Nuclear NAD Research Articles

Human Nuclear NAD+ADP-Ribosyltransferase(polymerizing): Organization of the Gene

Human nuclear NAD+: protein ADP-ribosyltransferase(polymerizing) [pADPRT; poly(ADP-ribose)poly-merase; EC 2.4.2.30] is a DNA-dependent protein-modifying enzyme composed of several domains important for DNA binding, automodification, and NAD binding. We report that the human pADPRT gene is 43 kb in length and is split into 23 exons. All the intron-exon boundaries correspond to a canonical splice consensus sequence. Each of the four metal coordinating sites putatively forming the two zinc fingers of the DNA-binding domain is encoded separately. The automodification domain and the NAD-binding domain are coded for by 4 and 12 exons, respectively.

Human nuclear NAD+ ADP-ribosyltransferase: localization of the gene on chromosome 1q41-q42 and expression of an active human enzyme in Escherichia coli.

The gene for human nuclear NAD+ ADP-ribosyltransferase [NAD+:poly(adenosine diphosphate D-ribose) ADP-D-ribosetransferase, EC 2.4.2.30; pADPRT] was localized to chromosome 1 at q41-q42 by in situ hybridization with a pADPRT-specific cDNA probe. Expression of a pADPRT cDNA under control of the lac promoter in Escherichia coli induces the synthesis of a group of related proteins that were immunoreactive with pADPRT antibody and that had catalytic properties very similar to those of the human enzyme. Purification of this enzymatic activity was performed essentially as described for the human enzyme. The Km, pH optimum, optimal reaction temperature, and inhibition by 3-aminobenzamide and 3-methoxybenzamide were found to be similar for the recombinant and the human enzymes. The purified recombinant enzyme consists of two major proteins of Mr 99,000 and Mr 89,000. Both proteins show pADPRT activity in activity gel analysis with [32P]NAD+ as substrate. Microsequencing of these two proteins isolated by denaturing gel electrophoresis and deletion mutagenesis of the pADPRT expression plasmid shows that the Mr 99,000 and Mr 89,000 proteins derive from initiation of translation at internal translational start signals located within the pADPRT cDNA.

Reversible inactivation of ribonucleases by ADPribosylation

The activity of purified bovine seminal RNAase and pancreatic RNAase A (EC 3.1.27.5) has been investigated following in vitro ADPribosylation in the presence of nuclear ADPribosyltransferase (EC 2.4.2.30) and NAD +. ADPribosylation of these enzymes was correlated with a significant decrease in their activities. Approximately three residues of ADPribose were present per mol of enzyme. Removal of the bound ADPribose restored enzyme activity to near normal levels. Similar results were obtained with nuclei isolated from bull seminal vesicles as an endogenous source of seminal RNAase and nuclear ADPribosyltransferase. The findings suggest that in vitro ADPribosylation has a reversible inactivating effect on ribonucleases.

Induction of Cardiac l-Ornithine Decarboxylase by Nicotinamide and Its Regulation by Putrescine

Repeated intraperitonal injection of nicotinamide (2 × 5 mmol/kg) in rats increases cardiac l-ornithine decarboxylase 36-fold in 4 h. This effect, which is dose dependent, is inhibited by cycloheximide. Cardiac l-ornithine decarboxylase decays with a t1/2 of 35 min. Nicotinic acid, at the same doses as nicotinamide, has only negligible effect on cardiac l-ornithine decarboxylase content. The calculated nicotinamide content of hearts is 11 mM, 30 min after the second injection of nicotinamide. Cardiac nicotinamide decays with a t1/2 of 3.1 h. Nicotinic acid increases cardiac nicotinamide content only to 1.1 mM. Injection of putrescine in doses which augment cardiac polyamine content to the same extent as nicotinamide-induced l-ornithine decarboxylase in vivo, inhibits the enzyme-inducing effect of nicotinamide within 30 min. The t1/2 of cardiac putrescine is 6.8 h. There is a temporal correlation between cardiac nicotinamide content, the cycle of induction of l-ornithine decarboxylase in 4 h, the subsequent decay of enzyme content and progressive inhibition of enzyme induction by putrescine. Induction of the protein inhibitor of l-ornithine decarboxylase, by repeated injections of putrescine, is a delayed second effect of the polyamine and has no kinetic relationship to the relatively rapid induction-decay cycle of l-ornithine decarboxylase elicited by nicotinamide. Within the experimental periods used, there is no effect of nicotinamide or putrescine on general protein synthesis in heart. Other inhibitors of poly(adenosine diphosphoribose) synthetase or nuclear NAD+ glycohydrolase (5′-methyl nicotinamide, thymidine or 3-isobutyl-1-methylxanthine) also induce l-ornithine decarboxylase. There is an apparent preferential inducing effect of nicotinamide on heart, whereas the other inhibitors act both on liver and heart to a varying extent.

