Km For NAD Research Articles

Rho-ADP-ribosylating exoenzyme from Bacillus cereus. Purification, characterization, and identification of the NAD-binding site.

The ADP-ribosyltransferase produced by a pathogenic strain of Bacillus cereus was purified to near homogeneity. The transferase is a 28,000 Da molecular mass enzyme with a pI of 10.3. The specific enzyme activity is 7.0 nmol of ADP-ribose min-1 mg-1 with a Km for NAD of 0.3 microM. Partial amino acid sequence analysis of the exoenzyme reveals no significant homology to Clostridium botulinum C3 nor to Clostridium limosum exoenzyme. The novel exoenzyme selectively modifies the small GTP-binding proteins of the Rho family presumably at the same acceptor amino acid (Asn-41) as determined for C3. Besides cellular Rho, recombinant RhoA and -B are substrates for the exoenzyme. However, recombinant Rac1 and CDC42, although belonging to the Rho family, are not modified. B. cereus exoenzyme was photolabeled with [carbonyl-14C]NAD resulting in inhibition of ADP-ribosyltransferase and NAD-glycohydrolase activity. A glutamic acid residue was identified as part of the NAD-binding site which corresponds to Glu-174 of C3. This glutamic acid is located in a domain which shows high homology with the C-terminal part of C3 exoenzyme, C. limosum exoenzyme, and Staphylococcus aureus EDIN and which probably represents the catalytic site of the transferases. The data indicate that B. cereus exoenzyme is a novel member of the family of C3-like ADP-ribosyltransferases which share the same substrate protein Rho and which have an identical highly conserved catalytic domain.

Cloning, Purification, and Properties of a Novel NADH Pyrophosphatase: EVIDENCE FOR A NUCLEOTIDE PYROPHOSPHATASE CATALYTIC DOMAIN IN MutT-LIKE ENZYMES

An Escherichia coli open reading frame containing significant homology to the active site of the MutT enzyme codes for a novel dinucleotide pyrophosphatase. The motif shared by these two proteins and several others is conserved throughout nature and may designate a nucleotide-binding or pyrophosphatase domain. The E. coli NADH pyrophosphatase has been cloned, overexpressed, and purified to near homogeneity. The protein contains 257 amino acids (M(r) = 29,774) and migrates on gel filtration columns as an apparent dimer. The enzyme catalyzes the hydrolysis of a broad range of dinucleotide pyrophosphates, but uniquely prefers the reduced form of NADH. The Vmax/Km for NADH (69 mumol min-1 mg-1 mM-1) is an order of magnitude higher than for any other dinucleotide pyrophosphate tested. In addition, the Km for NADH (0.1 mM) is 50-fold lower than the Km for NAD+. The hydrolysis of dinucleotide pyrophosphates requires divalent metal ions and yields two mononucleoside 5'-phosphates. The metals that most efficiently stimulate activity are Mg2+ and Mn2+. Although these metals support similar Vmax values at optimal metal concentration, the apparent Km for Mg2+ is 3.7 mM (at 1 mM NADH), whereas the apparent Km for Mn2+ at the same NADH concentration is 30 microM.

Involvement of serine 74 in the enzyme-coenzyme interaction of rat liver mitochondrial aldehyde dehydrogenase.

It has been suggested that the active site nucleophile in sheep liver aldehyde dehydrogenase was not a cysteine residue but was a serine located at position 74 [Loomes, K. M., Midwinter, G. G., Blackwell, L. F., & Buckley, P. D. (1990) Biochemistry 29, 2070-2080]. This enzyme form has not yet been cloned and expressed, but since the rat liver mitochondrial enzyme has been and shares 70% sequence homology with other cytosolic aldehyde dehydrogenases, the residue in the rat enzyme was converted into an alanine to test for the necessity of a hydroxyl group at that position. The recombinantly expressed mutant enzyme possessed 10% catalytic activity, but the Km for NAD increased from 10 to 1900 microM while the Kms for various aldehydes were unchanged. Kinetic analysis revealed that the dissociation constant for NAD also increased in the mutant as did k1, the on velocity for NAD binding. The mutant enzyme bound poorly to an AMP-Sepharose column and did not interact as well with NADH, as determined by fluorescence enhancement binding studies, or with ADP-ribose, a competitive inhibitor. Pulse-chase analysis showed that the mutant was as stable as was the recombinantly expressed native enzyme. It was less stable to heat denaturation at 50 degrees C (half-life of 1 min compared to 4). Converting the alanine to a cysteine or a threonine did not restore native-like properties of the enzyme. These mutants had kinetic properties very similar to those of the alanine mutant.(ABSTRACT TRUNCATED AT 250 WORDS)

Poly(ADP-ribose) polymerase in plant nuclei.

