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Scallop striated adductor muscle myosin is a regulatory myosin, its activity being controlled directly through calcium binding. Here, we show that millimolar concentrations of trifluoperazine were effective at removal of all regulatory light chains from scallop myosin or myofibrils. More important, 200 microM trifluoperazine, a concentration 10-fold less than that required for light-chain removal, resulted in the reversible elimination of actin-activated and intrinsic ATPase activities. Unlike desensitization induced by metal ion chelation, which leads to an elevation of activity in the absence of calcium concurrent with regulatory light-chain removal, trifluoperazine caused a decline in actin-activated MgATPase activity both in the presence and absence of calcium. Procedures were equally effective with respect to scallop myosin, myofibrils, subfragment-1, or desensitized myofibrils. Increased alpha-helicity could be induced in the isolated essential light chain through addition of 100-200 microM trifluoperazine. We propose that micromolar concentrations of trifluoperazine disrupt regulation by binding to a single high-affinity site located in the C-terminal domain of the essential light chain, which locks scallop myosin in a conformation resembling the off-state. At millimolar trifluoperazine concentrations, additional binding sites on both light chains would be filled, leading to regulatory light-chain displacement.

A neutral protease, mekratin, active in human hearts at end stage idiopathic dilated cardiomyopathy (IDC), mediates the breakdown of cardiac myosin LC2. Myosin purified from IDC heart tissue forms unusually short synthetic thick filaments. Therefore, determination of filament length and mekratin distribution in IDC heart muscle were initiated. Native thick filaments were prepared directly from control and IDC tissues and analyzed. Also, paraffin-embedded tissue sections were stained with a fluorescently-labeled anti-protease antibody to establish its distribution in myocardial tissues. Control sections had only very weak, background levels of fluorescence whereas IDC sections stained intensely throughout, indicating a wide ranging distribution of the protease within the myocyte cytoplasm. SDS-PAGE revealed LC2 to be present in stoichiometric amounts in control but greatly reduced in IDC heart muscle. Native thick filaments from control myocardium were structurally stable. They had a median length of 1.65 microm with well-defined bare zones and displayed the 43 nm helical periodicity typical of the relaxed arrangement of myosin heads close to the filaments' shafts. In contrast, native IDC filaments were less stable, and had a median length of 0.9 microm. These filaments were highly disordered: they had no surface periodicity and myosin heads were positioned away from the filaments' shafts. The shorter, less stable, aperiodic thick filaments from IDC hearts appear to result from depletion of LC2 caused by increased activity of mekratin in the IDC myocardium.

Calcium regulation in the human heart is impaired during idiopathic dilated cardiomyopathy (IDC). Here, we analyze the structural basis for impairment in the regulatory mechanism. Regulation of contractility was monitored by MgATPase and Ca2+-binding assays as a function of calcium. Myofibrillar proteolysis and expression of troponin T isoforms were established by gel electrophoresis and by Western blots. Myofibrillar ATPase assays in low salt however, revealed a drastic lowering of calcium sensitivity in IDC myofibrils as indicated by reductions in both activation by high calcium and in EGTA-mediated inhibition of MgATPase. Structural changes in myofilament proteins were found in most IDC hearts, specifically proteolysis of myosin light chain 2 (LC2), troponin T and I (TnT and TnI), and sometimes a large isoform shift in TnT. IDC did not induce mutations in LC2 and troponin C (TnC), as established by cDNA sequence data from IDC cases, thus, calcium binding to IDC myofibrils was unaffected. Reassociation of IDC myofibrils with native LC2 raised MgATPase activation at high Ca2+ to control levels, while repletion with intact, canine TnI/TnT restored inhibition at low Ca2+. A model, identifying possible steps in the steric blocking mechanism of regulation, is proposed to explain IDC-induced changes in Ca2+-regulation. Moreover, shifts in TnT isoforms may imply either a genetic or a compensatory factor in the development and pathogenesis of some forms of IDC.

