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The work deals with isolation of transketolase from the rat liver by means of ion-exchange chromatography and substrate elution of enzyme. Experimental data on the regulation of transketolase activity with thiamin pyrophosphate (TPP) and its anticoenzyme analogues are presented. The kinetics of dissociation of holo-TK at pH 4.0 and 5.0 and reactivation of apo-TK at a wide variation of the concentration of TPP and its derivatives with anticoenzyme properties has been studied. The dissociation of holo-TK into apoenzymes and coenzymes at the specified values of pH is characterised by most evident diphasic nature, both fast and slow process being observed. The most part of enzymic activity slowdown falls on the fast phase, while the remaining 20-30% take place within the slow phase. The kinetics research findings illustrate the nonidentity of enzyme active sites with respect to TPP binding with transketolase. The K(m) values for TPP both per the first and second active sites equalled 0.3-4.5 microM and 1.3-19.7 microM, accordingly.

Transketolase was isolated from rat liver tissue by ion exchange chromatography on Phosphocellulose. The enzyme was eluted by fructose-6-phosphate. The final product had a specific activity of 2.68 units/mg. The enzyme yield is 65% of its total content in the original extract. The possibility of regulation of transketolase activity by keto- and aldo-substrates has been studied; their interrelations are determined by the competition for relations with free and substituted forms of the enzyme.

Molecular forms of hepatic pyruvate kinase (PK) were separated by fractionating on DEAE-cellulose. 120-h food deprivation of rats entails a progressive decline in L-PK activity, but not the activity of M-type enzyme of the minor fraction. The rate of L-PK degradation depends on the fasting duration. A rapid inactivation phase is followed by a slower one with the speed constants 0.023 and 0.0065 h-1, respectively. To control the L-PK degradation rates in fasting diets, protein modification by phosphorylation can be employed.

Mechanisms of interaction between thiaminase I and cosubstrate were studied using thiazole-2-14C-thiamine and 14C-nicotinic acid. The dissociation constant of nicotinic acid in the presence of the enzyme was of an order of 1 x 10(-6) M. A mechanism of the prestationary phase of a thiaminase reaction at -10 degrees was studied. The low temperature step, which demonstrated specific characteristics, was involved in analytic estimation of total thiamine concentration in vitro. Native thiaminase I from the bivalve mollusca Anodonta cygnea proved to be a holoenzyme.

Properties of sorbitol dehydrogenase isolated from rat kidney were studied. Sorbitol dehydrogenase reactions followed the consecutive irregular order as shown in studies of kinetic patterns, inhibition by end products and thermoinactivation of the enzyme in the presence of substrates.

Radiometric assays revealed that thiamine deficiency in rats to whom hydroxythiamine was administered in variable doses, is concomitant with activation of gluconeogenesis from pyruvate in liver tissue. The most probable mechanism of this effect is the cAMP-dependent activation of key enzymes of intracellular glucose synthesis. This process is facilitated by the diminution of the ratio of free forms of NAD+ and NADPH in the cytoplasm.

Thiamine-deficiency in animals induced by everyday subcutaneous administration of oxythiamine in a dose of 4, 40 and 100 mg/kg of weight for 10 days results in a decrease of the total activity of pyruvate kinase in the liver tissue and does not affect the mentioned index in the kidney and heart tissues. It is shown that as a result of the enzyme fractionation in the column with DEAE-cellulose the total activity of pyruvate kinase in the liver tissue of rats with thiamine-deficiency decreases due to L-isoform while the content of M-isoform remains unchanged. Thiamine deficiency does not affect kinetic characteristics of the L-isoform, extracted from the liver and this shows the absence of changes in the degree of phosphorylation of pyruvate kinase L-isoform under these conditions.

Content of lactate in blood, liver and kidney tissues, skeletal muscle and in tumor tissue as well as some properties of lactate dehydrogenase (LDH), isolated from liver tissue, were studied in three groups of rats - intact rats, the tumor-bearing animals with sarcoma S-45 and the tumor-bearing rats treated with hydroxythiamin within 22 days. In skeletal muscle on the side of the tumor localization content of lactate was decreased as compared with the opposite side of the body. As shown by analysis of correlation between the content of lactate and the tumor weight and the lactate concentration in the tumor-bearing rat tissues studied, the tumor appears to be responsible for consumption but not for production of lactate. Hydroxythiamin altered distinctly the ratio of lactate content in various tissues and normalized the liver tissue LDH isoenzyme spectrum in tumor-bearing rats; the vitamin decreased 9- and 15-fold the enzyme specific activity in oxidation of lactate in presence of NAD+ and NADP, respectively. After the hydroxythiamin treatment the apparent KM value of the enzyme, isolated from the tumor-bearing rat liver tissue, was increased with pyruvate and decreased with lactate as substrate.

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.