We show that poly(ADP-ribose) polymerase is present in maize, pea and wheat nuclei. We have identified the enzyme product as poly(ADP-ribose) by purification and electrophoresis on a DNA sequencing gel. This reveals a polymer ladder consisting of up to 45 residues. The polymer product from maize, after digestion with snake venom phosphodiesterase, gave only 5'-AMP and (phosphoribosyl)-AMP; the mean chain length of the polymer was 5 and 11 residues in two separate experiments. The optimum pH of the plant enzyme is greater than pH 7.0 in pea, wheat and maize; the optimum temperature for enzyme activity is approximately 15 degrees C. The Km for NAD+ for the enzyme from maize is estimated to be approximately 50 microM under optimal conditions. Several compounds (nicotinamide, deoxythymidine, 3-aminobenzamide, 3-methoxybenzamide and 5-bromodeoxyuridine) that specifically inhibit the animal enzyme also inhibit the enzyme from plants. The ratio of the IC50 for 5-bromodeoxyuridine to the IC50 for 3-aminobenzamide in maize is similar to that of the animal enzyme indicating that the enzyme involved is poly(ADP-ribose) polymerase and not mono(ADP-ribosyl) transferase. SDS gel electrophoresis and gel filtration analysis of a crude extract of maize nuclei indicate a molecular mass for poly(ADP-ribose) polymerase of approximately 114 kDa.

Efficient independent activity of a monomeric, monofunctional dehydroquinate synthase derived from the N-terminus of the pentafunctional AROM protein of Aspergillus nidulans.

The dehydroquinate synthase (DHQ synthase) functional domain from the pentafunctional AROM protein of Aspergillus nidulans has previously been overproduced in Escherichia coli [van den Hombergh, Moore, Charles and Hawkins (1992) Biochem J. 284, 861-867]. We now report the purification of this domain to homogeneity and subsequent characterization. The monofunctional DHQ synthase was found to retain efficient catalytic activity when compared with the intact pentafunctional AROM protein of Neurospora crassa [Lambert, Boocock and Coggins (1985) Biochem J. 226, 817-829]. The apparent kcat. was estimated to be 8 s-1, and the apparent Km values for NAD+ and 3-deoxy-D-arabino-heptulosonate phosphate (DAHP) were 3 microM and 2.2 microM respectively. These values are similar to those reported for the intact N. crassa enzyme, except that the apparent Km for NAD+ reported here is 15-fold higher. The monofunctional DHQ synthase domain is inactivated by treatment with chelating agents in the absence of substrates and is re-activated by the addition of metal ions; among those tested, Zn2+ gave the highest kcat./Km value. The enzyme is inactivated by diethyl pyrocarbonate; both the substrate, DAHP, and the product phosphate protected against inactivation. Size-exclusion chromatography suggested an M(r) of 43,000 for the monofunctional domain, indicating that it is monomeric and compactly folded. The c.d. spectrum confirmed that the domain has a compact globular conformation; the near-u.v. c.d. of zinc- and cobalt-reactivated domains were superimposable.

Effects of changing glutamate 487 to lysine in rat and human liver mitochondrial aldehyde dehydrogenase. A model to study human (Oriental type) class 2 aldehyde dehydrogenase.

