[Hepatotoxic efects of acetaminophen. Protective properties of tryptophan-derivatives].

Rat intoxication with acetaminophen (APAP) (500-1500 mg/kg body weight intragastrically) caused a considerable dose-dependent decrease in reduced glutathione (GSH) level in both liver cellular cytoplasm and mitochondria (at the dose 1500 mg/kg body weight by 60% and 33%, respectively). The cytoplasmic GSH level decreased more pronounced by comparison with that in mitochondria. At the same time, we did not observe any inactivation of the mitochondrial enzymes: succinate dehydrogenase, alpha-ketoglutarate dehydrogenase, glutathione peroxidase despite of mitochondrial GSH consumption; also we did not observe any decrease in the respiratory activity of liver mitochondria isolated from APAP-intoxicated rats. A tryptophan derivative, melatonin (10 mg/kg body weight), did not prevent intramitochondrial GSH oxidation, but decreased the hepatoxity of APAP, diminishing the activities of AlT and AsT as well as bilirubin level in blood plasma of intoxicated rats. N-acetyl-nitrosotryptophan (a nitric oxide donor) did not exhibit any hepatoprotective effects.

[Hypoclorous acid modifies rat liver and heart enzymes of the pentose phosphate pathway and antioxidative defence in vitro].

Hypoclorous acid is an effective biological oxidant produced by activated neutrophils. HOCl plays a role of the major inflammation mediator in mammalian tissues. The aim of the present study was to investigate the mechanisms of hypochlorous acid-induced modification of antioxidant enzymes, which defence the cell under oxidative stress, and enzymes of the pentose phosphate pathway, which supply reducing equivalents in the cell. HOCl (100-1000 microM) in vitro inhibited considerably in a dose-dependent manner the activity of the enzymes of the pentose phosphate pathway in the rat liver postmitochondrial fraction. HOCI at a concentration of 100 nmol/mg protein inhibited transketolase activity by 65 +/- 5%, glucose-6-phosphate dehydrogenase--by 50 +/- 5% and 6-phosphogluconate dehydrogenase--by 55 +/- 5%. The activities of glutathione peroxidase and catalase slightly decreased. On the contrary, in the rat heart postmitochondrial fraction HOCl (100-1000 microM) inhibited considerably catalase, increased glutathione peroxidase activity and decreased significantly the activity of the key enzymes of the pentose phosphate pathway. The inhibition of the pentose phosphate pathway enzymes was accompanied by oxidation of intracellular reduced glutathione, oxidative protein modification (protein carbonyl group accumulation, mixed protein-glutathione disulphides and chloramine formation), and membrane lipid peroxidation. The sensitivity of rat heart cell components to oxidative damage by HOCl was higher in comparison with that of the liver.

[Effect of hydroxylated pyrimidine derivatives on activities of thiamine-dependent enzymes and some parameters of lipid metabolism in mice]. / Vliianie gidroksilirovannykh proizvodnykh pirimidina na aktivnost' tiaminzavisimykh fermentov i nekotorye pokazateli obmena lipidov u myshei.

It has been found that hydroxylated pyrimidine derivatives actively participate in metabolic proceeds related to functioning of vitamin B1-dependent enzymes (transketolase, 2-oxo acid dehydrogenase). Hydroxypyrimidines also induce a significant increase in the levels of total lipids and cholesterol in the mice liver, not changing the phospholipid content.

[Spectroscopic study of the structure and intramolecular mobility of yeast pyruvate decarboxylase]. / Spektroskopicheskie issledovaniia struktury i vnutrimolekuliarnoi podvizhnosti drozhzhevoi piruvatdekarboksilazy.

