Nicotinamide adenine dinucleotide glycohydrolase from rat liver nuclei. Isolation and characterization of a new enzyme.

A new type of nicotinamide adenine dinucleotide glycohydrolase (NADase) has been isolated from rat liver nuclei. When partially purified chromatin is passed through a Sephadex G-200 column in the presence of 1 M NaCl, enzyme activities catalyzing the liberation of nicotinamide from NAD elute in two peaks. One, which appears in the void volume fraction, hydrolyzes the nicotinamide-ribose linkage of NAD to produce nicotinamide and ADP-ribose in stoichiometric amounts. This activity is not inhibited by 5 mM nicotinamide. The other, which elutes much later, catalyzes the formation of poly(ADP-ribose) from NAD and is completely inhibited by 5 mM nicotinamide. The former, NADase, is DNase-insensitive and thermostable, has a pH optimum of 6.5 to 7, a Km for NAD of 28 muM, and a Ki for nicotinamide of 80 mM, and hydrolyzes NADP as well as NAD. The latter, poly(ADP-ribose) synthetase, is sensitive to DNase treatment and heat labile, has a pH optimum of 8 to 8.5, a Km for NAD of 250 muM and a Ki for nicotinamide of 0.5 mM and is strictly specific for NAD. Further, the former NADase is shown to lack transglycosidase activity, which has been documented to be a general property of NADases derived from animal tissues. These results indicate that the NAD-hydrolyzing enzyme newly isolated from nuclei is a novel type of mammalian NADase which catalyzes the hydrolytic cleavage of the nicotinamide-ribose linkage of NAD.     

Nicotinamide–adenine dinucleotide pyrophosphatase in the growing and aging mosquito

1. The disappearance of pyridine nucleotides during incubation with mosquito homogenates proceeds through the hydrolysis of the pyrophosphate linkage of these compounds as demonstrated by the formation of NMN and AMP from NAD(+). This reaction was also demonstrated by the loss in the coenzyme functioning property of NAD(+) (yeast alcohol dehydrogenase reaction) without a concomitant loss in reactivity towards cyanide. Transglycosidase activity was not observed in the mosquito homogenates, and low concentrations of nicotinamide did not inhibit the NAD(+) splitting activity of these homogenates. These observations are all in accord with the presence in these homogenates of a NAD(+) pyrophosphatase rather than a NADase. 2. The NAD(+) pyrophosphatase is destroyed by boiling, is not heat-activated, and has a pH optimum at pH8.75. In addition to NAD(+), other dinucleotides such as NADP(+), the 3-acetylpyridine and thionicotinamide analogues of NAD(+) and the thionicotinamide analogue of NADP(+), function as substrates in the hydrolysis catalysed by the pyrophosphatase. 3. A decrease in the specific activity of NAD(+) pyrophosphatase was observed during larval development, and a barely detectable activity was found in the pupa and adult. 4. Enzyme activity per organism increased in the larva but decreased to a very low value in the pupa and adult. These results indicate that the decrease in specific activity was due to a decrease in enzyme concentration rather than an increase in amounts of protein.     

Purification and properties of the soluble NAD glycohydrolase from Bungarus fasciatus venom

The NAD glycohydrolase (NADase) (EC 3.2.2.5) from Bungarus fasciatus (banded krait) venom was purified (1000-fold) to electrophoretic homogeneity through a 3-step purification procedure, the last step being affinity chromatography on Cibacron blue agarose. The purified NADase is a glycoprotein containing two subunits of Mr = 62,000 each. Nicotinamide and adenosine diphosphoribose were produced in a 1:1 stoichiometry and were the only products formed when the purified NADase was incubated with NAD. These results were confirmed by high performance liquid chromatography. The enzyme exhibited a brod pH profile with optimum pH for hydrolysis at 7.5 with very little change in Km from pH 6.0 to pH 8.5. The NADase is only slightly affected by changes in ionic strength. The enzyme studied titrimetrically at pH 7.5 and 38 degrees C exhibited a Km of 14 microM and a Vmax of 1380 mumol of NAD cleaved/min/mg of protein. The activation energy for the enzyme-catalyzed hydrolysis of NAD was 15.7 kcal/mol. In addition to NAD and NADP, a number of NAD analogs were shown to function as substrates for the enzyme. Product inhibition studies demonstrated nicotinamide to be a noncompetitive inhibitor with a KI of 1.5 mM and adenosine diphosphoribose a competitive inhibitor with a KI of 0.36 mM. Procion blue HB (Cibacron blue F3GA) was shown to be a competitive inhibitor with a KI of 33 nmol. The purified NADase catalyzed the pyridine base exchange reaction between 3-acetylpyridine and the nicotinamide moiety of NAD.     

