Hydrolysis Of NAD Research Articles

Enzymatic assays for NAD-dependent deacetylase activities

The NAD-dependent deacetylases are a new class of enzymes responsible for the removal of acetyl groups from lysines on proteins. Instead of water, the NAD-dependent deacetylases use a highly reactive ADP-ribose intermediate as a recipient for the acetyl group. The products of the reaction are nicotinamide, acetyl-ADP-ribose, and a deacetylated substrate. Many assays have been developed for the measurement of NAD-dependent deacetylase activity. In this review we present assays based on each of the two reactions catalyzed by these enzymes, deacetylation and NAD hydrolysis. First we describe methods for the production of acetylated protein and peptide substrates for use in deacetylation reactions. Then we describe four methods for assaying deacetylation, three of which directly measure the loss of acetyl groups from a protein or peptide substrate, and one that measures acetate production. We also describe two indirect methods for following enzyme activity, NAD hydrolysis and a novel NAD-nicotinamide exchange reaction. Finally, a quantitative method using a monoacetylated peptide as a substrate and HPLC to measure products is described.

Analysis of NAD(P) +-cofactors by redox-functionalized ISFET devices

Functional ISFET devices for the specific analysis of the NAD(P) +-cofactors are described. The functional ISFET devices consist of a pyrroloquinoline quinone (PQQ)-modified gate, to which the NAD(P) +-dependent enzymes, lactate dehydrogenase, LDH (for NAD + analysis), or alcohol dehydrogenase, AlcDH (for NADP + analysis), are covalently linked. In the presence of NAD + and lactate, or NADP + and ethanol, the biocatalyzed generation of NADH or NADPH occurs. The catalyzed oxidation of the generated NAD(P)H cofactors by PQQ and O 2 yields a steady-state concentration of PQQ/PQQH 2 on the gate interface. The ratio PQQ/PQQH 2 is controlled by the concentration of NAD(P)H, or by the parent oxidized NAD(P) +-cofactors. The functional ISFET devices allow the potentiometric analysis of NAD + with the lower detection limit of 2×10 −4 M, and a sensitivity of 23±2 mV per decade, and of NADP + with a detection limit of 1×10 −4 and sensitivity that corresponds to 35±2 mV per decade. The PQQ/LDH-functionalized ISFET device allows the kinetic analysis of the hydrolysis of NAD + by NADase ( k=2.0×10 −3 s −1) and by the cholera toxin, subunit A ( k=4.2×10 −4 s −1).

Unusual Enzymatic Hydrolysis of NAD by Solubilized Form of NAD+ Glycohydrolase.

Using solubilized form (sNADase) of membrane-bound porcine brain NAD(+) glycohydrolase (pNADase), the NADase-catalyzed hydrolysis and transglycosidation reactions of NAD (1) were examined. Unexpectedly, products in the reactions were found to be nicotinamide (5'-O-diphosphono)-beta-D-ribofuranoside (4) and adenosine (5). Adenosine 5'-diphosphate (ADP)-ribose (2) and nicotinamide (3) as well as a transglycosylated product, which are formed in a usual NAD/pNADase reaction system, were scarcely produced in the NAD/sNADase system. Setting aside the mechanical aspects of this unusual cleaving, it is quite interesting that the sNADase-catalyzed hydrolytic reaction of NAD resulted in the selective cleavage of the P-O bond of the adenosine side without the appreciable hydrolysis of the labile quaternary nicotinamide-ribose pyridinium linkage.

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.

Diphtheria toxin catalyzed hydrolysis of NAD(+): molecular dynamics study of enzyme-bound substrate, transition state, and inhibitor.

The mechanism of the diphtheria toxin-catalyzed hydrolysis of NAD(+) was investigated by quantum chemical calculations and molecular dynamics simulations. Several effects that could explain the 6000-fold rate acceleration (Delta Delta G(++) approximately 5 kcal/mol) by the enzyme were considered. First, the carboxamide arm of the enzyme-bound NAD(+) adopts a trans conformation while the most stable conformation is cis. The most stable conformation for the nicotinamide product has the amide carbonyl trans. The activation energy for the cleavage of the ribosidic bond is reduced by 2 kcal/mol due to the relaxation of this ground state conformational stress in the transition state. Second, molecular dynamics simulations to the nanosecond time range revealed that the carboxylate of Glu148 forms a hydrogen bond to the substrate's 2' hydroxyl group in E.S (approximately 17% of the time) and E.TS (approximately 57% of the time) complexes. This interaction is not seen in crystal structures. The ApUp inhibitor is held more tightly by the enzyme than the transition state and the substrate. Analysis of correlated motions reveals differences in the pattern of anticorrelated motions for protein backbone atoms when the transition state occupies the active site as compared to the E.NAD(+) complex.

