Effects of NAD or NADP on the stability of liver and pectoral muscle enzymes in 3-acetylpyridine treated quail by heat and trypsin

(1) The effects of long term treatment with 3-acetylpyridine on the stability of enzymes towards heat and trypsin treatment were studied. (2) In the liver NAD or NADP provided a similar degree of protection against heat inactivation at 55 degrees C for 6-phosphogluconate dehydrogenase (24%), glyceraldehyde-3-phosphate dehydrogenase (24%) and malic enzyme (20%), low level of protection of lactate dehydrogenase (13%) but didn't affect acetylcholinesterase at all. In the muscle, however, there was substantial protection against heat inactivation by coenzyme of glyceraldehyde-3-phosphate dehydrogenase (52%), an intermediate level of protection of lactate dehydrogenase (25%), low level of protection of 6-phosphogluconate dehydrogenase (17%) and malic enzyme (17%) and almost no protection of acetylcholinesterase. (3) In the susceptibility towards trypsin a low but similar degree of protection for dehydrogenases by coenzymes was observed in the liver whereas in the muscle there was substantial protection against trypsin inactivation by NAD of glyceraldehyde-3-phosphate dehydrogenase, an intermediate level of protection of 6-phosphogluconate dehydrogenase and malic enzyme and very little protection of lactate dehydrogenase but no protection of acetylcholinesterase. Among enzymes tested, glyceraldehyde-3-phosphate dehydrogenase showed the greatest protection against heat and trypsin inactivation by NAD. (4) The results suggest that the effect of 3-acetylpyridine treatment on the stability of muscle glyceraldehyde-3-phosphate dehydrogenase appears to be quite specific and selective.     

Study of the interaction of glyceraldehyde-3-phosphate dehydrogenase with DNA

Glyceraldehyde-3-phosphate dehydrogenase binds to homologous and heterologous single-stranded but not double-stranded DNA. Binding to RNA, poly(A) and poly(dA-dT) has also been observed. Enzyme binding to these nucleic acids leads to the formation of an insoluble complex which can be sedimented at low speed. The interaction of glyceraldehyde-3-phosphate dehydrogenase with DNA is strongly inhibited by NAD and NADH but not by NADP. Adenine nucleotides, which inhibit the dehydrogenase activity by competing with NAD for its binding site (Yang, S.T. and Deal, W.C., Jr. (1969) Biochemistry 8, 2806--2813), also inhibit enzyme binding to DNA, whereas glyceraldehyde-3-phosphate and inorganic phosphate are non-inhibitory. These results suggest that DNA interacts through the NAD binding sites of glyceraldehyde-3-phosphate dehydrogenase. In accordance with this idea, it was found that DNA also binds to lactate dehydrogenase, an enzyme containing a similar dinucleotide binding domain, and that this binding is inhibited by NADH. A study of the base specificity of the DNA-glyceraldehyde-3-phosphate dehydrogenase interaction using dinucleoside monophosphates shows that inhibition of DNA binding by the dinucleotides requires the presence of a 3'-terminal adenosine and is greater when the 5'-terminus contains a pyrimidine instead of a purine. These results suggest that the dinucleotides bind at the NAD site of the dehydrogenase and that the enzyme would interact preferentially with PypA dinucleotides present in the nucleic acid.     

Control of the shift from homolactic acid to mixed-acid fermentation in Lactococcus lactis: predominant role of the NADH/NAD+ ratio

During batch growth of Lactococcus lactis subsp. lactis NCDO 2118 on various sugars, the shift from homolactic to mixed-acid metabolism was directly dependent on the sugar consumption rate. This orientation of pyruvate metabolism was related to the flux-controlling activity of glyceraldehyde-3-phosphate dehydrogenase under conditions of high glycolytic flux on glucose due to the NADH/NAD+ ratio. The flux limitation at the level of glyceraldehyde-3-phosphate dehydrogenase led to an increase in the pool concentrations of both glyceraldehyde-3-phosphate and dihydroxyacetone-phosphate and inhibition of pyruvate formate lyase activity. Under such conditions, metabolism was homolactic. Lactose and to a lesser extent galactose supported less rapid growth, with a diminished flux through glycolysis, and a lower NADH/NAD+ ratio. Under such conditions, the major pathway bottleneck was most probably at the level of sugar transport rather than glyceraldehyde-3-phosphate dehydrogenase. Consequently, the pool concentrations of phosphorylated glycolytic intermediates upstream of glyceraldehyde-3-phosphate dehydrogenase decreased. However, the intracellular concentration of fructose-1,6-bisphosphate remained sufficiently high to ensure full activation of lactate dehydrogenase and had no in vivo role in controlling pyruvate metabolism, contrary to the generally accepted opinion. Regulation of pyruvate formate lyase activity by triose phosphates was relaxed, and mixed-acid fermentation occurred (no significant production of lactate on lactose) due mostly to the strong inhibition of lactate dehydrogenase by the in vivo NADH/NAD+ ratio.     

