Studies of self-inactivation of bovine seminal fluid NAD glycohydrolase

Bovine seminal fluid NAD glycohydrolase (NADase) was observed to be rapidly inactivated during catalytic hydrolysis of the substrate NAD. The first-order rate constant for the self-inactivation process was independent of enzyme concentration. The enzyme self-inactivation was a turnover-related process and the number of moles of NAD hydrolyzed required for inactivation was proportional to the enzyme concentration. A number of dinucleotides serving as substrates for the enzyme also promoted self-inactivation. The self-inactivation was an irreversible process having a different rate-limiting step from NAD hydrolysis and was not related to the reversible binding of products and substrate-competitive inhibitors. Modification of arginine residues of the enzyme resulted in the loss of NAD hydrolase activity with no differential effect on the self-inactivation process.     

Effects induced by rotenone during aerobic growth ofParacoccus denitrificans in continuous culture

Paracoccus denitrificans was grown aerobically in chemostat culture in the presence of rotenone. After 6 to 10 generation times, cells showed an oxygen uptake which was completely rotenone-insensitive after removal of rotenone by washing with bovine serum albumin containing medium.The H⁺/O ratio of these cells for endogenous substrates decreased from about 7.50 to 3.95. The latter ratio was similar to the value obtained for starved cells oxidizing exogenous succinate, indicating that site I phosphorylation was absent in these rotenone-insensitive cells.Membrane particles prepared from these cells showed an 80% decrease in activity of reduced nicotinamide adenine dinucleotide oxidase and reduced nicotinamide adenine dinucleotide-ferricyanide oxidoreductase, while also the kinetic behaviour of the reduced nicotinamide adenine dinucleotide dehydrogenase in the reduced nicotinamide adenine dinucleotide-ferricyanide oxidoreductase assay was changed. Moreover the reduced nicotinamide adenine dinucleotide oxidase activity was practically rotenone-insensitive.Electron paramagnetic resonance spectroscopy on membrane particles from rotenone-insensitive cells at 15 K revealed that the resonance lines atg _z 2.05 andg _y g _x 1.92 arising from iron-sulfur center 2 were undetectable. The intensities of the other electron paramagnetic resonance signals originating from reduced nicotinamide adenine dinucleotide dehydrogenase linked iron-sulfur centers were only slightly diminished.These observations confirm our previous suggestion that site I phosphorylation, rotenone sensitivity and the presence of iron-sulfur center 2 are correlated.Abbreviations EPR electron paramagnetic resonance - BSA bovine serum albumin - CCCP carbonylcyanide m-chlorophenylhydrazone - NAD nicotinamide adenine dinucleotide - NADP nicotinamide adenine dinucleotide phosphate - ATP adenosine triphosphate     

Evidence for a physiologically active nicotinamide phosphoribosyltransferase in cultured human fibroblasts

Nicotinamide phosphoribosyltransferase, (EC 2.4.2.12) was examined in extracts of diploid human fibroblasts grown in culture. The enzyme was found to have an apparent Km for nicotinamide of 1.6 × 10^?6M, to be specific for nicotinamide, stimulated by adenosine triphosphate (ATP) and inhibited by nicotinamide adenine dinucleotide (NAD). In these respects it is very similar to rat liver nicotinamide phosphoribosyltransferase but not like the enzyme previously observed in human tissue extracts which had a Km for nicotinamide of approximately 0.1 M and was insensitive to ATP. Discovery of this enzyme activity supports previous studies using radiolabeled nicotinamide which show that human fibroblasts can incorporate nicotinamide into NAD directly through nicotinamide mononucleotide.     

Partial purification and characterization of a coniferyl alcohol dehydrogenase fromRhodococcus erythropolis

A nicotinamide adenine dinucleotide (NAD)-dependent coniferyl alcohol dehydrogenase was enriched 1,200-fold from crude extracts ofRhodococcus erythropolis. The purification procedure involved ion exchange chromotography, gel filtration on Biogel A 1,5 and Sephadex G-200, and hydroxyapatite treatment. The enzyme had a molecular weight of approximately 200,000 and displayed maximal activity at pH 9.0. The apparentK _m values for NAD and coniferyl alcohol were, respectively, 0.22 and 0.645 mM. Nicotinamide adenine dinucleotide phosphate (NADP) could only partially replace NAD. The enzyme was active with vanillyl alcohol and aromatic alcohols bearing the ,-unsaturated side chain of coniferyl alcohols. These aromatic alcohols included the dilignols dehydrodiconiferyl alcohol and guaiacylglycerol--coniferyl ether.     

