Enzyme Changes in the Offspring of Female Rats due to Long-Term Administration of Cyclic AMP and Insulin before Pregnancy.

We studied the effects of insulin and cAMP on the offspring of female rats after daily treatment with these substances over 4 weeks. In adult offspring from cAMP-treated females, activities of pyruvate kinase and glucose-6-phosphate dehydrogenase decreased in the liver and brain and activities of NADP-dependent malate dehydrogenase and 6-phosphogluconate dehydrogenase decreased in the liver. In the offspring of insulin-treated females, we observed only activation of glucose-6-phosphate dehydrogenase and malate dehydrogenase in the liver and only in females. Enzyme activity probably correlates with their content, as no changes in their kinetic properties were observed under these conditions. Long-term hormone treatment before pregnancy can affect the expression of genes for some enzymes in the offspring due to transmission of epigenetic signals by the ovum. However, further studies are required to confirm this mechanism.

Changes in some properties of the aurochs heart pyruvate dehydrogenase complex induced by its partial phosphorylation.

Comparative studies on the properties of the dephosphorylated and partially phosphorylated (to 35% activity reduction) pyruvate dehydrogenase complex (PDC) from aurochs heart muscle have been made. Data have been obtained indicating that the partial phosphorylation of PDC abolishes the kinetic attributes of a positive cooperativity of the pyruvate binding sites (nH = 1.5) featuring at low substrate concentrations. In addition, the partially phosphorylated PDC is inactivated slower at 50 degrees C.

Some regulatory properties of the 2-oxoglutarate dehydrogenase complex from European bison heart.

Basic regulatory properties of the 2-oxoglutarate dehydrogenase complex (OGDC) isolated and purified from the heart muscle of European bison (Bison bonasus) were studied. Kinetic studies have shown that in the absence of phosphate ions OGDC exhibits kinetic attributes of negative cooperativity with respect to 2-oxoglutarate. ADP and phosphate lower S0.5 value of OGDC for 2-oxoglutarate without changing the maximum reaction rate. NADH inhibits OGDC versus both 2-oxoglutarate and NAD+. Moreover, bison heart OGDC shows negative kinetic cooperativity for NAD+ and positive kinetic cooperativity for CoA at low CoA concentrations. The latter property has not been observed in earlier studies on OGDC from bovine and pig heart and other tissues of these animals.

Basic properties of the pyruvate dehydrogenase complex isolated from aurochs heart.

The purified aurochs (Bison bonasus, European bison) heart pyruvate dehydrogenase complex (PDC) has a set of subunits typical of mammalian PDC. PDC from aurochs heart contains firmly bound tiamine pyrophosphate in the amount providing over 50% of the maximal activity of the complex. The apparent value for activation energy of PDC is 60 kJ/mol. The Michaelis constant values for aurochs heart PDC are 22.4 +/- 1.0, 3.3 +/- 0.1 and 24.4 +/- 3.6 microM for pyruvate, CoA and NAD, accordingly. Acetyl-CoA is a competitive inhibitor with respect to CoA (Ki = 14.2 +/- 0.4 microM), whereas NADH gives the same inhibition with respect to NAD (Ki = 46.9 +/- 10.0 microM). The Km for CoA and NAD of the aurochs heart PDC are lower than that of domestic animals PDC.

[Kinetic properties of lipoamide dehydrogenase of the oxoglutarate dehydrogenase complex of the human heart]. / Kineticheskie svoistva lipoamid-degidrogenazy oksoglutaratdegidrogenaznogo kompleksa serdtsa cheloveka.

Kinetics of lipoamide dehydrogenase catalyzed reaction is described by Michaelis-Menten equation if concentrations of NAD and dihydrolipoamide (DLA) varied. Effective Km values were equal to 0.11 mM for NAD and 0.50 mM for DLA, respectively. Kinetic indications of positive cooperation between sites binding both NAD and DLA were manifested in presence of NADH. Apparent Ki value for NADH constituted 0.88-0.10 mM, thus demonstrating the effective regulation of the lipoamide dehydrogenase activity by end products.

Participation of adenosine diphosphate in regulation of the 2-oxoglutarate dehydrogenase complex from human heart.

Adenosine diphosphate (ADP) increases the activity of the highly purified 2-oxoglutarate dehydrogenase complex (OGDC) from human heart. The degree of activation is higher at low 2-oxoglutarate concentrations. The OGDC-catalyzed reaction rate versus ADP concentration curve is S-shaped at unsaturating substrate concentration. This is a catalytic attribute of cooperativity of the sites for binding of the allosteric effector ADP.

Interaction of the pyruvate dehydrogenase complex from human heart with thiamine pyrophosphate.