Polyol metabolism by a caries-conducive Streptococcus: purification and properties of a nicotinamide adenine dinucleotide-dependent mannitol-1-phosphate dehydrogenase

The mannitol-1-phosphate dehydrogenase (M1PDH) (EC 1.1.1.17) from Streptococcus mutans strain FA-1 was purified to approximately a 425-fold increase in specific activity with a 29% recovery of total enzyme units, using a combination of (i) streptomycin sulfate and ammonium sulfate precipitation and (ii) diethyl-aminoethyl-cellulose (DE-52), agarose A 0.5M, and agarose-nicotinamide adenine dinucleotide (NAD) affinity column chromatography. Polyacrylamide gel electrophoresis of the purified enzyme preparation showed a single protein component that coincided with a band of M1PDH activity. The enzyme had a molecular weight of approximately 45,000 and was stable for long periods of time when stored at -80 degrees C in the presence of beta-mercaptoethanol. Its activity was not affected by mono- or divalent cations, and high concentrations of ethylenedia-minetetraacetic acid were not inhibitory. The M1PDH catalyzed both the NAD-dependent oxidation of mannitol-1-phosphate and the reduced NAD (NADH)-dependent reduction of fructose-6-phosphate. The forward reaction was highly specific for mannitol-1-phosphate and NAD, whereas the reverse reaction was highly specific for NADH and fructose-6-phosphate. The K(m) values for mannitol-1-phosphate and NAD were 0.15 and 0.066 mM, respectively, and the K(m) values for fructose-6-phosphate and NADH were 1.66 and 0.016 mM, respectively. The forward and reverse reactions catalyzed by the M1PDH from S. mutans appeared to be under cellular control. Both adenosine 5'-triphosphate and fructose-6-phosphate were negative effectors of the forward reaction, whereas adenosine 5'-diphosphate served as a negative effector of the reverse reaction catalyzed by the enzyme.     

Selective inactivation of glyceraldehyde-3-phosphate dehydrogenase by vinyl sulfones

Incubation of glyceraldehyde-3-phosphate dehydrogenase with vinyl sulfones resulted in a pseudo first-order loss of enzyme activity. The selective inactivation of the enzyme by vinyl sulfones is suggested from the structural requirement analysis and the enzyme susceptibility test. The enzyme inactivation was strongly reduced in the presence of NAD or glyceraldehyde-3-phosphate, and the prior treatment of the enzyme with 5,5'-dithio-bis-(2-nitrobenzoic acid) prevented the enzyme from the inactivation by vinyl sulfones (> or = 90%). Moreover, the early rapid phase of inactivation was much more responsive to L-cysteine reactivation, compared with the slower phase. Based on these results, it is proposed that vinyl sulfones inactivate the enzyme by inducing the oxidation of cysteine residue and/or covalent binding to cysteine residue in active site.     

Structure of 3-isopropylmalate dehydrogenase in complex with NAD+: ligand-induced loop closing and mechanism for cofactor specificity

