Purification and some properties of NAD(P)H dehydrogenase from Saccharomyces cerevisiae: Contribution to glycolytic methylglyoxal pathway

NAD(P)H dehydrogenase was purified approximately 480-fold from Saccharomyces cerevisiae with 6.5% activity yield. The enzyme was homogeneous on polyacrylamide gel electrophoresis. The molecular weight of the enzyme was estimated to be 40,000–44,000 by gel filtration on Sephadex G-150 column chromatography and SDS-polyacrylamide gel electrophoresis. The K_m values for NADPH and NADH were 7.3 μM and 0.1 mM, respectively. The activity of the enzyme increased approximately 4-fold with Cu²⁺. FAD, FMN and cytochrome c were not effective as electron acceptors, although Fe(CN)₆³⁻ was slightly effective. NADH generated by the reaction of lactaldehyde dehydrogenase in the glycolytic methylglyoxal pathway will be reoxidized by NAD(P)H dehydrogenase. NAD(P)H dehydrogenase thus may contribute to the reduction/oxidation system in the glycolytic methylglyoxal pathway to maintain the flux of methylglyoxal to lactic acid via lactaldehyde.     

Lactate dehydrogenase from gastrocnemius muscle of turtle

Five bands of lactate dehydrogenase (LDH) isoenzymes were seen by polyacrylamide gel electrophoresis in gastrocnemius muscle of the turtle (Kachuga smithi). The major band was of M2H2 type and was partially purified by gel filtration and affinity chromatography. The specific activity of the enzyme was 2.6 units/mg protein. The half-life of the enzyme at 4 degrees C, was about 7 days. The optimum temperature for enzyme activity was 30 degrees C and the enzyme was irreversibly inactivated at 40 degrees C. The optimum pH for the forward reaction (pyruvate to lactate) was 5.5, while for reverse reaction it was between 8.0 to 9.5. The apparent Km values for pyruvate, NADH, lactate and NAD+ were 0.20, 0.013, 25 and 0.333 mM, respectively. Oxalate was found to be the inhibitor of LDH with Ki of about 4.2 mM.     

Metabolism of L-fucose and L-rhamnose in Escherichia coli: aerobic-anaerobic regulation of L-lactaldehyde dissimilation.

L-Lactaldehyde is a branching point in the metabolic pathway of L-fucose and L-rhamnose utilization. Under aerobic conditions, L-lactaldehyde is oxidized to L-lactate by the enzyme lactaldehyde dehydrogenase, while under anaerobic conditions, L-lactaldehyde is reduced to L-1,2-propanediol by the enzyme propanediol oxidoreductase. Aerobic growth on either of the methyl pentoses induces a lactaldehyde dehydrogenase enzyme which is inhibited by NADH and is very stable under anaerobic conditions. In the absence of oxygen, the cell shifts from the oxidation of L-lactaldehyde to its reduction, owing to both the induction of propanediol oxidoreductase activity and the decrease in the NAD/NADH ratio. The oxidation of L-lactaldehyde to L-lactate is again restored upon a change to aerobic conditions. In this case, only the NAD/NADH ratio may be invoked as a regulatory mechanism, since both enzymes remain active after this change. Experimental evidence in the presence of rhamnose with mutants unable to produce L-lactaldehyde and mutants capable of producing but not further metabolizing it points toward L-lactaldehyde as the effector molecule in the induction of lactaldehyde dehydrogenase. Analysis of a temperature-sensitive mutation affecting the synthesis of lactaldehyde dehydrogenase permitted us to locate an apparently single regulator gene linked to the ald locus at 31 min and probably acting as a positive control element on the expression of the structural gene.     