Glucose-6-Phosphate Dehydrogenase from the Chemolithotroph Thiobacillus ferrooxidans

Glucose-6-phosphate dehydrogenase was partially purified from both glucose-grown and iron-glucose-grown Thiobacillus ferrooxidans. The enzyme possesses a dual nucleotide specificity for either nicotinamide adenine dinucleotide phosphate (NADP) or nicotinamide adenine dinucleotide (NAD) and has a molecular weight of 110,000 as determined by gel electrophoresis. Evidence is presented that T. ferrooxidans glucose-6-phosphate dehydrogenase is identical when isolated from cells grown mixotrophically (iron-glucose grown) or cells grown heterotrophically (glucose-grown cells). The enzyme is activated by Mg(2+), and to a lesser extent by low concentrations of Mn(2+). Reduced NAD inhibits the enzyme from T. ferrooxidans. No deviation from normal Michaelis-Menten kinetics was observed in velocity versus substrate concentration experiments. Adenosine triphosphate exerted a profound inhibition of the enzyme; the effect was 10 times more pronounced in the presence of NAD as compared to NADP. The physiological significance of this inhibition is discussed.     

Photodependent inhibition of rat liver NAD(P)H:quinone acceptor oxidoreductase by (A)-2-azido-NAD+ and (A)-8-azido-NAD

Two photoaffinity analogues of NAD+, (A)-2-azido-NAD+ [nicotinamide 2-azidoadenine dinucleotide] and (A)-8-azido-NAD+ [nicotinamide 8-azidoadenine dinucleotide], have been synthesized, and their reactivities with the rat liver NAD(P)H:quinone acceptor oxidoreductase have been investigated. The reduce nicotinamide nucleotide probes, (A)-2-azido-NADH and (A)-8-azido-NADH, were shown to be substrates of the quinone reductase. This enzyme was inhibited by (A)-8-azido-NADH, were shown to be substrates of the quinone reductase. This enzyme was inhibited by (A)-2-azido-NAD+ and (A)-8-azido-NAD+ in a photodependent manner, and the inhibition of the enzyme could be prevented by the presence of nicotinamide nucleotide substrates during photolysis. (A)-2-Azido-NAD+ was demonstrated to be a more potent inhibitor than (A)-8-azido-NAD+. In addition, the photodependent inhibition by (A)-8-azido-NAD+ increased when menadione, the substrate of the enzyme, was present during the photolysis, while menadione protected the enzyme from the photodependent inhibition by (A)-2-azido-NAD+. These results indicate that these two NAD+ analogues can be used to identify the nicotinamide nucleotide binding site of this quinone reductase and that they probably bind to the enzyme in different fashions.     

Uridine diphosphate D-glucose dehydrogenase of Aerobacter aerogenes

Uridine diphosphate d-glucose dehydrogenase (EC 1.1.1.22) from Aerobacter aerogenes has been partially purified and its properties have been investigated. The molecular weight of the enzyme is between 70,000 and 100,000. Uridine diphosphate d-glucose is a substrate; the diphosphoglucose derivatives of adenosine, cytidine, guanosine, and thymidine are not substrates. Nicotinamide adenine dinucleotide (NAD), but not nicotinamide adenine dinucleotide phosphate, is active as hydrogen acceptor. The pH optimum is between 9.4 and 9.7; the K(m) is 0.6 mm for uridine diphosphate d-glucose and 0.06 mm for NAD. Inhibition of the enzyme by uridine diphosphate d-xylose is noncooperative and of mixed type; the K(i) is 0.08 mm. Thus, uridine diphosphate d-glucose dehydrogenase from A. aerogenes differs from the enzyme from mammalian liver, higher plants, and Cryptococcus laurentii, in which uridine diphosphate d-xylose functions as a cooperative, allosteric feedback inhibitor.     