BACKGROUND: The leucine biosynthetic enzyme 3-isopropylmalate dehydrogenase (IMDH) belongs to a unique class of bifunctional decarboxylating dehydrogenases. The two best-known members of this family, IMDH and isocitrate dehydrogenase (IDH), share a common structural framework and catalytic mechanism but have different substrate and cofactor specificities. IMDH is NAD(+)-dependent, while IDHs occur in both NAD(+)-dependent and NADP(+)-dependent forms. RESULTS: We have co-crystallized Thermus thermophilus IMDH with NAD+ and have determined the structure at 2.5 A resolution. NAD+ binds in an extended conformation. Comparisons with the structure in the absence of cofactor show that binding induces structural changes of up to 2.5 A in the five loops which form the dinucleotide-binding site. The adenine and diphosphate moieties of NAD+ are bound via interactions which are also present in the NADP(+)-IDH complex. Amino acids which interact with the NADP+ 2'-phosphate in IDH are substituted or absent in IMDH. The adenosine ribose forms two hydrogen bonds with Asp278, and the nicotinamide and nicotinamide ribose interact with Glu87 and Asp78, all unique to IMDH. CONCLUSIONS: NAD+ binding induces a conformational transition in IMDH, resulting in a structure that is intermediate between the most 'open' and 'closed' decarboxylating dehydrogenase conformations. Physiological specificity of IMDH for NAD+ versus NADP+ can be explained by the unique interaction between Asp278 and the free 2'-hydroxyl of the NAD+ adenosine, discrimination against the presence of the 2'-phosphate by the negative charge on Asp278, and the absence of potential favorable interactions with the 2'-phosphate of NADP+.     

Comparative enzymatic properties of GapB-encoded erythrose-4-phosphate dehydrogenase of Escherichia coli and phosphorylating glyceraldehyde-3-phosphate dehydrogenase

GapB-encoded protein of Escherichia coli and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) share more than 40% amino acid identity. Most of the amino acids involved in the binding of cofactor and substrates to GAPDH are conserved in GapB-encoded protein. This enzyme shows an efficient non-phosphorylating erythrose-4-phosphate dehydrogenase activity (Zhao, G., Pease, A. J., Bharani, N., and Winkler, M. E. (1995) J. Bacteriol. 177, 2804-2812) but a low phosphorylating glyceraldehyde-3-phosphate dehydrogenase activity, whereas GAPDH shows a high efficient phosphorylating glyceraldehyde-3-phosphate dehydrogenase activity and a low phosphorylating erythrose-4-phosphate dehydrogenase activity. To identify the structural factors responsible for these differences, comparative kinetic and binding studies have been carried out on both GapB-encoded protein of Escherichia coli and GAPDH of Bacillus stearothermophilus. The KD constant of GapB-encoded protein for NAD is 800-fold higher than that of GAPDH. The chemical mechanism of erythrose 4-phosphate oxidation by GapB-encoded protein is shown to proceed through a two-step mechanism involving covalent intermediates with Cys-149, with rates associated to the acylation and deacylation processes of 280 s-1 and 20 s-1, respectively. No isotopic solvent effect is observed suggesting that the rate-limiting step is not hydrolysis. The rate of oxidation of glyceraldehyde 3-phosphate is 0.12 s-1 and is hydride transfer limiting, at least 2000-fold less efficient compared with that of erythrose 4-phosphate. Thus, it can be concluded that it is only the structure of the substrates that prevails in forming a ternary complex enzyme-NAD-thiohemiacetal productive (or not) for hydride transfer in the acylation step. This conclusion is reinforced by the fact that the rate of oxidation for erythrose 4-phosphate by GAPDH is 0.1 s-1 and is limited by the acylation step, whereas glyceraldehyde 3-phosphate acylation is efficient and is not rate-determining (>/=800 s-1). Substituting Asn for His-176 on GapB-encoded protein, a residue postulated to facilitate hydride transfer as a base catalyst, decreases 40-fold the kcat of glyceraldehyde 3-phosphate oxidation. This suggests that the non-efficient positioning of the C-1 atom of glyceraldehyde 3-phosphate relative to the pyridinium of the cofactor within the ternary complex is responsible for the low catalytic efficiency. No phosphorylating activity on erythrose 4-phosphate with GapB-encoded protein is observed although the Pi site is operative as proven by the oxidative phosphorylation of glyceraldehyde 3-phosphate. Thus the binding of inorganic phosphate to the Pi site likely is not productive for attacking efficiently the thioacyl intermediate formed with erythrose 4-phosphate, whereas a water molecule is an efficient nucleophile for the hydrolysis of the thioacyl intermediate. Compared with glyceraldehyde-3-phosphate dehydrogenase activity, this corresponds to an activation of the deacylation step by >/=4.5 kcal.mol-1. Altogether these results suggest subtle structural differences between the active sites of GAPDH and GapB-encoded protein that could be revealed and/or modulated by the structure of the substrate bound. This also indicates that a protein engineering approach could be used to convert a phosphorylating aldehyde dehydrogenase into an efficient non-phosphorylating one and vice versa.     