Purification and properties of mitochondrial malate dehydrogenase from unfertilized eggs of the sea urchin, Anthocidaris crassispina

Purification and characterization of mitochondrial malate dehydrogenase [EC 1.1.1.37] from unfertilized eggs of the sea urchin, Anthocidaris crassispina, are described. The purification method consisted of dextran sulfate fractionation, Blue Dextran Sepharose chromatography, Phenyl-Sepharose hydrophobic chromatography and DEAE-cellulose chromatography. The enzyme was purified 771-fold with a 7% yield from the crude extract. The purified enzyme appeared homogeneous on polyacrylamide gel electrophoresis under both native and denatured conditions. After incubation at 45 degrees C for 50 min, the enzyme lost about 90% of its activity. In the presence of NADH, however, the enzyme was protected against the heat denaturation. The native enzyme had a molecular weight of about 65,000 and probably consisted of two identical subunits. In the reduction of oxaloacetate with NADH, a broad optimum pH ranging from 8.2 to 9.4 was found with 50 mM Tris-HCl and glycine-NaOH buffers. Sodium phosphate buffer apparently activated the enzyme. The apparent Km values for oxaloacetate and NADH were 19 microM and 30 microM, respectively. The optimum pH for malate oxidation with NAD+ was 10.2 in 50 mM NaHCO3-Na2CO3 buffer. The apparent Km values for malate and NAD+ were 7.0 mM and 0.6 mM, respectively. Zinc ion, sulfite ion, p-chloromercuriphenylsulfonate and adenine nucleotides strongly inhibited the enzyme.     

Production and Purification of Extracellular D-Xylose Isomerase from an Alkaliphilic, Thermophilic Bacillus sp

An alkaliphilic, thermophilic Bacillus sp. (NCIM 59) produced extracellular xylose isomerase at pH 10 and 50 degrees C by using xylose or wheat bran as the carbon source. The distribution of xylose isomerase as a function of growth in comparison with distributions of extra- and intracellular marker enzymes such as xylanase and beta-galactosidase revealed that xylose isomerase was truly secreted as an extracellular enzyme and was not released because of sporulation or lysis. The enzyme was purified to homogeneity by ammonium sulfate precipitation followed by gel filtration, preparative polyacrylamide gel electrophoresis, and ion-exchange chromatography. The molecular weight of xylose isomerase was estimated to be 160,000 by gel filtration and 50,000 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, indicating the presence of three subunits. The enzyme is most active at pH 8.0 and with incubation at 85 degrees C for 20 min. Divalent metal ions Mg, Co, and Mn were required for maximum activity of the enzyme. The K(m) values for D-xylose and D-glucose at 80 degrees C and pH 7.5 were 6.66 and 142 mM, respectively, while K(cat) values were 2.3 x 10 s and 0.5 x 10 s, respectively.     

15-Hydroxyprostaglandin dehydrogenase from human placenta. 1. Isolation and characterization]

15-Hydroxyprostaglandin dehydrogenase was isolated from human term placenta up to a final purification of 380-fold. A spec. act. of 2000 mU/mg of protein was reached. The preparation was not homogeneous as judged by analytical disc electrophoresis. The enzyme could be stored in the presence of 50% glycerol and 10mM 2-mercaptoethanol without any loss of activity for at least one year. A distinct single protein band stained after discontinuous polyacrylamide gel electrophoresis was shown by enzymatic activity staining to correspond to 15-hydroxyprostaglandin dehydrogenase activity. Thus no evidence for the exitstence of isoenzymes was obtained. The protein in the final preparation steps showed neither alcohol dehydrogenase, NAD reductase, nor NADH oxidase activity, nor enzymatic conversion of prostaglandin or 15-oxoprostaglandin in the absence of NAD and NADH. No spontaneous reactions between NAD and prostaglandin or NADH and 15-oxoprostaglandin were detectable in the absence of the enzyme. Ethanol and glycerol slightly inhibited the reaction. Various buffers (Tris/HC1, potassium phosphate, HEPES, and triethanolamine) and salts (ammonium chloride, ammonium sulfate, potassium chloride, and sodium chloride) had different effects on the reaction rate. The pH profile of the reaction shows a plateau between pH 7.0 and 7.8 and a steep maximum at pH 9.5. A linear Arrhenius plot was obtained for the temperature dependence of the reaction from 20 to 37 degrees C. The molar activation enthalpy of the reaction was calculated to be 13.1 kcal/mole. The molecular weight of 15-hydroxyprostaglandin dehydrogenase was estimated to be 32000 -/+ 3000 by gel filtration on Sephadex G-150 in the presence of 10mM mercaptoethanol.     