Gain-of-function of poly(ADP-ribose) polymerase-1 upon cleavage by apoptotic proteases: implications for apoptosis.

Poly(ADP-ribosyl)ation is an important mechanism for the maintenance of genomic integrity in response to DNA damage. The enzyme responsible for poly(ADP-ribose) synthesis, poly(ADP-ribose) polymerase 1 (PARP-1), has been implicated in two distinct modes of cell death induced by DNA damage, namely apoptosis and necrosis. During the execution phase of apoptosis, PARP-1 is specifically proteolyzed by caspases to produce an N-terminal DNA-binding domain (DBD) and a C-terminal catalytic fragment. The functional consequence of this proteolytic event is not known. However, it has recently been shown that overactivation of full-length PARP-1 can result in energy depletion and necrosis in dying cells. Here, we investigate the molecular basis for the differential involvement of PARP-1 in these two types of cellular demise. We show that the C-terminal apoptotic fragment of PARP-1 loses its DNA-dependent catalytic activity upon cleavage with caspase 3. However, the N-terminal apoptotic fragment, retains a strong DNA-binding activity and totally inhibits the catalytic activity of uncleaved PARP-1. This dominant-negative behavior was confirmed and extended in cellular extracts where DNA repair was completely inhibited by nanomolar concentrations of the N-terminal fragment. Furthermore, overexpression of the apoptotic DBD in mouse fibroblast inhibits endogenous PARP-1 activity very efficiently in vivo, thereby confirming our biochemical observations. Taken together, these experiments indicate that the apoptotic DBD of PARP-1 acts cooperatively with the proteolytic inactivation of the enzyme to trans-inhibit NAD hydrolysis and to maintain the energy levels of the cell. These results are consistent with a model in which cleavage of PARP-1 promotes apoptosis by preventing DNA repair-induced survival and by blocking energy depletion-induced necrosis.

ADP-ribose gating of the calcium-permeable LTRPC2 channel revealed by Nudix motif homology.

Free ADP-ribose (ADPR), a product of NAD hydrolysis and a breakdown product of the calcium-release second messenger cyclic ADPR (cADPR), has no defined role as an intracellular signalling molecule in vertebrate systems. Here we show that a 350-amino-acid protein (designated NUDT9) and a homologous domain (NUDT9 homology domain) near the carboxy terminus of the LTRPC2/TrpC7 putative cation channel both function as specific ADPR pyrophosphatases. Whole-cell and single-channel analysis of HEK-293 cells expressing LTRPC2 show that LTRPC2 functions as a calcium-permeable cation channel that is specifically gated by free ADPR. The expression of native LTRPC2 transcripts is detectable in many tissues including the U937 monocyte cell line, in which ADPR induces large cation currents (designated IADPR) that closely match those mediated by recombinant LTRPC2. These results indicate that intracellular ADPR regulates calcium entry into cells that express LTRPC2.

A Single Residue at the Active Site of CD38 Determines Its NAD Cyclizing and Hydrolyzing Activities

CD38 is a multifunctional enzyme involved in metabolizing two Ca(2+) messengers, cyclic ADP-ribose (cADPR) and nicotinic acid adenine dinucleotide phosphate (NAADP). When incubated with NAD, CD38 predominantly hydrolyzes it to ADP-ribose (NAD glycohydrolase), but a trace amount of cADPR is also produced through cyclization of the substrate. Site-directed mutagenesis was used to investigate the amino acid important for controlling the hydrolysis and cyclization reactions. CD38 and its mutants were produced in yeast, purified, and characterized by immunoblot. Glu-146 is a conserved residue present in the active site of CD38. Its replacement with Phe greatly enhanced the cyclization activity to a level similar to that of the NAD hydrolysis activity. A series of additional replacements was made at the Glu-146 position including Ala, Asn, Gly, Asp, and Leu. All the mutants exhibited enhanced cyclase activity to various degrees, whereas the hydrolysis activity was inhibited greatly. E146A showed the highest cyclase activity, which was more than 3-fold higher than its hydrolysis activity. All mutants also cyclized nicotinamide guanine dinucleotide to produce cyclic GDP. This activity was enhanced likewise, with E146A showing more than 9-fold higher activity than the wild type. In addition to NAD, CD38 also hydrolyzed cADPR effectively, and this activity was correspondingly depressed in the mutants. When all the mutants were considered, the two cyclase activities and the two hydrolase activities were correlated linearly. The Glu-146 replacements, however, only minimally affected the base-exchange activity that is responsible for synthesizing NAADP. Homology modeling was used to assess possible structural changes at the active site of E146A. These results are consistent with Glu-146 being crucial in controlling specifically and selectively the cyclase and hydrolase activities of CD38.

