Thiamine cardiotropism.

Thiamine administration (200 mg/kg 2 hours before start of the experiment) substantially reduced the myocardial ischaemic lesion in a model of experimental myocardial infarction in the rat. As shown by stereometric analysis of histological heart preparations, the cytoprotective action of vitamin B1 is mediated by increased cardiomyocyte resistance towards ischaemia, i.e., through an adequate increase in the relative volume of the protection zone. Experiments on a model of emotional-painful stress in the rat show that the antiischaemic action of vitamin B1 cannot be explained by a specific cardiotropism of thiamine (the presence of thiamine phosphate buffer depots excludes the manifestation of coenzymatic effects of thiamine under given conditions) but by an antistressor action of thiamine resulting in exclusion of an important pathogenetic link in the mechanism of stress-conditioned cardiopathy.

[Kinetic characteristics of 6-phosphogluconate dehydrogenase from bull adrenal cortex]. / Kineticheskie kharakteristiki 6-fosfogliukonatdegidrogenazy iz kory nadpochechnikov byka.

The hyperbolic dependence of the initial rate of 6-phosphogluconate dehydrogenase-catalyzed reaction on 6-phosphogluconate and NADP concentrations has been established. The Lineweaver-Burk plots of V0 against concentration of one substrate with constant unsaturating concentrations of another substrate cross left from the ordinate axis. The Km value for 6-phosphogluconate is equal ot 0.035 mM, for NADP--0.018 mM. It has been shown that NADPH inhibits 6-phosphogluconate dehydrogenase by the competitive type with respect to NADP and by the noncompetitive one with respect to 6-phosphogluconate. Ribulose-5-phosphate inhibits the reaction by the mixed type with respect to NADP and by the noncompetitive type with respect to 6-phosphogluconate. Kinetic data are in agreement with the consecutive mechanism of the reaction: the first substrate is NADP, the last product--NADPH. The Arrhenius plot for the reaction shows a break at 27 degrees C.

[Purification and properties of NADP-specific malate dehydrogenase from bovine adrenal cortex mitochondria]. / Oshistka i nekotorye svoistva NADP-spetsifichnoi malatdegidrogenazy iz mitokhondrii kory nadpochechnikov byka.

An electrophoretically homogeneous preparation of mitochondrial NADP-dependent malate dehydrogenase with a specific activity of 155 u./mg and a 67% yield has been obtained, using ammonium sulfate fractionation, gel filtration through Toyopearl HW-55 F, ion-exchange chromatography on DEAE-Toyopearl 650 M and affinity chromatography on 2',5'-ADP-Sepharose 4B. The molecular mass of native malate dehydrogenase is 260 kD; Mr of the SDS-treated enzyme is 61 kD, which is suggestive of a tetrameric structure of the protein. Malate dehydrogenase is active only in the presence of Mg2+ or Mn2+, but not Ca2+ or Ba2+. The Km' values for Mn2+ and Mg2+ are 50 and 66 microM, respectively. At low malate concentrations and NADP saturation, the enzyme is characterized by a sigmoidal kinetics which changes to hyperbolic at low concentrations of NADP. The Lineweaver--Burk plots for the dependence of the initial reaction rate on the concentration of one substrate at several fixed concentrations of the other substrate intersect to the left of the B-axis. NADPH competes with NADP:pyruvate inhibits malate dehydrogenase ++noncompetitively with respect to the coenzyme. NADPH and pyruvate inhibit the malate dehydrogenase-catalyzed reaction via a mixed type mechanism with respect to malate. The data obtained are consistent with a consecutive mechanism of reaction, whose first substrate is NADP and the last product is NADPH.

[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.

[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.

Effect of oxythiamine on adrenal thiamine pyrophosphate-dependent enzyme activities.

The effect of parenterally administered low doses of oxythiamine (0.5 mumol/100 g body weight every 12 h) on rat adrenal transketolase, pyruvate dehydrogenase ( PDC ) and 2-oxoglutarate dehydrogenase complex ( OGDC ) activities was studied. It was found that the transketolase and the PDC activities decreased after 12, 16, and 20 injections. When the activities were measured in the absence of exogenous thiamine pyrophosphate (TPP), the PDC inhibition was significant after 20 oxythiamine injections. The OGDC activity was resistant to oxythiamine administration. The oxythiamine effects seem to be due to its being phosphorylated to yield diphosphate ester which, as kinetic studies on highly purified PDC from bovine adrenals have shown, is a strong competitive inhibitor (Ki = 0.07 microM) with respect to TPP (Km = 0.11 microM). The data obtained provide evidence for the possibility of a partial selective in vivo inhibition of the adrenal TPP-dependent enzymes.

