Cyclic ADP-ribose production by CD38 regulates intracellular calcium release, extracellular calcium influx and chemotaxis in neutrophils and is required for bacterial clearance in vivo.

Cyclic ADP-ribose is believed to be an important calcium-mobilizing second messenger in invertebrate, mammalian and plant cells. CD38, the best-characterized mammalian ADP-ribosyl cyclase, is postulated to be an important source of cyclic ADP-ribose in vivo. Using CD38-deficient mice, we demonstrate that the loss of CD38 renders mice susceptible to bacterial infections due to an inability of CD38-deficient neutrophils to directionally migrate to the site of infection. Furthermore, we show that cyclic ADP-ribose can directly induce intracellular Ca++ release in neutrophils and is required for sustained extracellular Ca++ influx in neutrophils that have been stimulated by the bacterial chemoattractant, formyl-methionyl-leucyl-phenylalanine (fMLP). Finally, we demonstrate that neutrophil chemotaxis to fMLP is dependent on Ca++ mobilization mediated by cyclic ADP-ribose. Thus, CD38 controls neutrophil chemotaxis to bacterial chemoattractants through its production of cyclic ADP-ribose, and acts as a critical regulator of inflammation and innate immune responses.

Kinetic competence of the cADP-ribose-CD38 complex as an intermediate in the CD38/NAD+ glycohydrolase-catalysed reactions: implication for CD38 signalling.

CD38/NAD(+) glycohydrolase is a type II transmembrane glycoprotein widely used to study T- and B-cell activation and differentiation. CD38 is endowed with two different activities: it is a signal transduction molecule and an ectoenzyme that converts NAD(+) into ADP-ribose (NAD(+) glycohydrolase activity) and small proportions of cADP-ribose (cADPR; ADP-ribosyl cyclase activity), a calcium-mobilizing metabolite, which, ultimately, can also be hydrolysed (cADPR hydrolase activity). The relationship between these two properties, and strikingly the requirement for signalling in the formation of free or enzyme-complexed cADPR, is still ill-defined. In the present study we wanted to test whether the CD38-cADPR complex is kinetically competent in the conversion of NAD(+) into the reaction product ADP-ribose. In principle, such a complex could be invoked for cross-talk, via conformational changes, with neighbouring partner(s) of CD38 thus triggering the signalling phenomena. Analysis of the kinetic parameters measured for the CD38/NAD(+) glycohydrolase-catalysed hydrolysis of 2'-deoxy-2'-aminoribo-NAD(+) and ADP-cyclo[N1,C1']-2'-deoxy-2'-aminoribose (slowly hydrolysable analogues of NAD(+) and cADPR respectively) ruled out that the CD38-cADPR complex can accumulate under steady-state conditions. This was borne out by simulation of the prevalent kinetic mechanism of CD38, which involve the partitioning of a common E.ADP-ribosyl intermediate in the formation of the enzyme-catalysed reaction products. Using this mechanism, microscopic rate conditions were found which transform a NAD(+) glycohydrolase into an ADP-ribosyl cyclase. Altogether, the present work shows that if the cross-talk with a partner depends on a conformational change of CD38, this is most probably not attributable to the formation of the CD38-cADPR complex. In line with recent results on the conformational change triggered by CD38 ligands [Berthelier, Laboureau, Boulla, Schuber and Deterre (2000) Eur. J. Biochem. 267, 3056-3064], we believe that the Michaelis CD38-NAD(+) complex could play such a role instead.

Aldehyde dehydrogenase activity of Drosophila melanogaster alcohol dehydrogenase: burst kinetics at high pH and aldehyde dismutase activity at physiological pH.

The ability of Drosophila alcohol dehydrogenase (D-ADH) to catalyze the oxidation of aldehydes to carboxylic acids has been re-examined. Prior studies are shown to have been compromised by a nonenzymic reaction between the aldehydic substrates and amine-containing buffers, e.g., glycine or Tris, and an amine-catalyzed addition of aldehyde to NAD+. These reactions interfere with spectrophotometric assays for monitoring aldehyde oxidation and obscure the nature and scope of D-ADH-catalyzed aldehyde oxidation, particularly at physiological pH. Use of nonreactive buffers, such as pyrophosphate or phosphate, and 1H NMR spectroscopy to monitor all the components of the reaction mixture reveals the facile dismutation of aldehydes into equimolar quantities of the corresponding acids and alcohols at both neutral and high pH. At high pH, dismutation is accompanied by a small burst of NADH production to a steady-state concentration ( < 10 microM) that represents a partitioning between NADH dissociation and aldehyde reduction. The increase in A340 is therefore not a direct measure of the aldehyde oxidation reaction, and the resulting kinetic values cannot be compared to those for alcohol dehydrogenation. The present results for D-ADH, combined with data from the literature, establish that aldehyde oxidation, manifest as dismutation, is a widespread property of alcohol dehydrogenases with potential physiological importance in alcohol metabolism and aldehyde detoxification.