Preparations of a highly purified pyruvate dehydrogenase complex (PDC) from human heart contain endogenous thiamine pyrophosphate (TPP) in an amount accounting for about 10% of the maximum activity. At pH values of 7.5 and 8.0, the effective Michaelis constants with respect to exogenous TPP for the PDC apoenzyme form were 0.22 microM and 1.8 microM, respectively.

Kinetic characterization of the pyruvate and oxoglutarate dehydrogenase complexes from human heart.

The Michaelis constant values for the highly purified pyruvate dehydrogenase complex (PDC) from human heart are 25, 13 and 50 microM for pyruvate, CoA and NAD, respectively. Acetyl-CoA produces a competitive inhibition of PDC (Ki = 35 microM) with respect to CoA, whereas NADH produces the same type of inhibition with respect to NAD (Ki = 36 microM). The oxoglutarate dehydrogenase complex (OGDC) from human heart has active sites with two different affinities for 2-oxoglutarate ([S]0.5 of 30 and 120 microM). ADP (1 mM) decreases the [S]0.5 values by a half. The inhibition of OGDC (Ki = 81 microM) by succinyl-CoA is of a competitive type with respect to CoA (Km = 2.5 microM), whereas that of NADH (Ki = 25 microM) is of a mixed type with respect to NAD (Km = 170 microM).

[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.

[Characteristics of kinetics of the enzymatic oxoglutarate dehydrogenase reaction in a system with 2,6-dichlorophenolindophenol]. / Osobennosti kinetiki fermentativnoi oksoglutaratdegidrogenaznoi reaktsii v sisteme s 2,6-dikhlorfenolindofenolom.

The 2-oxoglutarate: 2,6-dichlorophenolindophenol (DCPIP)--oxidoreductase reaction catalyzed by the oxoglutarate dehydrogenase complex from bovine adrenal glands corresponds to the kinetic mechanism of a "ping-pong" type. There are signs of positive cooperativity of the oxoglutarate dehydrogenase interaction with the substrate and negative cooperativity of that with the electron acceptor. The half-maximal rate of the model reaction is provided by 0.01 mM concentrations of 2-oxoglutarate and DCPIP. The exceeding of the DCPIP optimum concentration (0.1 mM) results in the enzyme inhibition.

[Regulation of the activity of the oxoglutarate dehydrogenase complex of animal origin (review)]. / Reguliatsiia aktivnosti oksoglutaratdegidrogenaznogo kompleksa zhivotnogo proiskhozhdeniia (obzor).

Recent data on regulation of the multi-enzyme oxoglutarate dehydrogenase complex from pigeon breast muscle, porcine heart, bovine adrenal glands and kidney are reviewed. The most characteristic property of the complex consists in activation of the trigger oxoglutarate dehydrogenase component by and ATP. Action of these agents is more pronounced at low concentrations of 2-oxoglutarate and is directed towards alteration of the substrate half-saturation value SO.5. The adrenal oxoglutarate dehydrogenase complex exhibits positive cooperativity of allosteric ADP-binding sites which improved its sensitivity to variations in the effector concentration. The oxoglutarate dehydrogenase complex appears to be not only an important functional component but also is a regulatory unit of the Krebs cycle.

[The role of various amino acid residues in the functioning of NADP-specific isocitrate dehydrogenase from bovine adrenal cortex cytoplasm]. / Rol' nekotorykh aminokislotnykh ostatkov v funktsionirovanii NADP-spetsifichnoi izotsitratdegidrogenazy iz tsitoplazmy kory nadpochechnikov byka.

The modification of SH-groups in the native isocitrate dehydrogenase accessible to 5,5-dithiobis (2-nitrobenzoic acid) (DTNB) is accompanied by the enzyme inactivation. Isocitrate rather than NADP and MnCl2 protects two SH-groups of the enzyme from modification by DTNB and attendant inactivation. The isocitrate dehydrogenase inactivation by DTNB obeys pseudofirst-order reaction kinetics. The number of DTNB-titrated sulphydryl groups does not change after the isocitrate dehydrogenase denaturation by sodium dodecyl sulphate. In the presence of manganese ions isocitrate and to a lesser extent NADP protect isocitrate dehydrogenase from the inactivation induced by 2,3-butanedione, a specific modifier of arginine residues. It has also been shown that the methylene blue-sensitized photoinactivation of the enzyme associated with the photooxidation of histidine residues decreases in the presence of NADP. These data provide evidence for an essential role of the SH-groups, arginine residues and, probably, histidine in the functioning of NADP-dependent isocitrate dehydrogenase from adrenal cortex.

[Effect of thiol reagents on the activity of NADP-dependent malate dehydrogenase isolated from bovine adrenal cortex cytoplasm]. / Vliianie tiolovykh reagentov na aktivnost' NADP-zavisimoi malatdegidrogenazy, vydelennoi iz tsitoplazmy kory nadpochechnikov byka.

