Properties of smooth muscle meta-vinculin.

Quantitative studies show that meta-vinculin is ninefold more soluble in 0.6 M salt than in the 0.01 M salt buffers used to extract vinculin. Based on this finding, we have developed a protocol for the purification of meta-vinculin in 43% yield and 98% purity from a high salt extract of gizzard smooth muscle. In contrast to our earlier extraction studies, which were done on unfixed cryostat sections (30), the present studies done on tissue homogenates show that nonionic detergents are not required for solubilization of meta-vinculin. Furthermore, neither purified nor partially purified meta-vinculin binds to Triton X-114 micelles. Purified meta-vinculin is a monomeric, asymmetric molecule with a Stokes radius of 50.9 A, a sedimentation coefficient of 6.35S, and a frictional ratio of 1.46. The calculated molecular weight of meta-vinculin is 145,000. Meta-vinculin has two isoforms of pI 5.9 and 6.2, and is phosphorylated in vivo to eightfold greater specific activity than vinculin. On immunoblots of smooth muscle proteins, [125I]meta-vinculin binds specifically to talin and also to unidentified polypeptides of 180, 150, 95, 70, 68, and 45 kD. On two-dimensional peptide maps, iodinated vinculin and meta-vinculin have at least 95% of their major chymotryptic peptides in common, but each protein also has at least one highly labeled peptide that appears to be unique. Comparative peptide maps of high salt soluble meta-vinculin and the low salt soluble 152-kD protein (described by Feramisco, J.R., J.E. Smart, K. Burridge, D. Helfman, and G.P. Thomas, 1982, J. Biol. Chem., 257:11024-11031) demonstrate extensive similarities among the vinculin-like proteins but suggest a lack of complete identity. In vivo pulse-chase experiments show that meta-vinculin and vinculin do not have a precursor-product relationship. The biochemical and structural differences found between vinculin and meta-vinculin suggest that there is a unique function for meta-vinculin in smooth muscle.

Identification by monoclonal antibodies and characterization of human platelet caldesmon.

Actin-based gels were prepared from clarified high-salt extracts of human platelets by dialysis against physiological salt buffers. The gel was partially solubilized with 0.3 M KCl. Mice were immunized with the 0.3 M KCl extract of the actin gel, and hybridomas were produced by fusion of spleen cells with myeloma cells. Three hybridomas were generated that secrete antibodies against an 80-kD protein. These monoclonal antibodies stained stress fibers in cultured cells and cross-reacted with proteins in several tissue types, including smooth muscle. The cross-reacting protein in chicken gizzard smooth muscle had an apparent molecular weight of 140,000 and was demonstrated to be caldesmon, a calmodulin and actin-binding protein (Sobue, K., Y. Muramoto, M. Fujita, and S. Kakiuchi, Proc. Natl. Acad. Sci. USA, 78:5652-5655). No proteins of molecular weight greater than 80 kD were detectable in platelets by immunoblotting using the monoclonal antibodies. The 80-kD protein is heat stable and was purified using modifications of the procedure reported by Bretscher for the rapid purification of smooth muscle caldesmon (Bretscher, A., 1985, J. Biol. Chem., 259:12873-12880). The 80-kD protein bound to calmodulin-Sepharose in a Ca++-dependent manner and sedimented with actin filaments, but did not greatly increase the viscosity of F-actin solutions. The actin-binding activity was inhibited by calmodulin in the presence of calcium. Except for the molecular weight difference, the 80-kD platelet protein appears functionally similar to 140-kD smooth muscle caldesmon. We propose that the 80-kD protein is platelet caldesmon.

Epinemin: a new protein associated with vimentin filaments in non-neural cells.

