Inactivation of glutamine synthetases by an NAD:arginine ADP-ribosyltransferase

Glutamine synthetase from ovine brain has a critical arginine residue at the catalytic site (Powers, S. G., and Riordan, J.F. (1975) Proc. Natl. Acad. Sci. U.S. A. 72, 2616-2620). This enzyme is now shown to be a substrate for a purified NAD:arginine ADP-ribosyltransferase from turkey erythrocyte cytosol that catalyzes the transfer of ADP-ribose from NAD to arginine and purified proteins. The transferase catalyzed the inactivation of the synthetase in an NAD-dependent reaction; ADP-ribose and nicotinamide did not substitute for NAD. Agmatine, an alternate ADP-ribose acceptor in the transferase-catalyzed reaction, prevented inactivation of glutamine synthetase. MgATP, a substrate for the synthetase which was previously shown to protect that enzyme from chemical inactivation, also decreased the rate of inactivation in the presence of NAD and ADP-ribosyltransferase. Using [32P]NAD, it was observed that approximately 90% inactivation occurred following the transfer of 0.89 mol of [32P]ADP-ribose/mol of synthetase. The erythrocyte transferase also catalyzed the NAD-dependent inactivation of glutamine synthetase purified from chicken heart; 0.60 mol of ADP-ribose was transferred per mol of enzyme, resulting in a 95% inactivation. As noted with the ovine brain enzyme, agmatine and MgATP protected the chicken synthetase from inactivation and decreased the extent of [32P]ADP-ribosylation of the synthetase. These observations are consistent with the conclusion that the NAD:arginine ADP-ribosyltransferase modifies specifically an arginine residue involved in the catalytic site of glutamine synthetase. Although the transferase can use numerous proteins as ADP-ribose acceptors, some characteristics of this particular arginine, perhaps the same characteristics that are involved in its function in the catalytic site, make it a favored ADP-ribose acceptor site for the transferase.     

Inhibition of the transport of citrate during ADP-ribosylation of inner membrane proteins of the mitochondria

The ability of rat liver submitochondrial particles to catalyze NAD+ hydrolysis with a transfer of ADP-ribose residues to protein membranes has been demonstrated ADP-ribosylation is directly dependent on NAD+ concentration upon saturation with 1 mM NAD+ and is inhibited by physiological compounds (e.g., ATP, 10 mM; nicotinamide, 10 mM); besides, it is an artificial acceptor of ADP-ribose, arginine methyl ester. It was found that ADP-ribose is accepted by inner mitochondrial membrane protein, whose molecular masses amount to 25-30 kDa. The fact that 5'-AMP is a product of ADP-ribose degradation by snake venom phosphodiesterase suggests that the inner membrane vesiculate proteins are modified by mono(ADP-ribose). Covalent modification of membrane proteins by ADP-ribose leads to citrate transport inhibition in inner membrane vesicles the [14C]citrate uptake is significantly decreased thereby. The ability of ADP-ribosylation inhibitors to restore the citrate transport rate is suggestive of a direct regulatory effect of NAD+-dependent ADP-ribosylation on the activity of citrate-translocating system of inner mitochondrial membranes.     

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     

Amino acid specific ADP-ribosylation: substrate specificity of an ADP-ribosylarginine hydrolase from turkey erythrocytes

An ADP-ribosylarginine hydrolase, which catalyzes the degradation of ADP-ribosyl[14C]arginine to ADP-ribose plus arginine, was separated by ion exchange, hydrophobic, and gel permation chromatography from NAD:arginine ADP-ribosyltransferases, which are responsible for the stereospecific formation of alpha-ADP-ribosylarginine. As determined by NMR, the specific substrate for the hydrolase was alpha-ADP-ribosylarginine, the product of the transferase reaction. The ADP-ribose moiety was critical for substrate recognition; (phosphoribosyl) [14C]arginine and ribosyl[14C]arginine were poor substrates and did not significantly inhibit ADP-ribosyl[14C]arginine degradation. In contrast, ADP-ribose was a potent inhibitor of the hydrolase and significantly more active than ADP greater than AMP greater than adenosine. In addition to ADP-ribosyl[14C]arginine, both ADP-ribosyl[14C]guanidine and (2'-phospho-ADP-ribosyl)[14C]arginine were also substrates; at pH greater than 7, ADP-ribosyl[14C]guanidine was degraded more readily than the [14C]arginine derivative. Neither arginine, guanidine, nor agmatine, an arginine analogue, was an effective hydrolase inhibitor. Thus, it appears that the ADP-ribosyl moiety but not the arginine group is critical for substrate recognition. Although the hydrolase requires thiol for activity, dithiothreitol accelerated loss of activity during incubation at 37 degrees C. Stability was enhanced by Mg2+, which is also necessary for optimal enzymatic activity. The findings in this paper are consistent with the conclusion that different enzymes catalyze ADP-ribosylarginine synthesis and degradation. Furthermore, since the hydrolase and transferases possess a compatible stereospecificity and substrate specificity, it would appear that the two enzymatic activities may serve as opposing arms in an ADP-ribosylation cycle.     

