Biomedical subjects

N B Gusev

Publications and source records attributed to N B Gusev.

At least 55 records · Page 3Linked to original sources

PubMed1993

Phosphorylation by casein kinase II affects the interaction of caldesmon with smooth muscle myosin and tropomyosin.

Smooth muscle caldesmon was phosphorylated by casein kinase II, and the effects of phosphorylation on the interaction of caldesmon and its chymotryptic peptides with myosin and tropomyosin were investigated. The N-terminal chymotryptic peptide of caldesmon of molecular mass 27 kDa interacted with myosin. Phosphorylation of Ser-73 catalysed by casein kinase II resulted in a 2-fold decrease in the affinity of the native caldesmon (or its 27 kDa N-terminal peptide) for smooth muscle myosin. At low ionic strength, caldesmon and its N-terminal peptides of molecular masses 25 and 27 kDa were retarded on a column of immobilized tropomyosin. Phosphorylation of Ser-73 led to a 2-4-fold decrease in the affinity of caldesmon (or its N-terminal peptides) for tropomyosin. Thus phosphorylation of Ser-73 catalysed by casein kinase II affects the interaction of caldesmon with both smooth muscle myosin and tropomyosin.

Animals↗

PubMed1993

Isolation of human cardiac troponin T and localization of epitopes recognized by monoclonal antibodies to cardiac troponin T.

Human cardiac troponin T has been isolated and its properties compared with those of rabbit skeletal and bovine cardiac troponin T. Seven monoclonal antibodies to troponin T have been obtained. Two antibodies cross-reacted with both cardiac and skeletal troponin T and recognized epitopes located between residues 98-177 of bovine cardiac troponin T. Five other antibodies were specific for cardiac troponin T and recognized antigenic determinants located between residues 180-258 of bovine cardiac troponin T. Localization of antigenic determinants in the central part of troponin T seems to be due to the high hydrophilicity and flexibility of this part of the molecule. The monoclonal antibodies thus obtained may be used for diagnosing various types of human heart diseases.

Animals↗

PubMed1992

Caldesmon-phospholipid interaction. Effect of protein kinase C phosphorylation and sequence similarity with other phospholipid-binding proteins.

Recently published data [Vorotnikov & Gusev (1990) FEBS Lett. 277, 134-136] indicate that smooth muscle caldesmon interacts with a mixture of soybean phospholipids (azolectin). Continuing this investigation, we found that duck gizzard caldesmon interacts more tightly with acidic (phosphatidylserine) than with neutral (phosphatidylcholine) phospholipids. A high concentration of Ca2+ (50 microM) decreased the interaction of caldesmon with phosphatidylserine. Among chymotryptic peptides of caldesmon, only those having molecular masses of 45, 40, 23, 22 and 20 kDa were able to specifically interact with phospholipids. These peptides, derived from the C-terminal part of caldesmon, contained the sites phosphorylated by Ca2+/phospholipid-dependent protein kinase, and phosphorylation catalysed by this enzyme decreased the affinity of these peptides for phospholipids. In the presence of Ca2+, calmodulin competed with phospholipids for the interaction with the caldesmon peptides. The C-terminal part of caldesmon contains three peptides with a primary structure similar to that of the calmodulin- and phospholipid-binding site of neuromodulin. These sites may be involved in the interaction of caldesmon with calmodulin and phospholipids.

Amino Acid Sequence↗

PubMed1991

Identification of the site phosphorylated by casein kinase II in smooth muscle caldesmon.

Phosphorylation of avian gizzard caldesmon by casein kinase II was investigated. The enzyme incorporates about 1 mol of phosphate per mol of caldesmon. All sites of phosphorylation are located in short chymotryptic peptides with Mr 25-27 kDa or in the short N-terminal peptide formed after cleavage of chicken gizzard caldesmon at Cys153. The primary structure of the tryptic peptide containing the main site of duck gizzard caldesmon phosphorylation is S-E-V-N-A-Q-N-X-V-A-E-D-E-T-K, where X is an unidentified residue, presumed to be phosphoserine. Thus, Ser73 is the main site phosphorylated by casein kinase II in avian gizzard caldesmon.

Amino Acid Sequence↗

PubMed1991

Stabilization by a disulfide bond of the N-terminal domain of a mutant troponin C (TnC48/82).