Multisite topographic microfluorometry of intracellular and exogenous fluorochromes.

Abstract. Microspectro fluorometry combined with microinjection of metabolites allows the monitoring of pathways regulating the energy metabolism, biosynthetic or metabolizing activity of the intact living cell. The topographic scan of ˜ 100–150 adjacent regions within a cell or the spectral scan of fluorescence from a single cell region is completed within 60 ms. The in situ activity of intracellular enzymes and substrate concentration vs rate relationships are derived from the kinetic analysis of transients (e.g. NAD(P) ⇄ NAD(P)H) elicited by sequential injections with increasing doses of substrate (e.g. glucose‐6‐P, 6‐phosphogluconate). A method of approximation is used to predict the non‐linear behavior of whole biochemical systems (e.g. the sum of reactions involved in NAD(P) reduction‐reoxidation transients). Approximation to power law kinetics is validated by prediction of system behavior over a 10–100 fold variation in the input concentration of substrate.The NAD‐dependent glycolytic chain (faster rise kinetics) and NADPH‐dependent shunt pathway (slower rise kinetics) are triggered optionally, by enhanced demand for degradation, synthesis or metabo‐lization of exogenous compounds (hydrocarbons, aflatoxins). Compartmentation of NAD(P) pathways is suggested by differences in the reoxidation of cytoplasmic vs nuclear NAD(P)H. The intercellular transfer of molecules can be evaluated by microinjection of fluorescent tracers.The difference spectra ΔIF due to microinjection of substrate are resolved in terms of bound vs free NAD(P)H (with differentiation of other endogenous and exogenous fluorochromes).On the basis of the above, a tentative scheme of intra‐ and intercellular dynamics can be developed.

Isolation of cell nuclei from ischemic renal tissue: Biochemical characteristics of these nuclei

The molecular pathology of renal ischemia was examined in isolated nuclei. Nuclei of good quality in high yield were isolated from mouse kidneys which had been ischemic at 37°C for 60 min. Freezing the tissues did not impair the subsequent nuclear isolation by differential and isopyknic centrifugation in sucrose media. Morphological studies, chemical measurements of protein, RNA, and DNA, and the measurement of cytoplasmic “marker” enzymes suggested that contamination of nuclei by cytoplasmic components was limited. Renal ischemia caused by renal artery ligation markedly decreased RNA synthesis by isolated nuclei. Ischemia produced by incubating the kidney in vitro similarly reduced RNA synthesis. On release of arterial occlusion the amount of RNA synthesis was restored to a normal level 4 hr after release of occlusion. The effects of in vitro renal ischemia on nuclear RNA synthesis were temperature dependent. Renal ischemia did not significantly alter nuclear NAD pyrophosphorylase. There was no significant difference between normal and ischemic nuclei demonstrated by RNA/DNA and protein/DNA ratios or by phasecontrast microscopy.

Study of the interaction of soluble and particle-bound nicotinamide adenine dinucleotide pyrophosphorylase with cytoplasmic ribosomes

The cytoplasm of cells infected with EMC virus contains new structures which possess activity of the nuclear enzyme NAD pyrophosphorylase [14]. An attempt was made to understand the mode of formation of these structures in the infected cell. It was found that soluble NAD pyrophosphorylase manifests a strong affinity for cytoplasmic ribosomes, sedimenting at 90S. When cytoplasmic ribosomes were dissociated to the 60S and 40S subunits, the enzyme was found to be adsorbed only to the 60S unit. In extracts of rat liver nuclei, NAD pyrophosphorylase is associated with 35S particles, composed mainly of protein and DNA. The bond between enzyme and particle is of a loose nature. When ribosomes are mixed with 35S nuclear particles, most of the enzyme activity is transferred from the nuclear particles to the ribosomes, thus forming particles with an average sedimentation coefficient of 90S. Similar structures are obtained when either soluble NAD pyrophosphorylase or 35S nuclear particles are mixed with preparations of cytoplasm isolated from non-infected cells. The results of these experiments suggest that the 90S cytoplasmic structures found in virus-infected cells could result from an association between either free or particle-bound NAD pyrophosphorylase with cytoplasmic ribosomes.

