Biomedical subjects

N B Gusev

Publications and source records attributed to N B Gusev.

At least 37 records · Page 2Linked to original sources

PubMed1997

Utilization of troponin C as a model calcium-binding protein for mapping of the calmodulin-binding sites of caldesmon.

Troponin C, a structural analogue of calmodulin, was used for mapping the calmodulin-binding sites of caldesmon. The apparent Kd values for the formation of the caldesmon-calcium-binding-protein complex as determined by native gel electrophoresis were 0.5, 1.2 and 3.9 microM for calmodulin, rabbit skeletal muscle troponin C and bovine cardiac troponin C respectively. Troponin C induced a 4-6 nm blue shift of the Trp fluorescence of caldesmon without affecting the amplitude of fluorescence. In the presence of Ca2+, troponin C induced partial displacement of caldesmon from actin tropomyosin complexes. Addition of 5,5'-dithiobis(nitrobenzoic) acid to an equimolar complex of caldesmon and troponin C induced disulphide cross-linking between Cys-98 of rabbit skeletal muscle troponin C and the single Cys residue of duck gizzard caldesmon, located in a position analogous to Cys-580 of the chicken gizzard protein. The cross-linked caldesmon-troponin C complex was ineffective in inhibiting actomyosin ATPase activity. It is concluded that Cys-580 of caldesmon can be located close to both the central helix of calcium-binding proteins and the C-terminal domain of actin. This may be important for the regulation of actomyosin ATPase activity by caldesmon.

Actins↗

PubMed1997

Troponin I is released in bloodstream of patients with acute myocardial infarction not in free form but as complex.

Fourteen monoclonal antibodies (mAbs) against human cardiac troponin I (cTnI) were generated by commonly used experimental techniques. All these antibodies, as well as antibody 414 (HyTest), were specific for human cTnI. Fifteen antibodies thus obtained were tested in a sandwich cTnI immunofluorescence assay (altogether 196 combinations). Ten pairs giving the highest sensitivity were selected for further investigation. The effect of TnI-TnC complex formation on antibody interaction with antigen was analyzed. The formation of TnI-TnC complex results in a significant decrease of the interaction of mAbs with TnI for seven of 10 analyzed pairs of antibodies. Using two pairs of cTnI-specific mAbs, one that recognized only free cTnI but not cTnI complexed with cTnC, and another that could be used for measurement of total cTnI (free cTnI and cTnI in complex with cTnC), we demonstrated that the main part of cTnI in serum collected from acute myocardial infarction patients is presented in the complex from. We concluded that effective and reliable immunological detection of TnI is possible only when antibodies used for assay development recognize both free TnI and TnI complexed with other troponin components.

Antibodies, Monoclonal↗

PubMed1996

Interaction of smooth muscle calponin and desmin.

Interaction of smooth-muscle calponin and desmin was analyzed by means of ultracentrifugation, fluorescent spectroscopy and affinity chromatography. At low and intermediate ionic strength (30-50 mM NaCl) calponin is cosedimented with desmin with an apparent dissociation constant 3-15 microM and stoichiometry of 1 calponin/4-6 desmin. Calmodulin decreases the quantity of calponin bound to desmin. Increase of ionic strength up to 150 mM weakens calponin-desmin interaction, but even at this ionic strength part of calponin remains bound to desmin. Calponin increases the rate and extent of fluorescence quenching induced by polymerization of 5-iodoacetamidofluorescein-labeled desmin. Affinity chromatography data indicate that desmin-binding sites are located in the N-terminal 22 kDa fragment of calponin. Since calponin interacts with desmin with an affinity comparable with that of, e.g., tropomyosin and myosin we suppose that calponin-desmin interaction may be important for cytoskeleton organization.

Animals↗

PubMed1996

Interaction of proteolytic fragments of calmodulin with caldesmon and calponin.