Mechanism of action of choleragen. Evidence for ADP-ribosyltransferase activity with arginine as an acceptor.

Choleragen catalyzed the hydrolysis of NAD to ADP-ribose and nicotinamide; nicotinamide production was dramatically increased by L-arginine methyl ester and to a lesser extent by D- or L-arginine, but not by other basic amino acids. Guanidine was also effective. Nicotinamide formation in the presence of L-arginine methyl ester was greatest under conditions previously shown to accelerate the hydrolysis of NAD by choleragen (Moss, J., Manganiello, V. C., and Vaughan, M. (1976) Proc. Natl. Acad. Sci. U.S.A. 73, 4424-4427). After incubation of [adenine-U14C]NAD and L[3H]arginine with coleragen, a product was isolated by thin layer chromatography that contained adenine and arginine in a 1:1 ratio and has been tentatively identified as ADP-ribose-L-arginine. Parallel experiments with [carbonyl-14C]NAD have demonstrated that formation of the ADP-ribosyl-L-arginine derivative was associated with the production of [carbonyl-14C]nicotinamide. As guanidine itself was active and D- and L-arginine was equally effective in promoting nicotinamide production, whereas citrulline, which possesses a ureido rather than a guanidino function, was inactive, it seems probable that the guanidino group rather than the alpha-amino moiety participated in the linkage to ADP-ribose. Based on the assumption that the ADP-ribosylation of L-arginine by choleragen is a model for the NAD-dependent activation of adenylate cyclase by choleragen, it is proposed that the active A protomer of choleragen catalyzes the ADP-ribosylation of an arginine, or related amino acid residue in a protein, which is the cyclase itself or is critical to its activation by choleragen.     

ADP-ribosyl transferase and NAD glycohydrolase activities in rat liver mitochondria

ADP-ribosyl transferase and NAD glycohydrolase activities have been estimated in mitochondria in mitoplasts as well as in other submitochondrial fractions. A high activity of these two enzymes was present in mitoplasts as compared to the outer membrane preparation or intermembrane compartment. Inhibitor studies provide strong evidence for the involvement of ADP-ribosyl transferase in the process of ADP-ribosylation of mitochondrial proteins. When NAD glycohydrolase was blocked by nicotinamide or 3-aminobenzamide, the incorporation of ADP-ribose into mitochondrial proteins still occurs. ADP-ribosyl transferase activity could also be detected when NAD glycohydrolase was separated by hydroxylapatite chromatography. The protein-linked ADP-ribose moiety appears to be an oligomer in mitochondria.     

An extracellular monoADP-ribosyl transferase activity in Entamoeba histolytica trophozoites

Due to the important role of monoADP-ribosyl transferases in physiological and pathological events, we investigated whether the protozoan parasite Entamoeba histolytica had monoADP-ribosyl transferase activity. Reactions were initiated using ameba-free medium as the source of both enzyme and ADP-ribosylation substrate(s) and [32P]NAD+ as source of ADP-ribose. Proteins were analyzed by electrophoresis, and [32P]-labeled proteins were detected by autoradiography. Using the crude extracellular medium, a major labeled product of Mr 37.000 was observed. The yield of this product was reduced markedly using medium from Brefeldin A-treated trophozoites, indicating that the extracellular monoADP-ribosyl transferase and/or its substrate depended on vesicular transport. The labeling of the 37-kDa substrate was dependent on reaction time, temperature, pH, and the ratio of unlabeled NAD+ to [32P]NAD+. After two purification steps, several new substrates were observed, perhaps due to their enrichment. The reaction measured ADP-ribosylation since [14C-carbonyl]NAD+ was not incorporated into ameba substrates and a 75-fold molar excess of ADP-ribose caused no detectable inhibition of the monoADP-ribosyl transferase reaction. On the basis of sensitivity to NH2OH, the extracellular monoADP-ribosyl transferase of E. histolytica may be an arginine-specific enzyme. These results demonstrate the existence in E. histolytica of at least one extracellular monoADP-ribosyl transferase, whose localization depends upon a secretion process.     