The pyridine-nucleotide cycle in tobacco Enzyme activities for the de-novo synthesis of NAD

The enzyme activities of the pyridine-nucleotide cycle, which transform nicotinic acid mononucleotide (NaMN) into NAD, have been characterized. The investigations were based on the extraction of protein, its purification on disposable gel-filtration columns, and determination of the enzymatic activities by high-performance liquid chromatography techniques. The latter technique avoided the synthesis and use of radioactive precursors. The NaMN-adenylyltransferase which converts NaMN into NaAD (nicotinic acid adenine dinucleotide) and NAD-synthetase which converts NaAD into NAD were characterized by their kinetic parameters and their specific activities in different tobacco tissues. This is the first report on NAD-synthetase from tissue of a higher plant. It was found that NAD-synthetase accepted both glutamine and asparagine for the amide transfer. Adenylyltransfer also occured with nicotinamide mononucleotide (NMN) which was transformed to NAD, whereas the glutamine-dependent amidation was only observed with NaAD. Thus, an additional route for the synthesis of NAD (NaMNNMNNAD) obviously does not exist. A comparison of the enzyme activities in tobacco tissues with different capacities for the synthesis of nicotine showed that, in contrast to quinolinic acid phosphoribosyltransferase whose activity was strictly correlated with the nicotine content, only NaMN-adenylyltransferase showed a smooth correlation, whereas NAD-synthetase was not affected at all.Abbreviations HPLC high-performance liquid chromatography - QA quinolinic acid - NaMN nicotinic acid mononucleotide - NaAD nicotinic acid adenine dinucleotide - NMN nicotinamide mononucleotide     

Cytochemical localization of nicotinamide adenine dinucleotide phosphatase (NADPase) in bovine Leydig cells

Summary The ultrastructural localization of nicotinamide adenine dinucleotide phosphatase (NADPase) in bovine Leydig cells has been studied and compared with the pattern of thiamine pyrophosphatase (TPPase) and acid phosphatase distribution in these cells. Using -nicotinamide adenine dinucleotide phosphate (-NADP⁺) as substrate, a marked staining is observed in the intermediate Golgi saccules with some focal extension to the trans aspect. Cisternae on the cis side and associated vesicles yielded only slightly positive reactions. The pattern of NADPase localization is clearly different from that of TPPase which consistently stains only the trans Golgi elements. The specifity of NADPase for its substrate, -NADP⁺, was clearly demonstrated by using substrates modified in either the nicotinamide region e.g. -nicotinamide adenine dinucleotide phosphate (-NADP⁺), -thionicotinamide adenine dinuclcotide phosphate (Thio-NADP⁺), in the attachment site of the monoester phosphate group to the molecule (e.g. 2 monophospho-adenosine 5-diphosphoribose (ATP-ribose) or adenosine-5-monophosphate (5AMP). With these substrates only weak or negative reactions were obtained in the Golgi apparatus of the bovine Leydig cell.     

NAD(P)H oscillation in relation to the rhythmic contraction in thePhysarum plasmodium

Summary ThePhysarum plasmodium shows rhythmic contractile activities with a period of a few min. Phases of the oscillation in the plasmodium migrating unindirectionally agreed sideways throughout at the frontal part. So, time course of an intracellular chemical component was determined by analyzing small pieces cut off successively from the frontal part of the large plasmodium. Intracellular NAD(P)H concentration oscillated with the same period as the rhythmic contraction but with a different phase advancing about 1/3 of the period. UV irradiation suppressed the rhythmic contraction without affecting the rhythmic variation of NAD(P)H. Thus, the NAD(P)H oscillator works independently of the rhythmic contractile system, but seems entraining with each other.Abbreviations UV ultraviolet - NADH nicotinamide adenine dinucleotide, reduced form - NADPH nicotinamide adenine dinucleotide phosphate, reduced form - ATP adenosine 5-triphosphate - cAMP cyclic adenosine 3, 5-monophosphate - FMNH₂ flavin mononucleotide, reduced form - TCA tricarboxylic acid - BSA bovine serum albumin - DTT dithiothreitol     

The cytotoxicity of chrysotile asbestos fibers to pulmonary alveolar macrophages I. Effects of inhibitors of ADP-ribosyl transferase