In this report I describe a new protein, defined by a monoclonal antibody, which is associated with vimentin filaments in a variety of cultured cells and in skeletal muscle. By immunofluorescence it is absent in smooth muscle, in cells without vimentin, and in neural vimentin containing cells. This protein has a molecular weight of 44,500, a pl of 5, a two-dimensional tryptic peptide fingerprint pattern different from vimentin, is unrelated to actin by Cleveland peptide analysis and by light and electron microscopy, and is not recognized by either a polyclonal antivimentin antibody (Frank, E.D., and L. Warren, 1981, Proc. Natl. Acad. Sci. USA, 78:3020-3024) or a monoclonal antibody against all classes of intermediate filaments (Pruss, R.M., R. Mirsky, M.C. Raff, R. Thorpe, A.J. Dowding, and B.H. Anderton, 1981, Cell, 27:419-428). The protein is resistant to nonionic detergent extraction, is soluble in high salt and can thus be removed from vimentin filaments, but fragments with vimentin in either low salt or anionic detergent and collapses with vimentin in colchicine-treated cells. By light microscopy, the distribution of the protein is indistinguishable from vimentin filaments and appears uniform along them. In contrast, immunoferritin electron microscopy reveals that the molecule is distributed in an intermittent pattern on vimentin filaments. Adopting the terminology of Granger and Lazarides (1980, Cell, 30:263-275), the molecule is called epinemin, meaning "upon filaments."

Interaction of alpha-actinin, filamin and tropomyosin with F-actin.

The abilities of alpha-actinin, filamin and tropomyosin to bind F-actin were examined by cosedimentation experiments. Results indicated that smooth muscle alpha-actinin and filamin can bind to actin filaments simultaneously with little evidence of competition. In contrast, tropomyosin exhibits marked competition with either filamin or alpha-actinin for sites on actin filaments.

The effect of caldesmon on actin-myosin interaction in skeletal muscle fibers.

The effects of caldesmon on structural and dynamic properties of phalloidin-rhodamine-labeled F-actin in single skeletal muscle fibers were investigated by polarized microphotometry. The binding of caldesmon to F-actin in glycerinated fibers reduced the alterations of thin filaments structure and dynamics that occur upon the transition of the fibers from rigor to relaxing conditions. In fibers devoid of myosin and regulatory proteins (ghost fibers) the binding of caldesmon to F-actin precluded structural changes in actin filaments induced by skeletal muscle myosin subfragment 1 and smooth muscle tropomyosin. These results suggest that the restraint for the alteration of actin structure and dynamics upon binding of myosin heads and/or tropomyosin evoked by caldesmon can be related to its inhibitory effect on actin-myosin interaction.

A hydrophobic region on myosin light chains modulated by divalent cations.

A hydrophobic region was detected on several types of myosin light chain by enhancement of the quantum yield of 1-anilino-8-naphthalenesulfonate (ANS) fluorescence. The character of this non-polar region was altered by the binding of Ca2+ or Mg2+ to the light chain, the quantum yield of the ANS being increased, and its emission maximum undergoing a blue-shift. These changes enabled the binding of divalent cations to the myosin light chains to be monitored. When Ca2+ was bound to gizzard regulatory light chain, a biphasic enhancement of light-chain-bound ANS fluorescence occurred, the first phase taking place in the micromolar range and the second in the millimolar range of free Ca2+ concentration. Enhancement of protein-bound ANS fluorescence as divalent cations were bound was also observed with other types of myosin light chain.

Diversity in smooth muscle thin filament composition.

Two classes of smooth muscle thin filament can be identified and separated based on their interaction with antibodies specific either to filamin or to caldesmon. One type is composed of actin, tropomyosin and filamin and the other of actin, tropomyosin and caldesmon.

Isolation and physico-chemical properties of blood platelet alpha-actinin.

A procedure for the isolation of alpha-actinin from human blood platelets is described. Typical yields were 10-13 mg from 48 g of frozen platelets. The purified platelet alpha-actinin has many physico-chemical properties (molecular weight in native state, molecular weight in denaturing conditions, Stokes radius, ellipticities at 208 and 221 nm) similar to those of muscle alpha-actinins. However, in contrast to muscle alpha-actinins, it is composed of isoforms containing subunits of slightly different molecular weights and its effect on actin gelation is calcium-sensitive. These two characteristics are common to other known non-muscle alpha-actinins.

Dinitrophenylation of phosphorylated gizzard myosin. Labelling of the subfragment 1 region.