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 p I of 4.9 and a K m value for NAD + of 26 μM. High activation of ADPR cyclase activity was observed in the presence of Ag + ions, corresponding to NADase inhibition.

NADP and NAD utilization in Haemophilus influenzae

Exogenous NAD utilization or pyridine nucleotide cycle metabolism is used by many bacteria to maintain NAD turnover and to limit energy-dependent de novo NAD synthesis. The genus Haemophilus includes several important pathogenic bacterial species that require NAD as an essential growth factor. The molecular mechanisms of NAD uptake and processing are understood only in part for Haemophilus. In this report, we present data showing that the outer membrane lipoprotein e(P4), encoded by the hel gene, and an exported 5'-nucleotidase (HI0206), assigned as nadN, are necessary for NAD and NADP utilization. Lipoprotein e(P4) is characterized as an acid phosphatase that uses NADP as substrate. Its phosphatase activity is inhibited by compounds such as adenosine or NMN. The nadN gene product was characterized as an NAD-nucleotidase, responsible for the hydrolysis of NAD. H. influenzae hel and nadN mutants had defined growth deficiencies. For growth, the uptake and processing of the essential cofactors NADP and NAD required e(P4) and 5'-nucleotidase. In addition, adenosine was identified as a potent growth inhibitor of wild-type H. influenzae strains, when NADP was used as the sole source of nicotinamide-ribosyl.

Metabolic fate of extracellular NAD in human skin fibroblasts.

Extracellular NAD is degraded to pyridine and purine metabolites by different types of surface-located enzymes which are expressed differently on the plasmamembrane of various human cells and tissues. In a previous report, we demonstrated that NAD-glycohydrolase, nucleotide pyrophosphatase and 5'-nucleotidase are located on the outer surface of human skin fibroblasts. Nucleotide pyrophosphatase cleaves NAD to nicotinamide mononucleotide and AMP, and 5'-nucleotidase hydrolyses AMP to adenosine. Cells incubated with NAD, produce nicotinamide, nicotinamide mononucleotide, hypoxanthine and adenine. The absence of ADPribose and adenosine in the extracellular compartment could be due to further catabolism and/or uptake of these products. To clarify the fate of the purine moiety of exogenous NAD, we investigated uptake of the products of NAD hydrolysis using U-[(14)C]-adenine-NAD. ATP was found to be the main labeled intracellular product of exogenous NAD catabolism; ADP, AMP, inosine and adenosine were also detected but in small quantities. Addition of ADPribose or adenosine to the incubation medium decreased uptake of radioactive purine, which, on the contrary, was unaffected by addition of inosine. ADPribose strongly inhibited the activity of ecto-NAD-hydrolyzing enzymes, whereas adenosine did not. Radioactive uptake by purine drastically dropped in fibroblasts incubated with (14)C-NAD and dipyridamole, an inhibitor of adenosine transport. Partial inhibition of [(14)C]-NAD uptake observed in fibroblasts depleted of ATP showed that the transport system requires ATP to some extent. All these findings suggest that adenosine is the purine form taken up by cells, and this hypothesis was confirmed incubating cultured fibroblasts with (14)C-adenosine and analyzing nucleoside uptake and intracellular metabolism under different experimental conditions. Fibroblasts incubated with [(14)C]-adenosine yield the same radioactive products as with [(14)C]-NAD; the absence of inhibition of [(14)C]-adenosine uptake by ADPribose in the presence of alpha-beta methyleneADP, an inhibitor of 5' nucleotidase, demonstrates that ADPribose coming from NAD via NAD-glycohydrolase is finally catabolised to adenosine. These results confirm that adenosine is the NAD hydrolysis product incorporated by cells and further metabolized to ATP, and that adenosine transport is partially ATP dependent.