Interaction of five tryptic fragments of calmodulin with caldesmon and calponin was analysed by native gel electrophoresis. In the presence of Ca2+ intact calmodulin interacts with caldesmon and calponin with apparent Kd values equal to 0.23 and 1.3 microM respectively. The interaction was abolished in the absence of Ca2+. Two large tryptic fragments of calmodulin obtained in the presence of Ca2+ (TR1C, residues 1-77, and TR2C, residues 78-148) interact with caldesmon with apparent Kd values of 11.9 and 4.6 microM. Affinity of TR2C to calponin (Kd 3.8 microM) was comparable with that of native calmodulin and was much higher than the corresponding value for TR1C (Kd 41 microM). The short C-terminal tryptic peptide of calmodulin obtained in the presence of EGTA (TR3E, residues 107-148) interacts with caldesmon and calponin with Kd values of 23.9 and 12.1 microM, whereas the large N-terminal peptide TR1E (residues 1-106) interacts with both caldesmon and calponin with a very low affinity (Kd 60 microM). Thus although both N- and C-terminal domains of calmodulin are involved in the interaction with caldesmon and calponin, the C-terminal part of calmodulin (residues 78-148) is of special importance and has the highest contribution for caldesmon and calponin binding.

Animals↗

PubMed1996

Identification of reactive carboxyl groups in troponin C.

Ca2+ regulates muscle contraction by reversible binding to troponin C (TnC), the Ca(2+)-binding subunit of troponin complex. In order to identify acidic amino acids exposed on its surface, carboxyl groups in TnC were activated with 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide, then labeled with dinitrophenylethylenediamine. Labeled protein was then digested with trypsin and thermolysin, and the resulting peptides were purified by HPLC. Modified peptides were detected by their specific absorbance at 360 nm, and labeled amino acids in these peptides were identified by sequence analysis. Although the total incorporation of label into TnC was only 2.6 mol/mol, we found that 14 of the 46 carboxyl groups of TnC were partially labeled. The labeled carboxyl groups were located in surface regions of the known three-dimensional structure of TnC which may interact with other components of the troponin complex.

Amino Acid Sequence↗

PubMed1995

Interaction of smooth muscle calponin with phospholipids.

Analyzing the primary structure we predicted that calponin may interact with phospholipids. In order to check this suggestion we investigated the interaction of calponin with phospholipids by ultracentrifugation, light scattering, vesicles leakage and differential scanning calorimetry. In agreement with our prediction calponin interacts with acidic phospholipids and the phospholipid-binding site was located in the short (13 kDa) N-terminal chymotryptic peptide of calponin. The apparent dissociation constant of calponin-phospholipids complex was less than 0.2 microM and calmodulin competes with phospholipids for calponin binding. Although the interaction of calponin with phospholipids decreases at high ionic strength, calponin binds phospholipids even in the presence of 100-150 mM of the salt. Under certain conditions calponin induced leakage of phospholipid vesicles and affected the cooperativity of lipid phase transition. It is concluded that both electrostatic and hydrophobic interactions provide for calponin-phospholipid complex formation.

Animals↗

PubMed1995

Computer-assistant prediction of phospholipid binding sites of caldesmon and calponin.

The primary structure of smooth muscle caldesmon and calponin was screened for the presence of amphiphilic alpha-helices which can participate in the formation of protein-lipid contacts. Only one caldesmon segment (residues 645-660) having a predominantly alpha-helical structure and high hydrophobic moment satisfies all criteria for a surface-seeking helix and is predicted to be involved in the caldesmon-phospholipid interaction. This prediction agrees with experimental results indicating that one of the caldesmon-phospholipid binding sites is located in the sequence 628-658 [Bogatcheva et al. (1994) FEBS Lett. 342, 176]. Two segments of calponin (residues 45-55 and 85-95) exhibit high hydrophobic moments and the sequence 85-95 is characterized by a high probability of alpha-helix formation. This may suggest that at least one of these segments could facilitate the calponin-phospholipid interaction and that calponin, as with many other actin binding proteins, is able to interact with membranes.

Binding Sites↗

PubMed1995

Interaction of smooth muscle caldesmon with calmodulin mutants.