Malic enzyme in human liver. Intracellular distribution, purification and properties of cytosolic isozyme

In human liver, almost 90% of malic enzyme activity is located within the extramitochondrial compartment, and only approximately 10% in the mitochondrial fraction. Extramitochondrial malic enzyme has been isolated from the post-mitochondrial supernatant of human liver by (NH4)2SO4 fractionation, chromatography on DEAE-cellulose, ADP-Sepharose-4B and Sephacryl S-300 to apparent homogeneity, as judged from polyacrylamide gel electrophoresis. The specific activity of the purified enzyme was 56 mumol.min-1.mg protein-1, which corresponds to about 10,000-fold purification. The molecular mass of the native enzyme determined by gel filtration is 251 kDa. SDS/polyacrylamide gel electrophoresis showed one polypeptide band of molecular mass 63 kDa. Thus, it appears that the native protein is a tetramer composed of identical-molecular-mass subunits. The isoelectric point of the isolated enzyme was 5.65. The enzyme was shown to carboxylate pyruvate with at least the same rate as the forward reaction. The optimum pH for the carboxylation reaction was at pH 7.25 and that for the NADP-linked decarboxylation reaction varied with malate concentration. The Km values determined at pH 7.2 for malate and NADP were 120 microM and 9.2 microM, respectively. The Km values for pyruvate, NADPH and bicarbonate were 5.9 mM, 5.3 microM and 27.9 mM, respectively. The enzyme converted malate to pyruvate (at optimum pH 6.4) in the presence of 10 mM NAD at approximately 40% of the maximum rate with NADP. The Km values for malate and NAD were 0.96 mM and 4.6 mM, respectively. NAD-dependent decarboxylation reaction was not reversible. The purified human liver malic enzyme catalyzed decarboxylation of oxaloacetate and NADPH-linked reduction of pyruvate at about 1.3% and 5.4% of the maximum rate of NADP-linked oxidative decarboxylation of malate, respectively. The results indicate that malic enzyme from human liver exhibits similar properties to the enzyme from animal liver.     

Purification and characterization of thermostable alpha-galactosidase from Ganoderma lucidum

Alpha-galactosidase was purified from a fresh fruiting body of Ganoderma lucidum by precipitation with ammonium sulfate and column chromatographies with DEAE-Sephadex and Con A-Sepharose. The purified enzyme was homogeneous on polyacrylamide gel electrophoresis. Its N-terminal amino acid sequence was similar to that of Mortierella vinacea alpha-galactosidase. The molecular mass of the enzyme was about 56 kDa by SDS-polyacrylamide gel electrophoresis, and about 249 kDa by gel filtration column chromatography. The optimum pH and temperature were 6.0 and 70 degrees C, respectively. The enzyme was fully stable to heating at 70 degrees C for 30 min. It hydrolyzed p-nitrophenyl-alpha-D-galactopyranoside (Km=0.4 mM) but hydrolyzed little o-nitrophenyl-alpha-D-galactopyranoside. It also hydrolyzed melibiose, raffinose, and stachyose. The enzyme catalyzed the transgalactosylation reaction which synthesized melibiose. The product was confirmed by various analyses.     

Thermostable alanine dehydrogenase from thermophilic Bacillus sphaericus DSM 462. Purification, characterization and kinetic mechanism