Many Oriental people possess a liver mitochondrial aldehyde dehydrogenase where glutamate at position 487 has been replaced by a lysine, and they have very low levels of mitochondrial aldehyde dehydrogenase activity. To investigate the cause of the lack of activity of this aldehyde dehydrogenase, we mutated residue 487 of rat and human liver mitochondrial aldehyde dehydrogenase to a lysine and expressed the mutant and native enzyme forms in Escherichia coli. Both rat and human recombinant aldehyde dehydrogenases showed the same molecular and kinetic properties as the enzyme isolated from liver mitochondria. The E487K mutants were found to be active but possessed altered kinetic properties when compared to the glutamate enzyme. The Km for NAD+ at pH 7.4 increased more than 150-fold, whereas kcat decreased 2-10-fold with respect to the recombinant native enzymes. Detailed steady-state kinetic analysis showed that the binding of NAD+ to the mutant enzyme was impaired, and it could be calculated that this resulted in a decreased nucleophilicity of the active site cysteine residue. The rate-limiting step for the rat E487K mutant was also different from that of the recombinant rat liver aldehyde dehydrogenase in that no pre-steady-state burst of NADH formation was found with the mutant enzyme. Both the rat native enzyme and the E487K mutant oxidized chloroacetaldehyde twice as fast as acetaldehyde, indicating that the rate-limiting step was not hydride transfer or coenzyme dissociation but depended upon nucleophilic attack. Each enzyme form showed a 2-fold activation upon the addition of Mg2+ ions. Substituting a glutamine for the glutamate did not grossly affect the properties of the enzyme. Glutamate 487 may interact directly with the positive nicotinamide ring of NAD+ for the Ki of NADH was the same in the lysine enzyme as it was in the glutamate form. Because of the altered NAD+ binding properties and kcat of the E487K variant, it is assumed that people possessing this form will not have a functional mitochondrial aldehyde dehydrogenase.

The NAD-glycohydrolase activity of the pertussis toxin S1 subunit. Involvement of the catalytic HIS-35 residue.

Pertussis toxin is a member of ADP-ribosylating bacterial toxins that are capable of catalyzing the cleavage of the N-glycosidic bond of NAD+ and the transfer of its ADP-ribose moiety to G proteins. The catalytic S1 subunit of pertussis toxin uses signal transducing G proteins as acceptor substrates but can also catalyze the transfer of the ADP-ribose moiety to water in the absence of G proteins. Site-directed mutagenesis followed by kinetic analyses of truncated soluble mutant proteins revealed that His-35 of S1 is a catalytic residue because alterations of this residue affect the turnover rate of NAD-glycohydrolysis by approximately two orders of magnitude without significantly affecting substrate binding. Replacement of the imidazole of His-35 by the side chain of glutamine maintained the highest residual activity. The pH dependence of the enzyme activity showed only slight variations over the experimental range with an optimum at pH 7.5 and an approximate pKa of 6.5 to 7. This pH dependence was abolished by the Gln substitution, which still retained significant activity, suggesting that His-35 probably does not act as a true base but rather as a proton acceptor. Direct catalytic roles for several other residues were ruled out. Ser-52 substitutions resulted in slight alterations of both kcat and Km for NAD+ suggesting an involvement in maintaining the local geometry of the active site rather than a direct role in catalysis for this residue. Kinetic studies on mutants with substitutions of Ser-40 indicate a role in NAD+ binding for this residue. In conjunction with previous findings, these studies suggest that the NAD-glycohydrolase activity of S1 utilizes 2 catalytic residues, His-35 and the previously identified Glu-129. The enzyme mechanism could therefore proceed through an activation by polarization of the acceptor substrate water or G protein by His-35, and the stabilization of an oxocarbonium-like transition state intermediate by Glu-129.

Purification and partial characterization of malate dehydrogenase (decarboxylating) from Tritrichomonas foetus hydrogenosomes

Malate dehydrogenase (decarboxylating) from Tritrichomonas foetus hydrogenosomes was purified close to homogeneity by a combination of differential centrifugation, zwitterionic detergent solubilization, Red-Sepharose chromatography and anion-exchange chromatography. The enzyme with apparent subunit size of 59 kDa and native molecular mass of 308 kDa utilized NAD+ preferentially to NADP+ as a cofactor and required Mn2+ or Mg2+ for its activity. Affinity curves for malate and coenzymes were hyperbolic. Km for malate was 100 microM and 458 microM in the presence of NAD+ and NADP+, respectively. Km for NAD+ and for NADP+ in the presence of malate was 18 microM and 207 microM, respectively. The enzyme is proposed to be a tetramer with a possible physiological role in the maintenance of an appropriate NAD+/NADH ratio in hydrogenosomes.

Evidence for a catalytic role of glutamic acid 129 in the NAD-glycohydrolase activity of the pertussis toxin S1 subunit.