Purification and biochemical properties of allelic forms of cytoplasmic glycerol-3-phosphate dehydrogenase from Drosophila virilis

Three homozygous allelic forms (alpha GPDHf, alpha GPDHm and alpha GPDHs) of NAD+-dependent glycerol-3-phosphate dehydrogenase (sn-glycerol-3-phosphate:NAD+ 2-oxidoreductase, EC 1.1.1.8) of Drosophila virilis were purified to homogeneity and their biochemical properties were compared. Although these three forms were mutually distinguishable by electrophoresis, no significant differences were found with respect to pH optima for both forward and reverse reactions (pH 6.0--6.5 for dihydroxyacetone phosphate reduction; pH 10.0--10.5 for glycerol 3-phosphate oxidation), native and subunit molecular weights (65 000 for native form; 35 000--37 000 for subunit) and Michaelis constants for NADH, glycerol 3-phosphate and NAD+ (5.3--6.0 microM for NADH; 1.8--1.9 mM for glycerol 3-phosphate; 100--110 microM for NAD+). Significant differences among three forms were observed in thermostability at 35 degrees C and inhibition by excess of dihydroxyacetone phosphate. The alpha GPDHf form was found to be most thermolabile and the alpha GPDHs form most susceptible to the inhibition.     

Bismuth carbomer foam enemas for active chronic pouchitis: a randomized, double-blind, placebo-controlled trial

One biological effect of nitric oxide (NO) has been believed to be exerted through induction of the ADP-ribosyltransferase activity of glyceraldehyde-3-phosphate dehydrogenase (GAPDH). Though this notion is based on the finding that NO increases the auto-ADP-ribosylation of GAPDH, controversial data have also been reported. To determine whether or not NO really activates ADP-ribosylation, we re-examined the NO-induced modification of GAPDH with NAD+. GAPDH was modified equally with [adenosine-14C]NAD+ and [carbonyl-14C]NAD+, indicating that the glycoside bond of NAD+ between ADP-ribose and nicotinamide is intact. The release of nicotinamide from NAD+ was not evident during incubation of GAPDH with [carbonyl-14C]NAD+. Thus, the modification of GAPDH is apparently not ADP-ribosylation. In addition, we found that basal and glyceraldehyde-3-phosphate-induced modifications of GAPDH, both of which have also been explained as ADP-ribosylation, were not ADP-ribosylation, and that the modification of GAPDH in the absence and presence of NO or GA3P was distinct in the dithiothreitol effect or resistance to HgCl2.     

Isolation of protease-free alcohol dehydrogenase (ADH) from Drosophila simulans and several homozygous and heterozygous Drosophila melanogaster variants

The enzyme alcohol dehydrogenase (ADH) from several naturally occurring ADH variants of Drosophila melanogaster and Drosophila simulans was isolated. Affinity chromatography with the ligand Cibacron Blue and elution with NAD+ showed similar behavior for D. melanogaster ADH-FF, ADH-71k, and D. simulans ADH. Introduction of a second Cibacron Blue affinity chromatography step, with gradient elution with NAD+, resulted in pure and stable enzymes. D. melanogaster ADH-SS cannot be eluted from the affinity chromatography column at a high concentration of NAD+ and required a pH gradient for its purification, preceded by a wash step with a high concentration of NAD+. Hybrid Drosophila melanogaster alcohol dehydrogenase FS has been isolated from heterozygous flies, using affinity chromatography with first elution at a high concentration NAD+, directly followed by affinity chromatography elution with a pH gradient. Incubation of equal amounts of pure homodimers of Drosophila melanogaster ADH-FF and ADH-SS, in the presence of 3 M urea at pH 8.6, for 30 min at room temperature, followed by reassociation yielded active Drosophila melanogaster ADH-FS heterodimers. No proteolytic degradation was found after incubation of purified enzyme preparations in the absence or presence of SDS, except for some degradation of ADH-SS after very long incubation times. The thermostabilities of D. melanogaster ADH-71k and ADH-SS were almost identical and were higher than those of D. melanogaster ADH-FF and D. simulans ADH. The thermostability of D. melanogaster ADH-FS was lower than those of D. melanogaster ADH-FF and ADH-SS. D. melanogaster ADH-FF and ADH-71k have identical inhibition constants with the ligand Cibacron Blue at pH 8.6, which are two times higher at pH 9.5. The Ki values for D. simulans ADH are three times lower at both pH values. D. melanogaster ADH-SS and ADH-FS have similar Ki values, which are lower than those for D. melanogaster ADH-FF at pH 8.6. But at pH 9.5 the Ki value for ADH-FS is the same as at pH 8.6, while that of ADH-SS is seven times higher. Kinetic parameters of Drosophila melanogaster ADH-FF, ADH-SS, and ADH-71k and Drosophila simulans ADH, at pH 8.6 and 9.5, showed little or no variation in K(m)eth values. The K(m)NAD values measured at pH 9.5 for Drosophila alcohol dehydrogenases are all lower than those measured at pH 8.6. The rate constants (kcat) determined for all four Drosophila alcohol dehydrogenases are higher at pH 9.5 than at pH 8.6. D. melanogaster ADH-FS showed nonlinear kinetics.     