Alanine dehydrogenase (L-alanine: NAD+ oxidoreductase, deaminating) was simply purified to homogeneity from a thermophile, Bacillus sphaericus DSM 462, by ammonium sulfate fractionation, red-Sepharose 4B chromatography and preparative slab gel electrophoresis. The enzyme had a molecular mass of about 230 kDa and consisted of six subunits with an identical molecular mass of 38 kDa. The enzyme was much more thermostable than that from a mesophile, B. sphaericus, and retained its full activity upon heating at 75 degrees C for at least 60 min and with incubation in pH 5.5-9.5 at 75 degrees C for 10 min. The enzyme can be stored without loss of its activity in a frozen state (-20 degrees C, at pH 7.2) for over 5 months. The optimum pH for the L-alanine deamination and pyruvate amination were around 10.5 and 8.2, respectively. The enzyme exclusively catalyzed the oxidative deamination of L-alanine in the presence of NAD+, but showed low amino acceptor specificity; hydroxypyruvate, oxaloacetate, 2-oxobutyrate and 3-fluoropyruvate are also aminated as well as pyruvate in the presence of NADH and ammonia. Initial velocity and product inhibition studies showed that the reductive amination proceeded through a sequential mechanism containing partially random binding. NADH binds first to the enzyme, and then pyruvate and ammonia bind in a random fashion. The products are sequentially released from the enzyme in the order L-alanine then NAD+. A dead-end inhibition by the formation of an abortive ternary complex which consists of the enzyme, NAD+ and pyruvate was included in the reaction. A possible role of the dead-end inhibition is to prevent the enzyme from functioning in the L-alanine synthesis. The Michaelis constants for the substrates were as follows: NADH, 0.10 mM; pyruvate, 0.50 mM; ammonia, 38.0 mM; L-alanine, 10.5 mM and NAD+, 0.26 mM.     

NAD-dependent formate dehydrogenase from methylotrophic bacterium, strain 1. Purification and characterization.

1. NAD-dependent formate dehydrogenase was isolated from gram-negative methylotrophic bacteria, strain 1, grown on methanol. The purification procedure involved ammonium sulfate fractionation, ion-exchange chromatography and preparative isotachophoresis or gel filtration; it resulted in a yield of 40%. 2. The final enzyme preparations were homogeneous as judged by sedimentation in an ultracentrifuge. Formate dehydrogenase purified in the presence of EDTA reveals two bands on electrophoresis in polyacrylamide gel both after protein and activity staining. Two components are transformed into a single one after prolonged storage in the presence of 2-mercaptoethanol. 3. Formate dehydrogenase is a dimer composed of identical or very similar subunits. The molecular weight of the enzyme is about 80 000. 4. Amino acid composition and some other physico-chemical properties of the enzyme were studied. 5. Formate dehydrogenase is specific for formate and NAD as electron acceptor. The Michaelis constant was 0.11 mM for NAD and 15 mM for formate (pH 7.0, 37 degrees C). 6. Formate dehydrogenase was rapidly inactivated in the absence of -SH compounds. The enzyme retained full activity upon storage at ambient temperature in solution for half a year in the presence of 2-mercaptoethanol or EDTA.     

Properties of glutamate dehydrogenase purified from Bacteroides fragilis

The dual pyridine nucleotide-specific glutamate dehydrogenase [EC 1.4.1.3] was purified 37-fold from Bacteroides fragilis by ammonium sulfate fractionation, DEAE-Sephadex A-25 chromatography twice, and gel filtration on Sephacryl S-300. The enzyme had a molecular weight of approximately 300,000, and polymeric forms (molecular weights of 590,000 and 920,000) were observed in small amounts on polyacrylamide gel disc electrophoresis. The molecular weight of the subunit was 48,000. The isoelectric point of the enzyme was pH 5.1. This glutamate dehydrogenase utilized NAD(P)H and NAD(P)+ as coenzymes and showed maximal activities at pH 8.0 and 7.4 for the amination with NADPH and with NADH, respectively, and at pH 9.5 and 9.0 for the deamination with NADP+ and NAD+, respectively. The amination activity with NADPH was about 5-fold higher than that with NADH. The Lineweaver-Burk plot for ammonia showed two straight lines in the NADPH-dependent reactions. The values of Km for substrates were: 1.7 and 5.1 mM for ammonium chloride, 0.14 mM for 2-oxoglutarate, 0.013 mM for NADPH, 2.4 mM for L-glutamate, and 0.019 mM for NADP+ in NADP-linked reactions, and 4.9 mM for ammonium chloride, 7.1 mM for 2-oxoglutarate, 0.2 mM for NADH, 7.3 mM for L-glutamate, and 3.0 mM for NAD+ in NAD-linked reactions. 2-Oxoglutarate and L-glutamate caused substrate inhibition in the NADPH- and NADP+-dependent reactions, respectively, to some extent. NAD+- and NADH-dependent activities were inhibited by 50% by 0.1 M NaCl. Adenine nucleotides and dicarboxylic acids did not show remarkable effects on the enzyme activities.     