A neutral protease with an estimated Mr of about 26 kD and responsible for cleavage ofmyosin LC2 was isolated from hamster skeletal muscle. Complementary DNAs were generated by RT-PCR using total hamster muscle RNA and degenerate oligonucleotide primers based on the sequences of two internal peptides. The nucleotide sequences of the resultant cDNAs were subsequently determined and the complete amino acid sequence of the protease deduced. Although the hamster protein shared 63-85% identity in nucleotide and amino acid sequences with rat and mouse mast cell proteases, it had a higher degree of specificity for myosin LC2 than mast cell proteases which also digested myosin LC1 and myosin heavy chains. As a result, the hamster protease was designated mekratin because of its unique enzymatic specificities to distinguish it from other mast cell proteases. A polyclonal antibody was raised specific to the hamster muscle and human cardiac muscle mekratins without apparent cross-reaction with rat mast cell proteases. We have earlier demonstrated the presence in excess of a neutral protease that specifically cleaves LC2 in human hearts obtained at end stage idiopathic dilated cardiomyopathy (IDC). Western analyses revealed that heart tissue from patients with IDC contained 5-10 fold more mekratin than control samples. Furthermore, the level of the protease in human IDC tissues was similar to that seen in myopathic hamster skeletal muscle. No bands were recognized by the antibody when IDC myofibrils were probed due to the removal of soluble proteins during sample preparation. Thus, these results strongly suggest that the anti-mekratin antibody will provide positive identification of IDC in many cases and diagnosis by exclusion may be replaced.

The primary structures of light chains isolated from the human myocardium with idiopathic dilated cardiomyopathy (IDC) were determined and compared with the sequence structures of myosin light chains obtained from control human heart myosin. Sequences were determined by chemical analysis and the identity of N-terminal residues established by mass spectrometry. The N-terminal residues in essential (ELC) and regulatory (RLC) light chains were blocked and were identified to be trimethyl alanine. The amino acid sequences of ELC and RLC from control human myosin revealed a high degree of homology with those purified from rat and chicken cardiac myosin. Comparison with a published partial chemical sequence of the human heart myosin light chains revealed significant variations. However, there was very good agreement with published sequences obtained by molecular biological techniques. Sequences of the light chains from cardiomyopathic myosin revealed no difference in the primary structures when compared with control human heart myosin light chains indicating IDC had no influence on, nor was caused by, altered myosin light chain gene expression.

Electron microscopic and physical-chemical properties of one- and two-chain tissue plasminogen activator (t-PA) were studied. The molecular weight of one-chain t-PA obtained by both sedimentation equilibrium and SDS-PAGE was estimated to be about 65,000, while both chains in the reduced two-chain form were in the range of 35,000-40,000. Sedimentation coefficients were identical for both forms of t-PA (S(0)20,w = 4.12). The two forms of t-PA were indistinguishable by electron microscopic analysis, which confirmed the sedimentation results, and showed that they were ellipsoidal and relatively compact. The major and minor axes were approx. 13 nm and approx. 10 nm and f/f0 was 1.36. The individual domains of t-PA are relatively small and are folded within the molecule, so that the overall appearance is globular.