Auto-ADP-ribosylation of NAD glycohydrolase from Neurospora crassa

NAD glycohydrolase (NADase; EC 3.2.2.5) is an enzyme that catalyzes hydrolysis of NAD to produce ADP-ribose and nicotinamide. We recently demonstrated that self-inactivation of NADase from rabbit erythrocytes was due to an auto-ADP-ribosylation. In the present study, a mechanism of self-inactivation of NADase from Neurospora crassa by its substrate was investigated by using intact mycelia of N. crassa and purified NADase, which had molecular characteristics different from mammalian NADases. The results suggested that inactivation of NADase from N. crassa was also due to an auto-ADP-ribosylation. These findings indicate that the auto-modification of NADase is one of the universal phenomena to regulate enzyme functions.

Transition-state structure for the ADP-ribosylation of recombinant Gialpha1 subunits by pertussis toxin.

Pertussis toxin ADP-ribosylates a specific Cys side chain in the alpha-subunit of several G-proteins. Recombinant Gialpha1-subunits were rapidly ADP-ribosylated in the absence of betagamma-subunits, with a Km of 800 microM and a kcat of 40 min-1. Addition of betagamma-subunits decreases Km to 0.3 microM with little change of kcat. Kinetic isotope effects established the transition-state structure for ADP-ribosylation of Gialpha1 subunits. The transition state is dissociative, with a 2.1 A bond to the nicotinamide leaving group and a bond of 2.5 A to the sulfur nucleophile. The nucleophilic participation of Gialpha1 at the transition state is greater than that for water in the hydrolysis of NAD+by pertussis toxin. Crystal structures for Gialpha1 show the Cys nucleophile in a disordered segment or inaccessible for attack on NAD+. Therefore, transition-state formation requires an altered Gialpha1 conformation to expose and ionize Cys. The transition state has been docked into the crystal structure of pertussis toxin in a geometry required for transition state formation.

Transition State Structure for the Hydrolysis of NAD Catalyzed by Diphtheria Toxin.

Diphtheria toxin (DTA) uses NAD(+) as an ADP-ribose donor to catalyze the ADP-ribosylation of eukaryotic elongation factor 2. This inhibits protein biosynthesis and ultimately leads to cell death. In the absence of its physiological acceptor, DTA catalyzes the slow hydrolysis of NAD(+) to ADP-ribose and nicotinamide, a reaction that can be exploited to measure kinetic isotope effects (KIEs) of isotopically labeled NAD(+)s. Competitive KIEs were measured by the radiolabel method for NAD(+) molecules labeled at the following positions: 1-(15)N = 1.030 ± 0.004, 1'-(14)C = 1.034 ± 0.004, (1-(15)N,1'-(14)C) = 1.062 ± 0.010, 1'-(3)H = 1.200 ± 0.005, 2'-(3)H = 1.142 ± 0.005, 4'-(3)H = 0.990 ± 0.002, 5'-(3)H = 1.032 ± 0.004, 4'-(18)O = 0.986 ± 0.003. The ring oxygen, 4'-(18)O, KIE was also measured by whole molecule mass spectrometry (0.991 ± 0.003) and found to be within experimental error of that measured by the radiolabel technique, giving an overall average of 0.988 ± 0.003. The transition state structure of NAD(+) hydrolysis was determined using a structure interpolation method to generate trial transition state structures and bond-energy/bond-order vibrational analysis to predict the KIEs of the trial structures. The predicted KIEs matched the experimental ones for a concerted, highly oxocarbenium ion-like transition state. The residual bond order to the leaving group was 0.02 (bond length = 2.65 Å), while the bond order to the approaching nucleophile was 0.03 (2.46 Å). This is an A(N)D(N) mechanism, with both leaving group and nucleophilic participation in the reaction coordinate. Fitting the transition state structure into the active site cleft of the X-ray crystallographic structure of DTA highlighted the mechanisms of enzymatic stabilization of the transition state. Desolvation of the nicotinamide ring, stabilization of the oxocarbenium ion by apposition of the side chain carboxylate of Glu148 with the anomeric carbon of the ribosyl moiety, and the placement of the substrate phosphate near the positively charged side chain of His21 are all consistent with the transition state features from KIE analysis.