Pulmonary alveoler macrophages exposedto very short chrysotile asbestos fibers present a typical cytotoxic response: extracellular releases of lactate dehydrogenase and -galactosidase, and a decrease in cellular ATP content. The objective of this study was to determine if nicotinamide and 3-aminobenzamide, two inhibitors of the ADP-ribosyl transferase, could modify the in vitro toxicity of chrysotilee fibers. After 30 min of pre-exposure with each of the two inihibitors, pulmonary alveolar macrophage monolayers were concominantly exposed for 18 hours to 50g of fibers. It was observed that, in a dose-effect relationship (5 to 30 mM), nicotinamide was very effective in reducing the extracellular liberation of the marker enzymes. At 30 mM, the enzyme releases in the medium had returned to control values; the restoration of cell viability was confirmed by ATP levels. Up to 5 mM 3-aminobenzamide did not provide any protection against chrysotile cytotoxicity. Nicotinic acid, a structural analogue of nicotinamide, but not an inhibitor of the ADP-ribosyl transferase, also showed no protective effect. Nicotinamide and 3-aminobenzamide increased the intracellular NAD⁺ pools, respectively by 350% and 250%. However, with or without additives, the chrysotile fibers caused a constant and significant decrease in NAD⁺ levels (40–55 pmoles). These results suggest that the inhibition of the nuclear ADP-ribosyl transferase is not the major mechanism by which nicotinamide protects pulmonary alveolar macrophages against the chrysotile asbestos fibers.Abbreviations 3-AB 3-aminobenzamide - ADPRT ADP-ribosyl transferase - -GAL -galactosidase - DTT dithiothreitol - FBS fetal bovine serum - FMN flavin mononucleotide - HEPES N-2-hydroxyethyl piperazine-N-2-ethanesulfonic acid - LDH lactate dehydrogenase - NAD⁺ nicotinamide adenine dinucleotide (oxidized form) - NADH nicotimide adenine dinucleotide (reduced forms) - NADPH nicotimide adenine dinucleotide phosphate (reduced form) - NAM nicotinamide - NIC nicotinic acid - ORS oxygen radical species - PAM pulmonary alveolar macrophages - S.E. standard error of the mean - TAPS tris (hydroxymethyl) methylamino-propane sulfonic acid - TRIS tris (hydroxymethyl) aminomethane - VSF very short chrysotile fibers     

Regulation of Tryptophan Pyrrolase Activity in Xanthomonas pruni

Tryptophan pyrrolase was studied in partially purified extracts of Xanthomonas pruni. The dialyzed enzyme required both heme and ascorbate for maximal activity. Other reducing agents were able to substitute for ascorbate. Protoporphyrin competed with heme for the enzyme, suggesting that the native enzyme is a hemoprotein. The enzyme exhibited sigmoid saturation kinetics. Reduced nicotinamide adenine dinucleotide (NADH), reduced nicotinamide adenine dinucleotide phosphate (NADPH), nicotinic acid mononucleotide, and anthranilic acid enhanced the sigmoid kinetics and presumably bound to allosteric sites on the enzyme. The sigmoid kinetics were diminished in the presence of alpha-methyltryptophan. NAD, NADP, nicotinic acid, nicotinamide, nicotinamide mononucleotide, and several other related compounds were without effect on the activity of the enzyme. These data indicate that the activity of the enzyme is under feedback regulation by the ultimate end products of the pathway leading to NAD biosynthesis, as well as by certain intermediates of this pathway.     

Immobilized-enzyme continuous-flow reactor incorporating continuous electrochemical regeneration of NAD

Electrochemical regeneration of the cofactor nicotinamide adenine dinucleotide (NAD) from its reduced form (NADH) has been coupled with the alcoholdehydrogenation reaction which consumes NAD and produces NADU using alcohol dehydrogenase bound to alumina. Alcohol (reactant) is added directly to the system while aldehyde (product) leaves the system through an ultrafiltration membrane which prevents loss of the cofactor. This system provides a continuous-flow process for carrying out a cofactor-requiring enzymatic reaction with no net loss or consumption of enzyme or cofactor and without the use of reagents for regenerating the cofactor. Although the process shown here is not economically practical, it may be a harbinger of useful and technically feasible chemical reaction systems based on immobilized enzymes requiring cofactors.     

Characterization of a pyridine nucleotide-nonspecific glutamate dehydrogenase from Bacteroides thetaiotaomicron.