Mechanistic implications of cyclic ADP-ribose hydrolysis and methanolysis catalyzed by calf spleen NAD+glycohydrolase.

Calf spleen NAD+glycohydrolase, besides its well known reactions, was shown to catalyze hydrolysis and methanolysis (with retention of configuration) of cyclic ADP-ribose, indicating that this classical NADase also belongs to the class of cyclic ADP-ribose hydrolases. No formation of cyclic ADP-ribose could be detected during the hydrolysis of beta-NAD+; moreover, the kinetic parameters of cyclic ADP-ribose seem to rule out that it is a kinetically competent reaction intermediate in the conversion of NAD+ into ADP-ribose by this enzyme.

NAD hydrolysis: chemical and enzymatic mechanisms.

The pyridine nucleotides have important non-redox activities as cellular effectors and metabolic regulators [1-3]. The enzyme-catalyzed cleavage of the nicotinamide-ribosyl bond of NAD+ and the attendant delivery of the ADPRibosyl moiety to acceptors is central to these many diverse biological activities. Included are the medically important NAD-dependent toxins associated with cholera, diphtheria, pertussis, and related diseases [4]; the reversible ADPRibosylation-mediated biological regulatory systems [5,6]; the synthesis of poly(ADPRibose) in response to DNA damage or cellular division [7]; and the synthesis of cyclic ADPRibose as part of an independent, calcium-mediated regulatory system [8]. As will be presented in this chapter, all evidence points to both the chemical and enzyme-catalyzed cleavage of the nicotinamide-ribosyl bond being dissociative in character via an oxocarbenium intermediate.

The oxidation of aldehydes by horse liver alcohol dehydrogenase.

A lag phase in the spectrophotometric assay progress curve of aldehyde oxidation by HL-ADH was observed and characterised. The aldehyde oxidation and aldehyde dismutation reactions were shown to be related, and a mechanism to explain net aldehyde oxidation was proposed. The spectrophotometric assay was shown to be unsuitable for measurement of kinetic parameters for aldehyde oxidation by HL-ADH, and kinetic constants previously determined were shown to be in error. Existing data on the aldehyde dismutation reaction are insufficient to discount a role for HL-ADH in aldehyde transformation in vivo.

Horse liver alcohol dehydrogenase-catalyzed oxidation of aldehydes: dismutation precedes net production of reduced nicotinamide adenine dinucleotide.

The oxidation of aldehydes by horse liver alcohol dehydrogenase (HL-ADH) is more complex than previously recognized. At low enzyme concentrations and/or high aldehyde concentrations, a pronounced lag in the assay progress curve is observed when the reaction is monitored for NADH production at 340 nm. When the progress of the reaction is followed by 1H NMR spectroscopy, rapid dismutation of the aldehyde substrate into the corresponding acid and alcohol is observed during the lag phase. Steady-state production of NADH commences only after aldehyde concentrations drop below 5% of their initial value; thereafter, NADH production occurs with continuous adjustment of the equilibrium between aldehyde, alcohol, NADH, and NAD+. The steady-state NADH production exhibits normal Michaelis-Menten kinetics and is in accord with earlier studies using much higher enzyme concentrations where no lag phase was reported. These results establish that the ability of HL-ADH to oxidize aldehydes is much greater than previously thought. The relationship between aldehyde dismutase and aldehyde dehydrogenase activities of HL-ADH is discussed.

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

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

Slow-binding inhibition of NAD+ glycohydrolase by arabino analogues of beta-NAD.

Modifications at the 2'-position of the nicotinamide-ribosyl moiety influence dramatically the nature of the interactions of the modified beta-NAD+ with calf spleen NAD+ glycohydrolase (EC 3.2.2.6), an enzyme that cleaves the nicotinamide-ribose bound in NAD(P)+. Nicotinamide arabinoside adenine dinucleotide (ara-NAD+) and nicotinamide 2'-deoxy-2'-fluoroarabinoside adenine dinucleotide (araF-NAD+) are not hydrolyzed at measurable rates and are the first documented examples of reversible slow binding inhibitors of this class of enzyme. The kinetic data obtained are consistent with both slow kon and koff rate constants in the formation of an enzyme-inhibitor complex, i.e. the association rate constants are about 10(4) and 10(6) slower than diffusion rates, respectively, for araF-NAD+ and ara-NAD+, and the half-life of the complex is about 3-10 min for both analogues. The kinetic model does not account for a slow turnover of an ADP-ribosyl-enzyme intermediary complex. AraF-NAD+ is one of the most potent inhibitors described for NAD+ glycohydrolase.