Purification and partial characterization of a methylglyoxal reductase from goat liver

An enzyme which catalyzes the reduction of methylglyoxal to lactaldehyde has been isolated and purified from goat liver to apparent homogeneity. NADH was found to be a better substrate than NADPH for methylglyoxal reduction. Stoichiometrically equivalent amounts of lactaldehyde and NAD are formed from methylglyoxal and NADH. Enzyme activity was located only in the soluble supernatant fractions of liver cells. Of the various carbonyl compounds tested, methylglyoxal was found to be the best substrate. The pH optimum of the enzyme was found to be 6.5, and Km for methylglyoxal was 0.4 mM. The molecular weight of the enzyme was found to be 89000 by gel filtration on a Sephadex G-200 column. Electrophoresis on sodium dodecyl sulfate-polyacrylamide gel revealed that the enzyme is composed of two subunits. The enzyme is highly sensitive to sulfhydryl group reagents. The inactivation by p-chloromercuribenzoate could be substantially protected by methylglyoxal in combination with NADH, indicating a possible involvement of one or more sulfhydryl group(s) at the active site of the enzyme.     

Purification and properties of 5,10-methenyltetrahydromethanopterin cyclohydrolase from Methanosarcina barkeri.

The 5,10-methenyltetrahydromethanopterin cyclohydrolase from Methanosarcina barkeri was purified 313-fold to a specific activity of 470 mumol min-1 mg-1 at 37 degrees C and pH 7.8. At this stage, the enzyme was pure as judged from polyacrylamide gel electrophoresis. The monofunctional enzyme was oxygen stable, but the presence of a detergent proved to be essential for its stability. Like the cyclohydrolase purified from Methanobacterium thermoautotrophicum (A. A. Dimarco, M. I. Donnelly, and R. S. Wolfe, J. Bacteriol. 168:1372-1377, 1986), the protein showed an apparent Mr of 82,000, and it is composed of two identical subunits as was concluded from nondenaturating and denaturating polyacrylamide gel electrophoresis. The enzymes from M. thermoautotrophicum and M. barkeri markedly differ with respect to the hydrolysis product of 5,10-methenyltetrahydromethanopterin: 5-formyl- and 10-formyltetrahydromethanopterin, respectively. The apparent Km for 5,10-methenyltetrahydromethanopterin was 0.57 mM at 37 degrees C and pH 7.8.     

L-fucose metabolism in mammals. Purification of pork liver 2-keto-3-deoxy-L-fuconate:NAD+ oxidoreductase by NAD+-Agarose affinity chromatography.

Pork liver has previously been reported to contain a soluble enzymatic pathway which converts L-fucose to 2-keto-3-deoxy-L-fuconate and D-arabinose to 2-keto-3-deoxy-D-arabonate. We now report the isolation from pork liver of a soluble NAD+-dependent dehydrogenase which acts on both 2-keto-3-deoxy-L-fuconate and 2-keto-3-deoxy-D-arabonate. This enzyme has been purified to homogeneity by a five-step procedure; the final step involved affinity chromatography on NAD+-agarose. A purification factor of about 3000-fold was achieved with a yield of over 20%. The enzyme was homogeneous on polyacrylamide gel electrophoresis at pH 9.1 and 7.0 and on the basis of sedimentation equilibrium analysis with the ultracentrifuge. The molecular weight of the native enzyme is about 100,000 while disc gel electrophoresis in the presence of sodium dodecyl sulfate and thiol showed the presence of a polypeptide of molecular weight 26,800; these results suggest that the enzyme is a tetramer. The enzyme has an isoelectric point of 5.4. The enzyme is unstable in the dilute state and in the absence of thiol but can be kept for 2 years at -70 degrees at a protein concentration of 4 mg per ml and in the presence of 1 mM dithiothreitol.     