Transition State Structure of the Solvolytic Hydrolysis of NAD+

The transition state structure has been determined for the pH-independent solvolytic hydrolysis of NAD+. The structure is based on kinetic isotope effects (KIEs) measured for NAD+'s labeled in various positions of the ribose ring and in the leaving group nitrogen. The KIEs for reactions performed at 100 °C in 50 mM NaOAc (pH 4.0) were as follows: 1-15N, 1.020 ± 0.007; 1‘-14C, 1.016 ± 0.002; [1-15N,1‘-14C], 1.034 ± 0.002; 1‘-3H, 1.194 ± 0.005; 2‘-3H, 1.114 ± 0.004; 4‘-3H, 0.997 ± 0.001; 5‘-3H, 1.000 ± 0.003; 4‘-18O, 0.988 ± 0.007. The transition state structure was determined using bond energy/bond order vibrational analysis to predict KIEs for trial transition state models. The structure that most closely matches the experimental KIEs defines the transition state. A structure interpolation method was developed to generate trial transition state structures and thereby systematically search reaction coordinate space. Structures are generated by interpolation between reference structures, reactant NAD+ and ...

Pertussis toxin: transition state analysis for ADP-ribosylation of G-protein peptide alphai3C20.

Pertussis toxin from Bordetella pertussis is one of the ADP-ribosylating toxins which are the cytotoxic agents of several infectious diseases. Transition state analogues of these enzymes are expected to be potent inhibitors and may be useful in therapy. Pertussis toxin catalyzes the ADP-ribosylation of a cysteine in the synthetic peptide alphai3C20, corresponding to the C-terminal 20 amino acids of the alpha-subunits of the G-protein Gi3. A family of kinetic isotope effects was determined for the ADP-ribosylation reaction, using 3H-, 14C- and 15N-labeled NAD+ as substrates. Primary kinetic isotope effects were 1.050 +/- 0.006 for [1'N-14C] and 1.021 +/- 0.002 for [1N-15N], the double primary effect of [1'N-14C,1N-15N] was 1.064 +/- 0.002. Secondary kinetic isotope effects were 1.208 +/- 0.014 for [1'N-3H], 1.104 +/- 0.010 for [2'N-3H], 0.989 +/- 0.001 for [4'N-3H], and 1.014 +/- 0.002 for [5'N-3H]. Isotope trapping experiments yielded a commitment factor of 0.01, demonstrating that the observed isotope effects are near intrinsic. Solvent D2O kinetic isotope effects are inverse, consistent with deprotonation of the attacking Cys prior to transition state formation. The transition state structure was determined by a normal mode bond vibrational analysis. The transition state is characterized by a nicotinamide leaving group bond order of 0.14, corresponding to a bond length of 2.06 A. The incoming thiolate nucleophile has a bond order of 0.11, corresponding to 2.47 A. The ribose ring has strong oxocarbenium ion character. Pertussis toxin also catalyzes the slow hydrolysis of NAD+ in the absence of peptides. Comparison of the transition states for NAD+ hydrolysis and for ADP-ribosylation of peptide alphai3C20 indicates that the sulfur nucleophile from the peptide Cys participates more actively as a nucleophile in the reaction than does water in the hydrolytic reaction. Participation of the thiolate anion at the transition state provides partial neutralization of the cationic charge which normally develops at the transition states of N-ribohydrolases and transferases. Thus, the presence of the peptide provides increased SN2 character in a loose transition state which retains oxocarbenium character in the ribose.

Transition State Analysis of NAD+Hydrolysis by the Cholera Toxin Catalytic Subunit

The transition state for NAD+ (oxidized nicotinamide adenine dinucleotide) hydrolysis by the cholera toxin A1 polypeptide (CTA) has been characterized by multiple V/K kinetic isotope effects (KIEs) using labeled NAD+ as the substrate. CTA causes cholera by catalyzing the ADP-ribosylation of the signal-transducing Gsα protein. In vitro, CTA catalyzes the ADP-ribosylation of several simple guanidino compounds as well as the slow hydrolysis of NAD+ (kcat = 8 min-1, Km = 14 mM) to form ADP−ribose and nicotinamide. KIEs for NAD+ hydrolysis are the following: primary 14C = 1.030 ± 0.005, primary 15N = 1.029 ± 0.004, α-secondary 3H = 1.186 ± 0.004, β-secondary 3H = 1.108 ± 0.004, γ-secondary 3H = 0.986 ± 0.003, δ-secondary 3H = 1.020 ± 0.003, and primary double = 1.052 ± 0.004. On the basis of steady-state kinetic parameters for CTA-catalyzed NAD+ hydrolysis, as well as a comparison with KIEs measured for NAD+ solvolysis, the enzymatic KIEs are near-intrinsic and describe a transition state that is relatively d...

