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N B Gusev

Publications and source records attributed to N B Gusev.

89 records · Page 5Linked to original sources
PubMed1982

Biological activities of bovine cardiac-muscle troponin C C-terminal peptide (residues 84-161).

1. Bovine cardiac-muscle troponin C was digested at cysteine residues 35 and 84, and the C-terminal peptide (residues 84-161) was isolated. 2. The C-terminal peptide contains two Ca2+-binding sites. These sites bind Ca2+ with a binding constant of 2.0 X 10(8) M-1. In the presence of 2 mM-Mg2+ the binding constant for Ca2+ is decreased to 3.7 X 10(7) M-1. The corresponding constants for native troponin C are 5.9 X 10(7) M-1. and 2.9 X 10(7) M-1 respectively. 3. Electrophoretic mobility of the C-terminal peptide is increased in the presence of 0.1 mM-CaCl2 as compared with the mobility in the presence of 2mM-EDTA. The same phenomenon was observed when electrophoresis was performed in the presence of 6 M-urea or 0.1% sodium dodecyl sulphate. 4. When saturated with Ca2+, the C-terminal peptide forms complexes with bovine cardiac-muscle troponin I both in the absence and in the presence of 6 M-urea. This complex is dissociated on removal of Ca2+. 5. The data suggest that the C-terminal peptide of troponin C contains two Ca2+/Mg2+-binding sites and interacts with troponin I. Thus, despite the 30% difference in amino acid composition, the properties of bovine cardiac-muscle troponin C C-terminal peptide are similar to those of rabbit skeletal-muscle troponin C C-terminal peptide.

Amino Acids↗
PubMed1981

[Troponin T kinase: possible relationship to casein kinases of the G type].

Troponin T kinase utilizes ATP and GTP as substrates for the protein kinase reaction. When phosvitin is used as substrate, the enzyme activity increases with an increase in the ionic strength up to 0.2-0.3 M KCl. The pH optimum for the enzyme lies at 8-9. Ultracentrifugation in sucrose density gradient showed that the sedimentation coefficient of troponin T kinase is 9.5 S. The Stocks radius determined by gel-filtration is equal to 49 A. The molecular weight of the enzyme calculated from the given values of the Stocks radius and sedimentation coefficient is 184,000. This is indicative of the oligomeric structure of the enzyme and suggests that the stoicheiometry of the enzyme subunits having mol. weights of 50,000, 46,000 and 31,000 is other than 1:1:1. Troponin T kinase is capable of autophosphorylation; the phosphorylation process involves only the protein with mol. weight of 31,000. The data obtained suggest that troponin T kinase can be referred to casein kinases of G type and may participate in translation processes.

Adenosine Triphosphate↗
PubMed1981

[Troponin from th bovine heart: isolation and study of cation-binding properties using a 3-dimethylaminonaphtheurodine fluorescent probe].

A method for isolation of bovine heart troponin has been developed. The troponin complex was separated into constituent components. The fluorescent probe 3-dimethylaminonaphtheurodine binds to heart troponin: the fluorescence of the troponin--dimethylaminonaphtheurodine complex is increased with a rise in Ca2+ concentration from pCa 8 to pCa 5. The apparent constant for Ca2+ binding to heart troponin calculated from the changes in fluorescence of the troponin-dimethylaminonaphtheurodine complex is equal to 2,1 . 10(6) M(-1). The corresponding constant for Sr2+ binding by heart troponin is 5 . 10(5) M(-1), Mg2+ have no effect on Ca2+ and Sr2+ binding. The results obtained suggest that heart troponin contains Ca-specific binding sites and that the binding of Ca2+ to these sites evokes conformational changes, which are registered by means of the fluorescent probe. The Hill coefficient for the conformational changes of heart troponin induced by Ca2+ and Sr2+ measured with the probe is equal to unity. The use of 3-dimethylaminonaphtheurodine as a marker of conformational changes allows to compare the cation-binding properties of troponin from heart and skeletal muscles.

Animals↗
PubMed1980

Phosphorylase kinase phosphorylation of skeletal-muscle troponin T.