The interaction of avian smooth muscle caldesmon with calmodulin (CaM) was investigated by studying the ability of selected mutant calmodulins to induce fluorescence changes in caldesmon. Different types of CaM mutants were used including point charge mutants, cluster mutations, and mutations which alter the calcium binding of CaM. The caldesmon binding properties were only slightly affected by E84K-CaM or by the double mutation E84Q/E120Q-CaM. Affinity of calmodulin to caldesmon was decreased 2-4 times by point mutation G33V-CaM, double mutation E84K/E120K-CaM, deletion of residues 82-84, and by cluster mutations DEE118-120-->KKK or EEE82-84-->KKK. Mutations of the first (E31A-CaM) and the second (E67A-CaM) calcium binding sites reduced the affinity of calmodulin to caldesmon by at least 5-fold; in addition these calmodulin mutants exhibited smaller changes in the fluorescence spectra of caldesmon. Simultaneous mutation of the two negatively charged clusters of calmodulin EEE82-84-->KKK and DEE118-120-->KKK resulted in a more than 15-fold decrease in the affinity of calmodulin for caldesmon. The data indicate that charged and uncharged amino acids in both halves of CaM play an important role in the binding of calmodulin to caldesmon, and that Ca2+ binding must be maintained in the amino-terminal sites for maximal interaction with caldesmon.

Animals↗

PubMed1995

A new method of human cardiac troponin I and troponin T purification.

New rapid and effective procedure for simultaneous purification of human cardiac troponin I and troponin T has been developed. Affinity chromatography on immobilized monoclonal antitroponin I antibody C5 was used for purification of the whole troponin complex with a yield of 120 mg from 100 g of tissue. Isolated troponin I and troponin T (about 20 and 35 mg from 100 g tissue) were obtained by conventionally used ion-exchange chromatography. Antibody C5 recognizes conservative epitope of troponin I, therefore the method is applicable for purification of skeletal and cardiac troponin from a number of different animal species.

Antibodies, Monoclonal↗

PubMed1995

[Isolation and study of certain properties of caldesmon from cattle aorta].

A modified method for bovine aorta caldesmon isolation has been developed. When isolated from large vessels, caldesmon is copurified with connective tissue proteins whose molecular weight is similar to that of caldesmon. Separation of these proteins can be achieved by stepwise anion- (Q-Sepharose) and cation (phosphocellulose)-exchange chromatography. Bovine aorta and duck gizzard caldesmons have similar apparent molecular weights, absorption at 280 nm and one-dimensional peptide maps. Casein kinase II transfers about one mol of phosphate per mol of bovine aorta or duck gizzard caldesmon. In both cases, the sites of phosphorylation are located in the N-terminal peptides of apparent molecular weights of 26-28 kDa. It is concluded that there are no substantial differences between the structures and properties of avian gizzard and mammalian vessel caldesmons.

Animals↗

PubMed1995

[Study of the structure and mechanism of action of troponin C and calmodulin by "protein" engineering].

The structure, properties and action mechanisms of troponin C and calmodulin are reviewed. The primary and tertiary structures of calcium-binding proteins and the mechanisms of Ca2+ binding are analyzed. The methods used for investigating the functional activity of Ca-binding proteins are compared. Molecular biology approaches for analyzing the role of various ligands in Ca2+ binding are described. The role of alpha-helices in the maintenance of the overall structure, Ca2+ binding and calmodulin and troponin C interaction with target proteins is discussed. Mutations directed at the change of the electric charge and hydrophobicity of calmodulin and troponin C are described and compared. Data on the incorporation of Cys residues into calmodulin and troponin C structure are presented. The use of Cys-containing mutants of Ca-binding proteins for the study of conformational changes and protein-protein interaction is analyzed.

Amino Acid Sequence↗

PubMed1994

Localization of phospholipid-binding sites of caldesmon.

