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The activity of the key enzymes of the salvage synthesis and degradation of NAD (nicotinic acid mononucleotide pyrophosphorylase and NAD glycohydrolase) was studied in Saccharomyces cerevisiae mutants selected earlier as potential producers of NAD. An enhanced NAD-producing ability of the mutants was shown to be related to their reduced NAD-degrading activity: thus, NAD glycohydrolase in mutants 110, 118, and 206 was inactive in all growth phases regardless of the level of NAD biosynthesis.

Nicotinic acid and nicotinamide inhibit in vitro the acetyl-CoA-carboxylase activity of partially purified enzyme from chicken liver. The incorporation of 10, 20, 50 and 100 mkmoles of nicotinic acid or nicotinamide into the incubation medium (0,9 ml) leads to the inhibition of the enzyme activity by 19, 45, 70 and 100% and by 39, 51, 60 and 78%, respectively. NADH+ and NADP+ at concentrations by one order of magnitude lower than those of nicotinic acid and nicotinamide decrease the enzyme activity in a similar manner. The constants of inhibition by the above-mentioned compounds were calculated with respect to ATP, acetyl-CoA and citrate.

The rate of [14C]NAD incorporation into chicken liver nuclear histones was studied under conditions of DNA damage by N-methyl-N-nitrosourea and pancreatic DNAase I. With an increase in N-methyl-N-nitrosourea concentration from 8.5 X 10(-2) to 34.0 X 10(-2) mM, the ADP ribosylation of histones increases by 20% as compared to the control. In DNAase I-treated nuclei, the binding by histones of [14C]NAD sharply increases, reaching its maximum (18.3 X 10(-8) mM) at 30% cleavage of DNA. When 50% of DNA was cleaved, the rate of [14C]NAD incorporation into the histones was 8.0 X 10(-8) mM as compared to 6.1 X 10(-8) mM/mg protein in control samples. The poly(ADPR)polymerase activity was increased in both cases. It was shown that the NAD-pyrophosphorylase activity in chicken liver nuclei treated with N-methyl-N-nitrosourea does not differ from the control one, while in DNAase I-treated nuclei the maximum of the NAD-pyrophosphorylase activity was achieved, as well as the maximum of [14]NAD incorporation into the histones within the range of DNA damage of 25-35%, being equal to 37 X 10(-8) mM NAD/min/mg protein as compared to 26.0 X 10(-8) mM/min/mg protein in the control. At different degrees of DNA damage, the average length of the poly-ADP-ribose chain did not practically alter, thus suggesting the increase in the number of polymer binding sites in the histones.

The content of NAD and DNA, the activity of DNA-polymerase the velocity of NAD pyrophosphorolysis have been studied in liver nuclei of 8, 14, 18 day-old chicken embryos and 1--2 month- and 6 month-old chickens. It has been found that during ontogenesis the NAD content in chicken liver nuclei is increased, whereas the DNA content is decreased, the correlation coefficient being--0,93. The DNA-polymerase activity is the highest in the liver nuclei of 8--14 day-old embryos. During ontogenesis the DNA-polymerase activity is decreased. The excess of inorganic pyrophosphate shifts the NAD synthesis reaction to the left and activates the NAD pyrophosphorolytic degradation. During chicken ontogenesis the maximal NAD pyrophosphorolytic degradation is observed during the embrionic period.

A protein which specifically binds [3H]retinol in vitro and has a sedimentation coefficient of 2S was isolated from the soluble fraction of chicken glandular stomach intermediate zone mucosa cells and purified to homogeneity. The purification procedure included gel filtration through Sephadex G-75 and G-50 and ion-exchange chromatography on DEAE-cellulose. The properties of the protein were compared to those of the retinol-binding protein from rat liver.

It was shown that nicotinamide and NAD inhibit the specific binding of [3H]flunitrazepam to benzdiazepine receptors without causing a direct influence of gamma-aminobutyric acid (GABA) receptors. The GABA-benzdiazepine complex was separated by solubilization with 0.5% lubrol PX. The solubilized preparations contain the binding sites for [3H]flunitrazepam alone (Kd = 5.9 nm). The Kd value for the membrane-bound benzdiazepine receptor is 2.9 nM. NAD inhibited the specific binding of [3H]flunitrazepam to the solubilized membrane preparation when used at concentrations by several orders of magnitude lower than that of nicotinamide. Using gel filtration on Sepharose 6B-CL, the molecular mass of the soluble benzdiazepine receptor protein was determined.

A kinetic analysis of the activity of acetyl-CoA carboxylase from chicken liver upon alimentary activation of lipogenesis and inhibition of this reaction by nicotinic acid was performed. It was found that the affinity of the enzyme isolated from chicken liver with stimulated lipogenesis is decreased by nicotinic acid for HCO3- but remains unchanged for ATP. The value of Vmax for ATP and the amount of the ATP used in this reaction remain unaffected. At the same time the enzyme affinity for acetyl-CoA is increased with a simultaneous decrease of Vmax. It is assumed that nicotinic acid inhibits the first step of the acetyl-CoA carboxylase-catalyzed reaction.