Critical Role for NAD Glycohydrolase in Regulation of Erythropoiesis by Hematopoietic Stem Cells through Control of Intracellular NAD Content

NAD glycohydrolases (NADases) catalyze the hydrolysis of NAD to ADP-ribose and nicotinamide. Although many members of the NADase family, including ADP-ribosyltransferases, have been cloned and characterized, the structure and function of NADases with pure hydrolytic activity remain to be elucidated. Here, we report the structural and functional characterization of a novel NADase from rabbit reticulocytes. The novel NADase is a glycosylated, glycosylphosphatidylinositol-anchored cell surface protein exclusively expressed in reticulocytes. shRNA-mediated knockdown of the NADase in bone marrow cells resulted in a reduction of erythroid colony formation and an increase in NAD level. Furthermore, treatment of bone marrow cells with NAD, nicotinamide, or nicotinamide riboside, which induce an increase in NAD content, resulted in a significant decrease in erythroid progenitors. These results indicate that the novel NADase may play a critical role in regulating erythropoiesis of hematopoietic stem cells by modulating intracellular NAD.     

Mouse T-cell antigen rt6.1 has thiol-dependent NAD glycohydrolase activity

Mouse Rt6.1 and Rt6.2, homologues of rat T-cell RT6 antigens, catalyze arginine-specific ADP-ribosylation. Without an added ADP-ribose acceptor, Rt6.2 shows NAD glycohydrolase (NADase) activity. However, Rt6.1 has been reported to be primarily an ADP-ribosyltransferase, but not an NADase. In the present study, we obtained evidence that recombinant Rt6.1 catalyzes NAD glycohydrolysis but only in the presence of DTT. The NADase activity of Rt6.1 observed in the presence of DTT was completely inhibited by N-ethylmaleimide (NEM). Native Rt6.1 antigen, immunoprecipitated from BALB/c mouse splenocytes with polyclonal antibodies generated against recombinant RT6.1, also exhibited NADase activity in the presence of DTT. Compared with Rt6.2, Rt6.1 has two extra cysteine residues at positions 80 and 201. When Cys-80 and Cys-201 in Rt6.1 were replaced with the corresponding residues of Rt6.2, serine and phenylalanine, respectively, Rt6.1 catalyzed the NADase reaction even in the absence of DTT. Conversely, replacing Ser-80 and Phe-201 in Rt6.2 with cysteines, as in Rt6.1, converted the thiol-independent Rt6.2 NADase to a thiol-dependent enzyme. Kinetic study of the NADase reaction revealed that the affinity of Rt6.1 for NAD and the rate of catalysis increased in the presence of DTT. Moreover, the NADase activity of Rt6.1 expressed on COS-7 cells was stimulated by culture supernatant from activated mouse macrophages, even in the absence of DTT. From these observations, we conclude that t!he Rt6.1 antigen has thiol-dependent NADase activity, and that Cys-80 and Cys-201 confer thiol sensitivity to Rt6.1 NADase. Our results also suggest that upon the interaction of T-cells expressing Rt6.1 with activated macrophages, the NADase activity of the antigen will be stimulated.     

Nicotinamide adenine dinucleotide (NAD) and its metabolites inhibit T lymphocyte proliferation: role of cell surface NAD glycohydrolase and pyrophosphatase activities