The S1 subunit of pertussis toxin is an ADP-ribosyl-transferase capable of transferring the ADP-ribose moiety of NAD+ to nucleotide-binding signal-transducing proteins of the Gi/G(o) family. In the absence of G proteins, the enzyme also catalyzes the hydrolysis of NAD+. Glu-129 was previously shown to be critical for both enzymatic activities. In this study, site-directed mutagenesis was used to make the conservative substitution of aspartate for Glu-129. The recombinant wild type and mutant proteins were purified to near homogeneity and used for enzymatic analyses. Kinetic experiments showed that the kcat of the mutant protein was about 200 times less than that of the wild type enzyme, whereas the Km for NAD+ of the two proteins were very similar, suggesting that Glu-129 is a catalytic residue for the NAD-glycohydrolase reaction of S1. This hypothesis was confirmed by a less than 2-fold change in Kd as measured by fluorescence quenching studies, indicating that the binding of NAD+ is not affected in the mutant protein in any important way. In another experiment, the replacement of Glu-129 by cysteine resulted in a disulfide bridge between Cys-129 and Cys-41 in rS1d-E129C, suggesting that the folding of the polypeptide chain is such that the catalytic Glu-129 residue is close to the amino-terminal NAD-binding site of S1. These findings imply that Glu-129 plays a key role in catalysis of the NAD-glycohydrolase reaction, possibly by electrostatically stabilizing a cationic transition state intermediate, or by serving as a general base to deprotonate the ADP-ribosyl acceptor substrates.

Cytoplasmic Poly(ADP-Ribose)Polymerase from Mouse Plasmacytoma Free Messenger Ribonucleoprotein Particles: Purification and Characterization

A cytoplasmic poly(ADP-ribose)polymerase (PARP) was purified from mouse plasmacytoma free messenger ribonucleoprotein particles using chromatography on 3-aminobenzamide affigel-10. The purified protein showed one band at 116 kDa on SDS-polyacrylamide gel electrophoresis and shared similar antigenic sites to the nuclear PARP. An apparent Km for NAD of 100.5 ± 6.3 μM and a Vmax of 174 ± 40 nmoles of ADP-ribose incorporated/min/mg protein were observed. RNA was detected in the enzyme preparation and the enzymatic activity was not DNA dependent.

Niacin deficiency and cancer in women.

A new interest in the relationship between niacin and cancer has evolved from the discovery that the principal form of this vitamin, NAD, is consumed as a substrate in ADP-ribose transfer reactions. Poly(ADP-ribose) polymerase, an enzyme activated by DNA strand breaks, is the ADP-ribosyltransferase of greatest interest with regard to effects on the niacin status of cells since its Km for NAD is high, and its activity can deplete NAD. Studies of the consequences of DNA damage in cultured mouse and human cells as a function of niacin status have supported the hypothesis that niacin may be a protective factor that limits carcinogenic events. To test this hypothesis in humans, we used a biochemical method based on the observation that as niacin nutriture decreases, NAD readily declines and NADP remains relatively constant. This has been demonstrated in both fibroblasts and in whole blood from humans. Thus, we use "niacin number," (NAD/NAD+NADP) x 100% from whole blood, as a measure of niacin status. Healthy control subjects showed a mean niacin number of 62.8 +/- 3.0 compared to 64.0 for individuals on a niacin-controlled diet. Analyses of women in the Malmö Diet and Cancer Study showed a mean niacin number of 60.4 with a range of 44 to 75. The distribution of niacin status in this population was nongaussian, with an unpredictably large number of individuals having low values.

Monomers of human beta 1 beta 1 alcohol dehydrogenase exhibit activity that differs from the dimer

A previously unreported enzymatic activity is described for monomers of the beta 1 beta 1 isoenzyme of human alcohol dehydrogenase that were prepared from dimeric enzyme by freeze-thaw in liquid nitrogen. Whereas the dimeric enzyme has optimal activity at low substrate concentrations (2.5 mM ethanol, 50 microM NAD+; "low Km" activity), the monomer has its highest activity at high substrate concentrations (1.5 M ethanol, 2.5 mM NAD+; "high Km" activity). While the activity of the monomer does not appear to be saturated at 1.5 M ethanol, its maximal activity at this high ethanol concentration exceeds the Vmax of the dimer by about 3-fold. The apparent Km of NAD+ with monomers is 270 microM, and no activity could be detected with nicotinamide mononucleotide as cofactor. During gel filtration the high Km activity elutes at a lower apparent molecular weight position than the dimer. The kinetics of monomer-to-dimer reassociation are consistent with a second-order process with a rate constant of 240 M-1 s-1. The reassociation rate is markedly enhanced by the presence of NAD+. During refolding of beta 1 beta 1 following denaturation in 6 M guanidine hydrochloride, an enzyme species with high Km activity and spectral properties similar to the freeze-thaw monomer is observed, indicating that a catalytically active monomer is an intermediate in the refolding pathway. The enzymatic activity of the monomer implies that the intersubunit contacts of beta 1 beta 1 are not crucial in establishing a catalytically competent enzyme. However, the differences in specific activity and Km between monomer and dimer suggest that dimerization may serve to modulate the catalytic properties.