Involvement of conserved glycine residues, 229 and 234, of Vibrio harveyi aldehyde dehydrogenase in activity and nucleotide binding

The involvement of two conserved glycine residues (Gly229 and Gly234) in activity and nucleotide binding in Vibrio harveyi aldehyde dehydrogenase (ALDH) have been investigated. Each of the glycine residues has been mutated to alanine and the mutant ALDHs have been expressed in Escherichia coli and specifically labelled with [35S]methionine. The G229A mutant was inactive with either NADP+ or NAD+ as coenzyme and did not bind to 2',5'-ADP Sepharose, indicating a complete loss of nucleotide affinity. In contrast, the G234A mutant showed a high affinity for 2',5'-ADP Sepharose. Purified G234A mutant showed similar kinetic properties to the native enzyme including a pre-steady-state burst of NADPH; however, the Michaelis constants for NAD+ and NADP+ were increased by 3- to 9-fold, showing that the mutation had an effect on saturation of the enzyme with NAD(P)+. These data are consistent with the structure for the nucleotide binding domain of Vh.ALDH being similar to that of class 3 or class 2 mammalian ALDHs which differ from the classical nucleotide binding domain found in most dehydrogenases.     

Inhibition of immunoglobulin production stimulating activity of glyceraldehyde-3-phosphate dehydrogenase by nucleotides

We have previously identified glyceraldehyde-3-phosphate dehydrogenase as an immunoglobulin production stimulating factor (IPSF) which facilitated immunoglobulin production by hybridomas and lymphocytes. The IPSF activity of this enzyme was suppressed by the coexistence of some sorts of nucleotides. We now report that the IPSF effect of GAPDH was suppressed by the coexistence of DNA, the inhibiting effect of degraded DNA being inferior to that of long-chain DNA. Both single-stranded and double-stranded synthetic polyribonucleotides also inhibited the IPSF activity of GAPDH. Moreover, nicotinamide adenine dinucleotide (NAD+) repressed the IPSF effect.     

Inhibition of glyceraldehyde-3-phosphate dehydrogenase in post-ischaemic myocardium

OBJECTIVE: Myocardial reperfusion following brief period of ischaemic is associated with prolonged, reversible periods of metabolic dysfunction. As the glycolytic enzyme glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is inhibited in vitro by reactive oxygen species, we hypothesized that production of reactive oxygen species during reperfusion would lead to inhibition of GAPDH in post-ischaemic myocardium. METHODS: Anaesthetized closed-chest-dogs were subjected to 20 min balloon occlusion of the left anterior descending coronary artery. Biopsy samples were taken after 3 and 24 h of reperfusion, to determine the activity of GAPDH and the concentrations of glycolytic intermediates in post-ischaemic and remote, non-ischaemic territories. RESULTS: A significant reduction in GAPDH activity was observed in post-ischaemic relative to remote tissue after 3 h reperfusion (4.8 +/- 0.5 vs. 2.9 +/- 0.2 mumol/min/mg protein; P < 0.01). Western blotting revealed no reduction in the levels of GAPDH protein. Analysis of enzyme kinetics showed the loss of activity to be associated with decreased Vmax (5.9 +/- 0.5 vs. 3.2 +/- 0.2 mumol/min/mg protein; P < 0.01) with no significant change in the Km for glyceraldehyde-3-phosphate (GAP). Incubation of the inhibited enzyme under both mild and strong reducing conditions failed to reactivate the enzyme. The acute reduction in enzyme activity in post-ischaemic tissue was accompanied by regional differences in glycolytic intermediates, notably a twofold accumulation of GAP (P < 0.05), and a reduction in the glucose metabolic rate (GMR) determined by positron emission tomography and [18F]2-fluorodeoxyglucose. By 24 h reperfusion, no regional differences in GAPDH activity, reaction Vmax or Km, GAP concentrations or GMR were detectable. CONCLUSIONS: These results suggest that inhibition of GAPDH activity may represent an important point at which glycolysis is limited during reperfusion, and further, that the mechanisms of enzyme inhibition do not involve simple oxidation or S-thiolation of critical active site thiol groups.     

