on amino and thiol groups in myosins from different sources.

Myosins from rabbit white and red skeletal, rabbit heart, fish skeletal and chicken gizzard muscles, as well as from human platelets were subjected to trinitrophenylation by trinitrobenzene sulfonate and alkylation by N-ethylmeleimide which affected their amino and thiol groups, respectively. The blocking of amino groups was carried out in the presence or in the absence of Mg-ADP and was followed both spectrophotometrically and enzymatically. Essential amino groups, whose modification throughly changes the enzymic characteristics of myosin, were found in heart and in all skeletal muscle myosins but were absent in myosins from chicken gizzard muscle and from human platelets. The reaction of these amino groups was highly retarded in the presence of Mg-ADP. Alkylation of thiols led to loss of the K+-activated ATPase (ATP phosphohydrolase, EC 3.6.1.3) in all myosins. However, the rate of loss of activity varied from one myosin to another and, for a given myosin, was affected by the presence of nucleotides and by the value of the ionic strength. The change in Ca(2+)-activated ATPase activity (ATP phosphohydrolase, EC 3.6.1.3) on alkylation was influenced by the presence of Mg - ADP during the reaction. In the absence of this nucleotide, the Ca(2+)-ATPase activity increased and reached a plateau as a consequence of modification. The extent of activation largely depended on the origin of the myosin. When alkylation was carried out in the presence of Mg-ADP, the Ca(2+)-ATPase activity as a function of time exhibited a maximum but the descending part of the curve was absent in myosins from heart and gizzard muscles.     

Electron microscopy of synthetic myosin filaments. Evidence for cross- bridge. Flexibility and copolymer formation

Electron micrographs of negatively stained synthetic myosin filaments reveal that surface projections, believed to be the heads of the constituent myosin molecules, can exist in two configurations. Some filaments have the projections disposed close to the filament backbone. Other filaments have all of their projections widely spread, tethered to the backbone by slender threads. Filaments formed from the myosins of skeletal muscle, smooth muscle, and platelets each have distinctive features, particularly their lengths. Soluble mixtures of skeletal muscle myosin with either smooth muscle myosin or platelet myosin were dialyzed against 0.1 M KC1 at pH 7 to determine whether the simultaneous presence of two types of myosin would influence the properties of the filaments formed. In every case, a single population of filaments formed from the mixtures. The resulting filaments are thought to be copolymers of the two types of myosin, for several reasons: (a) their length-frequency distribution is unimodal and differs from that predicted for a simple mixture of two types of myosin filaments; (b) their mean length is intermediate between the mean lengths of the filaments formed separately from the two myosins in the mixture; (c) each of the filaments has structural features characteristic of both of the myosins in the mixture; and (d) their size and shape are determined by the proportion of the two myosins in the mixture.     

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.     

The dynamics of the interaction between myosin subfragment 1 and pyrene-labelled thin filaments, from rabbit skeletal muscle

We have used actin labelled in Cys-374 with N-(1-pyrenyl)iodoacetamide to monitor the dynamics and equilibria of the interaction between myosin subfragment 1 and the actin-troponin-tropomyosin complex in the presence of calcium. These results are compared with those obtained for pure actin and myosin subfragment 1. The sensitivity of this fluorescent label allowed us to measure the binding affinity of myosin subfragment 1 for actin directly by fluorescence titration. The affinity of subfragment 1 for actin is increased sixfold by troponin-tropomyosin in the presence of calcium. Kinetic studies of the interaction of subfragment 1 and actin have revealed an isomerization of the actin-subfragment 1 complex from a state in which actin is weakly bound (Ka = 5.9 X 10(4) M-1) to a more tightly bound complex (Ka = 1.7 X 10(7) M-1) (Coates, Criddle & Geeves (1985) Biochem. J. 232, 351). Results in the presence of troponin-tropomyosin show the same isomerization. The sixfold increase in affinity of subfragment 1 for actin is shown to be due to a decrease in the rate of dissociation of actin from the weakly bound complex.     

