NAD Glycohydrolases Research Articles

Snake venom NAD glycohydrolases: primary structures, genomic location, and gene structure.

NAD glycohydrolase (EC 3.2.2.5) (NADase) sequences have been identified in 10 elapid and crotalid venom gland transcriptomes, eight of which are complete. These sequences show very high homology, but elapid and crotalid sequences also display consistent differences. As in Aplysia kurodai ADP-ribosyl cyclase and vertebrate CD38 genes, snake venom NADase genes comprise eight exons; however, in the Protobothrops mucrosquamatus genome, the sixth exon is sometimes not transcribed, yielding a shortened NADase mRNA that encodes all six disulfide bonds, but an active site that lacks the catalytic glutamate residue. The function of this shortened protein, if expressed, is unknown. While many vertebrate CD38s are multifunctional, liberating both ADP-ribose and small quantities of cyclic ADP-ribose (cADPR), snake venom CD38 homologs are dedicated NADases. They possess the invariant TLEDTL sequence (residues 144–149) that bounds the active site and the catalytic residue, Glu228. In addition, they possess a disulfide bond (Cys121–Cys202) that specifically prevents ADP-ribosyl cyclase activity in combination with Ile224, in lieu of phenylalanine, which is requisite for ADPR cyclases. In concert with venom phosphodiesterase and 5′-nucleotidase and their ecto-enzyme homologs in prey tissues, snake venom NADases comprise part of an envenomation strategy to liberate purine nucleosides, and particularly adenosine, in the prey, promoting prey immobilization via hypotension and paralysis.

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.

Mitochondrial Dysfunction and NAD+ Metabolism Alterations in the Pathophysiology of Acute Brain Injury

Mitochondrial dysfunction is commonly believed to be one of the major players in mechanisms of brain injury. For several decades, pathologic mitochondrial calcium overload and associated opening of the mitochondrial permeability transition (MPT) pore were considered a detrimental factor causing mitochondrial damage and bioenergetics failure. Mitochondrial and cellular bioenergetic metabolism depends on the enzymatic reactions that require NAD(+) or its reduced form NADH as cofactors. Recently, it was shown that NAD(+) also has an important function as a substrate for several NAD(+) glycohydrolases whose overactivation can contribute to cell death mechanisms. Furthermore, downstream metabolites of NAD(+) catabolism can also adversely affect cell viability. In contrast to the negative effects of NAD(+)-catabolizing enzymes, enzymes that constitute the NAD(+) biosynthesis pathway possess neuroprotective properties. In the first part of this review, we discuss the role of MPT in acute brain injury and its role in mitochondrial NAD(+) metabolism. Next, we focus on individual NAD(+) glycohydrolases, both cytosolic and mitochondrial, and their role in NAD(+) catabolism and brain damage. Finally, we discuss the potential effects of downstream products of NAD(+) degradation and associated enzymes as well as the role of NAD(+) resynthesis enzymes as potential therapeutic targets.

The TRPM2 ion channel, an oxidative stress and metabolic sensor regulating innate immunity and inflammation

TRPM2 (transient receptor potential melastatin 2) is the unique fusion of a Ca(2+)-permeable pore with an enzymatic domain that binds the NAD(+)-metabolite ADP-ribose (ADPR), resulting in channel opening. ADPR formation is a metabolic corollary of cellular stress, but can also be elicited enzymatically through NAD glycohydrolases like CD38. TRPM2 thus functions as a metabolic and oxidative stress sensor and translates this information into ion fluxes that can affect Ca(2+) signaling and the membrane potential. TRPM2 is strongly represented in immune cells of the phagocytic lineage, themselves professional generators of oxidants. The recent characterization of TRPM2-deficient mouse models has revealed the involvement of this channel in various aspects of immunity. Monocytes lacking TRPM2 show reduced production of the CXCL2 chemokine, resulting in diminished neutrophilic influx to the colon in chemically induced colitis, and thus protection against tissue ulceration in TRPM2(-/-) mice. However, the insufficient production of proinflammatory cytokines leads to high morbidity and lethality of the TRPM2(-/-) mice following infection with the bacterial pathogen Listeria monocytogenes. In the context of endotoxin-induced pulmonary inflammation, TRPM2's absence was found to promote inflammation and ROS production. TRPM2 acts thereby as a negative feedback loop by interfering through membrane depolarization with ROS generation by NADPH oxidases. In dendritic cells, TRPM2 is a lysosomal Ca(2+)-release channel that promotes chemokine responsiveness and cell migration, which is reminiscent of CD38-mediated functions. The discovery of TRPM2 has unveiled an unsuspected signaling pathway and established ADPR as a novel second messenger. Understanding TRPM2's complex involvement in inflammation is crucial to evaluating the potential of manipulating TRPM2 activity and ADPR metabolism for therapeutic intervention.