Oxidoreductases involved in cell carbon synthesis of Methanobacterium thermoautotrophicum

Cell-free extracts of Methanobacterium thermoautotrophicum were found to contain high activities of the following oxidoreductases (at 60 degrees C): pyruvate dehydrogenase (coenzyme A acetylating), 275 nmol/min per mg of protein; alpha-ketoglutarate dehydrogenase (coenzyme A acylating), 100 nmol/min per mg; fumarate reductase, 360 nmol/min per mg; malate dehydrogenase, 240 nmol/min per mg; and glyceraldehyde-3-phosphate dehydrogenase, 100 nmol/min per mg. The kinetic properties (apparent V(max) and K(M) values), pH optimum, temperature dependence of the rate, and specificity for electron acceptors/donors of the different oxidoreductases were examined. Pyruvate dehydrogenase and alpha-ketoglutarate dehydrogenase were shown to be two separate enzymes specific for factor 420 rather than for nicotinamide adenine dinucleotide (NAD), NADP, or ferredoxin as the electron acceptor. Both activities catalyzed the reduction of methyl viologen with the respective alpha-ketoacid and a coenzyme A-dependent exchange between the carboxyl group of the alpha-ketoacid and CO(2). The data indicate that the two enzymes are similar to pyruvate synthase and alpha-ketoglutarate synthase, respectively. Fumarate reductase was found in the soluble cell fraction. This enzyme activity coupled with reduced benzyl viologen as the electron donor, but reduced factor 420, NADH, or NADPH was not effective. The cells did not contain menaquinone, thus excluding this compound as the physiological electron donor for fumarate reduction. NAD was the preferred coenzyme for malate dehydrogenase, whereas NADP was preferred for glyceraldehyde-3-phosphate dehydrogenase. The organism also possessed a factor 420-dependent hydrogenase and a factor 420-linked NADP reductase. The involvement of the described oxidoreductases in cell carbon synthesis is discussed.     

Coenzyme binding at different ionization states of cytoplasmic and mitochondrial malate dehydrogenase

pH-titrations with NADH show two ionizable groups in mitochondrial and cytoplasmic malate dehydrogenase, the first with a pKa in the range 6.8-8.3 for the mitochondrial and 6.4-7.8 for the cytoplasmic enzyme, the second with a lower limit at 10.2 resp. 11. Comparison with bis-(dihydronicotinamide)-dinucleotide and dihydronicotinamide-ribosyl-P2-ribose-pyrophosphate instead of NADH indicates that the second alkaline ionization is caused by a residue placed near the adenine binding site of the active centre of the two isoenzymes. Binding studies with NADH and NAD+ give evidence for the participation of a group in the mitochondrial enzyme with pKa 6.8, deprotonation of which is necessary for detectable association of NAD+. In contrast the fixation of NAD+ to the cytoplasmic enzyme is independent of pH.     