ADP-Ribosylation of Highly Purified Rat Brain Mitochondria

Highly purified synaptic and nonsynaptic mitochondria were prepared from rat brain, and their ADP-ribosyl transferase and NAD glycohydrolase activities were investigated. Data show that there is no significant difference in ADP-ribosyl transferase activity between these two types of subcellular preparations. However, NAD glycohydrolase activity appeared to be much higher in nonsynaptic mitochondria. The specific activity of both enzymes was investigated in the presence of the inhibitor nicotinamide or its analogue 3-aminobenzamide or other adenine nucleotides, such as ATP or ADP-ribose. The inhibitory effect of nicotinamide or 3-aminobenzamide on ADP-ribosyl transferase appears rather weak compared with their effect on NAD glycohydrolase activity. However, ADP-ribose and ATP appeared more effective in inhibiting ADP-ribosyl transferase. Our results provide evidence for the existence of ADP-ribosyl transferase activity in rat brain mitochondria. When NAD glycohydrolase was inhibited totally by nicotinamide, the transfer of ADP-ribose from NAD to mitochondrial proteins still occurred. The chain length determinations show that the linkage of ADP-ribose to mitochondrial proteins is oligomeric.     

Amino acid specific ADP-ribosylation: specific NAD: arginine mono-ADP-ribosyltransferases associated with turkey erythrocyte nuclei and plasma membranes

Turkey erythrocytes contain NAD:arginine mono-ADP-ribosyltransferases which, like cholera toxin and Escherichia coli heat-labile enterotoxin, catalyze the transfer of ADP-ribose from NAD to proteins, to arginine and other low molecular weight guanidino compounds, and to water. Two such ADP-ribosyltransferases, A and B, have been purified from turkey erythrocyte cytosol. To characterize further the class of NAD:arginine ADP-ribosyltransferases, the particulate fraction was examined; 40% of erythrocyte transferase activity was localized to the nucleus and cell membrane. Transferase activity in a salt extract of a thoroughly washed particulate preparation was purified 36,000-fold by sequential chromatography on phenyl-Sepharose, (carboxymethyl) cellulose, concanavalin A-Sepharose, and NAD-agarose. Subsequent DNA-agarose chromatography separated two activities, termed transferases C and A', which were localized to the membrane and nucleus, respectively. Transferase C, the membrane-associated enzyme, was distinguished from the cytosolic enzymes by a relative insensitivity to salt and histone; transferase C was stimulated 2-fold by 300 mM NaCl in contrast to a 20-fold stimulation of transferase A and a 50% inhibition of transferase B. Similarly, histones, which stimulate transferase A 20-fold, enhanced transferase C activity only 2-fold. Transferase A', the nuclear enzyme, was retained on DNA-agarose. It was similar to transferase A in salt and histone sensitivity. Gel permeation chromatography showed slight molecular mass differences among the group of enzymes: A, 24,300 daltons (Da); B, 32,700 Da; C, and A', 25,500 Da. The affinities of transferase C for NAD and agmatine were similar to those of the cytosolic transferases A and B.(ABSTRACT TRUNCATED AT 250 WORDS)     

Inhibition of the GTPase activity of transducin by an NAD+:arginine ADP-ribosyltransferase from turkey erythrocytes.