The presence of NAD-metabolizing enzymes (e.g., ADP-ribosyltransferase (ART)2) on the surface of immune cells suggests a potential immunomodulatory activity for ecto-NAD or its metabolites at sites of inflammation and cell lysis where extracellular levels of NAD may be high. In vitro, NAD inhibits mitogen-stimulated rat T cell proliferation. To investigate the mechanism of inhibition, the effects of NAD and its metabolites on T cell proliferation were studied using ART2a+ and ART2b+ rat T cells. NAD and ADP-ribose, but not nicotinamide, inhibited proliferation of mitogen-activated T cells independent of ART2 allele-specific expression. Inhibition by P2 purinergic receptor agonists was comparable to that induced by NAD and ADP-ribose; these compounds were more potent than P1 agonists. Analysis of the NAD-metabolizing activity of intact rat T cells demonstrated that ADP-ribose was the predominant metabolite, consistent with the presence of cell surface NAD glycohydrolase (NADase) activities. Treatment of T cells with phosphatidylinositol-specific phospholipase C removed much of the NADase activity, consistent with at least one NADase having a GPI anchor; ART2- T cell subsets contained NADase activity that was not releasable by phosphatidylinositol-specific phospholipase C treatment. Formation of AMP from NAD and ADP-ribose also occurred, a result of cell surface pyrophosphatase activity. Because AMP and its metabolite, adenosine, were less inhibitory to rat T cell proliferation than was NAD or ADP-ribose, pyrophosphatases may serve a regulatory role in modifying the inhibitory effect of ecto-NAD on T cell activation. These data suggest that T cells express multiple NAD and adenine nucleotide-metabolizing activities that together modulate immune function.     

Activity of NMN+, nicotinamide ribose and analogs in alcohol oxidation promoted by horse-liver alcohol dehydrogenase. Improvement of this activity and structural requirements of the pyridine nucleotide part of the NAD+ coenzyme

Kinetic constants of horse-liver alcohol-dehydrogenase-mediated oxidation of alcohol by nicotinamide mononucleotide and nicotinamide ribose were determined and the role of different adenine moieties complementing the reaction mixtures was investigated. Five nicotinamide ribose analogs were synthesized and their activities as NAD+ inhibitors and as cofactors in this dehydrogenase-mediated oxidation of alcohol were assayed. In the light of these results, structural requirements of the pyridine ribofuranosyl part of the NAD+ are discussed.     

Amino acid-specific ADP-ribosylation: structural characterization and chemical differentiation of ADP-ribose-cysteine adducts formed nonenzymatically and in a pertussis toxin-catalyzed reaction.

ADP-ribosylation is a posttranslational modification of proteins by amino acid-specific ADP-ribosyltransferases. Both pertussis toxin and eukaryotic enzymes ADP-ribosylate cysteine residues in proteins and also, it has been suggested, free cysteine. Analysis of the reaction mechanisms of cysteine-specific ADP-ribosyltransferases revealed that free ADP-ribose combined nonenzymatically with cysteine. L- and D-cysteine, L-cysteine methyl ester, and cysteamine reacted with ADP-ribose, but alanine, serine, lysine, arginine, N-acetyl-L-cysteine, 2-mercaptoethanol, dithiothreitol, and glutathione did not. The 1H NMR spectrum of the product, along with the requirement for both free sulfhydryl and amino groups of cysteine, suggested that the reaction produced a thiazolidine linkage. ADP-ribosylthiazolidine was labile to hydroxylamine and mercuric ion, unlike the ADP-ribosylcysteine formed by pertussis toxin and NAD in guanine nucleotide-binding (G-) proteins, which is labile to mercuric ion but stable in hydroxylamine. In the absence of G-proteins but in the presence of NAD and cysteine, pertussis toxin generated a hydroxylamine-sensitive product, suggesting that a free ADP-ribose intermediate, expected to be formed by the NADase activity of the toxin, reacted with cysteine. Chemical analysis, or the use of alternative thiol acceptors lacking a free amine, is necessary to distinguish the enzymatic formation of ADP-ribosylcysteine from nonenzymatic formation of ADP-ribosylthiazolidine, thereby differentiating putative NAD:cysteine ADP-ribosyltransferases from NAD glycohydrolases.     

NAD(P)(+)-glycohydrolase from human spleen: a multicatalytic enzyme

NAD(P)(+)-glycohydrolase (NADase, EC 3.2.2.6) was partially purified from microsomal membranes of human spleen after solubilization with Triton X-100. In addition to NAD+ and NADP+, the enzyme catalyzed the hydrolysis of several NAD+ analogues and the pyridine base exchange reaction with conversion of NAD+ into 3-acetylpyridine adenine dinucleotide. The enzyme also catalyzed the synthesis of cyclic ADP-ribose (cADPR) from NAD+ and the hydrolysis of cADPR to adenosine diphosphoribose (ADPR). Therefore, this enzyme is a new member of multicatalytic NADases recently identified from mammals, involved in the regulation of intracellular cADPR concentration. Human spleen NADase showed a subunit molecular mass of 45 kDa, a pI of 4.9 and a Km value for NAD+ of 26 microM. High activation of ADPR cyclase activity was observed in the presence of Ag+ ions, corresponding to NADase inhibition.     