Stereospecificity of hydrogen transfer by the NADP-linked alcohol dehydrogenase from the thermophilic bacterium Thermoanaerobium brockii

Class A and class B NAD(H)/NADP(H) coenzyme-dependent dehydrogenases distinguish between the diastereotopic hydrogens pro-R and pro-S at position 4 of the cofactor. We investigated the stereochemistry of hydride transfer in reactions catalyzed by an unusual thermophilic, zinc-containing, NADP-linked enzyme Thermoanaerobium brockii alcohol dehydrogenase (TBAD). Using proton NMR spectroscopy of monodeuterated alcohols and coenzymes we found that TBAD is a class A enzyme that transfers the pro-R hydrogen from the pyridine 4 position of the reduced coenzyme. This stereospecificity is stable over (a) a broad range of temperatures up to 70 degrees C, (b) different concentrations of the coenzyme (catalytic or stoichiometric) and (c) a wide scope of substrates. Although NAD+ is not an effective coenzyme for TBAD, NADP+ and its synthetic analogs, 3-acetylpyridine-ADP+ and thio-NADP+, can be used successfully.     

Purification and characterization of Clostridium difficile glutamate dehydrogenase

Recombinant Clostridium difficile glutamate dehydrogenase (L-glutamate:NAD oxidoreductase, EC 1.4.1.2) was purified 177-fold to electrophoretic homogeneity with a 62% recovery through a four-step procedure involving gel filtration and ion-exchange and dye affinity chromatography. The approximate molecular weights of the native enzyme by gel filtration and subunits by sodium dodecyl sulfate-polyacrylamide gel electrophoresis were consistent with a hexameric structure for the purified enzyme. The enzyme-catalyzed glutamate oxidation was an NAD-dependent sequential process in which NADP could not be substituted as coenzyme. Several dinucleotide analogs of NAD structurally altered in either the pyridine or the purine moiety were observed to function as coenzymes when substituted for NAD. Nicotinamide mononucleotide did not serve as a coenzyme for glutamate oxidation. Product inhibition by NADH was competitive with respect to NAD. In deadend inhibition studies, adenosine diphosphoribose was shown to be an effective coenzyme-competitive inhibitor.     

Purification, characterization, and sequence analysis of 2-aminomuconic 6-semialdehyde dehydrogenase from Pseudomonas pseudoalcaligenes JS45

2-Aminonumconic 6-semialdehyde is an unstable intermediate in the biodegradation of nitrobenzene and 2-aminophenol by Pseudomonas pseudoalcaligenes JS45. Previous work has shown that enzymes in cell extracts convert 2-aminophenol to 2-aminomuconate in the presence of NAD+. In the present work, 2-aminomuconic semialdehyde dehydrogenase was purified and characterized. The purified enzyme migrates as a single band on sodium dodecyl sulfate-polyacrylamide gel electrophoresis with a molecular mass of 57 kDa. The molecular mass of the native enzyme was estimated to be 160 kDa by gel filtration chromatography. The optimal pH for the enzyme activity was 7.3. The enzyme is able to oxidize several aldehyde analogs, including 2-hydroxymuconic semialdehyde, hexaldehyde, and benzaldehyde. The gene encoding 2-aminomuconic semialdehyde dehydrogenase was identified by matching the deduced N-terminal amino acid sequence of the gene with the first 21 amino acids of the purified protein. Multiple sequence alignment of various semialdehyde dehydrogenase protein sequences indicates that 2-aminomuconic 6-semialdehyde dehydrogenase has a high degree of identity with 2-hydroxymuconic 6-semialdehyde dehydrogenases.     

College students'' attitudes and knowledge of AIDS

Specialized cells within the aphid, Schizaphis graminum, contain intracellular, vesicle-enclosed eubacterial endosymbionts (Buchnera aphidicola). Using oligonucleotide probes derived from conserved sequences of the ATP synthase beta-subunit and glyceraldehyde-3-phosphate dehydrogenase, and the polymerase chain reaction (PCR), we have amplified, cloned, and sequenced three DNA fragments. Amino acid sequence similarity indicated that two of these fragments corresponded to endosymbiont and host genes potentially coding for the beta-subunit of ATP synthase. The host gene fragment contained two putative introns. The third DNA fragment corresponded to a portion of a gene coding for a glyceraldehyde-3-phosphate dehydrogenase that was highly related to one of the enzymes from Escherichia coli (GapA). These results indicate that B. aphidicola may have an ATP synthase and consequently could synthesize ATP from a proton motive force generated within the intracellular vesicles of host cells containing the endosymbionts. The detection of a gene fragment coding for a protein similar to glyceraldehyde-3-phosphate dehydrogenase suggests the presence of this glycolytic enzyme in the endosymbiont and its involvement in energy-yielding metabolism.