Thiol reduction of 3'-azidothymidine to 3'-aminothymidine: kinetics and biomedical implications.

The ability of thiols to reduce 3'-azidothymidine (AZT) to 3'-aminothymidine has been investigated. Incubation with glutathione, dithiothreitol (DTT), or mercaptoethanol at pH 7.2 and 37 degrees C leads to quantitative reduction of the azido moiety to an amine. The reaction is first order in AZT and first order in reducing agent (mono- or dithiol). The second-order rate constants are 2.77 x 10(-3), 6.55 x 10(-5), and 6.35 x 10(-6) M-1 sec-1 for the dithiothreitol, glutathione, and mercaptoethanol reductions, respectively. The thiol reduction of alkyl azide to amine under mild conditions is a synthetic method particularly suitable for water-soluble azido compounds that are sensitive to catalytic hydrogenation. The potential for the mono- or dithiol-mediated reduction of alkyl azides under biological conditions must be considered when conducting studies of azido drugs.

Enhanced bleomycin-mediated damage of DNA opposite charged nicks. A model for bleomycin-directed double strand scission of DNA.

The anticancer drug, bleomycin, causes both single and double strand scission of duplex DNA in vitro, with double strand scission occurring in excess of that expected from the random accumulation of single strand nicks. The mechanism of the preferential double strand scission of DNA by bleomycin has been investigated through the synthesis of a series of double hairpin and linear oligonucleotides designed to contain a single nick-like structure at a defined site to serve as models of bleomycin-damaged duplex DNA. The 3' and/or 5' hydroxyls flanking the nick have been phosphorylated to model the increased negative charge at a bleomycin-generated nick. The ability of bleomycin to cleave the intact strand opposite the nick was then determined by autoradiography. The results demonstrate that phosphorylation at either the 3' or 5' hydroxyl, and especially when both sites are phosphorylated, strongly enhances selective cleavage by bleomycin of the opposite strand. These experiments indicate that bleomycin-mediated double strand scission is a form of self-potentiation in which the high affinity of bleomycin for the initially generated nicked sites leads to a greatly enhanced probability of scission of the strand opposite those sites.

Pyridine coenzyme analogues. Synthesis and characterization of alpha- and beta-nicotinamide arabinoside adenine dinucleotides.

The synthesis and characterization of a new pyridine coenzyme analogue containing a nicotinamide arabinonucleotide moiety are reported. The redox potentials are -339 mV for beta-oxidized nicotinamide arabinoside adenine dinucleotide and -319 mV for alpha-oxidized nicotinamide adenine dinucleotide, and the lambda max is 346 and 338 nm for beta- and alpha-reduced nicotinamide arabinoside adenine dinucleotides (araNADH), respectively. Anomerization of the reduced analogues leads to a 5:1 ratio of alpha-araNADH to beta-araNADH at 90 degrees C. These results establish that the relative configuration of the 2'-hydroxyl to the base is the primary determinant for the configuration-dependent changes in lambda max, the redox potential of the pyridine nucleotides, and the preferred anomeric configuration of the reduced coenzymes. Comparison of the 1H and 31P NMR spectral data of the analogues with those for the ribo coenzymes is reported and the conformational analysis discussed. The coenzyme properties of the arabino analogues have been evaluated with yeast and horse liver alcohol dehydrogenases. Both the alpha- and beta-anomers are found to serve as coenzymes, and the stereochemistry of hydride transfer is identical for both anomers.

Copper-dependent cleavage of DNA by bleomycin.

DNA strand scission by bleomycin in the presence of Cu and Fe was further characterized. It was found that DNA degradation occurred readily upon admixture of Cu(I) or Cu(II) + dithiothreitol + bleomycin, but only where the order of addition precluded initial formation of Cu(II)--bleomycin or where sufficient time was permitted for reduction of the formed Cu(II)--bleomycin to Cu(I)--bleomycin. DNA strand scission mediated by Cu + dithiothreitol + bleomycin was inhibited by the copper-selective agent bathocuproine when the experiment was carried out under conditions consistent with Cu chelation by bathocuproine on the time scale of the experiment. Remarkably, it was found that the extent of DNA degradation obtained with bleomycin in the presence of Fe and Cu was greater than that obtained with either metal ion alone. A comparison of the sequence selectivity of bleomycin in the presence of Cu and Fe using 32P-end-labeled DNA duplexes as substrates revealed significant differences in sites of DNA cleavage and in the extent of cleavage at sites shared in common. For deglycoblemycin and decarbamoylbleomycin, whose metal ligation is believed to differ from that of bleomycin itself, it was found that the relative extents of DNA cleavage in the presence of Cu were not in the same order as those obtained in the presence of Fe. The bleomycin-mediated oxygenation products derived from cis-stilbene were found to differ in type and amount in the presence of added Cu vs. added Fe. Interestingly, while product formation from cis-stilbene was decreased when excess Fe was added to a reaction mixture containing 1:1 Fe(III) and bleomycin, the extent of product formation was enhanced almost 4-fold in reactions that contained 5:1, as compared to 1:1, Cu and bleomycin. The results of these experiments are entirely consistent with the work of Sugiura [Sugiura, Y. (1979) Biochem. Biophys. Res. Commun. 90, 375-383], who first demonstrated the generation of reactive oxygen species upon admixture of O2 and Cu(I)--bleomycin.