Purification and characterization of an ADP-ribosyltransferase produced by Clostridium limosum.

We purified a novel ADP-ribosyltransferase produced by a Clostridium limosum strain isolated from a lung abscess and compared the exoenzyme with Clostridium botulinum ADP-ribosyltransferase C3. The C. limosum exoenzyme has a molecular weight of about 25,000 and a pI of 10.3. The specific activity of the ADP-ribosyltransferase is 3.1 nmol/mg/min with a Km for NAD of 0.3 microM. Partial amino acid sequence analysis of the tryptic peptides revealed about 70% homology with C3. The novel exoenzyme modifies selectively the small GTP-binding proteins of the rho family in human platelet membranes presumably at the same amino acid (asparagine 41) as known for C3. Recombinant rhoA and rhoB serve as substrates for C3 and the C. limosum exoenzyme. Whereas recombinant rac1 protein is only marginally ADP-ribosylated by C3 or by the C. limosum exoenzyme in the absence of detergent, in the presence of 0.01% sodium dodecyl sulfate rac1 is modified by C3 but not by the C. limosum exoenzyme. Recombinant CDC42Hs protein is a poor substrate for C. limosum exoenzyme and is even less modified by C3. The C. limosum exoenzyme is auto-ADP-ribosylated in the presence of 0.01% sodium dodecyl sulfate by forming an ADP-ribose protein bond highly stable toward hydroxylamine. The data indicate that ADP-ribosylation of small GTP-binding proteins of the rho family is not unique to C. botulinum C3 ADP-ribosyltransferase but is also catalyzed by a C3-related exoenzyme from C. limosum.

Poly(ADP ribose) polymerase-defective mutant cell clone of mouse L1210 cells

By a sequential mutation and selection utilizing N-methyl-N'-nitro-N-nitrosoguanidine as a mutagen, we succeeded in separating a poly(ADP ribose) polymerase-defective mutant clone (Cl-3527) from a mouse L1210 cell clone (Cl-3). The enzyme activity per cell in Cl-3527 cells was only 8% of that in wild type L1210 (CCL 219) cells. Immunoblot analysis of the enzyme protein in crude extracts of the mutant and wild type cells revealed that the enzyme defect was manifested as the loss of a 113-kDa wild type enzyme band in Cl-3527. Further analysis of partially purified enzyme from Cl-3527 by immunoblotting revealed that the molecular size of the enzyme in Cl-3527 was 108 kDa and that the amount of the mutant enzyme protein was markedly decreased in Cl-3527. The mutant enzyme was much more heat-labile than the wild type enzyme but the Km for NAD+, requirements for Mg2+ and nicked DNA, and the inhibition by 3-aminobenzamide, a potent inhibitor of the enzyme, however, were not so different from those of wild type enzyme. The mutant cells showed prolonged doubling time, increased temperature-sensitivity, increased percentage of active enzyme on a treatment of cells at high temperature, and increased expression of plasma membrane NADase, compared to wild type cells. Introduction of wild type ADPR pol gene into Cl-3527 cells partially restored the ADPR pol activity and the heat-resistance.

Ethanol enhances ADP-ribosylation of protein in rat hepatocytes.