It was found that the progressive development of vitamin B1 deficiency in rats caused by varying doses of oxythiamine results in a sharp decrease of the free NAD/NADH ratio in the liver after injection of a high dose of the antivitamin. The value of this ratio was calculated from the intracellular concentrations of pyruvate, lactate and Keq for lactate dehydrogenase, whose activity remained practically unchanged throughout the experiments. The increase of the cAMP level in the liver caused by corresponding doses of oxythiamine was concomitant with a marked activation of cAMP-dependent processes.

Phosphopentose stimulation of nucleic acids biosynthesis for 3h after subcutaneous phosphopentose administration in doses of 18 and 27 mg per rat has been stated. Injections of phosphopentoses (ribose-5-phosphate, xylulose-6-phosphate, ribulose-5-phosphate in the ratio of 1.0:0.3:0.3) were followed by a two-fold increase in the rate of [2-14C] orothic acid incorporation into cytoplasmic RNA of the rat liver. It is supposed that rapidly exchanging types of RNA contribute most of all to the effect of the label incorporation increase and the stimulation mechanism is associated with a rise of phosphoribosyl pyrophosphate accessibility as a substrate and an allosteric regulator of key enzymes of the synthesis of purine and pyrimidine nucleotides.

B1-hypovitaminosis was simulated in white rats of 150-170 g body mass by means of hydroxythiamin administration at a daily dose of 4, 40 or 100 mg/kg within 10 days. Concentrations of NADP and NADPH, main cofactors of 6-phosphogluconate dehydrogenase, were dissimilarly altered in the animal liver tissue depending on the antimetabolite dose administered. Kinetic characteristics of 6-phosphogluconate dehydrogenase isozymes were shifted towards more active binding of substrates in the hypovitaminosis. Total pool of metabolites, realized via 6-phosphogluconate dehydrogenase catalytic reactions and regulated by concentration of substrates, was especially sensitive to alterations in the NADP/NADPH ratio. In severe B1-hypovitaminosis caused by hydroxythiamin the rate of oxidative reactions was increased 3-10-fold.

An analysis of steady-state kinetics of purified rat liver transketolase shows that the reaction proceeds according to a two-stroke substitution ("ping-pong") mechanism. Based on the kinetic data, a competitive relationship was shown to exist between xylulose-5-phosphate and ribose-5-phosphate for the sites of substrate binding by the substituted form of the enzyme with the formation of a non-productive abortive complex (kd = 125 microM). The values of constants of two monomolecular steps of the reaction (k2 = 42 s-1; k4 = 9.4 s-1) were determined. It was assumed that the maximum rate-limiting step of the transketolase reaction is the degradation of the substituted form of transketolase--ribose-5-phosphate complex having a rate constant of k4.

Using stepwise protein fractionation by (NH4)2SO4 and ion-exchange chromatography on CM-cellulose, DEAE-cellulose and SP-Sephadex, two isoforms of 6-phosphogluconate dehydrogenase, A and B, from rat liver were obtained. The method developed allows to achieve complete separation of these forms and to obtain preparative amounts of the protein with specific activities of 5.7 and 10.7, respectively. The native enzyme forms A and B have molecular weights of 107000 and are represented by dimers composed of subunits with identical molecular weights equal to 54000. Both isoforms have a pH optimum at 8.5 and reveal different sensitivity to MgCl2 and MnCl2. The tetrahedron-shaped ions (phosphate, molibdate, arsenate) inhibit the both molecular forms of the enzyme, The Arrhenius plots for the reaction rate are uninterrupted lines within the temperature range of 21-44 degrees; the values of activation energy and the temperature coefficient for isoforms A and B are 12750 and 13500 cal/mole and 2.05 and 2.15, respectively.

Modification of the method for transketolase purification in the rat liver was used to reveal the existence of two molecular forms of this enzyme. One of the forms does not react to development of avitaminosis in animals caused by oxythiamine in different doses. The method is developed for isolation of the basic transketolase form in the rat liver with the 50-80% yield of activity. Km values of two sites for coenzyme binding on protein do not depend on the extent of holoenzyme reconstruction from apoenzyme.

Kinetic patterns and thermostability of glucose-6-phosphate dehydrogenase, isolated from liver tissue of intact and thiamine-deficient rats, were analyzed. Distinct alterations were not found in the patterns studied. The data obtained suggest that a decrease in the enzymatic activity, observed after administration of oxythiamine into rats, occurred due to lowering in content of the protein active form.

Lactate dehydrogenase M4-isoform in the homogeneous state was isolated from the rat liver by successive application of sulphate-ammonium fractionation, phosphocellulose ion-exchange chromatography with high-affinity elution of 1 mM NADH and subsequent hydroxyl apatite fractionation. The method permits obtaining the preparation amounts of the enzymic protein with yield 37.5%, specific activity 386.8 units per 1 mg of protein. It is established that 1 mM NAD+, 10 mM pyruvate and 100 mM lactate are also effective as agents of the selective enzyme elution.