Objective: A comparative study of the kinetics of proteolysis of myosin S1 heavy chain was performed using dog ventricular and atrial S1 to distinguish between protease sensitive sites in S1 isotypes and to determine the binding sites on S1 heavy chain for LC1, LC2, and actin., Methods: Digestion of S1 as a function of actin was performed at 25 degrees C at a trypsin to S1 ratio (w/w) of 1:1500. Myofibrils were digested (trypsin/myofibrils w/w ratio = 1:300) in the presence of ATP, ADP, and under rigor conditions. Light chain and actin binding sites were identified by the gel overlay method., Results: Ventricular and atrial S1 were proteolysed at 0.13 min-1 and 0.04 min-1 respectively. Actin significantly reduced the cleavage rate of both S1 heavy chains by blocking hydrolysis at the 50/20 kD site. Myofibrillar myosin heavy chains from ventricles were also hydrolysed faster than those of the atria in the presence of 4 mM MgATP. The calculated rates were 0.42-0.50 and 0.17-0.19 min-1 for ventricular and atrial myofibrils respectively. MgADP 2 mM or absence of nucleotides reduced the cleavage rates to 0.04-0.07 (ventricular myofibrils) and 0.02-0.03 min-1 (atrial myofibrils) respectively. Gel overlay experiments showed that 125I labelled LC1 and LC2 bound to the 20 kD fragment and actin mainly to the 50 and 20 kD peptides., Conclusions: The 50/20 kD site in either ventricular or atrial S1 was blocked when actin was present, while proteolysis at the 25/50 site proceeded regardless of the presence of actin. However, the 25/50 site was less accessible to trypsin in the alpha myosin heavy chain, since the roughly threefold reduction in the rates of hydrolysis of atrial S1 heavy chain was also maintained in the myofibrils in rigor or in the presence of ADP. Although actin made contact with the 70 kD and the 25 kD fragments, the 50 kD and 20 kD fragments appeared to be the central "anchor" for binding of both light chains and actin.

The effect of LC1 on cardiac myosin structure and activity was investigated using as a model S1 prepared by papain digestion in the presence of Mg2+. The resulting S1 contained LC2 but a part of the N-terminal region of LC1 was cleaved. Sequencing the N-terminal part of the band migrating below LC1 on SDS gels revealed it to consist of alternating alanyl and prolyl residues thus establishing LC1 as the origin of this band. However, Western blots did not reveal any LC1 while radioimmunoassays indicated it to be present at the 5% level suggesting the anti-LC1 antibody used in these experiments did not recognize the C-terminal portion of LC1 still attached to Mg.S1. Mixing a 10-15 M excess of isolated light chains with Mg.S1 in the presence of 10 mM ATP, 12 mM MgCl2, 4.7 M NH4Cl allowed LC1 to recombine with LC1-deficient Mg.S1. Equilibrium ultracentrifugation analysis revealed a highly heterogeneous LC1-deficient S1 which upon recombination with intact LC1 became monodisperse as indicated by the superimposition of molecular weight averages all across the centrifuge cell. LC1-deficient Mg.S1 had a Vm of 0.4 s-1, Ka of 30 microM and a Kbind of 28 microM. In the presence of intact LC1, Vm rose to 0.8 s-1 while Ka and Kbind were reduced to 7.5 and 12 microM, respectively. The fourfold decrease in Ka strongly indicated an increased affinity for actin by Mg.S1 in the presence of uncleaved LC1. Also, Ca(2+)-regulation of dog heart myofibrils was suppressed when Ca(2+)-activated MgATPase assays, as a function of Ca2+, were performed in the presence of anti-LC1 antibodies. These observations suggest the presence of intact, uncleaved LC1 in S1 is required for the stability of S1 heavy chains and proper Ca(2+)-regulation.