Chymotrypsin and papain digestion of 3H-labelled dinitrophenylated chicken gizzard myosin, pretreated with the myosin light chain catalyzed phosphorylating system, released a subfragment 1 devoid of its light chains but containing all of the label associated with the thiols of the heavy chain region. This was also the case when myosin was incubated in the presence of the phosphorylating system but without ATP. Dinitrophenylation of a reconstituted actomyosin made from phosphorylated myosin occurred mainly on -SH groups of the heavy chains, in contrast to the predominant modification of the light chains of myosin from a control actomyosin that was treated similarly. Conformational changes in myosin may govern, in part, actin-myosin interaction through the phosphorylation of the light chain of Mr 20.000.

Identical myoglobin is present in both skeletal and smooth muscles of chicken.

Chicken gizzard has been considered to be an exceptional organ of smooth musculature in which a myoglobin is present. Since the characterization of the gizzard myoglobin has to date, been very incomplete, we studied the structures and functions in detail. The main component, which constituted roughly 90% of the protein, isolated by chromatofocusing, was homogeneous by electrophoretic and ultracentrifugal analyses. The molecular weight was consistently 1.8 X 10(4) by equilibrium sedimentation and iron analysis, and the isoelectric point was 7.8. Spectroscopic properties of the oxy-, carboxy- and deoxy-derivatives were typical of myoglobin. The oxygenation equilibria were also typical of myoglobin, showing neither homotropic nor heterotropic allosteric interactions, and the temperature-dependence (delta H0) was estimated as -16.6 kcal/mol. All these characteristics of the gizzard myoglobin were identical with those of the protein from the skeletal muscles. The amino acid composition and peptide mapping results also concluded that identical myoglobin was present in the gizzard, skeletal and probably cardiac muscles.

Structural analysis of the C-terminal region of chicken gizzard desmin.

The C-terminal sequence of chicken gizzard desmin has been examined by computer programs based on Chou-Fasman prediction of secondary structure, auto-correlation and Fourier analysis of hydrophobic residue distribution, and cross-correlation analysis of acidic and basic residues. These analyses indicate that, although parts of the desmin sequence are alpha-helical, the helical regions may not be as extensive as predicted previously. Detailed analysis of the distribution of the charged residues in the desmin sequence strongly suggests that it cannot form a tropomyosin-like coiled-coil double-helix because of the almost complete absence of stabilizing salt-bridge interactions between proximal pairs of acidic and basic residues. Although the desmin sequence does contain the tropomyosin-like hydrophobic residue distribution, differences exist in the overall distributions of hydrophobes, with clusters of these residues being found in certain regions of the desmin sequence. Analyses of the sequences of porcine desmin and vimentin reveal very similar structural characteristics for these molecules, although vimentin is predicted to have a different structure at its C-terminus.

Immune response of rabbits to native G-actins.

Actins are highly conserved proteins and are therefore claimed to be not very immunogenic without prior denaturation or chemical modification. We have obtained in rabbits high-titered antibodies to "native" G-actins from chicken and man, and assayed their cross-reaction using an enzyme immunoassay, Western blotting and immunohistochemistry. The antigens differ in their ability to induce antibody formation (chicken gizzard actin [(beta), gamma] greater than chicken skeletal actin [alpha] = human platelet actin [beta, (gamma)]). Antibodies to skeletal actin [alpha] are muscle-specific and mainly directed against the homologous region comprising the N-terminus (residues 1-226). Antibodies to gizzard actin [(beta), gamma] cross-react, to a lesser extent, with the alpha and beta, (gamma) isoforms. They show no regional specificity within the homologous antigen. Antibodies to the tryptic core fragment (residues 69-374) of skeletal actin react with fragments comprising the C-terminal part of muscular actins. Antibodies to platelet actin [beta, (gamma)] cross-react with muscular actins, recognizing not the native, but slightly degraded molecules. Platelet actin induces the formation of high-titered albumin antibodies for hitherto unknown reasons.

Properties of carboxypeptidase A-treated chicken gizzard tropomyosin.