Autotaxin Is an Exoenzyme Possessing 5′-Nucleotide Phosphodiesterase/ATP Pyrophosphatase and ATPase Activities

Autotaxin (ATX) is an extracellular enzyme and an autocrine motility factor that stimulates pertussis toxin-sensitive chemotaxis in human melanoma cells at picomolar to nanomolar concentrations. This 125-kDa glycoprotein contains a peptide sequence identified as the catalytic site in type I alkaline phosphodiesterases (PDEs), and it possesses 5'-nucleotide PDE (EC 3.1.4.1) activity (Stracke, M. L., Krutzsch, H. C., Unsworth, E. J., Arestad, A., Cioce, V., Schiffmann, E., and Liotta, L. (1992) J. Biol. Chem. 267, 2524-2529; Murata, J., Lee, H. Y., Clair, T., Krutsch, H. C., Arestad, A. A., Sobel, M. E., Liotta, L. A., and Stracke, M. L. (1994) J. Biol. Chem. 269, 30479-30484). ATX binds ATP and is phosphorylated only on threonine. Thr210 at the PDE active site of ATX is required for phosphorylation, 5'-nucleotide PDE, and motility-stimulating activities (Lee, H. Y., Clair, T., Mulvaney, P. T., Woodhouse, E. C., Aznavoorian, S., Liotta, L. A., and Stracke, M. L. (1996) J. Biol. Chem. 271, 24408-24412). In this article we report that the phosphorylation of ATX is a transient event, being stable at 0 degrees C but unstable at 37 degrees C, and that ATX has adenosine-5'-triphosphatase (ATPase; EC 3.6.1.3) and ATP pyrophosphatase (EC 3.6.1.8) activities. Thus ATX catalyzes the hydrolysis of the phosphodiester bond on either side of the beta-phosphate of ATP. ATX also catalyzes the hydrolysis of GTP to GDP and GMP, of either AMP or PPi to Pi, and the hydrolysis of NAD to AMP, and each of these substrates can serve as a phosphate donor in the phosphorylation of ATX. ATX possesses no detectable protein kinase activity toward histone, myelin basic protein, or casein. These results lead to the proposal that ATX is capable of at least two alternative reaction mechanisms, threonine (T-type) ATPase and 5'-nucleotide PDE/ATP pyrophosphatase, with a common site (Thr210) for the formation of covalently bound reaction intermediates threonine phosphate and threonine adenylate, respectively.

Inhibition of the pyridine nucleotide-linked mitochondrial Ca(2+) release by 4-hydroxynonenal: the role of thiolate-disulfide conversion.

Rat liver mitochondria contain a specific Ca(2+) release pathway which operates when intramitochondrial NAD(+) is hydrolyzed to ADPribose and nicotinamide. The molecular details of this pathway are incompletely understood. It has been reported that NAD(+) hydrolysis and therefore Ca(2+) release stimulated by t-butylhydroperoxide is prevented by 4-hydroxynonenal (HNE). The reason underlying inhibition by HNE, however, remained unclear. It has also been reported that NAD(+) hydrolysis and Ca(2+) release are stimulated when some vicinal thiols are cross-linked, as shown with phenylarsine oxide or gliotoxin (GT). We now show that HNE also prevents the GT-induced Ca(2+) release, but only when given before GT. Conversely, GT stimulates Ca(2+) release only when given before HNE. Inhibition of Ca(2+) release by HNE is reduced by its preincubation with thiol compounds, the effectiveness of which increases with decreasing pKa of their sulfhydryl group. Preincubation of HNE with glutathione at high, but not at low, pH similarly reduces inhibition of Ca(2+) release by HNE. These findings provide evidence that HNE inhibition of Ca(2+) release is due to a modification of mitochondrial thiolates in a way that their cross-linking is prevented, and give further insight into the regulation of Ca(2+) release from intact mitochondria.

Regulation of NAD+ glycohydrolase activity by NAD(+)-dependent auto-ADP-ribosylation.

NAD+ glycohydrolase (NADase; EC 3.2.2.5) is an enzyme that catalyses hydrolysis of NAD+ to produce ADP-ribose and nicotinamide. Its physiological role and the regulation of its enzymic activity have not been fully elucidated. In the present study, the mechanism of self-inactivation of NADase by its substrate, NAD+, was investigated by using intact rabbit erythrocytes and purified NADase. Our results suggest that inactivation of NADase was due an auto-ADP-ribosylation reaction. ADP-ribosylated NADase of rabbit erythrocytes was deADP-ribosylated when incubated without NAD+, and thus enzyme activity was simultaneously restored. These findings suggest that reversible auto-ADP-ribosylation of NADase might regulate the enzyme's activity in vivo.