The regulatory activity of troponin C is reversibly inhibited by a disulfide bridge between cysteine residues introduced by site-directed mutagenesis in positions 48 and 82 (TnC48/82) in the N-terminal domain of rabbit skeletal troponin C (sTnC; Grabarek, Z., Tan, R.-Y., Tao, T., and Gergely, J. (1990) Nature 345, 132-135). In the present work we have investigated the effects of the disulfide on structural properties of TnC48/82 monitored by CD spectroscopy and limited trypsinolysis. The CD spectra of the mutant protein in the oxidized form (oxTnC48/82) with and without Ca2+ are similar to the corresponding ones of the reduced and carboxamidomethylated form (CAMTnC48/82), indicating that the disulfide has essentially no effect on the overall secondary structure. The N-terminal domain of oxTnC48/82 is resistant to thermal unfolding, but that of CAMTnC48/82 is only slightly more stable than the corresponding domain of sTnC. In the presence of Ca2+ oxTnC48/82 is more resistant to trypsinolysis than sTnC whereas the rate of tryptic digestion of CAMTnC48/82 is the same as that of sTnC, indicating that peptide bonds adjacent to lysine residues at position 84 and 88, the sites of tryptic attack, are protected by the disulfide. The disulfide cross-linked N-terminal peptide of TnC48/82 does not bind TnI, unlike its reduced or carboxamidomethylated forms. Our data indicate that the disulfide between Cys48 and Cys82 stabilizes the structure of the N-terminal domain of TnC and blocks its ability to interact with TnI. The effects of the disulfide appear to be restricted to the N-terminal domain of TnC.

Animals↗

PubMed1991

Some properties of duck gizzard caldesmon.

The domain structure of duck gizzard caldesmon was investigated. A single thiol group is located in the vicinity of the C-terminus of the protein. A simple method for the purification of a short (21 kDa) C-terminal peptide formed after chemical cleavage of caldesmon at cysteine residues was evolved. The C-terminal peptide of caldesmon interacts with calmodulin with an affinity one order of magnitude higher than that of native caldesmon. The Ca2+/phospholipid-dependent protein kinase (protein kinase C) transfers about 2 mol of phosphate per mol of caldesmon. All sites phosphorylated by protein kinase C are located in the short (21 kDa) C-terminal peptide of caldesmon. Phosphorylation does not affect the interaction of caldesmon with calmodulin.

Animals↗

PubMed1991

[Caldesmon and calponin are proteins, participating in the regulation of myosin and actin interaction in nonmuscle and smooth muscle cells].

The structure and properties of two novel regulatory proteins of nonmuscle cells and smooth muscles are reviewed. The localization of the "light" caldesmon isoform in nonmuscle cells and the involvement of this protein in the formation of cytoskeleton structures are described. A detailed description of the "heavy" caldesmon isoform and of localization of sites of caldesmon interaction with actin, tropomyosin, myosin and calmodulin, is given. The data on caldesmon phosphorylation by protein kinases are reviewed. Some present-day concepts concerning the mechanism of caldesmon function during regulation of myosin interaction with actin are considered. The structure, properties and phosphorylation of calponin are described. The properties of calponin and caldesmon are compared with those of troponin components involved in the regulation of the contractile activity of striated muscles.

Actins↗

PubMed1991

[The effect of various low molecular weight compounds on the cation-binding properties of troponin with the heart].

Using models of various complexity (isolated troponin C, troponin C-troponin I complex, troponin complex, troponin-tropomyosin complex, myofibrils), the effects of several low molecular weight organic compounds on the Ca(2+)-binding properties of troponin C were investigated. Trifluoperazine, calmidazolium and substance 48/90 increased the affinity of Ca(2+)-specific sites of troponin C both in the case of isolated troponin and in all the complexes under study. Nicardipine had no effect on the cation-binding activity of isolated troponin C, but increased the affinity of the Ca(2+)-binding sites of troponin C in the complex with troponin I. The cardiotonic drugs APP 201-533 and DPI 201-106 had practically no effect on the cation-binding properties of isolated troponin C or of simple complexes of troponin C. At the same time APP 201-533 increased, whereas DPI, 201-106 decreased the affinity of the Ca(2+)-binding sites of troponin C in myofibrils. It is concluded that the effects of the drugs on the cation-binding properties of troponin C depend on the protein-protein interaction with the filament. Studies of physiological activity of low molecular weight organic compounds require a detailed analysis of their effects on the Ca(2+)-binding activity of troponin C included into protein complexes of different complexity.

Animals↗

PubMed1990

Interaction of smooth muscle caldesmon with phospholipids.