The bacterial toxins, choleragen and pertussis toxin, inhibit the light-stimulated GTPase activity of bovine retinal rod outer segments by catalysing the ADP-ribosylation of the alpha-subunit (T alpha) of transducin [Abood, Hurley, Pappone, Bourne & Stryer (1982) J. Biol. Chem. 257, 10540-10543; Van Dop, Yamanaka, Steinberg, Sekura, Manclark, Stryer & Bourne (1984) J. Biol. Chem. 259, 23-26]. Incubation of retinal rod outer segments with NAD+ and a purified NAD+:arginine ADP-ribosyltransferase from turkey erythrocytes resulted in approx. 60% inhibition of GTPase activity. Inhibition was dependent on both enzyme and NAD+, and was potentiated by the non-hydrolysable GTP analogues guanosine 5'-[beta gamma-imido]triphosphate (p[NH]ppG) and guanosine 5'-[beta gamma-methylene]triphosphate (p[CH2]ppG). The transferase ADP-ribosylated both the T alpha and T beta subunits of purified transducin. T alpha (39 kDa), after ADP-ribosylation, migrated as two distinct peptides with molecular masses of 42 kDa and 46 kDa on SDS/polyacrylamide-gel electrophoresis. T beta (36 kDa), after ADP-ribosylation, migrated as a 38 kDa peptide. With purified transducin subunits, it was observed that the GTPase activity of ADP-ribosylated T alpha, reconstituted with unmodified T beta gamma and photolysed rhodopsin, was decreased by 80%; conversely, reconstitution of T alpha with ADP-ribosyl-T beta gamma resulted in only a 19% inhibition of GTPase. Thus ADP-ribosylation of T alpha, the transducin subunit that contains the guanine nucleotide-binding site, has more dramatic effects on GTPase activity than does modification of the critical 'helper subunits' T beta gamma. To elucidate the mechanism of GTPase inhibition by transferase, we studied the effect of ADP-ribosylation on p[NH]pp[3H]G binding to transducin. It was shown previously that modification of transducin by choleragen, which like transferase ADP-ribosylates arginine residues, did not affect guanine nucleotide binding. ADP-ribosylation by the transferase, however, decreased p[NH]pp[3H]G binding, consistent with the hypothesis that choleragen and transferase inhibit GTPase by different mechanisms.     

NAD-dependent ADP-ribosylation of arginine and proteins by Escherichia coli heat-labile enterotoxin.

Escherichia coli heat-labile enterotoxin (labile toxin, LT) catalyzed the hydrolysis of NAD to ADP-ribose and nicotinamide and the ADP-ribosylation of arginine (Moss, J., and Richardson, S.H. (1978) J. Clin. Invest. 62, 281-285). Analysis of the product of the ADP-ribosylation of arginine by nuclear magnetic resonance spectroscopy indicated that the reaction was stereospecific and resulted in the formation of alpha-ADP-ribosyl-L-arginine. This reaction product rapidly anomerized to yield a mixture of the alpha and beta forms. In the presence of [adenine-U-14C]NAD, E. coli enterotoxin catalyzed the transfer of the radiolabel to proteins; the ADP-ribosylation of proteins was inhibited by arginine methyl ester, an alternative substrate. Digestion of the 14C-protein with snake venom phosphodiesterase released predominantly 5'-AMP. No product was obtained with a mobility similar to that of 2'-(5'-phosphoribosyl)-5'-AMP. This result is consistent with the covalent attachment by the enterotoxin of ADP-ribose rather than poly(ADP-ribose) to protein. Thus, LT is catalytically equivalent to choleragen, an enterotoxin of Vibrio cholerae, and activates adenylate cyclase through a similar stereospecific ADP-ribosylation reaction.     

Regulation of acetyl-CoA carboxylase by ADP-ribosylation

Because of certain similarities between acetyl-CoA carboxylase (ACC) and tubulin, and the recent demonstration of the ADP-ribosylation of tubulin by cholera toxin, we have investigated a potential role for ADP-ribosylation in the regulation of ACC activity. Incubation of purified rat liver ACC with cholera toxin in the presence of millimolar concentrations of [adenylate-32P]NAD results in a time-dependent incorporation of ADP-ribose into ACC of greater than 2 mol/mol of enzyme subunit, accompanied by a marked inactivation of enzyme activity. This effect is not mimicked by pertussis toxin, ADP-ribose, or ribose 5-phosphate. Incubation of labeled ACC with snake venom phosphodiesterase and alkaline hydrolysis release 32P-products tentatively identified by high-performance liquid chromatography as 5'-[32P]AMP and [32P]ADP-ribose, respectively. These data are consistent with a mono-ADP-ribosylation of ACC catalyzed by cholera toxin. Phosphodiesterase treatment of inactivated ACC partially restores enzyme activity. The effects of ADP-ribosylation of ACC are expressed both as a decrease in the enzyme Vmax and as an increase in the apparent Ka for citrate. These results suggest that ACC might be a substrate for endogenous ADP-ribosyltransferases and that this covalent modification could be an important regulatory mechanism for the modulation of fatty acid synthesis in vivo.     