Remote nitrogen-15 isotope effects on addition of cyanide to NAD.

The reversible reaction NAD + CN(-)----NAD-CN was examined for remote secondary 15N isotope effects caused by isotopic substitution at the ring nitrogen of the nicotinamide group. These were compared with analogous effects for dehydrogenase-catalyzed reactions, since both cyanide and the hydride ion add at the N-4 position of the nicotinamide ring. The 15N effects on the rate constants for the forward and reverse processes were examined directly by conducting both the normal and isotopic reactions simultaneously under carefully controlled conditions in the sample and reference cells of a dual-beam spectrophotometer. In both cases, the 15N kinetic isotope effect differed from 1.00 by considerably less than 0.01. The 15N equilibrium isotope effect, 15K, was obtained as the ratio of equilibrium constants measured separately with natural-abundance and labeled NAD by using a concentration jump procedure [1.004 +/- 0.002 (cyanide addition)]. A similar value for 15K of 1.010 +/- 0.008 was obtained in an analogous manner for the reaction catalyzed by lactate dehydrogenase: NAD + lactate----pyruvate + NADH + H+. The latter value is significantly smaller than a previously reported value obtained from kinetic studies [1.044 +/- 0.012; Cook, P. F., Oppenheimer, N. J., & Cleland, W. W. (1981) Biochemistry 20, 1817]. The present value also is smaller than might be expected for a change in bond order from 4 to 3 [Cleland, W. W. (1980) Methods Enzymol. 64, 104-125] on the basis of the canonical resonance structures for NAD and NADH.(ABSTRACT TRUNCATED AT 250 WORDS)

The stereospecificity of oxidation of alpha-[4R-2H]NADH by dehydrogenases.

The stereospecificity of the enzyme-dependent oxidation of alpha-[4R-2H]NADH has been determined for four dehydrogenases: two pro-R specific enzymes, pig heart malate dehydrogenase and yeast alcohol dehydrogenase; and two pro-S specific enzymes, rabbit muscle glycerol-3-phosphate dehydrogenase and Rhodopseudomonas spheroides 3-hydroxybutyrate dehydrogenase. In all cases, an enzyme-dependent and substrate-specific oxidation to alpha-NAD+ is observed with the stereochemistry of oxidation identical with that found for the oxidation of the correspondingly labeled beta-NADH. The ability of dehydrogenases from diverse sources to utilize alpha-NADH in a stereochemically competent fashion is discussed in relation to proposed interactions between the nicotinamide sugar moiety and active site residues or obligatory alignments of the pyridine and sugar moieties.

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.

Two-dimensional 1H NMR of three spin-labeled derivatives of bovine pancreatic trypsin inhibitor.

Three nitroxide spin-labeled monoderivatives of bovine pancreatic trypsin inhibitor were prepared with the amino-specific reagent succinimidyl 1-oxy-2,2,5,5-tetramethyl-3-pyrroline-3-carboxylate. The monoderivatives were purified by ion-exchange and affinity chromatography. Thin-layer maps of tryptic peptides of the monoderivatives showed that the spin-label was incorporated at either the alpha-amino group, Lys-15, or Lys-26. Two-dimensional J-correlated 1H NMR spectra of the monoderivatives were recorded. Spectra were also recorded after reduction by ascorbic acid of the nitroxide label to hydroxylamine. With the nitroxide label present, significant line-broadening effects on many of the cross peaks in the spectra were observed. The extent of line broadening for the C alpha H-NH cross peaks was qualitatively correlated with the distance between the labeled amino group and the average C alpha H-NH position in the crystal structure. The spin-label affects cross peaks of protons within approximately 15 A. This study suggests that it is feasible to accumulate sufficient intramolecular distances in order to determine protein solution structures with the aid of distance geometry algorithms.