Mitochondrial dysfunction and nicotinamide dinucleotide catabolism as mechanisms of cell death and promising targets for neuroprotection

Both acute and chronic neurodegenerative diseases are frequently associated with mitochondrial dysfunction as an essential component of mechanisms leading to brain damage. Although loss of mitochondrial functions resulting from prolonged activation of the mitochondrial permeability transition (MPT) pore has been shown to play a significant role in perturbation of cellular bioenergetics and in cell death, the detailed mechanisms are still elusive. Enzymatic reactions linked to glycolysis, the tricarboxylic acid cycle, and mitochondrial respiration are dependent on the reduced or oxidized form of nicotinamide dinucleotide [NAD(H)] as a cofactor. Loss of mitochondrial NAD(+) resulting from MPT pore opening, although transient, allows detrimental depletion of mitochondrial and cellular NAD(+) pools by activated NAD(+) glycohydrolases. Poly(ADP-ribose) polymerase (PARP) is considered to be a major NAD(+) degrading enzyme, particularly under conditions of extensive DNA damage. We propose that CD38, a main cellular NAD(+) level regulator, can significantly contribute to NAD(+) catabolism. We discuss NAD(+) catabolic and NAD(+) synthesis pathways and their role in different strategies to prevent cellular NAD(+) degradation in brain, particularly following an ischemic insult. These therapeutic approaches are based on utilizing endogenous intermediates of NAD(+) metabolism that feed into the NAD(+) salvage pathway and also inhibit CD38 activity.

Hydrolysis of the phosphoanhydride linkage of cyclic ADP-ribose by the Mn 2+-dependent ADP-ribose/CDP-alcohol pyrophosphatase

Cyclic ADP-ribose (cADPR) metabolism in mammals is catalyzed by NAD glycohydrolases (NADases) that, besides forming ADP-ribose, form and hydrolyze the N 1-glycosidic linkage of cADPR. Thus far, no cADPR phosphohydrolase was known. We tested rat ADP-ribose/CDP-alcohol pyrophosphatase (ADPRibase-Mn) and found that cADPR is an ADPRibase-Mn ligand and substrate. ADPRibase-Mn activity on cADPR was 65-fold less efficient than on ADP-ribose, the best substrate. This is similar to the ADP-ribose/cADPR formation ratio by NADases. The product of cADPR phosphohydrolysis by ADPRibase-Mn was N 1-(5-phosphoribosyl)-AMP, suggesting a novel route for cADPR turnover.

Regulatory Role of Arginine 204 in the Catalytic Activity of Rat Alloantigens ART2a and ART2b

ART2a (RT6.1) and ART2b (RT6.2) are NAD glycohydrolases (NADases) that are linked to T lymphocytes by glycosylphosphatidylinositol anchors. Although both mature proteins possess three conserved regions (I, II, III) that form the NAD-binding site and differ by only ten amino acids, only ART2b is auto-ADP-ribosylated and only ART2a is glycosylated. To investigate the structural basis for these differences, wild-type and mutant ART2a and ART2b were expressed in rat mammary adenocarcinoma (NMU) cells and released with phosphatidylinositol-specific phospholipase C. All mutants were immunoreactive NADases. Arginine 204 (Arg204), NH2-terminal to essential glutamate 209 in Region III, is found in ART2b, but not ART2a. Replacement of Arg204 in ART2b with lysine, tyrosine, or glutamate abolished auto-ADP-ribosylation. Unlike wild-type ART2a, ART2a(Y204R) was auto-ADP-ribosylated. The tryptophan mutant ART2b(R204W) was auto-ADP-ribosylated and exhibited enhanced NADase activity. Incubation with NAD and auto-ADP-ribosylation decreased the NADase activities of wild-type ART2b and ART2b (R204W), whereas activity of ART2b(R204K), which is not auto-modified, was unchanged by NAD. Facilitation of auto-ADP-ribosylation by tryptophan 204 suggests that the hydrophobic amino acid mimics an ADP-ribosylated arginine. Thus, Arg204 in ART2b serves as a regulatory switch whose presence is required for additional auto-ADP-ribosylation and regulation of catalytic activity.