Mutants of Neurospora deficient in nicotinamide adenine dinucleotide (phosphate) glycohydrolase.

A new screening technique has been developed for the rapid identification of Neurospora crassa mutants that are deficient in nicotinamide adenine dinucleotide glycohydrolase (NADase) and nicotinamide adenine dinucleotide phosphate glycohydrolase (NADPase) activities. Using this procedure, five single-gene mutants were isolated whose singular difference from wild type appeared to be the absence of NAD(P)ase (EC 3.2.2.6). All five mutants were found to be genetically allelic and did not complement in heterocaryons. This gene, nada [NAD(P)ase], was localized in linkage group IV. One of the nada alleles was found to specify an enzyme that was critically temperature sensitive and had altered substrate affinity. Mutations at the nada locus did not affect the genetic program for the expression of NAD(P)ase during cell differentiation, nor did they have a general effect on NAD catabolism. Nada mutations did not have simultaneous effects on other glycohydrolase activities. Tests of dominance (in heterocaryons) and in vitro mixing experiments did not provide evidence that nada mutations alter activators or inhibitors of NAD(P)ase. Thus, the nada gene appears to specify only the structure of N. crassa NAD(P)ase.     

Protein binding of nicotinamide adenine dinucleotide and regulation of nicotinamide adenine dinucleotide glycohydrolase activity in homogenates of rabbit skeletal muscle

Gel-permeation chromatography and ultrafiltration have been used to study the free and bound forms of NAD in crude extracts prepared from rabbit muscle. Both techniques indicate that over 80% of the endogenous NAD is free.Nicotinamide inhibits the destruction of NAD in muscle homogenates (50% inhibition at 1.6 mm nicotinamide). In the absence of nicotinamide, there is a rapid destruction of free NAD, but a more gradual destruction of bound NAD. The latter result confirms earlier findings that bound NAD is protected from the hydrolytic action of NADase. However, this protection is unlikely to constitute an important mechanism for controlling NADase activity in muscle homogenates because such a small proportion of the endogenous NAD is bound.In the absence of nicotinamide, NAD also disappears rapidly from minced muscle. Interestingly, the NAD/NADH ratio remains constant (NAD/NADH = 18.1–18.5) during the disappearance of NAD in minced muscle. Upon homogenization of the mince, the NAD/NADH ratio abruptly decreases, then slowly increases during subsequent incubation. The latter rise in NAD/NADH ratio appears to be independent of absolute changes in NAD concentration brought about by the action of NADase or the addition of exogenous NAD.     

Synthesis and biological activity of N6-(N[(4-azido-3,5,6-trifluoro)-pyridin-2-yl]-2-aminoethyl)-adenosine 5'-monophosphate, a new AMP photoactivatable derivative. Covalent modification of horse liver alcohol dehydrogenase

N6-(N-[(4-Azido-3,5,6-trifluoro)pyridin-2-yl]-2-aminoethyl)- adenosine 5'-monophosphate has been synthesized and evidence presented for its structural assignment by ultraviolet and 19F-NMR spectroscopies. Its photolysis was shown to occur within 5 min. This AMP derivative behaves as a competitive inhibitor of NAD+ in horse-liver-alcohol-dehydrogenase-promoted oxidation of ethanol, with a Ki (0.95 mM) comparable to the Ki of AMP (1.9 mM). Moreover it is an activator of the enzyme when nicotinamide ribose is used as the oxidation cofactor. This activation is as good as that promoted by AMP or by the well known 8-azido-AMP. Upon photolysis of this new derivative in the presence of horse liver alcohol dehydrogenase, a covalent enzyme--analogue complex was isolated and assayed as a catalyst in the oxidation of ethanol using nicotinamide ribose as the cofactor. The reaction took place without complementation of AMP, indicating clearly that the AMP analogue is mainly covalently bound in the AMP-binding site, and that the linkage formed between the enzyme and the azido derivative has not dramatically altered the active site of the enzyme. A similar experiment with 8-azido-AMP produced a completely inactive complex.     