Taking into account the perimembrane localization of caldesmon [(1986) Nature 319, 68] and its ability to participate in the regulation of receptor clusterization [(1989) J. Biol. Chem. 264, 496], we studied the interaction of duck gizzard caldesmon with soybean phospholipids (azolectin). By using four independent methods, i.e. light scattering, gel-electrophoresis, gel-filtration and ultracentrifugation, we showed a Ca-independent complex formation between caldesmon and azolectin. Interacting with caldesmon, calmodulin is shown to dissociate the caldesmon-azolectin complex. It is supposed that the caldesmon-phospholipid interaction may affect caldesmon phosphorylation by Ca-phospholipid-dependent protein kinase. This effect may be important for various cell motility processes.

Animals↗

PubMed1990

Interaction of lactate dehydrogenase with skeletal muscle troponin.

Rabbit muscle troponin complex covalently bound to CNBr-activated Sepharose 4B was shown to interact with soluble lactate dehydrogenase with a stoichiometry of 2 mol lactate dehydrogenase/mol of troponin. The presence of Ca2+ influenced the strength of association (the KD values of 0.73 and 2.3 microM were determined in the presence of 200 microM EGTA or 100 microM Ca2+, respectively). In the absence of Ca2+, the affinity of lactate dehydrogenase to troponin was strongly pH-dependent, reaching a maximum in the region of pH 6.0-7.0. No change of catalytic activity was observed as a result of interaction between lactate dehydrogenase and troponin, the enzyme appeared capable of functioning in the bound form.

Animals↗

PubMed1990

Competitive binding of the troponin T-specific pool of caldesmon antibodies and tropomyosin to skeletal troponin T and smooth muscle caldesmon.

The fraction of polyclonal caldesmon antibodies cross-reacting with rabbit skeletal troponin T are shown to compete with smooth muscle tropomyosin for caldesmon and troponin T, as revealed by ELISA method. The epitope recognized by these antibodies was also found in Mr 77 kDa non-muscle caldesmon. These results provide functional confirmation for the suggestion that the regions of amino acid sequence homology in caldesmon isoforms and troponin T belong to the tropomyosin binding sites.

Animals↗

PubMed1990

Drug interaction with cardiac and skeletal muscle troponin C.

The effects of certain drugs on the binding of divalent cations to Ca(2+)-specific and Ca/Mg sites on cardiac and skeletal troponin C molecules were investigated by fluorescence and circular dichroism spectroscopy. Nicardipine and two new cardiotonic agents, APP 201-533 and DPI 201-106, interact with the N-terminal domain of troponin C, and either do not affect or, in the case of DPI 201-106, slightly increase the affinity of the Ca(2+)-specific site of troponin C for Ca2+ ion. Compound 48/80 seems to interact with the central alpha-helix of skeletal troponin C and affects the cation-binding properties of both the Ca(2+)-specific and the Ca/Mg sites of the protein. Trifluoperazine and calmidazolium (R24571) interact with the C-terminal domain of troponin C and increase the affinity of the Ca/Mg sites located there. At high concentrations these compounds interact with the N-terminal domain of troponin C and increase the affinity of the Ca(2+)-specific site. According to fluorescence spectroscopy data, R24571 induces conformational changes in troponin C similar to those evoked by troponin I. The experimental data suggest that the drugs studied act in similar ways with troponin C and calmodulin.

Allosteric Site↗

PubMed1989

Interaction of smooth muscle caldesmon with S-100 protein.

The interaction of caldesmon with certain Ca-binding proteins was investigated by means of electrophoresis under non-denaturating conditions. In the presence of Ca2+ calmodulin, troponin C and S-100 protein form a complex with caldesmon. No complex formation takes place in the absence of Ca2+. Lactalbumin and pike parvalbumin (pI4.2) do not interact with caldesmon independently of Ca-concentration. Both S-100 protein and calmodulin effectively inhibit phosphorylation of caldesmon by Ca-phospholipid-dependent protein kinase. At low ionic strength S-100 protein reverses the inhibitory action of caldesmon on the skeletal muscle acto-heavy meromyosin ATPase more effectively than calmodulin. It is supposed that in certain tissues and cell compartments the proteins belonging to the S-100 family are able to substitute for calmodulin in the caldesmon-dependent regulation of actin and myosin interaction.

Animals↗

PubMed1989

Caldesmon150, caldesmon77 and skeletal muscle troponin T share a common antigenic determinant.

