[Nonphysiological redox-agents are reduced at the binding center of NADP(H) glutathione reductase]. / Nefiziologicheskie redoks-agenty vosstanavlivaiutsiia v tsentre sviazyvaniia NADP(H) glutationreduktazy.

Studies of the acceptor reductase reaction of yeast glutathione reductase (EC 1.6.4.2) revealed that the competitive inhibitors for NADPH, 2',5'-ADP and Br- decrease the rate constants for the enzyme oxidation by ferricyanide, phenanthrene quinone, and juglone. A similar effect is observed when NADH which does not bind to the reduced enzyme is used as substrate. These observations support the hypothesis that non-physiological redox agents are reduced at the NADP(H)-binding center of glutathione reductase and that NADP(H) binding stimulates the reaction by displacing tyrosine-197 which protects FAD from the solvent.

[An ellipsometric study of the antigen-antibody interaction in the interphase solid/solution area]. / Ellipsometricheskoe izuchenie vzaimodeistvii antigen-antitelo v mezhfaznoi oblasti tverdoe telo/rastvor.

Ellipsometric studies have proved that monoclonal immunoglobulin G(IgG) against gamma-interferon (gamma-INF) and immunoglobulin fraction (Ig-fraction) of rabbit blood serum against human serum albumin (HSA) are adsorbed according to the Langmuir model on the surfaces of mirror plates of covalently modified gamma-INF or HSA, respectively. The maximum surface concentrations (Tmax) and equilibrium adsorption constants (K) for IgG and Ig-fraction are equal to 2.57 pmol/cm2 and 2 x 10(7) M-1, 3.3 mg/m2 and 0.1 cm3/micrograms, respectively. The additional treatment of gamma-INF modified surfaces with Tween-20 leads to an increase of K IgG ut to 2.7 x 10(-7) M-1 while Tmax decreases up to 1.12 pmol/cm2 which is conditioned by the blocking of protein non-specific binding sites. The role of specific and non-specific interactions of IgG and Ig-fraction with covalently immobilized antigens was studied at antibody-antigen mixture adsorption. The necessity to apply this method to quantitative determination of gamma-IHF and HSA in solutions was proved.

[Stimulation of the NADPH:adrenodoxin reductase diaphorase reaction by adrenodoxin]. / Stimulirovanie diaforaznoi reaktsii NADPH:adrenodoksinreduktazy adrenodoksinom.

The diaphorase activity of NADPH: adrenodoxin reductase (EC 1.18.1.2) is stimulated by adrenodoxin. The latter prevents the reductase inhibition by NADPH; the Line-weaver-Burk plots are characterized by a biphasic dependence of the reaction rate on the oxidizer concentration. At pH 7.0 the maximal rate of the first phase is 20s-1; that for the second phase at saturating concentrations of adrenodoxin is 5 s-1. Since the second phase rate is equal to that of the adrenodoxin-linked cytochrome c reduction by reductase it is concluded that this phase reflects the reduction of the oxidizers via reduced adrenodoxin. Quinones are reduced by adrenodoxin in an one-electron way; the logarithms of their rate constants depend hyperbolically on their single-electron reduction potentials (E7(1]. The oxidizers interact with a negatively charged domain of adrenodoxin. The depth of the adrenodoxin active center calculated from the Fe(EDTA)- reduction data is 5.9 A.

[The use of surface electromagnetic waves of IR range for recording the protein sorption on the phase boundary and optic biosensors on this basis]. / Ispol'zovanie poverkhnostnykh élektromagnitnykh voln IK-diapazona dlia registratsii sorbtsii belkov na granitsu razdela faz i optobiosensory na étoi osnove.

Monomolecular protein films adsorbed on the copper and aluminium surfaces were studied by the IR surface electromagnetic waves laser spectroscopy. The thickness of the protein films was measured. A model of IR biosensors was proposed and its sensitivity limits were estimated.

[Adsorption of fibrinogen on silicon oxide with controlled hydrophobic/hydrophilic properties]. / Adsorbtsiia fibrinogena na okisi kremniia s kontroliruemymi gidrofobno/gidrofilnymi svoistvami.

