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.

[Different properties of pyruvate kinase from rabbit and hare muscles].

Some catalytic and kinetic properties of pyruvate kinase (PK, EC 2.7.1.40) isolated from the heart and skeletal muscles of rabbits and hares with a 9-16-fold purification were studied. The initial specific activity of the enzyme in hare heart homogenates was 66% and in skeletal muscles 25% as high as in respective rabbit tissues. Temperature optimums and thermostability of PK from hare tissues were higher as compared with those in rabbits. From the comparison of K(M) (S0.5) values it follows that hare skeletal muscle PK exhibits a highest affinity to phosphoenol pyruvate, but lowest to ADP, as compared with rabbit skeletal muscle PK. Moreover, PK from both hare tissues exhibits a positive kinetic cooperativity (Hill coefficient > 1.35) of the phosphoenol pyruvate and ADP binding sites. In contrast to PK from rabbit tissues, the enzyme from the hare heart and muscles PK is presented by its allosteric isoform which might by advantageous under extreme conditions of the hare's habitation.

Effect of anabolic steroid nandrolone decanoate on the properties of certain enzymes in the heart, liver, and muscle of rats, and their effect on rats' cardiac electrophysiology.

Nandrolone decanoate (ND) is an anabolic steroid, modified to enhance anabolic rather than androgenic actions. The physiological effects of ND treatment are often used in various aspects of medical practice. In this investigation we have tried to establish whether a single, high dose of ND (20 mg/kg) would cause any anabolic effects. Moreover, we have attempted to correlate the eventual effects with changes in the activity and kinetic properties of anabolic- and bioenergetic-involved enzymes in different tissues of rats, along with the rats' ECG parameters. The body and liver weights of the rats were unchanged, but heart weight had increased 10 days after ND injection. Electrocardiographic data showed a small prolongation of the QRS complex 3, 6, and 10 days after ND treatment. It was established that ND causes activation of glucose-6-phosphate and 6-phosphogluconate dehydrogenases, malic enzyme, and NADP-linked isocitrate dehydrogenase in rat hearts. Moreover, 6-phosphogluconate dehydrogenase from the hearts of ND-treated rats showed higher affinity to its substrate, in comparison with control. Activation of transketolase by ND in the liver was accompanied by inhibition of glucose-6-phosphate and 6-phosphogluconate dehydrogenases. We observed an increase of glucose 6-phosphate dehydrogenase and NAD-linked malate dehydrogenase in the muscle of ND treated rats. It may be concluded that ND in a single high dose exhibits cardiotrophic action, especially towards the increase of heart dehydrogenases activity which generates NADPH and supplies ribose phosphate for the biosynthesis of nucleotides and nucleic acids. On the other hand, ND may cause activation of ATP synthesis in muscle by enhanced malate-aspartate shuttle action.

Short-term regulation of the alpha-ketoglutarate dehydrogenase complex by energy-linked and some other effectors.

The question of regulation of alpha-ketoglutarate dehydrogenase complex (KGDHC) has been considered in the biochemical literature very rarely. Moreover, such information is not usually accurate, especially in biochemical textbooks. From the mini-review of research works published during the last 25 years, the following basic view is clear: a) animal KGDHC is very sensitive to ADP, P(i), and Ca2+; b) these positive effectors increase manifold the affinity of KGDHC to alpha-ketoglutarate; c) KGDHC is inhibited by ATP, NADH, and succinyl-CoA; d) the ATP effect is realized in several ways, probably mainly via opposition versus ADP activation; e) NADH, besides inhibiting dihydrolipoamide dehydrogenase component competitively versus NAD+, decreases the affinity of alpha-ketoglutarate dehydrogenase to substrate and inactivates it; f) thioredoxin protects KGDHC from self-inactivation during catalysis; g) bacterial and plant KGDHC is activated by AMP instead of ADP. These main effects form the basis of short-term regulation of KGDHC.