Decreases in hepatocyte NAD+ produced by ethanol are only partially explained by the increased conversion of NAD+ to NADH and NADP+. The purpose of this study was to determine whether a mechanism for the ethanol-induced decrease in NAD+ is its increased use in ADP-ribosylation. Exposure of hepatocytes in culture for 2 hr to 100 mmol/L ethanol increased the incorporation of 14C-ribose from prelabeled NAD+ into 14C-ribosylated proteins. Poly (ADP-ribose) polymerase activity was increased by exposure of isolated hepatocytes to 100 mmol/L ethanol for 10 min. In hepatocyte culture, increases in poly (ADP-ribose) polymerase were not detected after exposure to 100 mmol/L ethanol for 10 min or 2 hr but rather occurred at 24 hr. Ethanol exposure of hepatocytes in culture for 2 hr, however, decreased the Km for NAD+ of poly (ADP-ribose) polymerase. Both nicotinamide and 5-aminobenzamide, which are inhibitors of poly (ADP-ribose) polymerase, prevented the decrease in NAD+ produced by 2-hr exposure of hepatocytes in culture to 100 mmol/L ethanol. The effect of ethanol in decreasing DNA synthesis on days 3 and 4 of culture was not reversed by the inhibitors of poly (ADP-ribose) polymerase. These results indicate that increased ADP-ribosylation of hepatocyte proteins is a mechanism for the effect of ethanol in decreasing NAD+.

ADP-ribosylation of gelsolin-actin complexes by clostridial toxins.

ADP-ribosylation of the 1:1 (G-A) and 1:2 (G-A-A) gelsolin-actin complexes by Clostridium perfringens iota toxin and Clostridium botulinum C2 toxin was studied. Iota toxin ADP-ribosylated actin in the G-A complex from human platelets as effectively as skeletal muscle actin. The Km for NAD (4 microM) was identical for both substrates. C2 toxin ADP-ribosylated actin in the G-A complex with lower efficacy than nonmuscle actin from platelet cytosol. In the G-A-A complex both actin molecules were ADP-ribosylated by iota toxin. The G-A complex bound ADP-ribosylated actin (Ar) to form the G-A-Ar complex in which the weakly bound actin is ADP-ribosylated. Vice versa, ADP-ribosylated 1:1 gelsolin-actin complex (G-Ar) was able to bind unmodified actin to yield the G-Ar-A complex. ADP-ribosylation did not change the nucleation activity of either the G-Ar complex or the G-Ar-A complex. When monomeric actin was added to the G-A-Ar complex, polymerization of actin was delayed by about 10 min. According to a quantitative kinetic analysis, the delay of polymerization corresponded to the rate of dissociation of ADP-ribosylated actin from the G-A-Ar complex. This suggests that the nucleation activity of the G-A-A complex is inhibited by ADP-ribosylation of the weakly bound actin and that the inhibition can be removed by dissociation of ADP-ribosylated actin from the G-A-Ar complex.

Expression and mutagenesis of human poly(ADP-ribose) polymerase as a ubiquitin fusion protein from Escherichia coli

The cDNA of human poly(ADP-ribose) polymerase (pADPRP), encoding the entire protein, was subcloned into the Escherichia coli expression plasmid pYUb. In this expression system, the carboxyl terminus of ubiquitin is fused to the amino terminus of a target protein, in this case pADPRP, stabilizing the accumulation of the cloned gene product. Following induction of the transformed cells, the sonicated extract contained a unique protein immunoreactive with both pADPRP and ubiquitin antibodies and corresponding to the predicted mobility of the fusion protein in SDS-PAGE. Fusion of ubiquitin to pADPRP increased the yield of pADPRP approximately 10-fold compared to that of the unfused enzyme. The resulting recombinant fusion protein had catalytic properties which were nearly identical to those of native pADPRP obtained from mammalian tissues. These properties included specific activity, Km for NAD, response to DNA strand breaks, response to Mg2+, inhibition by 3-aminobenzamide, and activity in activity gel analysis. An initial analysis by deletion mutagenesis of pADPRP's functional domains revealed that deletions in the NAD binding domain eliminated all activity; however, partial polymerase activity resulted from deletion in the DNA binding or automodification domains. The activities were not enhanced by breaks in DNA. We further report a colony filter screening procedure designed to identify functional polymerase molecules which will facilitate structure/function studies of the polymerase.

Purification and characterization of a molluscan egg-specific NADase, a second-messenger enzyme.