Steady-state and time-resolved fluorimetry were used to study the properties of holo- and apopyruvate decarboxylase (EC 4.1.1.1, PDC) from Brewer's yeast after interaction with substrate (pyruvate), cofactor (thiamine diphosphate, ThDP) and Mg2+ ions. The analysis of the enzyme's intrinsic fluorescence as well as of its complex with the probe 2-(p-toluidinylnaphthalene)-6-sulphonate (TNS) revealed that ThDP was found at the polar region of the PDC active sites, inducing a decrease in the mobility of the protein's nearest surroundings. The fluorescent probe had three different sites of binding to the protein apoform, two of which being located at the catalytic site and having different rotation freedom. The study of the PDC complex with thiochrome pyrophosphate, a ThDP structural analogue, pointed to the occurrence of a non-polar region of the enzyme active site for pyruvate absorption besides the polar region. The binding of pyruvate to the protein does not depend upon the cofactor's binding. On the basis of the fluorescent studies a model of the ThDP and pyruvate arrangement at the PDC active site is suggested.

[Coenzyme-binding sites of the brain pyruvate dehydrogenase complex]. / Izuchenie kofermentsviazyvaiushchikh tsentrov piruvatdegidrogenaznogo kompleksa mozga.

It is shown that the relative amount of the holoenzyme in the highly purified pyruvate dehydrogenase complex from the bovine brain is higher when the enzyme activity is assayed in the reaction of nonoxidative formation of acetaldehyde as compared to the pyruvate: NAD+ reductase reaction. The S0.5 values for thiamine pyrophosphate are as following: (TPP) (0.314 +/- 0.22) x 10(-7) M with reaction of nonoxidative formation of acetaldehyde, (0.188 +/- 0.08) x 10(-6) M and (1.65 +/- 1.16) x 10(-6) M in case of the pyruvate: NAD+ reductase reaction. TPP in the concentration of (0.5-6.0) x 10(-7) M completely protects the sites of nonoxidative formation of acetaldehyde from modification by the coenzyme analogs, 4'-oxythiamine pyrophosphate and tetrahydrothiamine pyrophosphate. However, the pyruvate: NAD+ reductase activity of the pyruvate dehydrogenase complex is inhibited in this case by 30-34%. The data obtained suggest that in contrast to the pyruvate: NAD+ reductase reaction the conversion of pyruvate to acetaldehyde occurs by the sites which tightly bound TPP.

[Antivitamin activity of oxythiamine disulfide nicotinate]. / Antivitaminnaia aktivnost' oksitiamindisul'fidnikotinata.

The B1-antivitamin activity of oxythiamine disulphide nicotinate has been determined in experiments on albino mice and it is shown that in the liver this derivative exerts the equal action while in the blood and heart--a more profound and prolonged inhibitory action on the transketolase activity in comparison with oxythiamine disulphide. Like the initial compound oxythiamine disulphide nicotinate does not penetrate through hemato-encephalic barrier and does not inhibit the brain transketolase.

[Features of thiamine diphosphate-dependent enzyme inhibition by oxodihydrothiochrome and its phosphorylated derivatives]. / Osobennosti ingibirovaniia tiamindifosfatzavisimykh fermentov oksodigidrotiokhromom i ego fosforilirovannymi proizvodnymi.

Anticoenzyme action of new derivatives of thiamine: oxodihydrothiochrome and its mono- and diphosphoric esters has been studied in the experiments on mice. It is shown that the given compounds exert an inhibiting action on transketolase and pyruvate dehydrogenase and do not change activity of 2-oxoglutarate dehydrogenase in the animal organism. Antivitamin effect of the studied inhibitors is observed with the lower doses and in the earlier terms as compared with the other known inhibitors of thiamine-diphosphate-dependent enzymes. The preparations inhibit activity of the yeast pyruvate-decarboxylase by the mixed (with respect to thiamine-diphosphate) type (Ki for oxodihydrothiochrome and its mono- and diphosphoric esters: 2.3 x 10(-3), 7.2 x 10(-4), 5.6 x 10(-5) M, respectively). Possible mechanisms of the action of the mentioned compounds as thiamine antimetabolites are discussed.

[Interaction of pyruvate dehydrogenase complex from the heart muscle with thiamine diphosphate and its derivatives]. / Vzaimodeistvie piruvatdegidrogenaznogo kompleksa iz serdechnoi myshtsy s tiamindifosfatom i ego proizvodnymi.