NAD-dependent inhibition of the NAD-glycohydrolase activity in A549 cells.

NAD glycohydrolases are enzymes that catalyze the hydrolysis of NAD to produce ADP-ribose and nicotinamide. Regulation of these enzymes has not been fully elucidated. We have identified a NAD-glycohydrolase activity associated with the outer surface of the plasma membrane in human lung epithelial cell line A549. This activity is negatively regulated by its substrate beta-NAD but not by alpha-NAD. Partial restoration of NADase activity after incubation of the cells with arginine or histidine, known ADP-ribose acceptors, suggests that inhibition be regulated by ADP-ribosylation. A549 do not undergo to apoptosis upon NAD treatment indicating that this effect be likely mediated by a cellular component(s) lacking in epithelial cells.

Synthesis of pyridine nucleotide analogs consisting of nicotinoylamino acids by means of transglycosidation reactions catalyzed by mammalian pyridine nucleotide transglycosidases.

Besides isonicotinic acid analogs of pyridine nucleotides, 24 novel pyridine nucleotide cofactors that have an amino acid residue at the carbonyl carbon of the nicotinamide moiety have been prepared by means of transglycosidation reactions catalyzed by rabbit spleen and guinea pig spleen pyridine nucleotide transglycosidases. Their chemical properties were characterized by means of proton NMR, Fab-mass, and UV spectral measurement and phosphodiesterase digestion. Except for the isonicotinic acid ones, these nicotinoylamino acid analogs were shown to function as substrates for both the hydrolysis and the transglycosidation reactions catalyzed by the mammalian NAD glycohydrolases, though their substrate activities were lower than those with the original pyridine nucleotides (NMN, NAD, and NADP). They were inactive in regard to yeast alcohol dehydrogenase- and Thermoanaerobium brockii alcohol dehydrogenase (NADP dependent)-oxidation.

Characterization of Mouse Rt6.1 NAD:Arginine ADP-ribosyltransferase

Rat RT6 proteins, and perhaps mouse Rt6, identify a set of immunoregulatory T lymphocytes. Rat RT6.1 (RT6.1) and rat RT6.2 (RT6. 2) are NAD glycohydrolases, which catalyze auto-ADP-ribosylation, but not ADP-ribosylation of exogenous proteins. Mouse Rt6.1 (mRt6.1) also catalyzes auto-ADP-ribosylation. The activity of mouse cytotoxic T lymphocytes is reportedly inhibited by ADP-ribosylation of surface proteins, raising the possibility that mRt6 may participate in this process. The reactions catalyzed by mRt6, would, however, need to be more diverse than those of the rat homologues and include the ADP-ribosylation of acceptors other than itself. To test this hypothesis, mRt6.1 and rat RT6.2 were synthesized in Sf9 insect cells and rat mammary adenocarcinoma (NMU) cells. mRt6.1, but not rat RT6.2, catalyzed the ADP-ribosylation of guanidino-containing compounds (e.g. agmatine). Unlike RT6.2, mRt6.1 was a weak NAD glycohydrolase. In the presence of agmatine, however, the ratio of [adenine-14C]ADP-ribosylagmatine formation from [adenine-14C]NAD to [carbonyl-14C]nicotinamide formation from [carbonyl-14C]NAD was approximately 1.0, demonstrating that mRt6.1 is primarily a transferase. ADP-ribosylarginine hydrolase, which preferentially hydrolyzes the alpha-anomer of ADP-ribosylarginine, released [U-14C]arginine from ADP-ribosyl[U-14C]arginine synthesized by mRT6.1, consistent with the conclusion that mRt6.1 catalyzes a stereospecific Sn2-like reaction. Thus, mRt6.1 is an NAD:arginine ADP-ribosyltransferase capable of catalyzing a multiple turnover, stereospecific Sn2-like reaction.

