Interaction of muscle glycogen phosphorylase b reconstituted from apoenzyme and analogs of pyridoxal-5'-phosphate with specific ligands.

Phosphorylase b from rabbit skeletal muscles was reconstituted with analogs of PLP containing residues -CH(2)-CH(2)-COOH, trans-CH=CH-COOH or -C=-COOH at position 5. Replacing native coenzyme in the phosphorylase molecule with any PLP analog tested leads to the decrease in the enzyme affinity for the allosteric inhibitor, FMN. Phosphorylase b reconstituted with analogs of PLP shows the greater ability for association in tetramers in the presence of 1 mM AMP than native enzyme.

[Reconstruction of muscle glycogen phosphorylase b from an apoenzyme and pyridoxal-5'-phosphate and its analogs. Interaction of apophosphorylase and the reconstructed enzyme with specific ligands]. / Rekonstruktsiia myshechnoi glikogenfosforilazy b iz apofermenta i piridoksal'-5'-fosfata i ego analogov. Vzaimodeistvie apofosforilazy i rekonstruirovannogo fermenta so spetsificheskimi ligandami.

Sedimentation methods were used to study the effects of modification of the pyridoxal-5'-phosphate (PLP) molecule at the 5th position on the affinity of reconstituted muscle glycogen phosphorylase b for the substrate (glycogen) and the allosteric inhibitor (FMN) as well as on the enzyme capacity to association induced by AMP. Reconstituted phosphorylase b was obtained with PLP analogs containing at the 5th position -CH2-CH2-COOH (analog I), trans-CH=CH-COOH (analog II) or -C identical to COOH (analog III) residues. Reconstitution of phosphorylase b is accompanied by the recovery of the enzyme quaternary structure. Phosphorylase b reconstituted with PLP or analogs I, II and III is not distinguished practically from the native enzyme in its affinity for glycogen. Substitution of the native coenzyme in the phosphorylase molecule with any tested PLP analog leads to lower enzyme affinity for FMN. Microscopic dissociation constants of the FMN-enzyme complexes increase in the following order: enzyme.I < enzyme.II < enzyme.III. Phosphorylase b reconstituted with analogs I, II and III differs substantially from the native enzyme in its capacity to association in the presence of 1 mM AMP: the reconstituted enzyme is represented practically by only the tetrameric form.

[The effect of ubiquinone-10 on the development of D-galactosamine-induced hepatitis in rats]. / Vliianie ubikhinona-10 na razvitie D-galaktozaminovogo gepatita u krys.

It has been shown that prophylactic administration of ubiquinone protects rats liver from the toxic damage by D-galactosamine both on ultrastructural and on cell levels. Ubiquinone administration prevents necrosis in hepatocytes and preserves their ability for compensatory reactions expressed in activation of protein-synthesis regulating structures in the cell. Ubiquinone decreases hyperfermentemia and hyperbilirubinemia as well as prevents the decrease in liver protein content caused by galactosamine. Ubiquinone exerts an antioxidant effect, blocking the induction of lipid peroxidation both in intact and hepatic rats.

[Interaction of muscle glycogen phosphorylase B with F-actin]. / Vzaimodeistvie myshechnoi glikogenfosforilay B s F-aktinom.

The binding of rabbit muscle glycogen phosphorylase b to F-actin has been studied by sedimentation in analytical centrifuge in 10 mM Tris-acetate buffer pH 6.8 at 20 degrees C. The adsorption capacity of F-actin is equal to (7.8 +/- 0.9) X 10(-7) mole of glycogen phosphorylase b per 1 g of F-actin; the microscopic dissociation constant for the glycogen phosphorylase-F-actin complex is (5.4 +/- 0.5) X 10(-7) M. It was found that the allosteric activator, AMP, facilitates the adsorption of glycogen phosphorylase b on F-actin, whereas the substrate, Pi, and the inhibitor, ATP, cause an opposite effect.

[Supramolecular organization of glycolytic enzymes]. / Nadmolekuliarnaia organizatsiia fermentov glikoliza.

On the basis of the analysis of the data on adsorption of glycolytic enzymes to structural proteins of skeletal muscle and to erythrocyte membranes, the data on enzyme-enzyme interactions and the data on the regulation of activity of glycolytic enzymes by cellular metabolites the structure of glycolytic enzyme complex adsorbed to a biological support has been proposed. The key role in the formation of the multienzyme complex belongs to 6-phosphofructokinase. The enzyme molecule has two association sites, one of which provides the fixation of 6-phosphofructokinase on the support and another is saturated by fructose-1,6-bisphosphate aldolase. The multienzyme complex fixed on structural proteins of skeletal muscle contains one tetrameric molecule of 6-phosphofructokinase and at two molecules of other glycolytic enzymes. Hexokinase is not involved in the complex composition. The molecular mass of the multienzyme complex is about 2,6 X 10(6) Da. The formation of the multienzyme complex leads to the compartmentation of the glycolytic process. The problem of integration of physico-chemical mechanisms of enzyme activity regulation (allosteric, dissociative and adsorptive mechanisms) is discussed.