An addition of alpha-tocopherol (I) and its synthetic derivatives (alpha-tocopheryl quinone (II), its short-chained analog (III), alpha-tocopherol lactone (IV), and short-chained alpha-tocopheryl acetate (V)) to the homogenized liver of vitamin E deficient rats resulted in a significant increase of ubiquinone after 2 hour incubation. Activity of the above derivatives (II-V) was not associated directly with their transformation into I or with a noticeable increase of the I content. There is a certain correlation between the chemical structure and the level of vitamin E activity of alpha-tocopherol derivatives that led to an increase in the ubiquinone content and prevented the decrease of tissue respiration and termination of pregnancy in rats.

A thiamine-binding protein (ThBP) with a specific activity of 8.21 nmoles/mg protein was isolated from rat brain synaptosomes by affinity chromatography and gel filtration on Sephadex G-200. The protein was purified 746-fold with a 40.5% yield. ThBP was homogeneous during sodium dodecyl sulfate gel electrophoresis; its molecular mass was determined by gel filtration on Sephadex G-200 and by sodium dodecyl sulfate gel electrophoresis and was equal to 107 and 103 kD, respectively. The pH optimum for the binding is 8.35. When the ability of ThBP to bind thiamine phosphates was tested, the latter decreased in the following order: thiamine monophosphate greater than thiamine triphosphate greater than greater than thiamine diphosphate.

The rate of incorporation of nicotinamide-[adenosine-U-14C]adenine dinucleotide [( Ado-U-14C]NAD) into histones and the poly(ADPR) polymerase activity of chromatin suggest that the NAD-dependent ADP-ribosylation of histones depends on the rate of NAD hydrolysis by glycohydrolase in chicken liver nuclei. With a rise in the NAD-glycohydrolase activity after treatment of nuclei with Triton X-100 the synthesis of poly(ADP-ribose) via the poly(ADPR)polymerase reaction is augmented, as a result of which the rate of [Ado-U-14C]NAD incorporation into total histones is increased. On the contrary, the decrease of NAD-glycohydrolase hydrolysis after treatment of nuclei with SDS lowers the poly(ADPR)polymerase activity and [Ado-U-14C]NAD incorporation into histones. Under these conditions, i. e. different rates of glycohydrolase hydrolysis of NAD in the nuclei, some redistribution of [Ado U-14C]NAD incorporation into individual histones occurs.

The binding of [14C]NAD to rat brain synaptic membranes is reversible and depends on incubation time, temperature and protein concentration in the reaction mixture. The value of the rate constant for [14C]NAD binding to the synaptic membranes at 24 degrees C (kl) is 1.1 X 10(-6) M-1 S-1, the rate constant for dissociation of the [14C]NAD-receptor complex (k-1) is 3.3 X 10(-3) S-1. The value of the constant for the ligand dissociation from this complex (Kd) is 3.0 nmole. Treatment of the experimental results in the Scatchard plots for the equilibrium binding of [14C]NAD to the synaptic membranes demonstrated that the receptor sites with high and low affinities for the ligand (Kd1 = 3.3 nmol, Kd2 = 14.4 nmole) and with binding capacities of 44 and 77 pmole of [14C]NAD, respectively. It was found that the synaptosomal membrane components which bind the labelled NAD have a protein nature. Data from [14C]NAD and [nicotinamide-3H]NAD binding suggest that brain synaptic membranes bind NAD at the nicotinamide and adenylic moieties.

The dependence of ADP-ribosylation of chicken liver nuclear histones on NAD concentration in the nuclei was studied under conditions of stimulation of coenzyme synthesis by the nicotinamide and nicotinic acid as well as upon addition of various concentrations of the [Ado-U-14C]NAD nuclei to the incubation mixture. In the first case, the rate of [Ado-U-14C]NAD incorporation into the histones was decreased due to the dilution of the label by the de novo synthesized NAD. The amount of the latter formed under effects of nicotinic acid and nicotinamide increased, correspondingly, from 2,2 X 10(-5) mmol up to 4.1 X 10(-5) and 7.0 X 10(-5) mmol per mg of nuclear protein. The incorporation of [Ado-U-14C]NAD into the histones decreased from 12.0 X 10(-8) mmol after incubation of liver slides with nicotinic acid and nicotinamide down to 8.0 X 10(-8) and 7.0 X 10(-8) mmol, respectively. With a rise in the concentration of exogenous [Ado-U-14C]NAD, the level of ADP-ribosylation of nuclear histones increased, the plot [14C]NAD incorporation at the labeled coenzyme concentration of 25 X 10(-7) mM/mg of histone had a plateau. Changes in the labeled substrate concentration brought about corresponding changes in the average length of the histone-linked poly-(ADP-ribose) chain.