Background: A number of parameters reflecting the effects of idiopathic dilated cardiomyopathy (IDC) on the structure and function of myosin from the human myocardium were analyzed., Methods and Results: The content of the regulatory light chain, LC2, was reduced in myopathic heart myosin in contrast to the controls in which it was present in stoichiometric amounts relative to the essential light chain, LC1. In IDC hearts, the absence or significant reduction in amount of LC2 was related to the presence of an active protease, which was isolated and purified about 130-fold. The protease exhibited a significant degree of specificity: It cleaved LC2 almost totally (but not the heavy chains) in human control heart myosin but only partially cleaved LC2 in canine heart or in rabbit skeletal muscle myosins. The protease was present at a very low level or was inactive in control heart tissue. When the LC1/LC2 molar ratio was calculated, it was found to be 1:1.0 in control heart myosin and remained constant in various samples analyzed, whereas in myopathic myosin from different individuals, this ratio varied from 1:0.1 to 1:0.69. The rates of ATP binding to control and myopathic myosins were similar, whereas the Vm of actin-activated ATPase of myopathic myosin was about 25% less than that of the control. However, ATP binding and its hydrolysis by control S1, i.e., the myosin head, were faster by a factor of 2 than that of the myopathic S1. In addition, control myosin synthetic thick filament length as well as turbidity in solution, measured by light scattering, were twice as large as those of the myopathic heart myosin. These effects induced by myopathy in both filament assembly and turbidity were reversed upon reassociation of IDC myosin with LC2., Conclusions: The changes in myosin structure and function were linked to a protease-mediated cleavage of LC2 in myosin; a possible role for the protease in the degenerative effects of idiopathic dilated cardiomyopathy is thus defined.

The participation of cardiac myosin hinge in contractility was investigated by in vitro motility and ATPase assays and by measurements of sarcomere shortening. The effect on contractile activity was analyzed using an antibody directed against a 20-amino acid peptide within the hinge region of myosin. This antibody bound specifically at the hinge at a distance of 55 nm from the S1/S2 junction, was specific to human, dog, and rat cardiac myosins, did not crossreact with gizzard or skeletal myosin, and had no effect on ATPase activity of purified S1 and myofibrils. However, it completely suppressed the movement of actin filaments in in vitro motility assays and reduced active shortening of sarcomeres of skinned cardiac myocytes by half. Suppression of motion by the anti-hinge antibody may reflect a mechanical constraint imposed by the antibody upon the mobility of the S2 region of myosin. The results suggest that the steps in the mechanochemical energy transduction can be separately influenced through S2.

The size and shape of Plasmodium lophurae histidine-rich protein have been determined by analytical centrifugation and electron microscopy. From the partial specific volume of 0.72 cc/g, the molecular weight was determined to be 43,000. The sedimentation velocity studies indicated a coefficient of 1.32 S in 0.9 M acetic acid (pH 3.5), monodispersity and significant asymmetry. Darkfield electron microscopy revealed the major species to be compact oblate spheroids 12 nm in width and extended filamentous particles of average length 35 nm by 1.5 nm. Analysis of the sequence of the protein by the method of Garnier et al. (J. Mol. Biol. (1978) 120, 97-120) predicted that 82% of its residues would be found in three long alpha-helices. The protein's CD spectrum has a strong resemblance to that of poly(L-histidine) at pH 4-5, where the homopolymer is thought to be in a right-handed alpha-helical form. A single helix containing 300 residues would be 45 nm long, the largest length found by electron microscopy. From the electron-microscopic data, sedimentation coefficients of 1.6 and 1.95 S, respectively, were calculated for flexible-coil and extended-rod models, in closer agreement with the measured value of 1.3 S than the value calculated for a spherical model. Thus, the major species in acetic acid is probably an incompletely extended rod which, as the pH is increased to neutrality, condenses to form spherical molecular aggregates seen in the malaria parasite.

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.

Incubation of myosin with myopathic hamster protease results in substantial (more than 80%) removal of light chain 2 (LC2) with limited breakdown of the heavy chains. LC2-deficient myosin, purified by ion exchange chromatography, migrates as a single, monodisperse boundary in the analytical ultracentrifuge. The Ca2+- and EDTA-activated ATPases of LC2-deficient myosin are similar to those of the control and LC2-recombined myosins indicating that no denaturation occurred in its preparation. Double reciprocal plots for LC2-deficient, control, and LC2-recombined myosins reveal a biphasic behavior i.e. at actin concentrations above 11 microM, there is a sharp break in the 1/V versus 1/[actin] plots for all samples. The Vm values for LC2-deficient myosin are 50% lower (at low actin, Vm = 3.0 s-1, and at high actin, Vm = 4.2 s-1) than those for control myosin (Vm = 5.3 s-1 at low actin and 8.3 s-1 at high actin). Readdition of LC2 to LC2-deficient myosin restores the actin-activated ATPase to control levels. Electron microscopy of shadow cast preparations reveals a subtle difference between LC2-deficient myosin, and control or recombined myosin. In control and recombined myosins, S1 heads appear "pear"-shaped, whereas in LC2-deficient myosin, the S1 heads are rounder and display a "thinning" of mass in the "neck" region, suggesting that LC2 binds at the S1/S2 junction. Furthermore, removal of LC2 apparently influences the assembly of myosin into minifilaments, as revealed to a certain degree, by an increase in the width of the bare zone, accompanied by a decrease in the stability of these minifilaments.

