[Phenothiazines are slowly oxidizable substrates of horseradish peroxidase].

Reactions of peroxidase oxidation of triftazine and thioproperazine have been investigated in the presence of horseradish peroxidase using steady state kinetic methods. It has been shown that phenothiazines are slowly oxidizable substrates for horseradish peroxidase. k(cat) and K(m) values have been determined in the range of pH from 4.5 to 7.5. The study of co-oxidation of phenothiazines and o-dianisidine (ODN) revealed that in the presence of aminazine and ODN in the reaction medium both substances follow sequential oxidation. ODN oxidation was not observed until full conversion of aminazine. At pH 4.5-5.5 thioproperazine bound to the enzyme-substrate complex and caused a nticompetitive inhibition of peroxidase. At pH>5.5 sequential substrate oxidation with preferential thioproperazine conversion occurred. In the range of pH from 4.5 to 7.5 triftazine did not influence ODN oxidation.

[Kinetic regulations of dihydroquercetin oxidation with horseradish peroxide].

The steady-state kinetics of horseradish peroxidase-catalyzed dihydroquercetin oxidation was studied. Dihydroquercetin was shown to be a slowly oxidized substrate of horseradish peroxidase. Two dihydroquercetin isoforms (cis and trans forms) that were selectively involved in peroxidase-induced oxidation were found in water-alcohol and buffer solutions. The k(cat) and K(m) were determined in the pH range of 4.5-8.0.

[The antioxidant system of wheat seeds during germination]. / Antioksidantnaia sistema v prorastanii semian pshenitsy.

The dynamics of peroxidase activity and antioxidant contents in wheat seeds were studied in the course of 24-hour swelling at 5 degrees C (group 1) and 23 degrees C (group 2). Both parameters were 1.5 times higher in seeds of the first group. In the same seeds, peroxidase activity in the endosperm and seed coat increased by factors of 1.5 and 1.8, respectively. Catalytic constants of wheat seed peroxidase were determined in the reactions of o-dianisidine and ascorbic acid peroxidation. In the pH range studied (pH 5-7), Km proved to change only slightly. In seedlings, an increase in the lipid peroxidation rate was accompanied by an increase in the content of antioxidants. Peroxidase activity increased as the content of antioxidants decreased, and vice versa. Thus, the reciprocal influence of peroxidase and low-molecular antioxidants during seed germination in wheat was revealed.

[Change in the reaction of the antioxidant system of wheat sprouts after UV-irradiation of seeds]. / Izmenenie reaktsii antioksidantnoi sistemy prorostkov pshenitsy posle UF-oblucheniia semian.

The response of the antioxidant system of sprouts of wheat Triticum aestivum L. to preliminary irradiation of seeds with UV light was studied. The dependence of lipid peroxidation and the extent of antioxidant activity on the duration of irradiation was studied. It was shown that low doses of UV radiation (5-15 min) stimulate the antioxidant protection of green wheat sprouts grown for eight days. Increasing the irradiation time to 30-60 min leads to the inhibition of lipid peroxidation by the antioxidant system. A more prolonged irradiation of seeds with UV light (for 1-6 h) led to an increase in the level of lipid peroxidation in sprouts. However, 1-2-day-old sprouts from seeds irradiated for 5-6 h, adapted themselves to the influence due to the compensatory mechanisms. By the 8th day of germination of preliminarily irradiated seeds, the content of antioxidants and malone dialdehyde returned to the norm. The dynamics of activity of peroxidase in seeds irradiated with low doses of UV light for 30 min was studied. It was found that on the third day of seed germination, a decrease in peroxidase activity followed by its slight increase occurred. The maximum activity of the enzyme in the endosperm was observed on day 5-6, and in roots and green sprouts, on day 3-5 of germination. It was concluded that antioxidants and peroxidase are involved in the compensatory mechanisms of inhibition of free radicals formed upon UV irradiation of seeds.

Effect of strophanthin G on peroxidase oxidation kinetics of slowly oxidizable peroxidase substrates.

Steady-state kinetics of thioproperazine, triftazine, aminazine, and o-dianisidine oxidation with hydrogen peroxide catalyzed by horseradish peroxidase were studied in the presence of strophanthin G. Values of the inhibition and activation constants (Ki, Ka) were determined in the pH range 5.0-7.5. At acidic pH, strophanthin G activated peroxidase during the oxidation of thioproperazine by the uncompetitive mechanism, and when triftazine was oxidized, the inhibition was noncompetitive. At pH > 6.0, the patterns of activation and inhibition changed to mixed-type during the peroxidase oxidation of thioproperazine and triftazine and to competitive inhibition of peroxidase with strophanthin G during the oxidation of aminazine. These effects are suggested to be due to an ionizable enzyme group of pK approximately 6.0. Strophanthin G inhibited free-radical oxidation of o-dianisidine via binding to the enzyme-substrate complex, preventing the generation of a stable semi-oxidized product of o-dianisidine, and thus inhibiting the enzyme by the anticompetitive mechanism. Mechanisms of oxidation of slowly and rapidly oxidizable substrates of peroxidase in the presence of strophanthin G are suggested.