The amount of fibrinogen irreversibly adsorbed on silicon dioxide does not exceed 3.6 pmol/cm2 and depends on the protein concentration, solution pH and surface hydrophobic/hydrophilic properties. Electrostatic interactions determine the fibrinogen adsorption rate. Partial denaturation of fibrinogen takes place in its adsorption form diluted solutions with the pH value lower than the protein isoelectric point.

[The relation of glutathione reductase and diaphorase activity of glutathione reductase from Saccharomyces cerevisiae]. / Sootnosheniia mezhdu glutationreduktaznoi i diaforaznoi aktivnost'iu glutationreductazy iz Saccharomyces cerevisiae.

Glutathione reductase from S. cerevisiae (EC 1.6.4.2) catalyzes the NADPH oxidation by glutathione in accordance with a "ping-pong" scheme. The catalytic constant kcat) is 240 s-1 (pH 7.0, 25 degrees C); kcat for the diaphorase reaction is 4-5 s-1. The enzyme activity does not change markedly at pH 5.5-8.0. At pH less than or equal to 7.0, NADP+ acts as a competitive inhibitor towards NADPH and as a noncompetitive inhibitor towards glutathione. NADP+ increases the diaphorase activity of the enzyme. The maximal activity is observed, when the NADP+/NADPH ratio exceeds 100. At pH 8.0, NADP+ acts as a mixed type inhibitor during the reduction of glutathione. High concentrations of NADP+ also inhibit the diaphorase activity due to the reoxidation of the reduced enzyme by NADP+ at pH 8.0. The redox potential of glutathione reductase calculated from the inhibition data is--306 mV (pH 8.0). Glutathione reductase reduces quinoidal compounds in an one-electron way. The hyperbolic dependence of the logarithm of the oxidation constant on the one electron reduction potential of quinone is observed. It is assumed that quinones oxidize the equilibtium fraction of the two-electron reduced enzyme containing reduced FAD.

[Inhibition of adrenodoxin reductase by NADP+ and NADPH]. / Ingibirovanie adrenodoksinreduktazy NADP+ i NADPH.

Adrenodoxin reductase (EC 1.18.1.2) catalyzes the oxidation of NADPH by 1.4-benzoquinone. The catalytic constant of this reaction at pH 7.0 is equal to 25-28 s-1. NADP+ acts as the mixed-type nonlinear inhibitor of enzyme increasing Km of NADPH and decreasing catalytic constant. NADP+ and NADPH act as mutually exclusive inhibitors relative to reduced adrenodoxin reductase. The patterns of 2',5'-ADP inhibition are analogous to that of NADP+. These data support the conclusion about the existence of second nicotinamide coenzyme binding centre in adrenodoxin reductase.

[Electrochemical regulation of the dehydrogenase process]. / Elektrokhimicheskoe upravlenie degidrogenaznym protsessom.

On changing stepwise the polypyrrole (PP) electrode potential a reverse change in the equilibrium concentration of a reduced coenzyme is observed in 0.1 M KNO3 solution containing nicotinamide adenine dinucleotide (NAD+), ethyl alcohol, alcohol dehydrogenase and electrode system based on PP-modified platinum. Direct measurements of the solution pH and calculations verify the conclusion mode that the electrochemical control of dehydrogenase process proceeds via the pH change of the solution.

[Giant Raman scattering of alkaline phosphatase, horseradish peroxidase and lactoperoxidase on silver electrodes]. / Gigantskoe kombinatsionnoe rasseianie shchelochnoi fosfatazy, peroksidazy iz khrena i laktoperoksidazy na serebrianykh elektrodakh.

Surface enhanced Raman scattering of three enzymes--alkaline phosphatase, horseradish peroxidase and lactoperoxidase is studied. The intensity of normal vibrations of definite amino acids is determined by their orientation on the surface and depends on the electrode potential. Alkaline phosphatase and lactoperoxidase make a complex with silver ions.

[Stabilization of native and immobilized urease]. / Stabilizatsiia nativnoi i immobilizovannoi ureazy.

The stability of native and immobilized urease isolated from Staphylococcus saprophyticus was studied at 4 degrees and 25 degrees C. The activity yield was 20% and 1.4% on the enzyme immobilization in albumin gel and latex membrane, respectively. Inactivation of native microbial urease proceeded 10 times slower in the solution containing 1 mM EDTA and 30 mM sodium sulfite. This solution contributed to a great extent to stabilization of immobilized urease both during storage in the phosphate buffer solution and in case of lyophilization.