Degradation of NAD by Synaptosomes and Its Inhibition by Nicotinamide Mononucleotide: Implications for the Role of NAD as a Synaptic Modulator

We have found NAD to be rapidly degraded by extracellular enzymes present on intact rat brain synaptosomes. The enzyme involved had the specificity of an NADase cleaving the molecule at the nicotinamide-glycoside linkage and was inhibited by nicotinamide mononucleotide (NMN). This inhibitor did not displace specific binding of NAD to rat brain membranes or affect electrical activity in the guinea pig hippocampus. Therefore, inclusion of NMN in binding assays allowed unambiguous demonstration of two specific NAD binding sites on rat brain synaptosomal membranes (KD1, 82 nM, KD2, 1.98 microM). The depressant action of NAD on the evoked synaptic activity of the guinea pig hippocampus was not blocked after inhibition of NAD degradation with NMN. The physiological implications of these results for the function of NAD as a neurotransmitter or neuromodulator in the CNS are discussed.     

Synthesis and properties of photoaffinity labels for the pyridine dinucleotide binding site of NAD glycohydrolase.

Two new photoactive analogues of oxidized nicotinamide adenine dinucleotide (NAD+) which are resistant to cleavage by NAD glycohydrolase were synthesized and characterized. The beta-D-ribonucleotide ring of the nicotinamide riboside moiety of NAD+ was replaced with a 2,3-dihydroxycyclopentane ring forming a carbocyclic dinucleotide analogue. Photoreactivity was achieved by the incorporation of an azido group at the 8-position of the adenosyl ring. The previously published synthesis of carbocyclic pyridine dinucleotide analogues [Slama, J. T., & Simmons, A. M. (1988) Biochemistry 27, 183] was modified by resolving the carbocyclic 1-aminoribose analogues and producing optically pure (+)-(1S)- or (-)-(1R)-4 beta-amino-2 alpha,3 alpha-dihydroxy-1 beta-cyclopentanemethanol. Each of these was converted to the corresponding carbocyclic nicotinamide 5'-nucleotide analogue and coupled with 8-azidoadenosine 5'-monophosphate. Two photoactive and isomeric NAD+ analogues were thus prepared. 8-Azidoadenosyl carba-NAD is the analogue in which D-dihydroxycyclopentane is substituted for the D-ribose of the nicotinamide nucleoside moiety. 8-Azido-adenosyl pseudocarba-NAD contains the L-carbocycle in place of the D-ribotide ring. 8-Azidoadenosyl carba-NAD was shown to inhibit the NAD glycohydrolase from Bungarus fasciatus venom competitively with an inhibitor dissociation constant of 187 microM. 8-Azidoadenosyl pseudocarba-NAD was shown to inhibit the same enzyme competitively with a Ki of 73 microM. The superior NADase inhibitor, 8-azidoadenosyl pseudocarba-NAD, was characterized kinetically and shown to fulfill the criteria required of a specific active site directed photoaffinity probe. Irradiation of mixtures of the photoprobe and NAD glycohydrolase with short-wave ultraviolet light resulted in the rapid and irreversible loss of enzyme activity.(ABSTRACT TRUNCATED AT 250 WORDS)     

Calf-spleen nicotinamide-adenine dinucleotide glycohydrolase. Properties of the active site.

The interaction between the nicotinamide adenine dinucleotide binding domain of calf spleen NAD glycohydrolase and its ligands has been studied. The use of competitive inhibitors, structurally related to different portions of the NAD molecule (i.e. adenosine and nicotinamide moieties), revealed the considerable importance of the binding between the pyrophosphate linkage and probably an arginyl residue of the active site. This interaction allows the positioning of the substrate in a conformation which permits catalysis to occur. The binding between the 2'-hydroxyl of the adenosine moiety and a residue of the active site, which exists in NAD-linked dehydrogenases, is probably missing in the calf spleen NAD glycohydrolase, based on the inhibition by salicylates, 2'-deoxyadenosine 5'-monophosphate and the hydrolysis of the 2'-deoxyadenosine analogue of NAD. The NAD glycohydrolase could be completely inactivated by 2,3-butanedione, an arginyl-modifying reagent. The reaction followed pseudo-first-order kinetics and the modification was found to be reversible. Woodward's reagent K, a reagent for carboxyl residues, partially inactivated the enzyme, which resulted in a change of the NAD glycohydrolase kinetic parameters Km and V. The inactivation rate was complicated by a parallel decomposition of the reagent.     

Pyridine nucleosidase in bull semen. II. Biochemical properties.