Turkey gizzard myosin was found to self-assemble into new polymorphic forms as detected by thin-section electron microscopy. In high ionic strength buffers (0.3 mM KCl, pH 6.0), aggregates of sidepolar filaments were produced. Electron microscopy of thin sections revealed individual filaments with a 13.5 nm axial repeat. Under a number of conditions, with varying ionic strength, pH, MgCl2 and ATP, the filaments assembled through the head region with the tail portion projecting out radially from the aggregate. The regions corresponding to heads and tails within the aggregates were established by immunoelectron microscopy using anti-S1 and anti-LMM antibodies coupled to gold. These filaments often interacted to produce bilayer sheets, which, when cut perpendicular to the plane of the sheet, appeared as ladders. A hitherto unreported structure was obtained at 0.2 M KCl (pH 8.0): myosin aggregated to generate a three-dimensional quasi-crystalline lattice with a 270 nm period. In these aggregates, myosin was arranged in an antiparallel fashion, stacked on one another, producing ribbon-like strips stabilized through non-covalent interactions between heads, thereby producing a crystalline lattice. Neither Mg2+ nor ATP were required for this form. Phosphorylation of the regulatory light chains or the cleavage of the heavy chains at a single site in the head region prevented myosin from assembling in the 3-D lattice form. Generally, unphosphorylated myosin produced periodic paracrystals at low ionic strength in the presence of 10 mM MgCl2, but as the ionic strength was increased the regular 3-D lattice became the predominant form. Some paracrystalline forms could be obtained at high ionic strength without magnesium with phosphorylated myosin.

Female protein (FP), a serum protein present in normal female hamsters was found to be similar to acute-phase reactant, C-reactive protein (CRP) and serum amyloid P component (SAP) in the following ways: (a) hamster FP complexed with phosphorylcholine (PC) in a Ca++-dependent fashion as shown by its isolation from serum by affinity chromatography with PC-Sepharose and selective elution with free PC or EDTA; (b) electron microscopy of FP indicated a pentameric structure similar in size and appearance to other pentraxins; (c) the parent molecule of FP (150,000 mol wt) was composed of five noncovalantly assembled subunits of 30,000 mol wt; and (d) the amino acid analysis and terminal NH2 sequence of FP clearly showed homology with SAP-CRP. Although FP evolved from an ancestral gene common to SAP and CRP, and shares functional, morphological and structural properties with these acute-phase proteins, the biological homology of FP appears quite diverse as this protein is a prominent serum constituent (1-2 mg/ml) of normal female hamsters and under hormonal control (testosterone suppression) in males.