Cloning and Characterization of a Novel Membrane-associated Lymphocyte NAD:Arginine ADP-ribosyltransferase

Mono-ADP-ribosylation is a post-translational modification of proteins in which the ADP-ribose moiety of NAD is transferred to proteins and is responsible for the toxicity of some bacterial toxins (e.g. cholera toxin and pertussis toxin). NAD:arginine ADP-ribosyltransferases cloned from human and rabbit skeletal muscle and from mouse lymphoma (Yac-1) cells are glycosylphosphatidylinositol-anchored and have similar enzymatic and physical properties; transferases cloned from chicken heterophils and red cells have signal peptides and may be secreted. We report here the cloning and characterization of an ADP-ribosyltransferase (Yac-2), also from Yac-1 lymphoma cells, that differs in properties from the previously identified eukaryotic transferases. The nucleotide and deduced amino acid sequences of the Yac-1 and Yac-2 transferases are 58 and 33% identical, respectively. The Yac-2 protein is membrane-bound but, unlike the Yac-1 enzyme, appears not to be glycosylphosphatidylinositol-anchored. The Yac-1 and Yac-2 enzymes, expressed as glutathione S-transferase fusion proteins in Escherichia coli, were used to compare their ADP-ribosyltransferase and NAD glycohydrolase activities. Using agmatine as the ADP-ribose acceptor, the Yac-1 enzyme was predominantly an ADP-ribosyltransferase, whereas the transferase and NAD glycohydrolase activities of the recombinant Yac-2 protein were equivalent. The deduced amino acid sequence of the Yac-2 transferase contained consensus regions common to several bacterial toxin and mammalian transferases and NAD glycohydrolases, consistent with the hypothesis that there is a common mechanism of NAD binding and catalysis among ADP-ribosyltransferases.

Structure and function of eukaryotic mono-ADP-ribosyltransferases.

ADP-ribosylation of proteins has been observed in numerous animal tissues including chicken heterophils, rat brain, human platelets, and mouse skeletal muscle. ADP-ribosylation in these tissues is thought to modulate critical cellular functions such as muscle cell development, actin polymerization, and cytotoxic T lymphocyte proliferation. Specific substrates of the ADP-ribosyltransferases have been identified; the skeletal muscle transferase ADP-ribosylates integrin alpha 7 whereas the chicken heterophil enzyme modifies the heterophil granule protein p33 and the CTL enzyme ADP-ribosylates the membrane-associated protein p40. Transferase sequence has been determined which should assist in elucidating the role of ADP-ribosylation in cells. There is sequence similarity among the vertebrate transferases and the rodent RT6 alloantigens. The RT6 family of proteins are NAD glycohydrolases that have been shown to possess auto-ADP-ribosyltransferase activity whereas the mouse Rt6-1 is also capable of ADP-ribosylating histone. Absence of RT6+ T cells has been associated with the development of an autoimmune-mediated diabetes in rodents. Humans have an RT6 pseudogene and do not express RT6 proteins. The reversal of ADP-ribosylation is catalyzed by ADP-ribosylarginine hydrolases, which have been purified and cloned from rodent and human tissues. In principle, the transferases and hydrolases could form an intracellular ADP-ribosylation regulatory cycle. In skeletal muscle and lymphocytes, however, the transferases and their substrates are extracellular membrane proteins whereas the hydrolases described thus far are cytoplasmic. In cultured mouse skeletal muscle cells, processing of the ADP-ribosylated integrin alpha 7 was carried out by phosphodiesterases and possibly phosphatases, leaving a residual ribose attached to the (arginine)protein. Several bacterial toxin and eukaryotic mono-ADP-ribosyltransferases, and perhaps other NAD-utilizing enzymes such as the RT6 alloantigens share regions of amino acid sequence similarity, which form, in part, the catalytic site. The catalytic cleft, found in the bacterial toxins that have been studied thus far, contains a critical glutamate and other amino acids that function to position NAD for nucleophilic attack at the N-glycosidic linkage, for either ADP-ribose transfer or NAD hydrolysis. Amino acid differences among the transferases at the active site may be required for accommodating the different ADP-ribose acceptor molecules.

NAD glycohydrolases and the metabolism of cyclic ADP-ribose

Cyclic ADP-ribose is a recently discovered metabolite of NAD that functions in cellular calcium signalling. The discovery that NAD glycohydrolases can catalyze the synthesis and hydrolysis of cyclic ADP-ribose has renewed interest in this class of ADP-ribose transferring enzymes that were discovered over 50 years ago.