It is most likely a single enzyme (NAD+ nucleosidase) present in semen from most bulls which hydrolyses the ribosyl pyridinium bond in both NAD and NADP. This conclusion is based on the following results: (i) each of 12 semen samples containing nucleosidase activity hydrolysed NAD at the same rate as NADP (r = 0.99); (ii) other untreated semen samples from different bulls which did not hydrolyse NAD were also inactive against NADP; (iii) enzyme denaturation produced by preliminary heating of semen filtrates for 15 min at varied temperatures or by heating at 55 degrees C for varied time intervals caused similar reductions in the rates of NAD and NADP hydrolysis; and (iv) nicotinamide inhibited enzyme activity to the same degree using either NAD or NADP as the substrate.     

Lipoamide dehyrogenase immobilized on porous glass.

Lipoamide dehydrogenase (NADH:lipoamide oxidoreductase, EC 1.6.4.3) isolate from pig heart and Escherichia coli was covalently coupled by both diazonium and amide bonds to controlled pore glass beads (96% silica). When the enzyme was immobilized in the presence of NAD+, the enzyme no longer exhibited its normal requirement for NAD+ for full activity. If the immobilized enzyme was then treated with NADase, the requirement for NAD+ was restored. Enzyme immobilized in the absence of NAD+ exhibited normal NAD+ dependence both prior to an after NADase treatment. These results are discussed in terms of co-immobilization of NAD+ at or near the allosteric site of the enzyme.     

Purification and comparative properties of the glycoprotein nicotinamide adenine dinucleotide glycohydrolase from rat liver microsomal and plasma membranes.

NAD glycohydrolase, or NADase (NAD+ glycohydrolase, EC 3.2.2.5) was solubilized with porcine pancreatic lipase from isolated fractions of microsomes and plasma membranes obtained from rat livers. The enzyme from each organelle was further purified by DEAE-cellulose chromatography, gel filtration and isoelectric focusing. The solubilized, partially purified enzymes had similar molecular weights, pH-activity profiles and Km values. Marked charge heterogeneity was observed for the microsomal enzyme on isoelectric focusing between pH 6 and 8 with maximum activity focusing at pH 8.0. Plasma membrane NADase displayed a single peak at pH 6.7. Treatment of the partially purified microsomal or plasma membrane enzyme with neuraminidase resulted in a single peak of activity on isoelectric focusing (pH 3.5--10) with a pI of 9.2. Polyacrylamide gel electrophoresis of either NADase revealed a periodate-Schiff positive band which was coincident with enzyme activity. Compositional analyses of the microsomal enzyme focusing at pH 8.0 confirmed the presence of hexoses, hexosamines and sialic acid. Differences in carbohydrate composition might be important in determining the subcellular distribution of this enzyme.     

NAD Glycohydrolases: A possible function in calcium homeostasis

NAD glycohydrolases are the longest known enzymes that catalyze ADP-ribose transfer. The function of these ubiquitous, membrane-bound enzymes has been a long standing puzzle. The NAD glycohydrolase are briefly reviewed in light of the discovery by our laboratory that NAD glycohydrolases are bifunctional enzymes that can catalyze both the synthesis and hydrolysis of cyclic ADP-ribose, a putative second messenger of calcium homeostasis.Abbreviations NADase nicotinamide adenine dinucleotide glycohydrolase - NAD nicotinamide adenine dinucleotide - ADP-ribose adenosine diphosphoribose - cADPR cyclic adenosine diphosphoribose     

Polypeptide synthetase activity of chromatin from eukaryotic cells]

It is shown that the polypeptide synthetase activity (PS-activity) of chromatin from rat liver is increased 9--21 hrs after partial hepatectomy. Among 9 amino acids studied alanine, methionine, lysine, tyrosine and arginine are not incorporated into the system in question. The highest rate of polymerization is observed in case of glutamic acid. The rate of glutamine, asparagine and glycine incorporation is 7--8 times slower. The PS-activity of chromatin is enhanced by chromatin preincubation with NAD (but not with its analogs). The activation is prevented by thymidine and nicotinamide. Storage of chromatin for 18 hrs at 2--4 degrees C results in a complete loss of PS-activity. Ability of "old" chromatin to incorporate of amino acids may be restored by its preincubation with NAD. Storage of chromatin in the presence of 5 mM cAMP does not decrease the PS-activity. It is assumed that in the system described poly-ADP ribose is an energy source for amino acid activation.