A hydrodynamic model of the high molecular weight platelet glycoprotein thrombospondin is formulated from physical solution measurements of molecular weight, partial specific volume, sedimentation coefficient, and intrinsic viscosity. Simultaneous sedimentation equilibrium analysis of thrombospondin in buffered saline prepared in H2O and D2O yielded values of 420,000 and 0.714 ml/g for the molecular weight and partial specific volume, respectively. A sedimentation coefficient of 8.6 S was found to be independent of the protein concentration over the range 0-6 mg/ml. The intrinsic viscosity was determined to be 40 ml/g at 20 degrees C for native thrombospondin and 52 ml/g for thrombospondin in the presence of 6 M guanidine-HCl. Based on these values thrombospondin is best described as a prolate ellipsoid with an axial ratio of 9.3. This model agrees well with the electron microscopic image of thrombospondin as a nodular rod (7 X 65 nm)-shaped molecule with an axial ratio of 10. Sedimentation equilibrium analysis and sodium dodecyl sulfate-polyacrylamide gel electrophoresis indicated that thrombospondin is comprised of three 140,000-dalton polypeptide chains. The percentage of residues in alpha-helix was calculated to be only 3% from the circular dichroism spectrum.

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.

A number of motile functions of macrophages are thought to be mediated by myosin. We have observed that myosin from rabbit alveolar macrophages is heterogeneous with respect to its 20 kDa light chain: two species of 20 kDa light chain are identified by one-dimensional and two-dimensional polyacrylamide gel electrophoresis, in a ratio of 2:1. Native myosin, analyzed on non-denaturing gels, is also composed of two species, in a ratio of 2:1. These results indicate that macrophages contain at least two different myosins, which might have different physiological functions.

alpha-Actinin exists in several polymorphic forms which appear to be characteristic of the muscle type from which it is isolated. In order to determine the possible physiological role of this structural protein in cardiac muscle, we describe and compare here the physico-chemical properties of cardiac alpha-actinin from two different mammalian species, rat (fast contracting muscle) and dog (slow contracting muscle). Purification of cardiac alpha-actinin was achieved by chromatography on DEAE-cellulose and hydroxyapatite columns. The alpha-actinins isolated were different in their electrophoretic mobility (SDS-polyacrylamide gel electrophoresis), molecular size and alpha-helical content. However, their shape as revealed by electron microscopy and their activating effect on Mg2+-ATPase activity of actomyosin appear to be similar. These studies suggest that the rat and dog cardiac alpha-actinin are structurally different but functionally similar proteins.

At an intermediate stage in the hydrolysis of magnesium adenosine 5'-phosphate (MgATP) by myosin or actomyosin, there is an exchange of oxygen between water and the P gamma group of enzyme-bound nucleotide. Starting with [P gamma-18O]ATP as substrate, the exchange is revealed in the [18O]Pi species that are ultimately released as product into the reaction medium. An analysis of the distribution of these labeled Pi species, which contain 3, 2, 1, or none of the 18O atoms originally on the P gamma of ATP, is used to probe intermediate stages of the hydrolytic mechanism. In recent years, studies of this kind by several groups have shown that more than one pathway of hydrolysis operates. The work reported here demonstrates that two of these pathways are spurious; one is a "nonexchanging MgATPase" that is present in fresh myosin preparations; the other is an induced slow exchange that develops in myosin during storage (-20 degrees C) and subsequent aging (4 degrees C). However, after correction for these artifacts, two normal pathways for actomyosin hydrolysis remain. These normal pathways differ in the mode of interaction between actin and myosin in the course of hydrolysis; one is the Lymn-Taylor pathway where oxygen exchange occurs at a stage when actin and myosin are dissociated; the other is a pathway in which actin and myosin are associated during oxygen exchange. Each of these two pathways contributes an equal amount of Pi to the product pool. Thus, on average, each myosin head uses each of these pathways half the time. The findings suggest, e.g., that during contraction, myosin can dissociate from the actin filament only during every other cycle of MgATP hydrolysis or that only half the heads, at any one time, can exchange oxygen while free of the actin filament.