[Inhibition of horseradish peroxidase by N-ethylamide of o-sulfobenzoylacetic acid]. / Ingibirovanie peroksidazy khrena N-étilamidom o-sul'fobenzoiluksusnoikisloty.

The carboxylic groups of horseradish peroxidase were modified by 1-cyclohexyl-3-(2-morpholinoethyl)carbodiimide metho-p-toluenesulfonate by the Koshland method. The catalytic properties of the native and modified peroxidase were studied in the presence of N-ethylamide of o-sulfobenzoylacetic acid (EASBA) at pH 5.0-7.5. In the oxidation of o-dianisidine, EASBA is a competitive inhibitor of the carbidiimide-modified peroxidase, and it increases both K(m) and Vm in the case of the native enzyme. These data show that at least one of the carboxylic groups modified with carbodiimide is located at the area of the peroxidase active site.

[Mechanism of combined oxidation of ascorbic acid and hydroquinone in the presence of horseradish peroxidase]. / Mekhanizm sovmestnogo okisleniia askorbinovoi kisloty i gidrokhinona v prisutstvii peroksidazy khrena.

A steady-state kinetics of peroxidase cooxidation of ascorbic acid and hydroquinone catalyzed by horseradish peroxidase was studied. Ascorbic acid and hydroquinone were shown to be oxidized successively, and hydroquinone promoted the oxidation of ascorbic acid. Excess ascorbic acid inhibited peroxidase in the cooxidation of the substrates at pH 5-7. The values of catalytic constants, (kcat, K(m), and Ka) were determined. A possible activation mechanism of the peroxidation of ascorbic acid in the presence of hydroquinone was suggested, and its biological significance was considered.

Effect of antioxidants (digoxin, quercetin, and ascorbic acid) on catalytic properties of horseradish peroxidase.

Antioxidants (digoxin, quercetin, and ascorbic acid) inhibited the oxidation of o-dianisidine catalyzed by horseradish peroxidase. Digoxin bound with the enzyme-substrate complex which included a stable semioxidized product of o-dianisidine and inhibited horseradish peroxidase by the anticompetitive pattern, while the enzyme inhibition by quercetin followed the mixed pattern. The oxidation of ascorbic acid and o-dianisidine with their combined presence in the reaction mixture was differentiated. o-Dianisidine was oxidized after the oxidation of 90-100% of ascorbic acid was completed. The rate of peroxidase oxidation of ascorbic acid in the presence of o-dianisidine was more than two orders higher than the rate of its individual oxidation and 1.5-2.0 times higher than the rate of o-dianisidine oxidation. Feasible inhibition mechanisms of peroxidase oxidation of o-dianisidine are discussed.

[Possible mechanisms of regulating glucose-6-phosphate dehydrogenase activity by an excess of substrate and coenzyme]. / Vozmozhnye mekhanizmy regulirovaniia aktivnosti gliukozo-6-fosfat- degidrogenazy izbytkom substrata i kofermenta.

The kinetics of glucose 6-phosphate oxidation by glucose 6-phosphate dehydrogenase from wheat seeds was studied at pH 6-11 within a broad interval of the glucose 6-phosphate and NADH concentrations. A high substrate concentration (over 4-6 mM) activated the enzyme within the pH range studied; the excess of NADH inhibited glucose 6-phosphate dehydrogenase at pH 6.0-9.5. The enzyme's active site groups with pK of 7.2, 8.0, and 9.0 were shown to be involved in the substrate binding in the enzyme-substrate complexes. The ionization of the groups with pK 8.0 and 9.5 appeared to affect the catalytic activity of the enzyme. The deprotonation of the group with pK 9.5 increased the reaction rate, whereas its protonation favored enzyme inhibition by high NADH concentrations.

[Effect of intersubunit interaction in horse liver alcohol dehydrogenase on the kinetics of ethanol oxidation]. / Vliianie mezhsub'edinichnogo vzaimodeistviia v alkogol'degidrogenaze iz pecheni loshadi na kinetiku okisleniia étanola.

The kinetics of enzymatic oxidation of ethanol in the presence of alcohol dehydrogenase within a wide range of ethanol and NAD concentrations (pH 6.0--11.5) were studied. It was shown that high concentrations of ethanol (greater than 0.7--5 mM, depending on pH) and NAD (greater than 0.4--0.8 mM) activate alcohol dehydrogenase from horse liver within the pH range of 6.0--7.9. A mechanism of activation based on negative cooperativity of ADH subunits for binding of ethanol and NAD was proposed. The catalytic and Michaelis constants for alcohol dehydrogenase were calculated from ethanol and NAD at all pH values studied. The changes resulting from the subunit cooperativity were revealed. The nature of ionogenic groups of alcohol dehydrogenase, which affect the formation of complexes between the enzyme and NAD and ethanol, and the rate constants for catalytic oxidation of ethanol was assumed. The biological significance of the enzyme capacity for activation by high concentrations of ethanol within the physiological range of pH in the blood under excessive use of alcohol is discussed.