The interaction of phosphatidylserine with intact smooth muscle caldesmon and caldesmon fragments obtained by bacterial expression was investigated by means of light scattering. Among these fragments only those derived from the C-terminal part of caldesmon (so-called domain 4) were able to interact with phospholipids. Fragments 606C (residues 606-756), H7 (566-710) and H2 (626-710) form tight complexes with phosphatidylserine, whereas fragments H8 (658-737), H9 (669-737) and fragment H4 (566-624) interact with phospholipids less effectively. It is concluded that the phospholipid-binding site is located in the sequence 626-710 of caldesmon. This sequence contains calmodulin-binding sites and serine residues phosphorylated by protein kinase C and pro-directed protein kinases. This could explain the effects of calmodulin and phosphorylation on the caldesmon-phospholipid interaction described earlier.

Actins↗

PubMed1994

Location of two contact sites between human smooth muscle caldesmon and Ca(2+)-calmodulin.

We measured Ca(2+)-calmodulin binding to expressed human caldesmon fragments by three techniques: tryptophan fluorescence enhancement, change in fluorescence of TA-calmodulin, and cosedimentation with calmodulin-Sepharose. Ca(2+)-calmodulin bound with similar affinity to peptide M73 (C714SMWEKGNVFSSPGF727, N terminus of domain 4b), to all the fragments of caldesmon containing this peptide, and also to H9 (Thr726-Val793), which did not contain this peptide (Kd = 0.2-0.8 microM). We conclude that Ca(2+)-calmodulin binds at two sites on caldesmon; site A is the sequence 715MWEKGNVFS723 previously identified by Zhan et al. (Zhan, Q., Wong, S. S., and Wang, C.-L.A. (1991) J. Biol. Chem. 266, 21810-21814), and site B is located nearer the C terminus of caldesmon. Ca(2+)-calmodulin binding at site B is coupled to reversal of caldesmon inhibition of actin-tropomyosin activated myosin MgATPase, while calmodulin binding at site A has no detectable function. H9 did not displace M73 from Ca(2+)-calmodulin, while the other fragments did. High concentrations of M73 (> 1000 x Kd) could not displace H9 bound to Ca(2+)-calmodulin-Sepharose. Thus sites A and B in calmodulin are functionally separate. Analysis of overlapping expressed fragments indicates that site B is located in the sequence Thr726-Leu767, which includes Trp749. The minimal Ca(2+)-calmodulin binding sequence could be 744SRINEWLTK752.

Actins↗

PubMed1994

Filamin and gelsolin influence Ca(2+)-sensitivity of smooth muscle thin filaments.

Sheep aorta thin filaments were prepared by ultracentrifugation of an ATP-containing extract in the presence of different concentrations of ethanediol. Thin filaments prepared without ethanediol contained small quantities of tropomyosin (0.027 Tm:actin) and caldesmon (0.017 CD:actin) and activated the MgATPase of skeletal myosin independently of Ca2+. Ultracentrifugation in the presence of 10-20% ethanediol resulted in preparation of thin filaments with increased content of tropomyosin (0.17 Tm:actin) and caldesmon (0.04 CD:actin). These thin filaments possessed high Ca(2+)-sensitivity in activation of skeletal muscle myosin ATPase. Besides actin, tropomyosin and caldesmon, thin filaments contained gelsolin and filamin. Gelsolin content (0.007 gelsolin:actin) was independent of the presence of ethanediol. The filamin content decreased from 0.015 to 0.007 mol:mol actin when the ethanediol concentration was increased from 0 to 20%, and was negatively correlated with the Ca2+ sensitivity of thin filaments. In a reconstituted system, pure filamin or gelsolin affected caldesmon regulation of actomyosin ATPase. Gelsolin (0.01:actin) reduced the inhibition of actomyosin ATPase caused by caldesmon and increased the potency of Ca(2+)-calmodulin in reversing this inhibition. Filamin (0.007:actin) also decreased the inhibitory action of caldesmon on actin-activated myosin ATPase and also potentiated the reversal of this inhibition by calmodulin. We conclude that minor components of smooth muscle thin filaments (gelsolin and filamin) significantly modify caldesmon mediated regulation of actomyosin ATPase. We suggest a tropomyosin-mediated mechanism by which filamin or gelsolin could exert similar effects.