Cooperative regulation of myosin-actin interactions by a continuous flexible chain I: actin-tropomyosin systems

We present a model for cooperative myosin binding to the regulated actin filament, where tropomyosins are treated as a weakly-confined continuous flexible chain covering myosin binding sites. Thermal fluctuations in chain orientation are initially required for myosin binding, leaving kinked regions under which subsequent myosins may bind without further distortion of the chain. Statistical mechanics predicts the fraction of sites with bound myosin-S1 as a function of their affinities. Published S1 binding curves to regulated filaments with different tropomyosin isoforms are fitted by varying the binding constant, chain persistence length nu (in actin monomers), and chain kink energy A from a single bound S1. With skeletal tropomyosin, we find an S1 actin-binding constant of 2.2 x 10(7) M(-1), A = 1.6 k(B)T and nu = 2.7. Similar persistence lengths are found with yeast tropomyosin. Larger values are found for tropomyosin-troponin in the presence of calcium (nu = 3.7) and tropomyosins from smooth muscle and fibroblasts (nu = 4.5). The relationship of these results to structural information and the rigid-unit model of McKillop and Geeves is discussed.     

Characterization of caldesmon binding to myosin

Caldesmon inhibits the binding of skeletal muscle subfragment-1 (S-1).ATP to actin but enhances the binding of smooth muscle heavy meromyosin (HMM).ATP to actin. This effect results from the direct binding of caldesmon to myosin in the order of affinity: smooth muscle HMM greater than skeletal muscle HMM greater than smooth muscle S-1 greater than skeletal muscle S-1 (Hemric, M. E., and Chalovich, J. M. (1988) J. Biol. Chem. 263, 1878-1885). We now show that the difference between skeletal muscle HMM and S-1 is due to the presence of the S-2 region in HMM and is unrelated to light chain composition or to two-headed versus single-headed binding. Differences between the binding of smooth and skeletal muscle myosin subfragments to actin do not result from the lack of light chain 2 in skeletal muscle S-1. In the presence of ATP, caldesmon binds to smooth muscle myosin filaments with a stoichiometry of 1:1 (K = 1 x 10(6) M-1). Similar results were obtained for the binding of caldesmon to smooth muscle rod as well as the binding of the purified myosin-binding fragment of caldesmon to smooth muscle myosin. The binding of caldesmon to intact myosin is ATP sensitive. The interaction of caldesmon with myosin is apparently specific and sensitive to the structure of both proteins.     

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.     

Myosin types in cultured muscle cells

Fluorescent antibodies against fast skeletal, slow skeletal, and ventricular myosins were applied to muscle cultures from embryonic pectoralis and ventricular myocadium of the chicken. A number of spindle-shaped mononucleated cells, presumably myoblasts, and all myotubes present in skeletal muscle cultures were labeled by all three antimyosin antisera. In contrast, in cultures from ventricular myocardium all muscle cells were labeled by anti-ventricular myosin, whereas only part of them were stained by anti-slow skeletal myosin and rare cells reacted with anti-fast skeletal myosin. The findings indicate that myosin(s) present in cultured embryonic skeletal muscle cells contains antigenic determinants similar to those present in adult fast skeletal, slow skeletal, and ventricular myosins.     

Primary structure of myosin heavy chain from fast skeletal muscle of Chum salmon Oncorhynchus keta

The nucleotide sequence of the cDNA encoding myosin heavy chain of chum salmon Oncorhynchus keta fast skeletal muscle was determined. The sequence consists of 5,994 bp, including 5,814 bp of translated region deducing an amino acid sequence of 1,937 residues. The deduced sequence showed 79% homology to that of rabbit fast skeletal myosin and 84-87% homology to those of fast skeletal myosins from walleye pollack, white croaker and carp. The putative binding-sites for ATP, actin and regulatory light-chains in the subfragment-1 region of the salmon myosin showed high homology with the fish myosins (78-100% homology). However, the Loop-1 and Loop-2 showed considerably low homology (31-60%). On the other hand, the deduced sequences of subfragment-2 (533 residues) and light meromyosin (564 residues) showed 88-93% homology to the corresponding regions of the fish myosins. It becomes obvious that several specific residues of the rabbit LMM are substituted to Gly in the salmon LMM as well as the other fish LMMs. This may be involved in the structural instability of the fish myosin tail region.     