Supramolecular organization of glycolytic enzymes.

On the basis of the analysis of the data on adsorption of glycolytic enzymes to structural proteins of skeletal muscles and to the erythrocyte membranes, the data on enzyme-enzyme interactions and the data on the regulation of activity of glycolytic enzymes by cellular metabolites, the structure of the glycolytic enzymes complex adsorbed to a biological support has been proposed. The key role in the formation of multienzyme complex belongs to 6-phosphofructokinase. The enzyme molecule has two association sites, one of which provides the fixation of 6-phosphofructokinase on the support and another is saturated by fructose-1,6-bisphosphate aldolase. The multienzyme complex contains one tetrameric molecule of 6-phosphofructokinase and two molecules of each of other glycolytic enzymes. Hexokinase is not a part of the complex. The molecular mass of the multienzyme complex is about 2.6 X 10(6) daltons. The multienzyme complex has symmetry axis of second order. The formation of the multienzyme complex leads to the compartmentation of glycolytic process. The problem of integration of physico-chemical mechanisms of enzyme activity regulation (allosteric, dissociative and adsorptive mechanisms) is discussed.

Turbidimetric method for determination of glycogen phosphorylase activity and its use for estimation of equilibrium position of enzymic reaction.

A turbidimetric method has been developed for the continuous monitoring of the enzyme reaction catalyzed by glycogen phosphorylase. This method is based on the registration of the turbidity of glycogen solution at wavelengths above 300 nm. It has been shown that the increase in the turbidity is strictly proportional to the quantity of glucose 1-phosphate formed during the enzyme reaction. The method has the advantage of continuity, and it is suitable for determining the initial rate of catalytic synthesis or degradation of glycogen in a relatively simple and fast way. The kinetic experiments may be carried out under various conditions. The method of calculation of the overall equilibrium constant of the enzyme reaction catalyzed by glycogen phosphorylase has been elaborated. This method is based on the analysis of the dependence of the initial rate of the enzymic reaction on the proportion of the substrate of the forward reaction: [Pi]/( [Pi] + [G-1-P] ).

[Regulation of ox liver glutamate dehydrogenase activity by coenzymes]. / Reguliatsiia aktivnosti glutamatdegidrogenazy iz pecheni byka kofermentami.

The effects of coenzymes NAD(P) and NAD(P)H on the kinetics of the ox liver glutamate dehydrogenase reaction have been studied. The oxidized coenzymes were shown to activate alpha-ketoglutarate amination at inhibiting concentrations of NADH and NADPH. The reduced coenzymes, NADH and NADPH, inhibit glutamate deamination with both NAD and NADP as coenzymes. The data obtained are discussed in terms of literature data on the mechanisms of the coenzyme effects on the glutamate dehydrogenase activity and are inconsistent with the theory of direct ligand--ligand interactions. It was shown that the peculiarities of the glutamate dehydrogenase kinetics can easily be interpreted in the light of the two state models.

[Regulation of enzyme activity in adsorptive enzyme systems. III. Interaction of pig muscle lactate dehydrogenase with F-actin]. / Reguliatsiia aktivnosti fermentov v adsorbtsionnykh fermentnykh sistemakh. III. Vzaimodeistvie laktatdegidrogenazy iz myshts svin'i s F-aktinom.

The binding of pig skeletal muscle lactate dehydrogenase by F-actin has been studied using the sedimentation method in 10 mM Tris-acetate buffer, pH 6.0 at 20 degrees C. Adsorption capacity of F-actin is equal to (1 +/- 0.1) . 10(-5) moles of lactate dehydrogenase per 1 g of actin. NADH decreases the affinity of F-actin with respect to lactate dehydrogenase. The binding of lactate dehydrogenase by F-actin in diminishing the rate of enzymatic reduction of alpha-ketoglutarate. The microscopic dissociation constant for the complex of the enzyme with F-actin which is estimated from the dependence of the enzymatic reaction rate of F-actin concentration at saturating NADH concentrations is equal (3.0 +2- 0.5) . 10(-7) M. It has been shown that the bound enzyme is characterized by the greater value of Km and the lower value of Vmax in comparison to the free enzyme.