Regulatory effect of thiamin pyrophosphate on pig heart pyruvate dehydrogenase complex.

The kinetic behavior of pig heart pyruvate dehydrogenase complex (PDC) containing bound endogenous thiamin pyrophosphate (TPP) was affected by exogenous TPP. In the absence of exogenous TPP, a lag phase of the PDC reaction was observed. TPP added to the PDC reaction medium containing Mg2+ led to a disappearance of the lag phase, inducing strong reduction of the Km value for pyruvate (from 76.7 to 19.0 microM) but a more moderate decrease of Km for CoA (from 12.2 to 4.3 microM) and Km for NAD+ (from 70.2 to 33.6 microM), with no considerable change in the maximum reaction rate. Likewise, thiamin monophosphate (TMP) decreased the Km value of PDC for pyruvate, but to a lesser extent (from 76.7 to 57.9 microM) than TPP. At the unsaturating level of pyruvate, the A50 values for TPP and TMP were 0.2 microM and 0.3 mM, respectively. This could mean that the effect of TPP on PDC was more specific. In addition, exogenous TPP changed the UV spectrum and lowered the fluorescence emission of the PDC containing bound endogenous TPP in its active sites. The data obtained suggest that TPP plays, in addition to its catalytic function, the important role of positive regulatory effector of pig heart PDC.

Different extent of inhibition of pyruvate dehydrogenase and 2-oxoglutarate dehydrogenase both containing endogenous thiamine pyrophosphate, by some anticoenzyme analogues.

Inhibitory effects of 4'-oxythiamine pyrophosphate (OTPP) and tetrahydrothiamine pyrophosphate (ThTPP) on the purified bison heart pyruvate dehydrogenase complex (PDC) semisaturated with endogenous thiamine pyrophosphate (TPP) and 2-oxoglutarate dehydrogenase complex (OGDC) saturated about 85% with endogenous TPP, were studied. It has been established that the thiamine derivatives strongly inhibit not only the PDC apoenzyme moiety, but also the PDC holoenzyme moiety. The apparent I50 values for the holoenzyme were 0.006 microM and 0.046 microM for OTPP and ThTPP, respectively. The inhibition of the PDC is reversible. After removal of the anticoenzyme analogues by gel filtration the endogenous TPP within the PDC is retained as it is evidenced by complete recovery of the enzyme activity without added TPP. In contrast with the PDC, OGDC holoenzyme form is weakly inhibited by the anticoenzyme analogues.

An investigation of the kinase activity of aurochs heart pyruvate dehydrogenase complex.

The purified aurochs heart pyruvate dehydrogenase complex (PDC) saturated to approximation 60% with endogenous thiamine pyrophosphate (TPP), was slowly and incompletely inactivated by its kinase in the presence of ATP. Exogenous TPP or ADP, but not pyruvate, strongly inhibited the kinase activity. The kinetic properties of the aurochs heart PDC kinase suggested the occurrence of two active sites each with different affinities for ATP (K'm - 1.7 microM, K''m = 48 microM).

Thiamine pyrophosphate as an effector of 2-oxoglutarate dehydrogenase complex from European bison heart.

The purified 2-oxoglutarate dehydrogenase complex (OGDC) from the European bison heart was near saturated with endogenous bound thiamine pyrophosphate (TPP). Exogenous TPP added to the full OGDC reaction medium decreased S0.5 for 2-oxoglutarate approximately 2.6-fold without any notable change in the maximum reaction rate. The TPP effect was observed in the presence of 1 mM ADP which alone is a strong positive allosteric effector of OGDC. At an unsaturating 2-oxoglutarate concentration the A50 value for TPP was approximately 0.05 mM. The ADP-like action of exogenous TPP was also found in the 2-oxoglutarate dehydrogenase (E1) reaction, determined in the presence of 2,6-dichlorophenoloindophenol as an electron acceptor.

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.