A novel approach to detect toxin-catalyzed ADP-ribosylation in intact cells: its use to study the action of Pasteurella multocida toxin

Certain microbial toxins are ADP-ribosyltransferases, acting on specific substrate proteins. Although these toxins have been of great utility in studies of cellular regulatory processes, a simple procedure to directly study toxin-catalyzed ADP-ribosylation in intact cells has not been described. Our approach was to use [2-3H]adenine to metabolically label the cellular NAD+ pool. Labeled proteins were then denatured with SDS, resolved by PAGE, and detected by flurography. In this manner, we show that pertussis toxin, after a dose-dependent lag period, [3H]-labeled a 40-kD protein intact cells. Furthermore, incubation of the gel with trichloroacetic acid at 95 degrees C before fluorography caused the release of label from bands other than the pertussis toxin substrate, thus, allowing its selective visualization. The modification of the 40-kD protein was ascribed to ADP-ribosylation of a cysteine residue on the basis of inhibition of labeling by nicotinamide and the release of [3H]ADP-ribose from the labeled protein by mercuric acetate. Cholera toxin catalyzed the [3H]-labeling of a 46-kD protein in the [2-3H]adenine-labeled cells. Pretreatment of the cells with pertussis toxin before the labeling of NAD+ with [2-3H]adenine blocked [2-3H]ADP-ribosylation catalyzed by pertussis toxin, but not that by cholera toxin. Thus, labeling with [2-3H]adenine permits the study of toxin-catalyzed ADP-ribosylation in intact cells. Pasteurella multocida toxin has recently been described as a novel and potent mitogen for Swiss 3T3 cell and acts to stimulate the phospholipase C-mediated hydrolysis of polyphosphoinositides. The basis of the action of the toxin is not known. Using the methodology described here, P. multocida toxin was not found to act by ADP-ribosylation.     

Effect of oxidative stress on in vivo ADP-ribosylation of eukaryotic elongation factor 2

Different lines of evidence indicate that eukaryotic elongation factor 2 (eEF2) can be ADP-ribosylated endogenously. The physiological significance of this reaction has, however, remained unclarified. In order to address this issue we investigated the in vivo ADP-ribosylation of eEF2 and the effect of oxidative stress thereon. The investigation revealed that the endogenous ADP-ribosylation of eEF2 is complex and can take place in K562 cell lysates either under the action of endogenous transferase from [adenosine-14C]NAD or by direct binding of free [14C]ADP-ribose. These two types of ADP-ribosylation were distinguished by use of different treatments based on the chemical stability of the respective bonds formed. Under standard culture conditions, in vivo labeling of eEF2 in the presence of [14C]adenosine was reversed to about 65% in the presence of diphtheria toxin and nicotinamide. This finding implied that the modification that took place under physiological circumstances was, mainly, of an enzymic nature. On the other hand, H2O2-promoted oxidative stress gave rise to a nearly two-fold increase in the extent of in vivo labeling of eEF2. This was accompanied by a loss of eEF2 activity in polypeptide chain elongation. Oxidative stress specifically inhibited the subsequent binding of free ADP-ribose to eEF2. The results thus provide evidence that endogenous ADP-ribosylation of eEF2 can also take place by the binding of free ADP-ribose. This nonenzymic reaction appears to account primarily for in vivo ADP-ribosylation of eEF2 under oxidative stress.     

De-ADP-ribosylation actin by Clostridium perfringens iota-toxin and Clostridium botulinum C2 toxin

The reverse reaction of the ADP-ribosylation of actin by Clostridium botulinum C2 toxin and Clostridium perfringens iota-toxin was studied. In the presence of nicotinamide (30-50 mM) C2 toxin and iota-toxin decreased the radioactive labeling of [32P]ADP-ribosylated actin and catalyzed the formation of [32P]NAD. The pH optima for both reactions were 5.5-6.0. Concomitant with the removal of ADP-ribose, the ability of actin to polymerize was restored and actin ATPase activity increased. Neither ADP-ribosylation nor removal of ADP-ribose was observed after treatment of actin with EDTA, indicating that the native structure of actin is required for both reactions. ADP-ribosylation of platelet actin by C2 toxin was reversed by iota-toxin, confirming recent reports that both toxins modify the same amino acid in actin. However, C. botulinum C2 toxin was not able to cleave ADP-ribose from skeletal muscle actin which had been incorporated by iota-toxin, corroborating the different substrate specificities of both toxins.     