A number of enzymes are currently in use for obtaining proteolytic subfragments of rabbit skeletal muscle myosin. Subfragment-1 can be obtained by papain digestion of polymeric myosin in the presence (Mg-S1) or absence (EDTA-S1) of divalent cations [Margossian, S.S., Lowey, S., & Barshop, B. (1975) Nature (London) 258, 163-166]. Subfragment-1 prepared by chymotrypsin is readily fractionated according to its alkali light-chain content into S1(A1) and S1(A2) [Weeds, A.G., & Taylor, R.S. (1975) Nature (London) 257, 54-56]. Digestion of soluble myosin by trypsin or chymotrypsin leads to heavy meromyosin (HMM) and light meromyosin (LMM). Many of these subfragments show extensive cleavages in the heavy- and/or light-chain region by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. In view of the widespread use of proteolytic subfragments in kinetics and structural studies, it was of interest to establish the extent of heterogeneity of these preparations under nondenaturing conditions by equilibrium centrifugation. Analysis of the fringe displacements by the computer programs of Roark & Yphantis [Roark, D.E., & Yphantis, D.A. (1969) Ann. N.Y. Acad. Sci. 164, 245-278] showed that for three initial loading concentrations, the molecular weight averages Mn, Mw, M2, were superimposable across the entire solution column for all S1 and HMM species. The same applied for the initial molecular weight averages of LMM and rod, except that with these highly asymmetric molecules, a small drop in molecular weight was observed toward the cell bottom as would be expected from excluded volume effects. We conclude that the subfragments of myosin are remarkably homogeneous in benign solvents, despite the existence of some cleavages in their primary structure.

Calcium-dependent self-association of rabbit skeletal troponin-C was investigated by equilibrium ultracentrifugation. Troponin-C which was homogeneous by the criterion of polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate was shown to undergo reversible dimerization (K2,app = 1.7 X 10(3) M-1) even in the presence of ethylene glycol bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA) (0.1-1.0 M KCl, 0.01 M imidazole, 0.005 M potassium phosphate, 0.4 mM EGTA (pH 7.0), 1.0 mM dithiothreitol). The existence of a monomer-dimer reaction under these conditions could be demonstrated as long as the effects of the Donnan equilibrium were taken into account. The value of the observed second virial coefficient was consistent with the value expected from troponin-C at this pH. The dimerization could be enhanced by the addition of calcium to 0.1 mM (K2,app = 1.2 X 10(4) M-1). A monomer molecular weight of 18,700 was estimated from a "two-species" plot of the data. The effect of calcium was completely reversible.

Structural properties of dog cardiac myosin and the influence of the regulatory light chain (LC2) on the shape of myosin heads were investigated by electron microscopy. LC2 was reversibly removed using a neutral protease from myopathic hamsters (Margossian, J. Biol. Chem. 260 (1985) 13747-54). The distribution of myosin head length centred around 17 nm with the mean length being 18.9 nm. Statistical analysis suggested that myosin heads became more globular upon removal of LC2. No extensive aggregation of myosin could be detected after LC2 was dissociated, either by sedimentation velocity or by gels run under non-denaturing conditions. The centre-to-centre distance between heads remained constant at about 21 nm, regardless of the presence or absence of LC2. The distribution of length of the globular region reveals two peaks at 7.5 and 9.5 nm, suggesting an extended and a shorter configuration of this region. The decrease in mass at the head/tail junction upon LC2 removal suggests that it is the binding site for the regulatory light chains. A bend at 57 nm from the head/tail joint was sometimes noticed, corresponding to the myosin hinge region. In high resolution micrographs individual particles revealed invaginations along the contours of the head, possibly delineating the boundaries of structural domains within the head. The conformation of arrowheads in actin decorated with either subfragment 1 (S1) or heavy meromyosin (HMM) was investigated in the presence and absence of LC2.