Kinetic analysis of the slow skeletal myosin MHC-1 isoform from bovine masseter muscle

Several heavy chain isoforms of class II myosins are found in muscle fibres and show a large variety of different mechanical activities. Fast myosins (myosin heavy chain (MHC)-II-2) contract at higher velocities than slow myosins (MHC-II-1, also known as beta-myosin) and it has been well established that ADP binding to actomyosin is much tighter for MHC-II-1 than for MHC-II-2. Recently, we reported several other differences between MHC-II isoforms 1 and 2 of the rabbit. Isoform II-1 unlike II-2 gave biphasic dissociation of actomyosin by ATP, the ATP-cleavage step was significantly slower for MHC-II-1 and the slow isoforms showed the presence of multiple actomyosin-ADP complexes. These results are in contrast to published data on MHC-II-1 from bovine left ventricle muscle, which was more similar to the fast skeletal isoform. Bovine MHC-II-1 is the predominant isoform expressed in both the ventricular myocardium and slow skeletal muscle fibres such as the masseter and is an important source of reference work for cardiac muscle physiology. This work examines and extends the kinetics of bovine MHC-II-1. We confirm the primary findings from the work on rabbit soleus MHC-II-1. Of significance is that we show that the affinity of ADP for bovine masseter myosin in the absence of actin (represented by the dissociation constant K(D)) is weaker than originally described for bovine cardiac myosin and thus the thermodynamic coupling between ADP and actin binding to myosin is much smaller (K(AD)/K(D) approximately 5 instead of K(AD)/K(D) approximately 50). This may indicate a distinct type of mechanochemical coupling for this group of myosin motors. We also find that the ATP-hydrolysis rate is much slower for bovine MHC-II-1 (19 s(-1)) than reported previously (138 s(-1)). We discuss how this work fits into a broader characterisation of myosin motors from across the myosin family.     

Interaction of myosin subfragments with F-actin.

The effect of ionic strength, temperature, and divalent cations on the association of myosin with actin was determined in the ultracentrifuge using scanning absorption optics. The association constant (Ka) for the binding of heavy meromyosin (HmM) to F-actin was 1 X 10(7) M-1 at 20 degrees C, in 0.10 M KCl, 0.01 M imidazole (pH 7.0), 5 MM potassium phosphate, 1 mM MgCl2, and 0.3 mM ethylene glycol bis(beta-aminoethyl ether)-N,N'-tetraacetic acid. Ka was the same for HMM prepared by trypsin or chymotrypsin. The affinity of subfragment 1 (S1) for actin under the same ionic conditions was 3 X 10(6) M-1. Varying the preparative procedure for S1 had little effect on Ka. The small difference in binding energy between HMM and S1 suggests that either only one head can bind strongly to actin at a time or that free energy is lost during the sterically unfavorable attachment of the two heads to actin.     

Characterisation of monoclonal antibodies raised against testosterone

Using the antigens testosterone-17 beta-hemisuccinate and testosterone-3-(o-carboxymethyl) oxime, each coupled to bovine serum albumin, we have produced 44 monoclonal antibodies to testosterone. Of the 17 monoclonal antibodies raised against the 17 beta-linked antigen 8 showed extremely low affinity for testosterone (Ka less than or equal to 8 X 10(7) M-1) and none had an affinity greater than 5 X 10(9) M-1. Of the 27 monoclonal antibodies raised against the 3-linked antigen 2 had affinities less than 8 X 10(7) M, 7 had affinities greater than 5 X 10(9) M-1 and one had an affinity (Ka = 9 X 10(10) M-1) greater than that of a high affinity rabbit antiserum (Ka = 6 X 10(10) M-1). The affinity constant (Ka = 5 X 10(9) M-1) measured in the serum of the mouse whose spleen gave rise to the greatest number of high affinity antibodies, was significantly higher than those measured in the sera of the remaining mice (Ka = 0.7 - 3 X 10(8) M-1). The cross-reactions of the monoclonal antibodies varied widely but none showed an overall improvement in specificity when compared with the corresponding rabbit antisera. Results suggest that as well as the structure of the steroid antigen careful selection of the spleen donor facilitates the development of monoclonal antibodies with good binding characteristics.