NADP-dependent malate dehydrogenase was rapidly inactivated in the presence of mercurous chloride. Titration of malate dehydrogenase by 5,5'-dithiobis (2-nitrobenzoic acid) (DTNB) in a solution of 8 M urea revealed 18 SH groups per molecule of the enzyme. Eight sulphydryl groups reacted with DTNB in native malate dehydrogenase and their modification was not accompanied by a loss of the enzyme activity. The interaction of p-chloromercury benzoate (PCMB) with malate dehydrogenase resulted in a 70% decrease in the enzyme activity. The binding of the thiol reagents by the malate dehydrogenase molecule appreciably increased the Michaelis constant value for the substrate. In the presence of magnesium ions, NADP and malate did not affect the process of malate dehydrogenase modification by DTNB and did not protect the enzyme from the inactivation by PCMB. It is suggested from the data obtained that the sulphyryl groups are involved in maintaining the active conformation of the enzyme.

[Kinetic properties of NADP-specific isocitrate dehydrogenase from bovine adrenals]. / Kineticheskie svoistva NADP-spetsifichnoi izotsitratdegidrogenazy iz nadpochechnikov byka.

At low concentrations of Mg2+ or Mn2+ the reaction catalyzed by isocitrate dehydrogenase from bovine adrenal cortex proceeds with a lag period which disappears as a result of the enzyme saturation with Mn2+ or Mg2+. The nu o versus D,L-isocitrate concentration curve is non-hyperbolic, which may be interpreted either by the presence of two active sites with different affinity for the substrate (K'mapp = 2.3 and 63 microM) within the enzyme molecule or by the "negative" cooperativity of these sites. The apparent Km value for NADP lies within the range of 3.6-9 microM. High concentrations of NADP inhibit isocitrate dehydrogenase (Ki = 1.3 mM). NADP.H inhibits the enzyme in a mixed manner with respect to NADP (Ki = 0.32 mM). In the presence of NADP.H the curve nu o dependence on NADP concentration shows a "negative" cooperativity between NADP binding sites. The reverse enzyme-catalyzed reaction of reductive carboxylation of 2-oxoglutarate does not exhibit any significant deviations from the Michaelis-Menten kinetics. The Km value for 2-oxoglutarate is 120 microM, while that for NADP.H is 10 microM.

[Purification and properties of NADP-dependent isocitrate dehydrogenase from the bovine adrenal cortex]. / Ochistka i svoistva NADP-zavisimoi izotsitratdegidrogenazy iz kory nadpochechnikov byka.

NADP-dependent isocitrate dehydrogenase (EC 1.1.42) was isolated and 430 times purified from the hyaloplasm fraction of bull adrenal cortex using fractionation by ammonium sulphate and acetone, heat treatment, chromatography on DEAE-Sephadex A-50, gel-filtration on Sephadex G-200 and affinity chromatography on 2',5'-ADP-sepharose 4B. The specific activity of homogeneous enzyme is 60 units per 1 mg of protein at 30 degrees C, yield--34%, pH optimum--8.0, molecular weight, determined by gel filtration on Sephadex G-200, is 96 kDa. The preparation electrophoresis in PAAG in the presence of DS-Na reveals one protein fraction with the mobility corresponding to that of protein having molecular weight of 46 kDa. The data obtained evidence for a dimer structure of the isocitrate dehydrogenase molecule from bull adrenals.

[Various kinetic and spectral-fluorescent properties of NADP-dependent malate dehydrogenase from bovine adrenal cortex cytoplasm]. / Nekotorye kineticheskie i spektral'no-fluorestsentnye svoistva NADP-zavisimoi malatdegidrogenazy iz tsitoplazmy kory nadpochechnikov byka.

Malate dehydrogenase from bovine adrenal cortex has been purified to homogeneity, using affinity chromatography on 2',5'-ADP-Sepharose 4B. The kinetic data do not contradict the consecutive mechanism of the reaction with the ordered addition of substrates: NADP binds first, then malate. The enzyme conformation initiated by NADP and malate binding is less thermostable. Malate dehydrogenase has intrinsic tryptophan fluorescence with the spectrum maximum at 335 +/- 1 nm, half-width of 50 +/- 1 nm and quantum yield of 0.08. The tryptophan residues belonging to class 1 (75%) and class 2 (25%) make the main contribution to the intrinsic fluorescence of malate. The binding of cofactors and substrates results in the quenching of enzyme fluorescence. The values of dissociation constants for malate dehydrogenase complexes with NADP (4 microM), with NADP . H (8 microM) and with pyruvate (2.5 mM) correlate with the corresponding values of Km. The shifts in pH of the medium induce changes in the fluorescence parameters which are probably related to conformational changes in the enzyme molecule. The changes in the fluorescence parameters correlate with the alterations of the malate dehydrogenase enzymatic activity.