Inhibitory effects of 23 thiamin derivatives on the bovine heart pyruvate dehydrogenase complex (PDC) were studied. Oxythiamin diphosphate and tetrahydroxythiamin diphosphate exhibited the most pronounced effect on the PDC activity, affecting the complex by a competitive type of inhibition for thiamin diphosphate (TDP). The apparent affinity of TDP and the anticoenzyme derivatives for apo PDC depended on presence of phosphate and divalent metal ions. Phosphate considerably increased the Km values for TDP (up to 0.17 microM) and the Ki values for oxythiamin diphosphate (0.40 microM) as well as for tetrahydroxythiamin diphosphate (0.23 microM). In presence of Mn2+, Km value for TDP was 3.5-fold lower as compared with Mg2+ containing medium.

[Metabolism of transketolase coenzyme in the rat liver]. / Obmen kofermenta v sostave transketolazy pecheni krysy.

Kinetic analysis permitted to determine two sites of hydroxythiamine diphosphate binding in apotransketolase. The Ki values for these sites differed significantly: (7-22) X 10(-9) M and (13.0-19.7) X 10(-8) M. The rate of thiamine diphosphate turnover within holotransketolase in rat liver tissue was studied by the radioisotope method, using [14C]thiamine as a labeled precursor. The absolute values of half-substitution time and the rate constant of coenzyme degradation in the transketolase molecule are close to those for the protein moiety of the enzyme and are 153 hours and 0.108 days-1, respectively. In vivo rat liver transketolase exists in a substituted alpha-carbanion form. Within the holoenzyme molecule substitution of thiamine diphosphate for hydroxythiamine diphosphate does not influence the formation of an intermediate alpha-carbanion form of the enzyme.

[Selective inhibition of pyruvate dehydrogenase in the liver and heart of mice by triphosphate esters of thiochrome and tetrahydrothiamine]. / Izbiratel'noe ingibirovanie piruvatdegidrogenazy pecheni i serdtsa myshei trifosfornymi éfirami tiokhroma i tetragidrotiamina.

In experiments with white mice it was shown that in contrast to hydroxythiamine and other known vitamin B1 antagonists, triphosphate esters of thiochrome and tetrahydrothiamine possess a selective anticoenzyme activity with respect to the only one of the thiamine pyrophosphate-dependent enzymes, i.e. pyruvate dehydrogenase.

[Interaction of thiamine pyrophosphate and some of its analogs with the pyruvate dehydrogenase complex from the adrenal cortex]. / Vsaimodeistvie tiaminpirofosfata i nekotorykh ego analogov s piruvatdegidrogenaznym kompleksom kory nadpochechnikov.

Highly purified preparations of the pyruvate dehydrogenase complex from bovine adrenals partially contain strongly bound thiamine pyrophosphate (TPP) which provides to 35% of the maximal activity measured under saturation with the exogenous TPP. The dependence of the complex-catalyzed reaction rate on the TPP concentration is described by Michaelis-Menten equation. The apparent value of Km for TPP without Mg2+ is 2.3 mumol. Magnesium ions reduce Km to 1.1 mumol. The constant of the TPP with the enzyme association rate calculated by the lag-period without Mg2+ is 3043 mol-1 s-1 in the presence of Mg2+ it is 9090 mol-1 . s-1. Phosphorus ethers of oxy- and tetrahydrothiamine produce a competitive type inhibition on the pyruvate dehydrogenase complex with respect to TPP. Oxythiamine pyrophosphate (Ki--0,07 microM) and tetrahydrothiamine pyrophosphate (Ki--0,1 microM) possess the highest inhibitory action.

[Specific and metabolic effect of oxythiamine]. / Spetsificheskie i metabolitnye éffekty oksitiamina.

Increasing doses of oxythiamine were studied as exerting the effect on transketolase inactivation in rat tissues. A conclusion is made that in the process of synthesis de novo there is a transient form of the enzyme accessible for interaction with oxythiamine pyrophosphate. Injection of oxythiamine in the increasing doses are accompanied by a decrease in the glycogen amount, increase in the intracellular level most of the studied intermediates of glycolysis and pentose cycle as well as cAMP. The probable biochemical mechanism of the oxythiamine action is connected with the activation of processes dependent on cAMP.