Rabbit skeletal-muscle troponin T was phosphorylated by a standard preparation of phosphorylase kinase [Cohen (1973) Eur. J. Biochem. 34, 1--14] and by fractions obtained after chromatography of phosphorylase kinase on phosphocellulose. The original preparation of phosphorylase kinase phosphorylated at least two sites, one of which was serine-1. The second and probably the third sites were presumably located in the peptide flanked by amino-acid residues 147 and 161 of troponin T. Fractions of phosphorylase kinase was adsorbed on phosphocellulose phosphorylated only the second site. Tightly adsorbed fractions possessed high troponin T kinase and phosvitin kinase activities and phosphorylated only serine-1 of troponin T. The results suggest that standard preparations of phosphorylase kinase are contaminated by troponin T kinase, which can phosphorylate serine-1 of troponin T.

Amino Acids↗
PubMed1980

Ca2+-induced conformational changes in the troponin complex detected by crosslinking.

In an attempt to detect Ca2+-induced conformational changes by crosslinking, rabbit muscle troponin complex was reacted with the bifunctional reagents 1,3-difluoro-4,6-dinitrobenzene, 4,4'-difluoro-3,3'-dinitrodiphenylsulfone and dimethyl suberimidate under various conditions and the products were analyzed by dodecyl sulfate gel electrophoresis. In the absence of divalent cations, with the two aromatic reagents at a low reagent/protein ratio, the main cross-link products were troponin T-I and I-C. With dimethyl suberimidate the only major crosslink product was conjugate T-I. Ca2+, alone as well as in the presence of Mg2+, prevented the formation of I-C crosslinks with both aromatic reagents, but it did not affect crosslinking with dimethyl suberimidate. Ca2+ also decreased the number of NH2 groups of troponin that are highly reactive towards 2,4,6-trinitrobenzene sulfonate. Both effects of Ca2+ can be interpreted in terms of a conformational change in the troponin complex elicited by the binding of the specific divalent cation.

Animals↗
PubMed1980

Identification of an intramolecular cross-link in troponin C after cross-linking the troponin complex with 1,3-difluoro-4,6-dinitrobenzene.

The troponin complex isolated from rabbit skeletal muscle was cross-linked with 1,3-difluoro-4,6-dinitrobenzene, subjected to tryptic hydrolysis, and the labelled peptides were separated. One peptide was purified to homogeneity; it proved to be peptide Gly(89)-Arg(100) of troponin C on the basis of amino acid composition and N-terminal analysis. The absorption spectrum of this peptide between 300 and 500 nm was very similar to that of 1-(S-cysteinyl)-5-(N epsilon-lysyl)-2,4-dinitrobenzene, which could only be due to cross-link formation between Lys-90 and Cys-98. This finding is interpreted in terms of the proposed tertiary structure of troponin C.

Chemical Phenomena↗
PubMed1980

Isolation and some properties of troponin T kinase from rabbit skeletal muscle.

A method for isolation of troponin T kinase (ATP-protein phosphotransferase, EC 2.7.1.37) from rabbit skeletal muscles in proposed. The method gives a 7000-10 000-fold purification and results in an enzyme with specific activity of 400-800-nmol x min-1 x mg-1 of protein. The molecular weight of tropin T kinase as determined by gel filtration exceeds 500 000. Electrophoresis in polyacrylamide gel in the presence of sodium dodecyl sulphate revealed that isolated preparations of the enzyme consisted of at least three distinct proteins with apparent mol.wt. of 50 000, 46 000 and 31 000. The enzyme phosphorylates isolated troponin T at a rate which exceeds the phosphorylation rates of casein, phosvitin, histones, phosphorylase b and protamine 5-30-fold. Within the whole troponin complex, only troponin T is phosphorylated by the enzyme. The enzyme phosphorylates only the N-terminal serine residue of troponin T, i.e. the site that is normally phosphorylated in the whole troponin complex isolated from rabbit skeletal muscles.

Animals↗
PubMed1978

[Skeletal muscle troponin and phosphorylation: a site of troponin T, that is phosphorylated by specific protein kinase].

A procedure is described of the isolation of protein kinase, which phosphorylates isolated troponin T with a rate, 5--30 fold exceeding the phosphorylation rate of other substrates (phosvitine, caseine, protamine sulphate, H1, H2A, H2b, H3, H4 histones). Troponin T-specific protein kinase transfers 0.85--0.95 moles of P per 1 mol of dephosphorylated troponin T. It phosphorylates only N-terminal acetylated serine residue, i. e. the site of troponin T structure, which is normally phosphorylated, when the whole troponin complex is isolated from skeletal muscles. Protein kinase is incapable to phosphorylate N-terminal serine residue in a mixture of triptic peptides of troponon T.