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 glycohydrolases 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.

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.

Purification and characterization of a pyridine nucleotide glycohydrolase from rabbit spleen.

A particulate NMN glycohydrolase of rabbit spleen was solubilized with Triton X100 and purified approximately 100-fold. The enzyme was shown to have a pH maximum of 6.5, a Km of 0.25 mM, a Vmax of 5.3 mumol/min/mg protein, an activation energy of 7.9 kcal/mol, and a molecular weight of approximately 400,000. Both of the purified and the particulate enzymes exhibited identical catalytic properties with respect to substrate specificity, activation energy, pH profile and exchange reaction with nicotinic acid, except that the purified enzyme was highly activated with Triton X100 as compared with the particulate enzyme; it appears that the purified enzyme possesses the same catalytic properties as the enzyme present in the tissue and that solubilization does not significantly alter the native protein. In addition to catalytic activity with NMN, the rabbit spleen enzyme catalyzed an irreversible hydrolysis with NAD and NADP, exhibiting catalyzing activity ratios of NMN:NAD:NADP = 1.00:1.45:0.44 and Vmax/Km ratios of 1.00:1.7:2.3, respectively. These ratios of activity remained constant throughout purification of the enzyme and no separation of these activities was detected. Mutually competitive inhibition of the enzyme with Ki values similar to Km, and identical rates of thermal denaturation of the enzyme and activity-pH profiles with NMN or NAD indicated the hydrolysis of the C-N glycosidic linkage of the pyridine nucleotides to be catalyzed by the same enzyme. The enzyme was less specific for the purine structure of the substrate dinucleotides but was stereospecific for the glycosidic linkage cleaved. Nicotinamide riboside, the nicotinic acid analogs and the reduced forms were not hydrolyzed. A linear noncompetitive inhibition of NMN hydrolysis with nicotinamide indicated an ordered Uni-Bi mechanism in which nicotinamide was the first product released from the enzyme. A property that the rabbit spleen enzyme appears to share with other NAD glycohydrolases is the transglycosidation reaction. The ratio of transglycosidation reaction vs. hydrolysis catalyzed by the enzyme in the presence of NMN and nicotinic acid indicated that the enzyme could function as a primary transglycosidase rather than a hydrolytic enzyme in vivo.

Inhibition of NAD glycohydrolase and ADP-ribosyl transferases by carbocyclic analogs of oxidized nicotinamide adenine dinucleotide

Analogues of oxidized nicotinamide adenine dinucleotide (NAD+) in which a 2,3-dihydroxycyclopentane ring replaces the beta-D-ribonucleotide ring of the nicotinamide riboside moiety of NAD+ have recently been synthesized [Slama, J. T., & Simmons, A. M. (1988) Biochemistry 27, 183]. Carbocyclic NAD+ analogues have been shown to inhibit NAD glycohydrolases and ADP-ribosyl transferases such as cholera toxin A subunit. In this study, the diastereomeric mixture of dinucleotides was separated, and the inhibitory capacity of each of the purified diastereomers was defined. The NAD+ analogue in which the D-dihydroxycyclopentane is substituted for the D-ribose is designated carba-NAD and was demonstrated to be a poor inhibitor of the Bungarus fasciatus venom NAD glycohydrolase. The diastereomeric dinucleotide pseudo-carbocyclic-NAD (psi-carba-NAD), containing L-dihydroxycyclopentane in place of the D-ribose of NAD+, was shown, however, to be a potent competitive inhibitor of the venom NAD glycohydrolase with an inhibitor dissociation constant (Ki) of 35 microM. This was surprising since psi-carba-NAD contains the carbocyclic analogue of the unnatural L-ribotide and was therefore expected to be a biologically inactive diastereomer. psi-Carba-NAD also competitively inhibited the insoluble brain NAD glycohydrolase from cow (Ki = 6.7 microM) and sheep (Ki = 31 microM) enzyme against which carba-NAD is ineffective. Sensitivity to psi-carba-NAD was found to parallel sensitivity to inhibition by isonicotinic acid hydrazide, another NADase inhibitor. psi-Carba-NAD is neither a substrate for nor an inhibitor of alcohol dehydrogenase, whereas carba-NAD is an efficient dehydrogenase substrate.(ABSTRACT TRUNCATED AT 250 WORDS)