An oxidized nicotinamide adenine dinucleotide phosphate/oxidized nicotinamide adenine dinucleotide (NADP+/NAD+) nonspecific L-glutamate dehydrogenase from Bacteroides thetaiotaomicron was purified 40-fold (NADP+ or NAD+ activity) over crude cell extract by heat treatment, (NH4)2SO2 fractionation, diethylaminoethyl-cellulose, Bio-Gel A 1.5m, and hydroxylapatite chromatography. Both NADP+- and NAD+-dependent activities coeluted from all chromatographic treatments. Moreover, a constant ratio of NADP+/NAD+ specific activities was demonstrated at each purification step. Both activities also comigrated in 6% nondenaturing polyacrylamide gels. Affinity chromatography of the 40-fold-purified enzyme using Procion RED HE-3B gave a preparation containing both NADP+- and NAD+-linked activities which showed a single protein band of 48,5000 molecular weight after sodium dodecyl sulfate-polyacrylamide gradient gel electrophoresis. The dual pyridine nucleotide nature of the enzyme was most readily apparent in the oxidative direction. Reductively, the enzyme was 30-fold more active with reduced NADP than with reduced NAD. Nonlinear concave 1/V versus 1/S plots were observed for reduced NADP and NH4Cl. Salts (0.1 M) stimulated the NADP+-linked reaction, inhibited the NAD+-linked reaction, and had little effect on the reduced NADP-dependent reaction. The stimulatory effect of salts (NADP+) was nonspecific, regardless of the anion or cation, whereas the degree of NAD+-linked inhibition decreased in the order to I- greater than Br- greater than Cl- greater than F-. Both NADP+ and NAD+ glutamate dehydrogenase activities were also detected in cell extracts from representative strains of other bacteroides deoxyribonucleic acid homology groups.     

Utilization and metabolism of NAD by Haemophilus parainfluenzae

The utilization of exogenous nicotinamide adenine dinucleotide (NAD) by Haemophilus parainfluenzae was studied in suspensions of whole cells using radiolabelled NAD, nicotinamide mononucleotide (NMN), and nicotinamide ribonucleoside (NR). The utilization of these compounds by H. parainfluenzae has the following characteristics. (1) NAD is not taken up intact, but rather is degraded to NMN or NR prior to internalization. (2) Uptake is carrier-mediated and energy-dependent with saturation kinetics. (3) There is specificity for the beta-configuration of the glycopyridine linkage. (4) An intact carboxamide groups is required on the pyridine ring. The intracellular metabolism of NAD was studied in crude cell extracts and in whole cells using carbonyl-14C-labelled NR, NMN, NAD, nicotinamide, and nicotinic acid as substrates in separate experiments. A synthetic pathway from NR through NMN to NAD that requires Mg2+ and ATP was demonstrated. Nicotinamide was found as an end-product of NAD degradation. Nicotinic acid mononucleotide and nicotinic acid adenine dinucleotide were not found as intermediates. The NAD synthetic pathway in H. parainfluenzae differs from the Preiss-Handler pathway and the pyridine nucleotide cycles described in other bacteria.     

Effects of calmodulin on the (Ca2+ + Mg2+)ATPase partially purified from erythrocyte membranes.

The stimulation of the (Ca²⁺ + Mg²⁺)ATPase of erythrocyte ghosts by calmodulin was observed not only in intact ghosts, but also in the solubilized (Triton X-100) and partially purified, reconstituted (phosphatidylserine liposomes) forms. Since the solubilized form of the enzyme migrated on Sepharose 6B at a position corresponding to a molecular weight of about 150,000, these results show that calmodulin stimulates by direct interaction with the ATPase complex. Additionally, the effects of calmodulin on erythrocyte ghosts prepared by the Dodge-EDTA method (hypotonic ghosts) and by the method of Ronner et al. (involving lysis followed by an isotonic wash repeated several times) were compared (P. Ronner, P. Gazzotti, and E. Carafoli, 1977, Arch. Biochem. Biophys. 179, 578–583). The (Ca²⁺ + Mg²⁺)ATPase of the hypotonic ghosts was low and was stimulated by added calmodulin while that of the isotonic ghosts was high and changed only slightly upon calmodulin addition; this difference in response to calmodulin persisted in the solubilized and reconstituted forms. Hypotonic ghosts bound ¹²⁵I-labeled calmodulin, while isotonic ghosts did not. This comparison of two types of ghosts showed that isotonic ghosts possess an intact calmodulin-(Ca²⁺ + Mg²⁺)ATPase complex, and that the calmodulin remained with the ATPase during solubilization and reconstitution. The isotonic preparation is a particularly useful method of preparing ghosts with an intact calmodulin-ATPase complex, since it requires no special equipment and produces an enzyme activity which is stable to freezing.     