Chicken gizzard tropomyosin was digested with carboxypeptidase A at the weight ratios of enzyme to substrate 1:200 and 1:50. Removal of about 16 C-terminal amino acid residues per tropomyosin molecule, at lower enzyme concentration, caused reversion of the effect on skeletal actomyosin ATPase activity from activating to inhibiting without an influence on polymerizability and actin-binding ability. Removal of about 26 C-terminal amino acid residues per molecule, at higher enzyme concentration, resulted in loss of polymerizability and actin binding ability. Digestion of gizzard tropomyosin with carboxypeptidase A has no dramatic effect on its binding to troponin T. The results show that not only the existence of head-to-tail overlapping regions but also their length is important for the functional properties of chicken gizzard tropomyosin.

Kinetic evidence for insertion of actin monomers between the barbed ends of actin filaments and barbed end-bound insertin, a protein purified from smooth muscle.

An actin polymerization-retarding protein was isolated from chicken gizzard smooth muscle. This protein copurified with vinculin on DEAE-cellulose and gel filtration columns. The polymerization-retarding protein could be separated from vinculin by hydroxylapatite chromatography. The isolated polymerization-retarding protein lost its activity within a few days, but was stable for weeks when it was not separated from vinculin. We termed the polymerization-retarding protein "insertin". Because of the instability of the isolated insertin, we investigated the effect of insertin-vinculin on actin polymerization. Insertin-vinculin retarded nucleated actin polymerization maximally fivefold. Polymerization at the pointed ends of gelsolin-capped actin filaments was not affected by insertin-vinculin, suggesting that insertin-vinculin binds to the barbed ends, but not to the pointed ends, of actin filaments. Retarded polymerization was observed even if the actin monomer concentration was between the critical concentrations of the ends of treadmilling actin filaments. As at this low monomer concentration the pointed ends depolymerize, monomers appeared to be inserted at the barbed ends between the terminal subunit and barbed end-bound insertin molecules. Insertin-vinculin was found not to increase the actin monomer concentration to the value of the pointed ends. These observations support the conclusion that insertin is not a barbed end-capping protein but an actin monomer-inserting protein. According to a quantitative analysis of the kinetic data, all observations could be explained by a model in which two insertin molecules were assumed to bind co-operatively to the barbed ends of actin filaments. Actin monomers were found to be inserted between the barbed ends and barbed end-bound insertin molecules at a rate of about 1 x 10(6) M-1 s-1. Insertin may be an essential part of the machinery of molecules that permit treadmilling of actin filaments in living cells by insertion of actin molecules between membranes and actin filaments.

Studies on the effect of phosphorylation of the 20,000 Mr light chain of vertebrate smooth muscle myosin.

Myosin was rapidly prepared from turkey gizzard muscle to a high level of purity, in high yield and in a non-phosphorylated state. It was consistently observed that the actin-activated Mg2+ ATPase activity of this myosin was dependent on the level of phosphorylation of the 20,000 Mr light chain, for example, in the non-phosphorylated state, the myosin Mg2+ ATPase activity was not activated by actin whereas, when the light chains were phosphorylated, the Mg2+ ATPase activity of the myosin was activated approximately ninefold by actin. Using the "desensitized" scallop myosin test system (Kendrick-Jones et al., 1976; Sellers et al., 1980) it was further demonstrated that phosphorylation of the 20,000 Mr gizzard light chain has a regulatory role. These results also suggest that the regulatory mechanisms mediated by smooth muscle myosin light chains and molluscan myosin regulatory light chains are similar, i.e. in the absence of Ca2+, both types of light chain inhibit myosin interaction with actin and this inhibition is relieved by either phosphorylation in smooth muscle or by direct calcium binding in molluscan myosins. The basis of regulation exerted by these light chains is therefore repression derepression. Using a variety of techniques, i.e. turbidity measurements, quantitative high speed centrifugation, electron microscopy and dark field light microscopy, it was observed that the stability of gizzard myosin filaments at approximately physiological conditions (0.15 M-NaCl, 1 mM-MgATP, pH 7.0) was dependent on the level of light chain phosphorylation. Using purified calmodulin-dependent light chain kinase and phosphatase, it was further shown that these gizzard myosin filaments can be reversibly assembled and disassembled as a result of phosphorylation-dephosphorylation of the 20,000 Mr light chain.

Hormonal regulation of a chicken oviduct messenger ribonucleic acid that shares a common domain with gizzard myosin light chain kinase.