ADP-ribosylation of membrane proteins by bacterial toxins in the presence of NAD glycohydrolase

The ADP-ribosylation of membrane G proteins is difficult to achieve in tissues that are rich in membranebound NAD glycohydrolase (NAD + glycohydrolase, EC 3.2.2.5). For many animal species this problem can be surmounted by inhibiting NAD hydrolysis with a combination of the anti-tuberculous drug, isonicotinic acid hydrazide, and the NAD analog, 3-acetylpyridine adenine dinucleotide, which act synergistically. In their presence, the ADP-ribosylation of cholera and pertussis toxin substrates reach plateau levels even with only 10 μM NAD. Although 3-acetylpyridine adenine dinucleotide acts as a weak substrate for the toxins, it is simple to estimate its contribution to the ADP-ribosylation and thus to determine the total amount of ADP-ribosylation substrate present in a tissue sample. NAD glycohydrolases that are insensitive to isonicotinic acid hydrazide are also less sensitive to 3-acetylpyridine adenine dinucleotide, but may be inactivated by dithiothreitol. Isonicotinic acid hydrazide adenine dinucleotide, the product of an exchange reaction eatalysed by NAD glycohydrolase, runs with NAD in most thin-layer chromatographic systems. It can be separated from NAD, and quantitated if the chromatographic solvent contains benzaldehyde. Isonicotinic acid hydrazide itself inhibits NAD glycohydrolase. It need not first be converted into isonicotinic acid hydrazide adenine dinucleotide.

Site-directed enzyme inactivation studies of 3-aminopyridine 1, N6-ethenoadenine dinucleotide

3-Aminopyridine 1, N 6-ethenoadenine dinucleotide (ϵ-AAD) was prepared from 3-aminopyridine and ϵ-NAD via the pyridine base exchange reaction catalyzed by purified Bungarus fasciatus venom NAD glycohydrolase. ϵ-AAD exhibited a fluorescence spectrum characteristic of the presence of the two fluorescing heterocyclic bases. ϵ-AAD was diazotized in nitrous acid and the resulting diazonium derivative was coupled with N-(1-naphthyl)ethylenediamine to form an azodye. Diazotized ϵ-AAD retained the fluorescence characteristics of the ethenoadenine moiety. Selective binding of ϵ-AAD to several dehydrogenases and NAD glycohydrolases was demonstrated through competitive inhibition studies. Diazotized ϵ-AAD rapidly and irreversibly inactivated yeast alcohol dehydrogenase at pH 7.0. The enzyme was protected from inactivation by the presence of NAD. Analysis of the fluorescing ethenoadenine moiety covalently attached to the inactivated dehydrogenase demonstrated the presence of 4 mol of ϵ-AAD bound per mole of enzyme (one per catalytic subunit). Fluorescence titration of the inactivated dehydrogenase by fluorescein mercuric acetate revealed the loss of four sulfhydryl groups per mole of enzyme during the inactivation process.

Isolation and properties of an NAD- and guanidine-dependent ADP-ribosyltransferase from turkey erythrocytes.

An NAD- and guanidine-dependent ADP-ribosyltransferase has been purified more than 500,000-fold from turkey erythrocytes with an 18% yield. The enzyme in the 100,000 X g supernatant fraction was bound to phenyl-Sepharose, eluted with 50% propylene glycol, and further purified by sequential chromatographic steps on carboxymethylcellulose, NAD-agarose and concanavalin A-agarose. The transferase was specifically eluted from concanavalin A-agarose with alpha-methylmannoside. The enzymatic activity was extremely labile following the first purification step. Both propylene glycol and NaCl stabilized the transferase; significant increases in enzyme recovery were obtained by conducting the NAD- and concanavalin A-agarose chromatography in buffer containing propylene glycol. The purified protein exhibits one predominant protein band on SDS-polyacrylamide gels with an estimated molecular weight of 28,300. On Ultrogel AcA54 chromatography, single coincident peaks of ADP-ribosyltransferase activity and protein were observed. Enzyme activity was independent of DNA; the highly purified transferase was inhibited by thymidine, nicotinamide, and theophylline. The specific activity of the purified enzyme (350 mumol of ADP-ribose transferred from NAD to arginine methyl estermin-1mg-1) is comparable to that reported for purified NAD glycohydrolases and poly(ADP-ribosyl)transferases.