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O A Stafeeva

Publications and source records attributed to O A Stafeeva.

11 recordsLinked to original sources
PubMed1995

[Preparation of monoclonal antibodies to E1-component and their use for studying the pyruvate dehydrogenase complex].

A monoclonal antibody against the E1-component of pigeon breast muscle has been prepared. The dissociation constant of the E1-mAb F7F10 complex was determined to be equal to 5.93.10(-8) M. The cross-reaction between mAb F7F10 and the E1-component of the pyruvate dehydrogenase complex from various species (including human beings) was established. The F7F10 antibody was shown to interact with both alpha- and beta-subunits of E1, which suggests that the amino acid residues in the both subunits are constituents of the antigenic determinant. Binding of the F7F10 antibody to the antigen had no effect on the enzymatic activity of E1 but induced rapid inactivation of the pyruvate dehydrogenase complex in the pyruvate:NAD oxidoreductase reaction. The competition between the F7F10 antibody and the E2-component of the pyruvate dehydrogenase complex for the binding to E1 was revealed by immunoenzymatic analysis. It was concluded that the antigenic determinant and the E1 site responsible for the E1-E2 interaction within the pyruvate dehydrogenase complex may overlap.

Animals↗
PubMed1984

[Amino acid composition of the alpha-ketoglutarate dehydrogenase from the breast muscle of the pigeon].

The amino acid composition of alpha-ketoglutarate dehydrogenase--a component of alpha-ketoglutarate dehydrogenase complex--from the pigeon breast muscle has been determined. A significant similarity of the enzyme amino acid composition isolated from systematically remote species has been revealed by comparison of the data obtained with the literary one concerning the alpha-ketoglutarate dehydrogenase from Escherichia coli and pig heart.

Amino Acids↗
PubMed1983

Essential arginine residues for catalytic and regulatory functions of alpha-ketoglutarate dehydrogenase from pigeon breast muscle.

The number of arginine residues of pigeon breast muscle alpha-ketoglutarate dehydrogenase modified by 2,3-butanedione and 2,4-pentanedione was determined. It was shown that two of the 40 arginine residues in the enzyme monomer (Mr = 86,000) are accessible to the action of dicarbonyl compounds and are functionally significant. The protective effect of alpha-ketoglutarate and ADP against enzyme modification by 2,3-butanedione suggests the participation of alpha-ketoglutarate dehydrogenase arginine residues in the binding of substrate and allosteric activator, ADP.

Animals↗
PubMed1982

[Sulfhydryl groups of alpha-ketoglutarate dehydrogenase from pigeon breast muscle].

Using various thiol reagents (DTNB, pCMB, NBD-chloride), the number and reactivity of SH-groups of the alpha-ketoglutarate dehydrogenase component (KGD) of the alpha-ketoglutarate dehydrogenase complex were established. The total number of SH-groups as determined in the presence of a detergent is 12 per monomer of KGD. In the native enzyme DTNB titrates 3 or 4 SH-groups which, according to their reactivity, can be divided into 3 types. pCMB and NBD-chloride modify 5 to 6 SH-groups of KGD. In the presence of alpha-ketoglutarate and its active analog, alpha-ketoadipate, two SH-groups of the enzymes, the most and the least reactive ones, become inaccessible to the action of DTNB but the titrated by a 30-100-fold molar excess of pCMB. Binding of alpha-ketoacids which are competitive inhibitors of the enzyme causes no masking of SH-groups of KGD. When 3-4 SH-groups of KGD are blocked, the enzyme activity is reduced by 30% due to modification of the same two SH-groups which are inaccessible to DTNB in the presence of substrate. The fluorescence spectra of S-NBD derivatives of KGD suggest that these two SH-groups have hydrophobic environment. In terms of the number and reactivity of the thiol groups the apoenzyme does not differ from the holoenzyme; however, an addition of alpha-ketoglutarate to the apoenzyme does not decrease the number of SH-groups interacting with DTNB or the rate of their modification. The masking of two SH-groups of the holoenzyme results from conformational changes accompanying the formation of a productive ternary enzyme -- coenzyme -- substrate complex. Possible localization of these groups in the proximity of the active site is discussed.