Actin Cytoskeleton↗

PubMed1994

Phosphorylation of aorta caldesmon by endogenous proteolytic fragments of protein kinase C.

Endogenous caldesmon kinase activity in sheep aorta smooth muscle was purified and characterized. The enzyme was identified as a proteolytic fragment of protein kinase C by cross-reactivity with anti-protein kinase C antibodies, autophosphorylation, substrate specificity and the primary structure of the sites of phosphorylation on caldesmon. The enzyme phosphorylated aorta caldesmon both in native thin filaments and in the isolated state. Up to 2.9 mols of phosphate per mol of caldesmon were transferred. Prolonged incubation of caldesmon with the kinase resulted in phosphorylation of Ser-127, Ser-587, Ser-600, Ser-657, Ser-686, and Ser-726 (numbering corresponds to chicken gizzard caldesmon sequence). Ser-600 and Ser-587 were the major sites of phosphorylation containing more than 30% of phosphate transferred. Phosphorylation did not significantly affect the interaction of caldesmon with Ca(2+)-calmodulin. However, phosphorylation of both intact caldesmon and of its C-terminal fragment (658C), containing residues 658-756, significantly decreased their ability to inhibit acto-heavy meromyosin ATPase. This seems to be partially due to a decrease in the binding of caldesmon and 658C to actin-tropomyosin and partly due to an uncoupling of the binding-inhibition relationship.

Actin Cytoskeleton↗

PubMed1993

Effect of 67 kDa calcimedin on caldesmon functioning.

Interaction of smooth muscle caldesmon with calmodulin, troponin C, S-100 protein and 67 kDa calcimedin was analyzed. Native gel electrophoresis and crosslinking revealed the complex formation between caldesmon and three EF-hand Ca-binding proteins, whereas calcimedin did not interact with caldesmon. In the presence of Ca2+, calcimedin binds to actin-tropomyosin without affecting the interaction of caldesmon with this complex. Although calcimedin reversed the inhibitory action of caldesmon on the actomyosin ATPase activity at a lower concentration than three other Ca-binding proteins, this effect only slightly depends on Ca2+ and was observed at the concentration of calcimedin comparable to that of actin. It is concluded that calcimedin itself cannot be responsible for Ca-dependent regulation of caldesmon functioning, but actin bundling induced by calcimedin (or by other actin binding proteins) decreases the inhibitory action of caldesmon on the actomyosin ATPase activity.

Actins↗

PubMed1993

Identification of casein kinase II as a major endogeneous caldesmon kinase in sheep aorta smooth muscle.

A caldesmon kinase activity was detected in an ATP extract of the myofibril-like pellet from sheep aorta. The enzyme was purified 745-fold and was identified as casein kinase II on the basis of molecular size, substrate specificity, and high sensitivity to heparin inhibition. Casein kinase II phosphorylated isolated caldesmon and caldesmon incorporated into native thin filaments, and transferred about 1 mol of phosphate per mol of caldesmon-h. Ser-73 was the main site phosphorylated by casein kinase II in chicken gizzard caldesmon. Phosphorylation of caldesmon reduced its affinity for smooth muscle myosin but had no effect upon the ability of caldesmon to inhibit the ATPase activity of actomyosin.

Animals↗

PubMed1993

The functional effects of mutations Thr673-->Asp and Ser702-->Asp at the Pro-directed kinase phosphorylation sites in the C-terminus of chicken gizzard caldesmon.

We expressed the C-terminal 99 amino acids of chicken gizzard caldesmon (658C) and two point mutants in which the preferred phosphorylation sites of MAP kinase and p34cdc2 kinase, Ser702 and Thr673 were altered to aspartic acid. The T673D mutant was indistinguishable from 658C but S702D was not phosphorylated by MAP kinase, was significantly less potent as an inhibitor of actin-tropomyosin activation of myosin MgATPase, and bound less actin-tropomyosin at low concentrations. Thus Ser702 is involved in the tropomyosin-dependent inhibitory mechanism of caldesmon, and its phosphorylation by MAP kinase or p34cdc2 kinase could modulate caldesmon function.

Actins↗