Transketolase was isolated from wet tissue of rat liver and purified by ammonium sulfate and CM-cellulose fractionation and by adsorption chromatography on hydroxylapatite. The native enzyme is made up of two subunits with molecular weight of 70 000, is electrophoretically homogeneous and has a specific activity of 2.8 u. per mg of protein (30 degrees). The enzymatic and fluorimetric assays revealed the presence of two moles of thiamine diphosphate per mole of protein. The Arrhenius plots for the rate of the transketolase reaction with a pentose phosphate mixture as substrate are continuous at 10-32 degrees; the activation energy is 89.9 cJ/mole, temperature index is 3.3. The curve for the reaction rate versus substrate concentration is S-shaped; the apparent Km value for xylulose 5-phosphate is 2.2 x 10(-5) M. The ions with a tetraedric structure (arsenate, phosphate, sulfate) act as competitive inhibitors of the transketolase-catalyzed reaction.

Tiaminase I (EC 2.5.1.2) catalyzed degradation of thiamin in the reaction mixture free of substrate-acceptor. Thiamin exhibited properties of both substrate-donor and -acceptor of the group transferred by thiaminase I. Identification of the products developed after thiaminase reaction involving thiamin was performed using chromatography and radiometry. Kinetics of the reaction is discussed.

Vitamin B1 deficiency in mongrel albino rats induced by oxythiamine is concomitant with an increase in the ratio of free forms of NAD+/NADH, NADP+/NADPH in liver mitochondria. This is accompanied with a rise in steady-state concentrations of isocitrate, 2-oxoglutarate and with a decrease of malate in liver tissue, which testifies to the prominent regulatory role of the 2-oxoglutarate dehydrogenase complex in the intracellular metabolism under vitamin B1 deficiency. Moderate physical load causes a 10-fold increase in the steady-state concentration of pyruvate and lactate in the blood of thiamine-deficient animals, which seems to be due to the stimulation of glycolysis in the maintenance of energy homeostasis. The observed increase in the excretion of pyruvate, lactate, 2-oxoglutarate (30-fold against control) and pentose phosphates (3-fold) with urine, depending on the degree of vitamin B1 deficiency, points to one of essential mechanisms of cell metabolism stabilization under the given pathological condition.

The rate of the production of thiazole, the first product of the reaction, catalyzed by thiaminase I (EC 2.5.1.2) from Anodonta cygnea muscles is characterized by an initial flash-up explained by a considerable difference in the rates of the enzyme-substrate complex formation and its further transformation. Basing on this phenomenon, a new method for measuring the total thiamine in vitro and in biological material has been developed. Optimal conditions for thiamine measurements in the blood and urine have been selected.

Inhibition of rat liver transketolase under conditions of hydroxythiamin or alimentary B1 avitaminosis was accompanied by an increase in intracellular concentration of 6-phosphogluconate and xylulose-5-phosphate. The alterations in their content was inversely correlated with the transketolase activity. The dehydrogenases involved in the pentose phosphate shunt exhibited various unspecific sensitivity to hydroxythiamin. The doses of the antimetabolites up to 0.4 mg per rat did not alter the activity of the pentose phosphate shunt dehydrogenases; an increase in the dose from 4 up to 20 mg led to an inhibition of the enzymes. Possible biochemical alteration, occurring in response to various degree of transketolase inhibitions, are discussed.

Thiamine deficiency in rats induced by oxythiamine is accompanied by an increase in the free NADP+/NADPH ratio in liver tissue, which results in multifold stimulation of the metabolite flux in the oxidation branch of the pentose cycle. The increase in the intracellular concentrations of isocitrate and alpha-ketoglutarate with a simultaneous decrease of malate in the liver of vitamin-deficient rats points to the inhibition of alpha-ketoglutarate dehydrogenase responsible for the anomalous metabolism under conditions of thiamine deficiency. The decrease of the functional activity of the tricarboxylic acid cycle is concomitant with the activation of conversions in the oxidation branch of the pentose cycle, glucuronate and glycolytic pathways of carbohydrate metabolism, which is directed at eliminating the energy deficiency in rats with B1-hypovitaminosis.

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.

Hydoxythiamine injections (400 mg/kg for 72 hours) resulted in the inhibition of transketolase activity and of 14C-orotic acid incorporation into RNA in rat liver tissue. At the same time, the decrease of RNA and phosphoribosylpyrophosphate levels has been observed, which suggests a regulation effect (via pentospohosphates) of hydroxythiamine on nucleotide and nucleic acid metabolism.

Patterns of intracellular glucose-6-phosphate dehydrogenase turnover were studied in liver tissue of intact and thiamin-deficient rats using a new procedure for the enzyme purification. The alternations, found in the half-life period, in constant rates of synthesis and degradation of the key enzyme of the pentosephosphate pathway, correlated with a decrease of the glucose-6-phosphate dehydrogenase activity in vitamin B1 deficiency.