Animals↗
PubMed1978

[Role of Ca-specific binding sites in the induction of conformational changes in troponin structure].

A new technique for the registration of troponin conformational changes by hydrophobic fluorescent dye, dimethylnaphteirodin, is described. The increase of Ca2+ concentration from pCa 8 to pCa 5 is accompanied by 4--5-fold increase of fluorescence of troponin--dimethylnaphteirodin complex and by the shift of the fluorescence maximum by 10 nm into the short-wave region. Isolated troponin components and troponin I--troponin T and troponin I--troponin C complexes do not bind dimethylnaphteirodin. The troponin T--TROPONIN C complex binds dimethylnaphteirodin, and fluorescence of the bound dye increases under the increase of Ca2+ concentration, as it does in the case of complete froponin complex. The apparent Ca2+-binding constant by the whole troponin complex (K=8.10(6) M(-1)) and by troponin T--troponin C complex (K=8.10(5) M(-1)), and the value of fluorescence of troponin- and troponin components-bound dimethylnaphteirodin do not depend on the magnesium concentration. Highly cooperative dependence of the fluorescence intensity of troponin-dimethylnaphteirodin on Ca2+ concentration (the Hill coefficient is 2.0--2.4) suggests, that the changes in the troponin structure, registered using dimethylnaphteirodin, take place only after Ca2+ binding to two Ca-specific sites.

Binding Sites↗
PubMed1976

[Study of reactivity of sulfhydryl groups of troponin].

The reactivities of SH-groups of troponin and its components were studied, using 4-chloro-7-nitrobenzo-2-oxa-1,3-diazole (NBD-Cl). At low concentrations of Ca2+ (pCa greater than 8) one rapidly- and two slowly-reacting SH-groups of troponin I and one SH-group of troponin C slowly reacting with NBD-chloride are titrated in the whole troponin complex. When Ca2+ concentration is increased up to pCa less than 5, only one slowly-reacting and one rapidly-reacting with NBD-chloride SH-groups of troponin I are titrated. The increase of Ca2+ concentration from pCa greater than 8 to pCa less than 5 results in a change of the environment polarity for the highly reactive SH-group of troponin I in the whole troponin complex. This phenomenon may suggest that the changes in troponin C structure during Ca2+ binding somehow induce changes in that of troponin I. The half-maximal change of troponin SH-groups reactivity is found at pCa 6.8. No cooperativity for Ca2+ binding by the troponin complex is observed using SH-groups titration by NBD-Cl.

4-Chloro-7-nitrobenzofurazan↗
PubMed1976

[Search for a protein kinase specific for treponin T].

A specific to troponin T (as compared to f1 histone) proteinkinase is isolated using affinity chromatography on Sepharose 4B with covalently bound dephosphorylated troponin T. Homogeneous proteinkinase has a molecular weight of 175.000 and phosphorylates troponin T at a rate exceeding that of histone phosphorylation, while crude proteinkinase preparation has troponin T phosphorylation rate 15--20 times as low as that of histone phosphorylation. The enzyme isolated is capable of phosphorylating troponin T, but not troponin I, in the whole troponin complex.

Animals↗
PubMedDate not supplied

[Effect of carnosine and anserine on excitation and contraction of fatigued skeletal muscle].

The effect of dipeptides (carnosine and anserine) on a neuromuscular preparation under fatigue or diplacine blocking is shown to result in a significant restoration of the muscle contraction. The analysis of this phenomenon evidences that synaptic processes are not responsible for the restoration, at least under the given experimental conditions. At the same time, the absence of any effect of the compounds studied on the maximal rate of isotonic shortening and on the tension of glycerinized fibres and ATPPase of native and desenitized actomyosine indicates that the contractile mechanism is not involved in the effect described. On the other hand, the dipeptides restore the value of the transmembrane potential depolarized by exhaustion, increase the amplitude of isometric twitch and the value of maximal load under isotonic conditions. Moreover, the time of the contractile cycle in the presence of the dipeptides is considcted to the processes of electromechanical coupling in skeletal muscles.

Actomyosin↗