Plasmodium falciparum Sir2 is an NAD+-dependent deacetylase and an acetyllysine-dependent and acetyllysine-independent NAD+ glycohydrolase

Sirtuins are NAD (+)-dependent enzymes that deacetylate a variety of cellular proteins and in some cases catalyze protein ADP-ribosyl transfer. The catalytic mechanism of deacetylation is proposed to involve an ADPR-peptidylimidate, whereas the mechanism of ADP-ribosyl transfer to proteins is undetermined. Herein we characterize a Plasmodium falciparum sirtuin that catalyzes deacetylation of histone peptide sequences. Interestingly, the enzyme can also hydrolyze NAD (+). Two mechanisms of hydrolysis were identified and characterized. One is independent of acetyllysine substrate and produces alpha-stereochemistry as established by reaction of methanol which forms alpha-1- O-methyl-ADPR. This reaction is insensitive to nicotinamide inhibition. The second solvolytic mechanism is dependent on acetylated peptide and is proposed to involve the imidate to generate beta-stereochemistry. Stereochemistry was established by isolation of beta-1- O-methyl-ADPR when methanol was added as a cosolvent. This solvolytic reaction was inhibited by nicotinamide, suggesting that nicotinamide and solvent compete for the imidate. These findings establish new reactions of wildtype sirtuins and suggest possible mechanisms for ADP-ribosylation to proteins. These findings also illustrate the potential utility of nicotinamide as a probe for mechanisms of sirtuin-catalyzed ADP-ribosyl transfer.     

Substrate specificity and kinetic mechanism of the Sir2 family of NAD+-dependent histone/protein deacetylases

The Silent information regulator 2 (Sir2) family of enzymes consists of NAD(+)-dependent histone/protein deacetylases that tightly couple the hydrolysis of NAD(+) and the deacetylation of an acetylated substrate to form nicotinamide, the deacetylated product, and the novel metabolite O-acetyl-ADP-ribose (OAADPR). In this paper, we analyzed the substrate specificity of the yeast Sir2 (ySir2), the yeast HST2, and the human SIRT2 homologues toward various monoacetylated histone H3 and H4 peptides, determined the basic kinetic mechanism, and resolved individual chemical steps of the Sir2 reaction. Using steady-state kinetic analysis, we have shown that ySir2, HST2, and SIRT2 exhibit varying catalytic efficiencies and display a preference among the monoacetylated peptide substrates. Bisubstrate kinetic analysis indicates that Sir2 enzymes follow a sequential mechanism, where both the acetylated substrate and NAD(+) must bind to form a ternary complex, prior to any catalytic step. Using rapid-kinetic analysis, we have shown that after ternary complex formation, nicotinamide cleavage occurs first, followed by the transfer of the acetyl group from the donor substrate to the ADP-ribose portion of NAD(+) to form OAADPr and the deacetylated product. Product and dead-end inhibition analyses revealed that nicotinamide is the first product released followed by random release of OAADPr and the deacetylated product.     

Endogenous ADP-ribosylation for eukaryotic elongation factor 2: evidence of two different sites and reactions

Eukaryotic elongation factor 2 can undergo ADP-ribosylation in the absence of diphtheria toxin under the action of an endogenous transferase. The investigation which aimed to gain insight into the nature of endogenous ADP-ribosylation revealed that this reaction may be, in some cases, due to covalent binding of free ADP-ribose to elongation factor 2. Binding of free ADP-ribose, and NAD- and endogenous transferase-dependent ADP-ribosylation were suggested to be distinct reactions by different findings. Free ADP-ribose could bind to elongation factor 2 previously subjected to ADP-ribosylation by diphtheria toxin or endogenous transferase. The binding of free ADP-ribose was inhibited by neutral NH2OH, L-lysine and picrylsulfonate, whereas endogenous ADP-ribosyltransferase was inhibited by NAD glycohydrolase inhibitors and L-arginine. The ADP-ribosyl-elongation factor 2 adduct which formed upon binding of free ADP-ribose was resistant to neutral NH2OH, but decomposed almost completely upon treatment with NaOH. The product of endogenous transferase-dependent ADP- ribosylation was partially resistant to NH2OH and NaOH treatment. Moreover, this reaction was reversed in the presence of diphtheria toxin and nicotinamide. Both types of endogenous ADP-ribosylation gave rise to inhibition of polyphenylalanine synthesis. This study thus provides evidence for the presence of two different types of endogenous ADP-ribosylation of eukaryotic elongation factor 2. The respective sites involved in these reactions are distinct from one another as well as from diphthamide, the site of attack by diphtheria toxin.     

Dependence of ADP-ribosylation of histones of chicken liver nuclei on the intranuclear content of NAD

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.     

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

NAD glycohydrolases are enzymes that catalyze the hydrolisis of NAD to produce ADP-ribose and nicotinamide. Regulation of these enzymes has not been fully elucidated. We have identified an 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 -NAD but not by -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.