D-glucose dehydrogenase from Bacillus megaterium M 1286: purification, properties and structure.

1) Glucose dehydrogenase from Bacillus megaterium has been purified to a specific activity of 550 U per mg protein. The homogeneity of the purified enzyme was demonstrated by gel electrophoresis and isoelectric focusing. 2) The amino acid composition has been determined. 3) The molecular weight of the native enzyme was found to be 116000 by gel permeation chromatography, in good agreement with the values of 120000 and 118000, which were ascertained electrophoretically according to the method of Hedrick and Smith and by density gradient centrifugation, respectively. 4) In the presence of 0.1% sodium dodecylsulfate and 8M urea, the enzyme dissociates into subunits with a molecular weight of 30000 as determined by dodecylsulfate gel electrophoresis. These values indicate that the native enzyme is composed of four polypeptide chains, each probably possessing one coenzyme binding site, which can be concluded from fluorescent titration of the NADH binding sites. 5) In polyacrylamide disc electrophoresis, samples of the purified enzyme exhibit three bands of activity, which present the native (tetrameric) form of glucose dehydrogenase and two monomeric forms (molecular weight 30000), arising under the conditions of pH and ionic strength of this method. 6) The enzyme shows a sharp pH optimum at pH 8.0 in Tris/HCl buffer, and a shift of the pH optimum to pH 9.0 in acetate/borate buffer. The limiting Michaelis constant at pH 9.0 for NAD is 4.5 mM and 47.5 mM for glucose. The dissociation constant for NAD is 0.69 mM. 7) D-Glucose dehydrogenase is highly specific for beta-D-glucose and is capable of using either NAD or NADP. The enzyme is insensitive to sulfhydryl group inhibitors, heavy metal ions and chelating agents.     

UDP-glucose-4-epimerase from Poterioochromonas malhamensis

UDP-glucose-4-epimerase of Poterioochromonas malhamensis, Peterfi has been purified to apparent electrophoretic homogeneity. The enzyme has an apparent MW of 120 000 as determined by gel filtration of the active enzyme. Sodium dodecylsulfate polyacrylamide gel electrophoresis gave a MW of 59 000, thus indicating a dimeric structure. The epimerase does not require external NAD for activity. The apparent K_m values for UDP-glucose and UDP-galactose were calculated to be 1.67 mM and 0.26 mM, respectively. The pH optimum is at pH 8.7 and the isoelectric point is at pH 5.1 ± 0.15.     

Purification and characterization of a thermostable beta-xylosidase from Thermoanaerobacter ethanolicus.

A highly thermostable beta-xylosidase, exhibiting similarly high activities for arylxylose and arylarabinose, was purified (72-fold) to gel electrophoretic homogeneity from the ethanologenic thermophilic anaerobe Thermoanaerobacter ethanolicus. The isoelectric point is pH 4.6; the apparent molecular weight is around 165,000 for the native enzyme (gel filtration and gradient polyacrylamide gel electrophoresis) and 85,000 for the two subunits (sodium dodecyl sulfate-polyacrylamide gel electrophoresis). The enzyme exhibited the highest affinity towards p-NO2-phenyl xyloside (pNPX) (substrate concentration for half-maximal activity = 0.018 mM at 82 degrees C and pH 5.0) but the highest specific activity with p-NO2-phenylarabinofuranoside. T(opt), 5 min, the temperature for the maximum initial activity in a 5-min assay of the purified enzyme, was observed around pH 5.9 and 93 degrees C; however at 65 and 82 degrees C, the pH optimum was 5.0 to 5.2, and at this pH the maximal initial activity was observed at 82 degrees C (pH 5.0 to 5.5). The pH curves and temperature curves for arylxylosides as substrates differed significantly from those for arylarabinosides as substrates. An incubation for 3 h at 82 degrees C in the absence of substrate reduced the activity to around 75%. At 86 degrees C the half-life was around 15 min. With pNPX as the substrate, an Arrhenius energy of 69 kJ/mol was determined. The N-terminal sequence did not reveal a high similarity to those from other published enzyme sequences.     