Probing Aplysia californica adenosine 5'-diphosphate ribosyl cyclase for substrate binding requirements: design of potent inhibitors.

Readily synthesized nicotinamide adenine dinucleotide (NAD(+)) analogues have been used to investigate aspects of the cyclization of NAD(+) to cyclic adenosine 5'-O-diphosphate ribose (cADPR) catalyzed by the enzyme adenosine 5'-O-diphosphate (ADP) ribosyl cyclase and to produce the first potent inhibitors of this enzyme. In all cases, inhibition of Aplysia californica cyclase by various substrate analogues was found to be competitive while inhibition by nicotinamide exhibited mixed-behavior characteristics. Nicotinamide hypoxanthine dinucleotide (NHD(+)), nicotinamide guanine dinucleotide (NGD(+)), C1'-m-benzamide adenine dinucleotide (Bp(2)A), and C1'-m-benzamide nicotinamide dinucleotide (Bp(2)N) were found to be nanomolar potency inhibitors with inhibition constants of 70, 143, 189, and 201 nM, respectively. However, NHD(+) and NGD(+) are also known substrates and are slowly converted to cyclic products, thus preventing their further use as inhibitors. The symmetrical bis-nucleotides, bis-adenine dinucleotide (Ap(2)A), bis-hypoxanthine dinucleotide (Hp(2)H), and bis-nicotinamide dinucleotide (Np(2)N), exhibited micromolar competitive inhibition, with Ap(2)A displaying the greatest affinity for the enzyme. 2',3'-Di-O-acetyl nicotinamide adenine dinucleotide (AcONAD(+)) was not a substrate for the A. californica cyclase but also displayed some inhibition at a micromolar level. Finally, inhibition of the cyclase by adenosine 5'-O-diphosphate ribose (ADPR) and inosine 5'-O-diphosphate ribose (IDPR) was observed at millimolar concentration. The nicotinamide aromatic ring appears to be the optimal motif required for enzymatic recognition, while modifications of the 2'- and 3'-hydroxyls of the nicotinamide ribose seem to hamper binding to the enzyme. Stabilizing enzyme/inhibitor interactions and the inability of the enzyme to release unprocessed material are both considered to explain nanomolar inhibition. Recognition of inhibitors by other ADP ribosyl cyclases has also been investigated, and this study now provides the first potent nonhydrolyzable sea urchin ADP ribosyl cyclase and cADPR hydrolase inhibitor Bp(2)A, with inhibition observed at the micromolar and nanomolar level, respectively. The benzamide derivatives did not inhibit CD38 cyclase or hydrolase activity when NGD(+) was used as substrate. These results emphasize the difference between CD38 and other enzymes in which the cADPR cyclase activity predominates.     

NAD deamidation “a new reaction” by an enzyme from <Emphasis Type="Italic">Aspergillus terreus</Emphasis> DSM 826

NAD deamidation is a non-previously recognized reaction. This reaction has been found to be catalyzed by extracts of Aspergillus terreus DSM 826. Conversion of NAD to the biosynthetic intermediate, deamido NAD, by these extracts, at the optimum pH and temperature did not exceed about 55 of the amount of the substrate added. Completion of the reaction was achieved when the extracts were pre-heated at 50 °C for 15 min in absence of the substrate. In a very similar manner, the extracts catalyzed hydrolytic cleavage of the amide linkages of different biomolecules such as nicotinamide, nicotinamide riboside, nicotinamide mononucleotide, L-glutamine, L-asparagine and acetamide. Polyacrylamide was also deamidated under the same conditions. In addition, complete dephosphorylation of the dinucleotide molecule was also effected by the same extracts. Separation of the NAD deamidating enzyme from the NAD dephosphorylating enzyme was achieved on using either DEAE - Sephadex A-25 or Sephadex G-200 column chromatography. The obtained phosphohydrolase-free-deamidase showed optimum activity at pH 8 of 0.1 M phosphate buffer and 50 °C. It exhibited broad substrate specificity and hyperbolic substrate saturation kinetics. It was isosterically inhibited by the product of its activity and this inhibition was prevented by heating the extracts at 50 °C for 15 min. Its activity was not affected in presence of sodium fluoride, partially inhibited in presence of magnesium chloride and was retained in the freezer for some months.     