An egg-specific NADase has been purified to homogeneity from the ovotestis of the opisthobranch mollusk Aplysia californica. Unlike other NADases, the Aplysia enzyme generates primarily cyclic-ADP-ribose (cADPR) rather than ADP-ribose from NAD. cADPR has been shown to stimulate the release of Ca2+ from microsomes prepared from sea urchin egg and, when injected into intact eggs, to activate the cortical reaction, multiple nuclear cycles, and DNA synthesis. The Aplysia enzyme was initially identified as an inhibitor of cholera and pertussis toxin-catalyzed ADP-ribosylation. By the use of an NADase assay, it was purified from the aqueous-soluble fraction of ovotestis by sequential column chromatography. The enzyme has an apparent molecular mass of 29 kDa, a Km for NAD of 0.7 mM, and a turnover rate of approximately 27,000 mol NAD.min-1.mol enzyme-1 at 30 degrees C. Monoclonal antibodies were generated to the NADase. Immunoblots of two-dimensional gels revealed multiple isoforms of the enzyme, with pls ranging from 8.1 to 9.8. The multiple isoforms were resolved with a cation exchange high-pressure liquid chromatography column and shown to generate cADPR. Immunohistochemical analysis of cryostat sections of Aplysia ovotestis shows that the enzyme is specific to the eggs and restricted to large 5- to 10-microns granules or vesicles. To date the cADPR-generating enzyme activity has been identified in various organisms, including mammals. The Aplysia enzyme is the first example in which the enzyme that generates cADPR has been purified. All of the available evidence indicates that this NADase is a second-messenger enzyme, implying that other NADases may serve a similar function.

Coenzyme A-acylating aldehyde dehydrogenase from Clostridium beijerinckii NRRL B592

Acetaldehyde and butyraldehyde are substrates for alcohol dehydrogenase in the production of ethanol and 1-butanol by solvent-producing clostridia. A coenzyme A (CoA)-acylating aldehyde dehydrogenase (ALDH), which also converts acyl-CoA to aldehyde and CoA, has been purified under anaerobic conditions from Clostridium beijerinckii NRRL B592. The ALDH showed a native molecular weight (Mr) of 100,000 and a subunit Mr of 55,000, suggesting that ALDH is dimeric. Purified ALDH contained no alcohol dehydrogenase activity. Activities measured with acetaldehyde and butyraldehyde as alternative substrates were copurified, indicating that the same ALDH can catalyze the formation of both aldehydes for ethanol and butanol production. Based on the Km and Vmax values for acetyl-CoA and butyryl-CoA, ALDH was more effective for the production of butyraldehyde than for acetaldehyde. ALDH could use either NAD(H) or NADP(H) as the coenzyme, but the Km for NAD(H) was much lower than that for NADP(H). Kinetic data suggest a ping-pong mechanism for the reaction. ALDH was more stable in Tris buffer than in phosphate buffer. The apparent optimum pH was between 6.5 and 7 for the forward reaction (the physiological direction; aldehyde forming), and it was 9.5 or higher for the reverse reaction (acyl-CoA forming). The ratio of NAD(H)/NADP(H)-linked activities increased with decreasing pH. ALDH was O2 sensitive, but it could be protected against O2 inactivation by dithiothreitol. The O2-inactivated enzyme could be reactivated by incubating the enzyme with CoA in the presence or absence of dithiothreitol prior to assay.

Photolabelling of mutant forms of the S1 subunit of pertussis toxin with NAD+

The S1 subunit of pertussis toxin catalyses the hydrolysis of NAD+ (NAD+ glycohydrolysis) and the NAD(+)-dependent ADP-ribosylation of guanine-nucleotide-binding proteins. Recently, the S1 subunit of pertussis toxin was shown to be photolabelled by using radiolabelled NAD+ and u.v.; the primary labelled residue was Glu-129, thereby implicating this residue in the binding of NAD+. Studies from various laboratories have shown that the N-terminal portion of the S1 subunit, which shows sequence similarity to cholera toxin and Escherichia coli heat-labile toxin, is important to the maintenance of both glycohydrolase and transferase activity. In the present study the photolabelling technique was applied to the analysis of a series of recombinant-derived S1 molecules that possessed deletions or substitutions near the N-terminus of the S1 molecule. The results revealed a positive correlation between the extent of photolabelling with NAD+ and the magnitude of specific NAD+ glycohydrolase activity exhibited by the mutants. Enzyme kinetic analyses of the N-terminal mutants also identified a mutant with substantially reduced activity, a depressed photolabelling efficiency and a markedly increased Km for NAD+. The results support a direct role for the N-terminal region of the S1 subunit in the binding of NAD+, thereby providing a rationale for the effect of mutations in this region on enzymic activity.