Properties of the exo-1,4-beta-xylosidase of Aspergillus niger 15

Properties of exo-1,4-beta-xylosidase from the fungus Aspergillus niger 15 were investigated. The enzyme was homogeneous during gel filtration, electrophoresis in polyacrylamide gel in the presence and absence of Na dodecyl sulfate, ultracentrifugation and isoelectric focusing. The enzyme had a temperature optimum at 70 degrees, pH optimum 3.8-4.0 for p-nitrophenyl-beta-D-xylopyranoside (p-NPX), was stable at pH 3-8, retained its 100% activity for 1 hour at 50 degrees and 42% activity at 60 degrees. Km was 0.23 mM for p-NPX and 0.67 mM for xylobiose. Xylose was a competitive inhibitor of exo-1,4-beta-xylodidase with Ki = 2.9 mM. The enzyme showed a transglycosilase activity. The aminoacid analysis of exo-1,4-beta-xylosidase showed that the enzyme molecule contained predominantly dicarboxylic and hydrophobic amino acids as well as serine. The enzyme contained no carbohydrates. Its activity was inhibited by p-chloromercury benzoate.     

Purification and characterization of the NAD-dependent glutamate dehydrogenase in the ectomycorrhizal fungus laccaria bicolor (Maire) orton

The NAD-dependent glutamate dehydrogenase (GDH) (EC 1.4.1.2) from Laccaria bicolor was purified 410-fold to apparent electrophoretic homogeneity with a 40% recovery through a three-step procedure involving ammonium sulfate precipitation, anion-exchange chromatography on DEAE-Trisacryl, and gel filtration. The molecular weight of the native enzyme determined by gel filtration was 470 kDa, whereas sodium dodecyl sulfate-polyacrylamide gel electrophoresis gave rise to a single band of 116 kDa, suggesting that the enzyme is composed of four identical subunits. The enzyme was specific for NAD(H). The pH optima were 7.4 and 8.8 for the amination and deamination reactions, respectively. The enzyme was found to be highly unstable, with virtually no activity after 20 days at -75 degrees C, 4 days at 4 degrees C, and 1 h at 50 degrees C. The addition of ammonium sulfate improved greatly the stability of the enzyme and full activity was still observed after several months at -75 degrees C. NAD-GDH activity was stimulated by Ca2+ and Mg2+ but strongly inhibited by Cu2+ and slightly by the nucleotides AMP, ADP, and ATP. The Michaelis constants for NAD, NADH, 2-oxoglutarate, and ammonium were 282 &mgr;M, 89 &mgr;M, 1.35 mM, and 37 mM, respectively. The enzyme had a negative cooperativity for glutamate (Hill number of 0.3), and its Km value increased from 0.24 to 3.6 mM when the glutamate concentration exceeded 1 mM. These affinity constants of the substrates, compared with those of the NADP-GDH of the fungus, suggest that the NAD-GDH is mainly involved in the catabolism of glutamate, while the NADP-GDH is involved in the catalysis of this amino acid. Copyright 1997 Academic Press. Copyright 1997 Academic Press     

Characterization of recombinant Drosophila melanogaster myo-inositol-1-phosphate synthase expressed in Escherichia coli

Cloned myo-inositol-1-phpsphate synthase (INOS) of Drosophila melanogaster was expressed in Escherichia coli, and purified using a His-affinity column. The purified INOS required NAD+ for the conversion of glucose-6-phosphate to inositol-1-phosphate. The optimum pH for myo-inositol-1-phosphate synthase is 7.5, and the maximum activity was measured at 40 degrees C. The molecular weight of the native enzyme, as determined by gel filtration, was approximately Mr 271,000 +/- 15,000. A single subunit of approximately Mr 62,000 +/- 5,000 was detected upon SDS-polyacrylamide gel electrophoresis. The Michaelis (Km) and dissociation constants for glucose-6-phosphate were 3.5 and 3.7 mM, whereas for the cofactor NAD+ these were 0.42 and 0.4 mM, respectively.