Purification and characterization of the pyruvate-ferredoxin oxidoreductase from Clostridium acetobutylicum

The pyruvate-ferredoxin oxidoreductase from Clostridium acetobutylicum was purified to homogeneity and partially characterized. A 9.2-fold purification was achieved in a three step purification procedure: ammonium sulfate fractionation, chromatography on Phenyl Sepharose and on Procion Blue H-EGN12. The pure enzyme exhibited a specfic activity of 25 U/mg of protein. Homogeneity of the pyruvate-ferredoxin oxidoreductase was confirmed by native polyacrylamide gel electrophoresis and sodium dodecylsulfate (SDS)-polyacrylamide gel electrophoresis. The molecular weight was determined to be 123,000/monomer. The subunit composition of the native enzyme could not be determined because of the instability of the pure enzyme. The pyruvate-ferredoxin oxidoreductase is sensitive to oxygen and dilution during purification. The dilution inactivation could be partially overcome by the addition of 300 M coenzyme A or 50% ethyleneglycol. A thiamine pyrophosphate content of 0.39 mol per mol of enzyme monomer was found, the iron and sulfur content was 4.23 and 0.91, respectively. The pH-optimum was at pH 7.5 and the temperature optimum was at 60°C. Kinetic constants were measured in the forward reaction. The apparent K _m for pyruvate and coenzyme A were 322 M and 3.7 M, respectively. With 2-ketobutyrate the pyruvate-ferredoxin oxidoreductase showed 12.5% of the activity compared to pyruvate. No activity was found with 2-ketoglutarate. Ferredoxin from Clostridium pasteurianum could be used as physiological electron acceptor.Non-standard abbreviations NAD(H) nicotinamide adenine dinucleotide (reduced) - NADP(H) nicotinamide adenine dinucleotide phosphate (reduced) - DTE dithioerythritol - PMS phenazine methosulfate - NBT nitro blue tetrazolium chloride - DMSO dimethyl sulfoxide - DCPIP dichlorophenolindophenol - MTT 3-(4,5-dimethylthiazolyl-2)-2,5-diphenyl-tetrazolium bromide - TTC triphenyltetrazolium chloride - FAD flavin adenine dinucleotide - FMN flavin mononucleotide     

Partial purification and properties of long-chain acyl-CoA hydrolase from rat brain cytosol

Long-chain acyl-CoA hydrolase (EC 3.1.2.2.) has been partially purified from the 100,000 × g supernatant fraction of rat brain tissue. The purification procedure included chromatography on gel filtration media, DEAE-cellulose, CM-cellulose, and hydroxyapatite. The partially purified enzyme had a specific activity of 7.1 mol/min-mg, and when analyzed by polyacrylamide gel electrophoresis, revealed one major and three minor bands of protein in the presence of dodecyl sulfate and two major bands of protein in the absence of dodecyl sulfate. The enzyme had a molecular weight of 65,000 and showed no evidence of aggregated or dissociated forms. The highest catalytic activity was exhibited with palmitoyl-CoA and oleoyl-CoA as substrates. Lower activity was found with decanoyl-CoA as the substrate and little or no activity was found with acetyl-CoA, malonyl-CoA, butyryl-CoA, or acetoacetyl-CoA. The enzyme was inhibited by CoA, various metal ions, including Mn²⁺, Mg²⁺ and Ca²⁺, and by bovine serum albumin. Heating the enzyme produced a loss of activity which corresponded to a first-order kinetic process, the rate of which was independent of the choice of substrate used to measure enzyme activity. This finding supports the idea that the purification procedure yields a single species of long-chain acyl-CoA hydrolase.