[Inhibition of kinase activity of the adrenal pyruvate dehydrogenase complex by the mitochondrial component]. / Tormozhenie kinaznoi aktivnosti piruvatdegidrogenaznogo kompleksa nadpochechnikov mitokhondrial'nym komponentom.

A component inhibiting the phosphorylation-linked inactivation of the adrenal pyruvate dehydrogenase complex in the presence of ATP was revealed during purification of the complex from bovine adrenal mitochondria. The degree of the kinase activity inhibition is greater at lower concentrations of ATP. It was assumed that the mitochondrial component screens the kinase active site or the phosphorylation sites of pyruvate dehydrogenase, thus limiting the ATP access to them. Proteins and lipids are incorporated into the component at a ratio 2:1, which is suggestive of its lipoprotein nature. The effect of the mitochondrial component on the kinase activity of the pyruvate dehydrogenase complex is somewhat specific and is unaffected by bovine serum albumin or blood serum lipoproteins.

[Pyruvate dehydrogenase complex in the rabbit adrenal glands: activation by administration of ACTH]. / Pyruvatdegidrogenaznyi kompleks nadpochechnykh zhelez krolikov: aktivatsiia pri vvedenii AKTG.

The pyruvate dehydrogenase complex (PDC) from the rabbit adrenal mitochondria was partially purified (18-fold) and its main kinetic parameters were determined: Michaelis' constants for pyruvate, CoA and NAD are 16, 10 and 140, respectively. The inhibitor constant for acetyl-CoA is 25 microM. Phosphate ions augment the PDC affinity for the substrate. After ACTH administration to rabbits the active PDC form in the adrenals rises simultaneously with the PDC phosphatase activity growth. The data obtained are indicative of the adrenal PDC involvement in mobilizing metabolism to provide intensive steroidogenesis.

[Enzyme activity of thiamine pyrophosphate in the rat after oxythiamine administration]. / Aktivnost' tiaminpirofosfatsoderzhashchikh krys pri vvedenii oksitiamina.

Intraperitoneal injection of hydroxythiamine to rats (1 mmol per kg bw) resulted after 2-4 h in a more than 4-fold decrease in the activity of the oxoglutarate dehydrogenase complex, pyruvate dehydrogenase complex and NADP-dependent isocitrate dehydrogenase in adrenal mitochondria. Inhibition of hyaloplasmic transketolase, 6-phosphogluconate dehydrogenase and NADP-dependent malate dehydrogenase occurred later. Based on the correlation of the time course of enzymatic activity in the adrenals and the decreased concentration of 11-hydroxycorticosteroids in the blood the paramount role in the maintenance of the steroidogenesis among thiamine pyrophosphate-containing enzymes is assigned to the oxoglutarate dehydrogenase and pyruvate dehydrogenase complexes.

[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.

[Lag period as affected by the adrenal pyruvate dehydrogenase complex containing residual endogenous thiamine pyrophosphate]. / Lag-period pri funktsionirovanii piruvatdegidrogenaznogo kompleksa nadpochechnikov, soderzhashchego ostatochnyi éndogennyi tiaminpirofosfat.

The pyruvate dehydrogenase complex from bovine adrenals does not completely lose thiamine pyrophosphate (TPP) in the course of purification. The preparations contain firmly bound residual TPP in the amount providing for one third of the maximal activity of the complex. A lag period (tau) is observed during catalysis by the pyruvate dehydrogenase complex with the residual TPP in the absence of exogenous coenzyme. The duration of the lag period depends on concentration of the complex: tau is decreased as the concentration increases. The decrease of the lag period occurs after preincubation of the complex with pyruvate, but it is the most remarkable after preincubation with pyruvate and CoA. The tetrahydrothiamine pyrophosphate at low concentrations completely inhibits the activity. However, the residual TPP is retained as is evidenced by the recovery of the complex activity after gel-filtration through Sephadex G-25. The development of the lag period may be interpreted in terms of partial limited dissociation of the residual TPP in the region of the catalytic center.

[Characterization of the kinase activity of bovine adrenal pyruvate dehydrogenase complex]. / Kharakteristika kinaznoi aktivnosti piruvatdegidrogenaznogo kompleksa iz nadpochechnikov byka.