Caldesmon and troponin are known to control actin-myosin interaction in smooth and striated muscle. We showed that polyclonal antibodies to smooth muscle caldesmon150 cross-react with non-muscle caldesmon77 and with skeletal muscle troponin T. The epitope recognized by caldesmon antibodies is located in the CB 2 peptide [residues 71-151] of troponin T. It is supposed that the observed cross-reactivity is due to the common structure of the tropomyosin-binding site of caldesmon and troponin T.

Animals↗

PubMed1989

[Phosphorylation of isolated components of the troponin complex of skeletal and cardiac muscle phosphorylase kinase from bird skeletal muscles].

Pigeon and chicken skeletal muscle phosphorylase kinase purified to a nearly homogeneous state is able to phosphorylate both cardiac and skeletal troponin I and T. After 1-hr incubation, the enzyme transfers up to 0.35 mole of phosphorus per mole of skeletal troponin I, up to 0.5 mole of cardiac troponin I and up to 0.1 mole of cardiac and skeletal troponin T. Avian muscle phosphorylase kinase does not phosphorylate the first serine residue of cardiac and skeletal troponin T, but catalyzes the phosphate incorporation into the site(s) of troponin T located in the central or C-terminal parts of the protein molecule. The rate of troponin phosphorylation by pigeon muscle phosphorylase kinase is pH-dependent: the 6.8/8.2 ratio for troponin I is close to 0,2, whereas that with troponin T varies in the range of 0.5-0.7. Troponin phosphorylation by avian phosphorylase kinase depends on the presence of Ca2+ in the incubation mixture. In the presence of 3 mM EGTA troponin I phosphorylation is inhibited by 70-90%, whereas that of troponin T--by 50%. The experimental results indicate that the phosphorylation of troponin I and T is catalyzed either by two different active centers or by different conformations of the single center of avian phosphorylase kinase.

Animals↗

PubMed1989

[Localization of segments, phosphorylated by Ca-phospolipid-dependent protein kinase in smooth muscle caldesmon].

Phosphorylation of caldesmon from duck gizzard by Ca-phospholipid-dependent protein kinase was investigated. Ca-phospholipid-dependent protein kinase transfers about 3.5 moles of phosphate per mole of caldesmon (140 kDa). Tropomyosin does not affect, while calmodulin strongly inhibits the phosphorylation of caldesmon by Ca-phospholipid-dependent protein kinase. Data from one-dimensional peptide mapping suggest that the sites phosphorylated by the enzyme are located in fragments with apparent molecular weights of 43 and 35 kDa, which are supposed to be located in the vicinity of N- or C-termini of the protein molecule and involved in the caldesmon interaction with actin and calmodulin.

Actins↗

PubMed1988

Ca2+-induced conformational changes in cardiac troponin C as measured by N-(1-pyrene)maleimide fluorescence.

Bovine cardiac troponin C was modified by N-(1-pyrene)maleimide at Cys-35 and Cys-84; the Ca2+-induced conformational changes were followed by measuring pyrene fluorescence. In isolated troponin C, the saturation of Ca2+, Mg2+-sites leads to a simultaneous increase in the pyrene monomer as well as to a decrease in the pyrene excimer fluorescence, whereas the saturation of Ca2+-specific sites results in a slight decrease in the fluorescence of pyrene monomer. Troponin T does not influence the dependence of pyrene-troponin C fluorescence on Ca2+ concentration. Within the equimolar complex of troponin C and troponin I, the saturation of Ca2+, Mg2+-sites has no effect on pyrene fluorescence, whereas the saturation of Ca2+-specific sites leads to a simultaneous decrease of both pyrene monomer and pyrene excimer fluorescence. It is supposed that troponin I diminishes the conformational changes in troponin C that are induced by the saturation of Ca2+, Mg2+-sites and enhances the conformational changes induced by the saturation of Ca2+-specific sites of troponin C.

Animals↗

PubMed1988

Phosphorylation of smooth muscle caldesmon by three protein kinases: implication for domain mapping.

Phosphorylation of duck gizzard caldesmon by Ca2+/phospholipid-dependent protein kinase, Ca2+/calmodulin-dependent protein kinase and casein kinase II has been investigated. The Ca2+/phospholipid-dependent protein kinase incorporates more than 3 mol phosphate per mol (140 kDa) caldesmon. All phosphorylation sites are localized in the actin- and calmodulin-binding peptide (40-45 kDa) supposed to be a part of the C-terminal domain of caldesmon. Casein kinase II phosphorylates only one site located in a short (25-27 kDa) peptide, presumably in the caldesmon N-terminal domain. The Ca2+/calmodulin-dependent protein kinase phosphorylates two sites located in the N- and C-terminal domains of caldesmon.

Animals↗