[Determination of glycogen phosphorylase B activity by the turbidimetric method in the presence of the forward and reverse reaction substrates]. / Opredelenie aktivnosti glikogenfosforilazy B turbidimetricheskim metodom pri odnovremennom prisutstvii substratov priamoi i obratnoi reaktsii.

The turbidimetric method based on monitoring changes in the turbidity of glycogen solution at wavelengths above 300 nm has been developed for the measurement of catalytic activity of rabbit skeletal muscle glycogen phosphorylase B. The dependences of the initial rate of enzymatic reaction on molar ratio of inorganic phosphate in the inorganic phosphate-glucose-1-phosphate mixture have been obtained at various concentrations of the allosteric activator, AMP, or in the presence of inhibitors (D-glucose, glucose 6-phosphate). It has been shown that the changes in glycogen phosphorylase B activity in the presence of these modifiers are not affected by variations in the molar ratios of the substrate. A theoretical expression for the dependence of the initial rate of the reversible reaction S in equilibrium P catalyzed by the enzyme has been deduced as a function of the substrate (S) molar ratio.

[Regulation of enzyme activity in adsorptive enzyme systems. II. Influence of dextran sulfate on catalytic properties of lactate dehydrogenase (isozyme M4)]. / Reguliatsiia aktivnosti fermentov v adsorbtsionnykh fermentnykh sistemakh. II. Vliianie dekstransul'fata na kataliticheskie svoistva laktatdegidrogenazy (izoferment M4).

It has been shown that the binding of pig skeletal muscle lactate dehydrogenase (isozyme M4) by dextran sulfate with weight-average molecular weight 500 000 is accompanied by a decrease of the rate of enzymatic reduction of pyruvate. The hyperbolic dependence of the enzymic reaction rate on NADH concentration observed for free lactate dehydrogenase is transformed in a sigmoidal curve in the case of adsorbed enzyme form (Hill's coefficient is equal to 2.1). The experimental data have been described quantitatively using the model of adsorptive enzyme system where the enzyme interacts reversibly with the support and co-operative interaction of substrate binding sites in the adsorbed enzyme molecule are realized. It is assumed that the value of microscopic dissociation constant for the complex of the substrate with adsorbed enzyme is being changed by a constant factor during saturation of the binding sites by the substrate in the enzyme molecule. The value of parameters of the model for the adsorptive enzyme system under study are determined.

[Denaturation of beef liver glutamate dehydrogenase under the action of guanidine hydrochloride and a study of the possibility of the enzyme renaturation]. / Denaturatsiia glutamatdegidrogenazy iz pecheni byka pod deistviem guanidinkhlorida i izuchenie vozmozhnosti renaturatsii fermenta.

It was shown that denaturation of beef liver glutamate dehydrogenase under the action of guanidine hydrochloride results in a diplacement of the protein fluorescence maximum from 332 to 349 nm, in a decrease of optical rotation of the protein at 233 nm and in an appearance of negative bands in the difference absorbance spectrum with extrema at 279 and 287 nm. The transition of native enzyme into a denaturated state is observed within a narrow interval of guanidine hydrochloride concentrations. The middle point of the transition corresponds to approximately 2,2 M guanidine hydrochloride. The inactivation kinetics for glutamate dehydrogenase coincide with those of the enzyme spectral properties alterations due to denaturation. The attempts at renaturation of glutamate dehydrogenase by diluting the denaturated enzyme solution or by a dialysis against a buffer solution were unsuccessful.

[Kinetic studies of the formation of abortive ternary complex lactate dehydrogenase (isoenzyme h4)-NAD-pyruvate]. / Kinetika Obrazovaniia Neproduktiznogo Troinogo Kompleksa Laktatdegidrogenaza (Izoferment N4) Nad-Piruvat

The rate constants and equilibrium constants for four stages of process of the abortive ternary complex lactate dehydrogenase (porcine isoenzyme H4)-NAD-pyruvate formation are determined. These stages are 1) the enolization of pyruvate, 2) the formation of binary complex enzyme-NAD, 3) the formation of "intermediate" ternary complex enzyme-NAD-enol and 4) the transformation of "intermediate" complex into "final" ternary one, accumulation of the latter being followed by spectrophotometric and fluorometric methods. The constants obtained were compared with the corresponding ones for porcine isoenzyme M4 determined in earlier work. It was shown that the greater stability of ternary complex and the greater initial rate of ternary complex formation in the case of H4 is due to greater affinity of isoenzyme H4 with respect to NAD, greater magnitude of reaction rate constant for transformation of "intermediate" ternary complex into "final" one and lesser magnitude of reaction rate constant for the reverse transition.