[Kinetics of adrenodoxin reductase oxidation by non-physiologic electron acceptors]. / Kinetika okisleniia adrenodoksinreduktazy nefiziologicheskimi aktseptorami élektronov.

The reactions of NADPH oxidation by quinones and inorganic complexes catalyzed by NADPH: adrenodoxin reductase were studied. The catalytic constant for the enzyme at pH 7.0 is 20-25 s-1; the oxidative constants for the quinones vary from 5 X 10(5) to 1.1 X 10(3) M-1 s-1 and show an increase with a rise in the one-electron acceptor reduction potential. The mode of adrenodoxin reductase interaction with oxyquinones differs from that of the enzyme interaction with alkyl-substituted quinones and inorganic complexes. NADPH competitively inhibits electron acceptors, whereas NADP+ is a competitive inhibitor of NADPH and a uncompetitive inhibitor of electron acceptors. (Ki = 25 microM). The depth of FAD incorporation into the enzyme molecule as calculated according to the outer sphere electron transfer theory is 6.1 A.

[Characteristics of the interaction of adrenal lipoamide dehydrogenase with physiological and quinone electron acceptors]. / Zakonomernosti vzaimodeistviia lipoamiddegidrogenazy nadpochechnikov s fiziologicheskimi i khinonovymi aktseptorami élektronov.

Lipoamide dehydrogenase (EC 1.6.4.3) from the ketoglutarate dehydrogenase complex of adrenals catalyzes the oxidation of NADH by lipoamide and quinone compounds according to the "ping-pong" scheme. The catalytic constants of these reactions are equal to 220 and 24 s-1, respectively (pH 7.0). The maximal quinone reductase activity is observed at pH 5.6, whereas the lipoamide reductase activity changes insignificantly at pH 7.5-5.5. The maximal dihydrolipoamide-NAD+ reductase activity is observed at pH 7.8. The oxidative constants of quinone electron acceptors vary from 6 X 10(6) to 4 X 10(2) M-1 s-1 and increase with their redox potential. The patterns of NAD+ inhibition in the quinone reductase reaction differ from that of lipoamide reductase reaction. The quinones are reduced by lipoamide dehydrogenase in the one-electron mechanism.

[Diaphorase reactions of lipoamide dehydrogenases from the adrenal ketoglutarate dehydrogenase complex]. / Diaforaznye reaktsii lipoamiddegidrogenazy iz oksoglutaratdegidrogenaznogo kompleksa nadpochechnikov.

Lipoamide dehydrogenase, a component of the bovine adrenal ketoglutarate dehydrogenase complex, catalyzes the oxidation of NADH by p-quinones and ferricyanide. The kinetics of oxidation obey the ping-pong mechanism. At pH 7.0, the constants for the active center oxidation by quinones (kox) are equal to 1.1 X 10(4)-5.3 X 10(5) M-1s-1 and increase as the acceptor potential rises. The values of kox for quinones change insignificantly within the pH range of 7.7-5.0, whereas that for ferricyanide increases 10-fold with a decrease of pH from 7.0 to 5.0. The value of the catalytic constant for the enzyme (kcat) reaches its maximum at pH 5.5. The quinones interact with the thiol groups of lipoamide dehydrogenase by inhibiting the fluorescence of FAD and diaphorease activity. The reaction is catalyzed by a basic amino acid (pK 6.7) within the composition of the enzyme.

[Effect of the redox state of glutathione on acetate kinase activity in E. coli]. / Vliianie okislitel'no-vosstanovitel'nogo sostoianiia glutationa na aktivnost' atsetatkinazy E. coli.

Oxidized glutathione inhibits acetate kinase (EC 2.7.2.1) of E. coli. The rate of inactivation depends on ATP concentration. The rate constant for the glutathione-induced inhibition is 0.17 min-1, Ki is 4.2 mM (pH 7.2, 25 degrees C). The inhibition of acetate kinase by glutathione is reversible, the equilibrium constant being equal to 4.4 or 0.09 at saturating concentrations of ATP (pH 8.0, 25 degrees C). The physiological level of reduced and oxidized glutathione can modulate the acetate kinase activity in vivo.