Inhibition of nuclear NAD nucleosidase and poly ADP-ribose polymerase activity from rat liver by nicotinamide and 5′-methyl nicotinamide

The effectiveness of nicotinamide and 5′-methyl nicotinamide as inhibitors of nuclear NAD nucleosidase and poly ADP-ribose polymerase from rat liver has been investigated. Nicotinamide inhibited the NAD nucleosidase and the poly ADP-ribose polymerase with K i 's of 5 · 10 −4 M and 2 · 10 −5 M, respectively, 5′-methyl nicotinamide with K i 's of 3.3 · 10 −4 M and 2 · 10 −4 M, respectively. As both nicotinamide and 5′-methyl nicotinamide are similarly effective inhibitors of these enzymes, inhibition at these sites cannot explain the different effects that nicotinamide and 5-methyl nicotinamide have on hepatic NAD synthesis (Clark and Pinder, 1969). It is suggested therefore that the poly ADP-ribose polymerase is more likely involved in the control of DNA synthesis than in NAD synthesis (as suggested elsewhere).

Effects of androgen in vivo on some properties of isolated rat ventral prostate nuclei

The effects of (1) the presence or absence of androgen in vivo, and (2) treatment with Triton-X-100 in vitro upon some chemical and biochemical properties of rat ventral prostate nuclei, isolated in hypertonic sucrose, are described and compared with results reported for rat liver nuclei. Light- and electron microscopic evidence of nuclear purity, hormone-induced changes in the content of homogenate and nuclear protein, RNA and DNA, and the effect of detergent-washing on the composition of isolated nuclei are presented. Nuclei obtained from castrated rats contained less protein and RNA than those from normal animals, while androgen restored the content of these macromolecules. The association of several marker enzymes with prostate nuclei was examined: nuclei washed with detergent appear to retain cytochrome oxidase, an acid phosphatase and glucose-6-phosphatase activity; with the methods employed lactic dehydrogenase and uricase were not detected. Nuclear RNA, NAD and putative protein synthesis were not adversely affected by detergent. The effect of androgen on this apparent nuclear protein synthesis is being investigated.

Localization and regulation of two NAD nucleosidases in Ehrlich ascites cells.

After differential centrifugation of Ehrlich ascites cells by the de Duve method, NAD nuclosidase activities were found in the nuclear and the microsomal fractions, but not in the lysosomal fraction. The nuclear NAD nucleosidase is inhibited 50% by 0.2 mM nicotinamide and is highly specific for β-NAD. Its activity is markedly decreased after DNase treatment; it is associated with chromatin and is very probably identical with an enzyme that polymerizes the adenosine diphosphate ribose moiety of NAD to an acid-insoluble polymer first described in rat liver nuclei by Chambon, Weill, and Mandel. The microsomal NAD nucleosidase is inhibited 50% by 1.2 mM nicotinamide, and splits NAD, NADP and NMN at a ratio of 1:0.5:0.2. NAD and NADP nucleosidase activities are reciprocally inhibited by NADP and NAD. Evidence is presented that the microsomal enzyme is active mainly at the surface of the cell. Nicotinamide concentration may be one factor regulating the nuclear but not the microsomal NAD nucleosidase. Within the cell, NAD is protected by membrane structure from degradation by the microsomal NAD nucleosidase.

Effect of hypophysectomy on liver diphosphopyridine nucleotide: the role of nicotinamide

The effectiveness of nicotinamide and 5′-methyl nicotinamide as inhibitors of nuclear NAD nucleosidase and poly ADP-ribose polymerase from rat liver has been investigated. Nicotinamide inhibited the NAD nucleosidase and the poly ADP-ribose polymerase with Ki's of 5 · 10−4 M and 2 · 10−5 M, respectively, 5′-methyl nicotinamide with Ki's of 3.3 · 10−4 M and 2 · 10−4 M, respectively. As both nicotinamide and 5′-methyl nicotinamide are similarly effective inhibitors of these enzymes, inhibition at these sites cannot explain the different effects that nicotinamide and 5-methyl nicotinamide have on hepatic NAD synthesis (Clark and Pinder, 1969). It is suggested therefore that the poly ADP-ribose polymerase is more likely involved in the control of DNA synthesis than in NAD synthesis (as suggested elsewhere).