The possible role of the regulatory light chains (LC2) in in vitro assembly of rabbit skeletal and dog cardiac myosins was examined by formation of minifilaments and synthetic thick filaments. After LC2 was removed, the resulting myosin preparations exhibited little aggregation in 0.5 M KCl and 0.05 M potassium phosphate (pH 6.5). Minifilaments migrated as a single, hypersharp peak during sedimentation velocity, but electron microscopic analysis revealed a more destabilized structure for LC2-deficient minifilaments. Thick filaments were formed in buffers containing 0.15 M KCl and the following: 20 mM imidazole; 20 mM imidazole, 5 mM ATP; or 20 mM imidazole, 5 mM ATP, and 5 mM MgCl2, all at pH 7.0. Skeletal and cardiac myosin filaments formed in imidazole buffer alone were bipolar, tapered at both ends, and about 1.6 micron long. Removal of LC2 resulted in the formation of shorter thick filaments (1.2 micron long). This effect could be reversed by reassociation with LC2. Inclusion of ATP in the buffer disrupted the filament structure, resulting in irregular, short filaments (less than 0.6 micron); addition of both ATP and MgCl2 largely reversed the effects of ATP alone. In cardiac myosin filaments, the bare zone diameter increased from 16 nm as measured in control and LC2-recombined samples to 20 nm in LC2-deficient myosin assemblies. These results implicate LC2 in an active role in controlling synthetic thick filament length in both skeletal and cardiac muscles.

Human endothelial cells release components into the growth medium that stimulate cell-substratum adhesion. Several macromolecular components were isolated by ultracentrifugation of the endothelial cell conditioned medium. The components were heterogeneous, consisting of several sizes when examined by sedimentation velocity and gel filtration. When the extracellular components were evaluated by electron microscopy, structurally discrete particles were observed. The extracellular components and the complexes mediated cell-substratum adhesion to both human umbilical and arterial endothelial cells. The majority of the extracellular components that promote endothelial cell adhesion were pelleted by ultracentrifugation. Although the complexes contained fibronectin, antibodies to fibronectin did not inhibit cell adhesion to the complexes. Significant inhibition of endothelial cell adhesion was observed in the presence of heparin and heparan sulfate. The supernatant fraction following ultracentrifugation of the growth medium contained a component that suppressed endothelial cell adhesion to culture dishes coated with fibronectin, type I collagen, and endothelial cell complexes. SDS-polyacrylamide gel electrophoresis indicated that the complexes contained several components, and the majority of the large-molecular-weight components were pelleted by ultracentrifugation. The conditioned medium from human endothelial cells contains specific complexes that promote cell-substratum adhesion and components that suppress cell-substratum adhesion.

The regulatory light chains of dog heart myosin were removed by digestion with myopathic hamster neutral protease. The heavy chains were also cleaved to an extent of 15%, but a homogeneous, rod-free LC2-deficient myosin was obtained by ion-exchange chromatography. A similar approach was used to prepare LC2-deficient heavy meromyosin. Neither Ca2+- nor K+-EDTA-activated ATPases were affected by LC2 removal. The Lineweaver-Burk plots for actin-activated ATPase in 25 mM KCl were biphasic giving a Vmax of 1.54 s-1 for control and LC2-recombined myosins and 1.08 s-1 for LC2-deficient myosin at low actin concentrations. At high actin concentrations, the Vmax for control and recombined myosins was 2.33 s-1 and 1.39 s-1 for LC2-deficient myosin. Increasing the KCl concentration in the reaction mixtures resulted in more linear plots without suppressing the 35-45% decrease in Vmax that accompanied LC2 removal. The results from assays with control and LC2-deficient heavy meromyosin performed in the absence of KCl, paralleled those obtained with myosin. The latter was also assayed in the presence of equimolar concentrations of C-protein in 50 mM KCl: C-protein induced a significant increase in the actin-activated ATPase of both control and LC2-recombined myosins, with no effect on LC2-deficient myosin. The Vmax for actin-activation in the presence of C-protein was 2.38 s-1, 0.83 s-1, and 1.71 s-1 for control, LC2-deficient, and recombined myosins, respectively. The enhancement of actin-activation in both the control and LC2-recombined myosins represents a possible role for C-protein in a LC2-mediated potentiation of actomyosin ATPase.