Changes in myosin light chain kinase (MLCK) and calmodulin (CaM) mRNAs have been evaluated during estrogen-mediated differentiation of the chicken oviduct. Also examined were acute changes that occur in oviduct RNA from animals stimulated with estrogen, withdrawn from hormone and then injected for 1, 2, and 4 days with synthetic estrogen [diethylstilbestrol (DES)], progesterone (P), or testosterone (T). Small changes were noted in both CaM and MLCK RNAs during primary stimulation when oviduct cells are actively dividing. On the other hand no significant changes were observed during secondary stimulation regardless of the steroid hormone injected. These data support the contention that CaM and MLCK are constitutively expressed but vary as a function of cell cycle. The MLCK mRNA is 5.5 kilobases (kb) but the MLCK cDNA also hybridizes to an oviduct RNA 2.7 kb long. This RNA species is acutely regulated by estrogen, P, and T but in a manner different from that of ovalbumin mRNA. The magnitude of stimulation of the 2.7 kb mRNA by diethylstilbestrol and T is greater than that of ovalbumin whereas changes in response to P are similar. The 12- to 16-fold increase of the 2.7 kb mRNA in response to T is the largest effect reported for this hormone acting on oviduct. The 2.7 kb mRNA encodes an unknown protein yet contains a 520 nucleotide segment that is highly homologous with the COOH-terminal coding portion of the MLCK mRNA. Since this homology does not include either catalytic or CaM-binding domains of MLCK, it is unlikely that the 2.7 kb mRNA encodes a CaM-dependent protein kinase.(ABSTRACT TRUNCATED AT 250 WORDS)

Vascular smooth muscle calponin. A novel troponin T-like protein.

In a search for additional Ca2+ regulatory components in vascular smooth muscle, a novel troponin T-like protein was purified from bovine aorta smooth muscle. The isolated protein was separated into several isoforms on isoelectric focusing. The major isoelectric variants were focused in the pH region of 8.4 to 9.1. The protein had slightly different molecular masses in the Mr range of 35,000 on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Its molar ratio relative to tropomyosin in the muscle extract was estimated to be 0.9:1.0. The novel protein bound to the immobilized calmodulin and exhibited a number of common physicochemical properties with gizzard (Mr = 34,000) calmodulin-binding and F-actin-binding protein. The aorta and gizzard proteins were immunologically cross-reactive. Both proteins shared a common antigenic determinant with COOH-terminal segments of rabbit skeletal and bovine cardiac troponin T and bound to the immobilized smooth muscle tropomyosin. Both proteins interacted with rabbit skeletal troponin C in the presence and absence of Ca2+, but they did not interact with troponin I. These results suggest that the novel protein, which is designated calponin, may be a specialized component of smooth muscle thin filament involved in the regulation of contractile apparatus.

The tissue form of chicken type VI collagen.

We have purified intact type VI collagen from chicken gizzard. The protein was found to consist of a 130 kDa, a 140 kDa and a 180-200 kDa subunit. The 130 kDa and 140 kDa subunits were obtained in equimolar amounts and identified as the alpha 2 (VI) and the alpha 1 (VI) chains, respectively. The third subunit was usually obtained in the form of 3-4 closely related polypeptides, which may represent different processing or modification products of the alpha 3 (VI) chain.

Vinculin localization in cardiac muscle.

Vinculin isolated from chicken cardiac muscle crossreacts with antibodies against smooth muscle vinculin. Antibodies to vinculin were used for localization of vinculin in cardiac muscle by indirect immunofluorescence method. In cardiac muscle vinculin was localized in intercalated discs and near plasma membrane at the cell periphery between external myofibrils and sarcolemma. It was suggested that vinculin plays an important role in myofibril-sarcolemma interaction in cardiac muscle.

Molecular cloning and sequencing of the chicken smooth muscle myosin regulatory light chain.

A cDNA probe was constructed from a chicken skeletal muscle regulatory light chain cDNA and was used to screen a chicken gizzard cDNA library. A clone containing the entire coding region of the chicken gizzard regulatory light chain was isolated and sequenced. The deduced protein sequence is identical to the most recently reported chemical sequence of the chicken smooth muscle regulatory light chain, and has homologies with other troponin C-like calcium-binding proteins.