[NADP-dependent malate dehydrogenase from bovine adrenal cortex cytoplasm. Isolation and properties]. / NADP-zavisimaia malatdegidrogenaza iz tsitoplazmy kory nadpochechnikov byka. Ochistka i nekotorye svoistva.

The NADP-dependent decarboxylating malate dehydrogenase was isolated from the cytoplasmic fraction of bovine adrenal cortex and purified 3530-fold by 3-fold ammonium sulfate fractionation, ion-exchange chromatography on DEAE-Toyopearl 650 M and DEAE-Sephadex A-50 with subsequent two-fold gel filtration through Toyopearl HW-55. The specific activity of homogeneous enzyme preparations was equal to 60 U/mg protein with a 30% yield. The enzyme molecular weight as determined by gel filtration on Sephadex G-20 was 155000. Upon polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate malate dehydrogenase dissociated into two subunits with Mr 77000. The Arrhenius plot for the reaction rate showed a break at 30 degrees C. The values of activation energy and temperature coefficient above and below the breakpoint were equal to 45049 and 147188 J X mol-1; 1.68 and 2.63, respectively. Within the temperature range of 26-40 degrees C, malate dehydrogenase exhibited hyperbolic kinetics with respect to the substrate. At 30 degrees C, Km for malate was equal to 250 microM, whereas at 40 degrees C it was 130 microM. The curve for the dependence of the initial reaction velocity versus NADP concentration was S-shaped. The Hill coefficient was 1.4, which testifies to positive cooperativity of NADP interaction with malate dehydrogenase.

[Characteristics of thiamine thiazolone diphosphate-induced inhibition of a pyruvate dehydrogenase complex in vitro and in intact mitochondria]. / Osobennosti ingibirovaniia tiamintiazolondifosfatom piruvatdegidrogenaznogo kompleksa in vitro i v sostave intaktnykh mitokhondrii.

Thiamine thiazolone diphosphate (TTPP) was capable of penetrating through the mitochondrial membrane and of inhibiting the pyruvate dehydrogenase complex (PDC) in intact mitochondria. TTPP depressed the activity of mammalian PDC in a mixed manner (Ki = 5.10(-8) M) and yeast pyruvate decarboxylase (Ki = 5.10(-6) M) via a competitive mechanism with respect to thiamine diphosphate. It was shown that decarboxylation of pyruvate in intact and disrupted mitochondria of rat liver and brain is less inhibited by TTPP than the overall activity of PDC determined by the formation of acetyl-CoA. It was assumed that TTPP as a transition state analog participates only in oxidative reactions (but not in simple decarboxylation of pyruvate).

[Some peculiarities of regulation of the adrenal pyruvate de- hydrogenase complex by acetyl-coenzyme A]. / Osobennosti reguliatsii nadpochechnikovogo piruvatdegidrogenaznogo kompleksa atsetil-kofermentom A.

Acetyl-CoA increased incorporation of 32P from [gamma-32P]ATP into the adrenal pyruvate dehydrogenase complex (PDC), but did not raise the rate of inactivation of the complex. This may be explained by the stimulation of phosphorylation of the centers which are not responsible directly for the PDC activity. At the same time the PDC phosphorylated in the presence of acetyl-CoA was reactivated slower by specific phosphatase.

[Purification and various properties of hyaloplasmic NADP-dependent isocitrate dehydrogenase from the rabbit adrenal gland]. / Ochistka i nekotorye svoistva gialoplazmaticheskoi NADP-zavisimoi izotsitratdegidrogenazy iz nadpochechnikov krolika.

NADP-dependent isocitrate dehydrogenase was isolated from the hyaloplasmic fraction of rabbit adrenal glands and purified by ammonium sulfate and polyethylene glycol fractionation and chromatography on DEAE-Sephadex A-50 to a specific activity of 26.8 U/mg with a 53% yield. Polyacrylamide gel electrophoresis revealed one distinct protein band with mobility corresponding to Mr approximately 50 000 in the presence of SDS. Data from gel filtration suggest that the detergent-untreated isocitrate dehydrogenase has a twice as great molecular mass, which is indicative of its dimeric structure of identical subunits. The pH optimum for the adrenal isocitrate dehydrogenase-catalyzed reaction is 7.5-7.7; the apparent activation energy is 61.3 kJ X mol-1. Mn2+ activate the enzyme more effectively than Mg2+. The curve for the dependence of the isocitrate dehydrogenase reaction rate versus D-isocitrate and NADP concentrations is S-shaped. At low substrate or coenzyme concentrations the Hill coefficient is 2.0 and 1.9, respectively, which serves as a kinetic attribute of positive cooperativity of their interaction with isocitrate dehydrogenase. The concentrations of D-isocitrate and NADP providing for the half-maximal rate of the reaction are 3.8 and 6.6 microM, respectively.