[Determination of kinase activity by using an enzyme electrode]. / Opredelenie aktivnosti kinaz pri pomoshchi fermentnogo élektroda.

A method for determining the enzymatic activity of hexokinases, acetate kinase and pyruvate kinase using an enzyme electrode was developed. The assay time is 2-3 min. The lower limit of the activity determining is 0,054 U/ml. The proposed method was applied to investigation of pyruvate kinase and acetate kinase reactivation under the action of mercaptoethanol.

[Determination of the biochemical oxygen demand in nutrient media using microbial flow-through electrodes]. / Opredelenie biokhimicheskoi potrebnosti v kislorode v nekotorykh pitatel'nykh sredakh s pomoshch'iu protochnykh mikrobnykh élektrodov.

Biochemical oxygen demand (BOD) in some culture media was determined by means of flow-through microbial electrodes, based on Hansenula anomala, Escherichia coli K-12, and activated sludge obtained from the All-Union Scientific-Research Institute of Applied Enzymology (Vilnius). The sensitivity of the electrodes was 112.1, 65.5 and 32.8% O2, respectively, in the presence of 1 mM glucose as substrate. A maximum sensitivity of the electrodes, based on E. coli and activated sludge, was observed on pyruvate oxidation--120 and 82.3% O2 in the presence of 1 mM pyruvate. The yeast electrode exhibited a minimum sensitivity for sucrose--9.3% O2/mM. The time of the electrode response varies from 8 to 35 min, depending on the substrate used. BOD depends on pH of the solution. The microbial electrodes retained the initial sensitivity for 33--38 days. The were used for estimation of BOD in the Rider's and Shopfer's media.

[Inhibition of cytochrome b2 by acrylamide]. / Ingibirovanie tsitokhroma b2 akrilamidom.

Acrylamide (0.4--0.9 M) irreversibly inhibits reduced (Ered) cytochrome b2 (L (+) -lactate: cytochrome c oxidoreductase) from the yeast Hansenula anomala (ki = 1,67 min-1 at 35 degrees in 0.73 M solution of acrylamide). Changes in fluorescence of FMN, which reflect the changes in protein structure occur symbatically to the inactivation. The rate of inactivation depends on concentration of acrylamide in a degree of 6.4. The inactivation of the oxidized enzyme occurs faster than that of th reduced one at concentrations less than 0.5 M. The inactivation of Ered by acrylonitrile and acrylic acid occurs 10 times slower and does not correlate with the rate of mercaptoethanol binding to the monomers. The inhibition of Ered is caused by specific effects of carylamide and modification of the enzyme active center.

[Enzymatic oxidation of glucose on modified electrodes]. / Fermentativnoe okislenie gliukozy na modifitsirovannykh élektrodakh.

Glucose oxidase (EC 1.1.3.4) entrapped at the paraelectrode layer of a glass-carbon electrode coated with tetracyano-p-quinodimethane, tetracyano-p-quinodimethane anion potassium salt, piocyanine. 9.10-phenantrenequinone, dichlorophenolindophenol or dextran-dopamine catalyzes electrochemical oxidation of glucose. The value of maximal current amounts to 67 micro A . cm-2 and depends on the enzyme concentration and the nature of the modifier. The compounds used are substrates for glucose oxidase. The electrocatalytical oxidation of glucose occurs in a mediator fashion.

[Determination of penicillins by means of an enzymatic electrode]. / Opredelenie penitsillinov fermentnym élektrodom.

Penicillin sensitive enzyme electrodes were prepared on the basis of native penicillinase (EC 3.5.2.6) and penicillinamidase (CE 3.5.1.11), as well as penicillinase entrapped in a complex of polyethylenimine and polyacrylic acid or linked with albumin and lattice entrapped penicillinase. The time of the electrode response is 2-10 minutes. The electrode potential change within a minute is linear at 1-20 mM of benzylpenicillin and depends on the electrode type. The electrodes prepared on the basis of the native enzymes lost their sensitivity within the first 10 days. Sensitivity of immobilized penicillinase did not change for 15 days. The electrodes prepared on the basis of albumin-linked penicillinase preserved their high stability for 60 days. The sensitivity of these electrodes is slightly dependent on the phosphate buffer concentration (from 0.001 to 9 mM).