In the presence of [gamma-32P]ATP the bovine adrenal pyruvate dehydrogenase complex accepts the label simultaneously and becomes inactivated. This suggests the existence of kinase in the composition of the complex as is typical of the complexes from other animal sources. The Pi is incorporated into the subunit with molecular weight of 42 000. The kinase activity of the adrenal pyruvate dehydrogenase complex is high: within the first 20 sec of incubation with ATP the inactivation is as high as 60%. The pH optimum for kinase is around 7.3. The apparent Km value for ATP with 50 mM KCl is 7 microM; that in the absence of KCl is 10 microM. ADP is a competitive inhibitor of kinase with respect to ATP (Ki = 100 microM), when K+ are present in the medium. Thiamine pyrophosphate and pyruvate decrease the rate of pyruvate dehydrogenase complex inactivation.

2-oxoglutarate dehydrogenase complex from bovine-adrenal-cortex mitochondria. Purification and partial characterization.

The 2-oxoglutarate dehydrogenase complex from bovine adrenal-cortex mitochondria has been purified by polyethylene glycol fractionation, ultracentrifugation through a layer of sucrose, isoelectric precipitation and gel filtration of Sepharose 4 B. The specific activity of the preparation obtained wa 9.9 U/mtg of protein; the sedimentation coefficient, S20, w, was 30 S. The results of sodium dodecyl sulphate polyacrylamide gel electrophoresis indicated decomposition of the 2-oxoglutarate dehydrogenase complex into 3 clear-cut protein fractions with mobilities corresponding to molecular weights of 51 000, 56 000 and about 110 000. Michaelis constants for the reactants of the 2-oxoglutarate dehydrogenase complex reactions were: 2-oxoglutarate = 200 micro M; CoA - 4,5 micro M; NAD - 25 micro M.

[Steady-state kinetics of the bovine adrenal pyruvate dehydrogenase complex-catalyzed reactions]. / Statsionarnaia kinetika reaktsii kataliziruemykh piruvatdegidrogenaznym kompleksom nadpochechnikov byka.

The hyperbolic dependence of the initial rate of the adrenal pyruvate dehydrogenase complex-catalyzed reactions on pyruvate, CoA and NAD concentrations was established. The Lineweaver--Burk plots of v0 against one substrate concentration at constant unsaturating concentrations of other substrates represent families of parallel lines. The Km values for pyruvate, CoA and NAD are 0,017, 0.005 and 0.030 mM, respectively. CoA-SAc was shown to compete with CoA (Ki=0.067 mM) whereas NADH--with NAD (Ki=0.023 mM). Both CoA-SAc and NADH are uncompetitive inhibitors with respect to pyruvate. The inhibition of CoA-SAc against NAD is of a mixed type, while that of NAD against CoA is non-competitive. The results obtained are in agreement with the kinetic model of a three-site "ping-pong" mechanism.

[Kinetic and regulatory properties of alpha-ketoglutarate dehydrogenase complex from bovine adrenals]. / Kineticheskie i reguliatornye svoistva al'fa-ketoglutaratdegidrogenaznogo kompleksa, vydelennogo iz nadpochechnikov byka.

The dependence of the rate of reactions catalyzed by the alpha-ketoglutarate dehydrogenase complex on the alpha-ketoglutarate, CoA and NAD+ concentration is described by Michaelis-Menten equation. Km is 0.190, 0.012 and 0.025 mM, respectively. ADP plays a role of a complex activator at low alpha-ketoglutarate concentrations. ATP, on the contrary, produces a mixed type inhibition on the alpha-ketoglutarate dehydrogenase complex with respect to CoA and NAD+. A conclusion is drawn that the adrenal alpha-ketoglutarate complex regulation by ATP/ADP ratio value is possible.

[Purification of the pyruvate dehydrogenase complex from bovine adrenal cortex mitochondria]. / Ochistka piruvatdegidrogenaznogo kompleksa iz mitokhondrii kory nadpochechnikov byka.

Isolation of the pyruvate dehydrogenase complex from bovine adrenal cortex and its purification including fractionation by polyethyleneglycol, ultracentrifugation and gel filtration on Sepharose 4B is described. The preparation obtained having the specific activity of 4,5 U/mg was purified 370-fold with a yield of 37%. Under polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate the pyruvate dehydrogenase complex is degraded into 4 protein fractions with the mobility corresponding to the molecular weights of 74 000, 56 000, 42 000 and 37 000.