Animals↗
PubMed1978

[Kinetic properties of the alpha-ketoglutarate dehydrogenase complex from pigeon breast muscle].

A complex kinetic behaviour of the alpha-ketoglutarate dehydrogenase component isolated from the pigeon breast muscle alpha-ketoglutarate dehydrogenase complex was established. The dependence curve of the reaction rate versus substrate concentration has an intermediate plateau and a maximum. The shape of the kinetic curve and specific activity depend on the enzyme concentration both in the reaction mixture and in the original solution of enzyme. The kinetic pattern of isolated alpha-ketoglutarate dehydrogenase suggests that the enzyme is a multi-step dissociating system characterized by different rates of oligomeric forms interconversions at various steps of dissociation. It is assumed that the process of alpha-ketoglutarate dehydrogenase oligomerization is a cooperative one. The kinetic behaviour of the complex and its constituent alpha-ketoglutarate dehydrogenase component is characterized by the same type of the v + [S] dependence curves as is the isolated enzyme. The correlation between the kinetic properties of the enzyme complex and its alpha-ketoglutarate dehydrogenase component and the kinetic behaviour of free alpha-ketoglutarate dehydrogenase suggest that the association-dissociation processes which are probably typical for the component within the complex determine the kinetic behaviour of the complex to a large extent. It is assumed that the anomalous kinetic behaviour of the alpha-ketoglutarate dehydrogenase component is due to the cooperative effects during substrate binding, which are mediated by a shift in the equilibrium between the oligomeric forms of enzyme.

Animals↗
PubMedDate not supplied

[Fluoroimmunometric method of determining digitoxin].

Digitoxin at concentrations up to 5 x 10(-10) M (therapeutic concentrations are 2 x 10(-8) M) can be reliable measured by a fluoroimmunometric method with coproporphyrin as a tracer. The use of nylon filters with immobilized digitoxin to remove an excess of labelled antibodies increases reliability and reduces the time of measurements, and thus simplifies the assay.

Chromatography, Ion Exchange↗
PubMedDate not supplied

[Obtaining immune sera to high-titre thyroxine for immunoassay].

A method of synthesis of L-thyroxine-protein conjugates was described. It included an additional step of acylation of thyroxine by succinic anhydride. The acylated derivative was activated by carbodiimide to produce a compound which could react with the protein amino-groups. Protein conjugates with a high thyroxine content (over 20 thyroxine residues per molecule) were synthesized with minimal protein cross-linking using this method. The excess of acylated thyroxine was easily dialized against alkaline buffer. The rabbits immunized monthly with such conjugates gave antisera with high titers (1:2000-1:4000 by the ELISA method) in 9-13 weeks after the first injection. The method can be applied to other thyroid hormones and some-molecular-weight antigens.

Acylation↗
PubMedDate not supplied

[Isolation and study of the properties of IgG from antisera to influenza virus proteins].

IgG to internal (NP, M) and external (HA, NA) proteins of influenza virus were isolated from immune rabbit sera using caprylic acid. The IgG retained their specificity and activity, worked in HI, lectin test, and enzyme-immunoassay. IgG migrated towards cathode in electrophoresis on acetate cellulose. Electrophoresis in polyacrylamide gel revealed a heavy chain (55,000) and a light chain (25,000) in IgG molecule. Different methods revealed changes in IgG structure after lyophilization. The possibility of recovering IgG from rat sera using caprylic acid was demonstrated. A comparative analysis of IgG recovered by means of caprylic acid as well as by polyethylene glycol or ammonium sulphate showed the IgG recovered with caprylic acid to contain less admixtures and to be suitable for use in immunological tests without additional purification.

Animals↗