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.     

Uronic acid dehydrogenase from Pseudomonas syringae. Purification and properties.

1. Uronic acid dehydrogenase was purified to homogeneity. After a 338-fold purification a yield of 16% was achieved with a specific activity of 81 mumol NADH formed min-1 mg protein-1. 2. The purity of the enzyme was controlled by disc electrophoresis, sodium dodecylsulfate electrophoresis and ultracentrifugation. 3. A molecular weight of 60 000 was determined by gel chromatography and by ultracentrifugation. 4. The native enzyme is composed of two subunits, their molecular weight being 30 000 as estimated by sodium dodecylsulfate electrophoresis. The subunits as such are inactive. 5. The absorption spectrum with a maximum at 278 nm shows no evidence for a prosthetic group. 6. For catalytic activity no SH groups and no metals seem to be necessary. 7. The Michaelis constants determined with the pure enzyme are for glucuronic acid Km = 0.37 mM, galacturonic acid Km = 54 muM and NAD+ (with glucuronic acid) Km = 80 muM. 8. A weak reverse reaction could be observed with glucaric acid lactones at acidic pH. 9. NADH is competitive with NAD+. The inhibitor constant is Ki = 60 muM. 10. The NAD+ binding site seems to be of lower specificity than the uronic acid binding site.     

Potato tuber succinate semialdehyde dehydrogenase: purification and characterization

Succinate semialdehyde dehydrogenase (SSADH) has been purified from potato tubers with 39% yield, 832-fold purification, and a specific activity of 6.5 units/mg protein. The final preparation was homogeneous as judged from native and sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Gel filtration on Sepharose 6B gave a relative molecular mass (Mr) of 145,000 for the native enzyme. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis gave a single polypeptide band of Mr 35,000. Thus the enzyme appears to be a tetramer of identical subunits. Chromatofocusing of the enzyme gave a pI of 8.7. The enzyme was maximally active at pH 9.0 in 100 mM sodium pyrophosphate buffer. In 100 mM Tris-HCl buffer, pH 9.0, the enzyme gave only 20% of the activity found in pyrophosphate buffer and had a shorter linear rate. The enzyme was specific for succinate semialdehyde (SSA) as substrate and could not utilize acetaldehyde, glyceraldehyde 3-phosphate, malonaldehyde, lactate, or ethanol as substrates. The enzyme was also specific for NAD+ as cofactor and NADP+ and 3-acetylpyridine adenine dinucleotide could not serve as cofactors. Potato SSADH had a Km of 4.6 microM for SSA when assayed in pyrophosphate buffer and was inhibited by that substrate at concentrations greater than 120 microM. The Km for NAD+ was found to be 31 microM. The enzyme required exogenous addition of a thiol compound for maximal activity and was inhibited by the thiol-directed reagents p-hydroxymercuribenzoate, dithionitrobenzoate, and N-ethyl-maleimide, by heavy metal ions Hg2+, Cu2+, Cd2+, and Zn2+, and by arsenite. These results indicate a requirement of a SH group for catalytic activity.     

Isolation and characterization of valine dehydrogenase from Streptomyces aureofaciens.

Valine dehydrogenase was purified to homogeneity from the crude extracts of Streptomyces aureofaciens. The molecular weight of the native enzyme was 116,000 by equilibrium ultracentrifugation and 118,000 by size exclusion high-performance liquid chromatography. The enzyme was composed of four subunits with molecular weights of 29,000. The isoelectric point was 5.1. The enzyme required NAD+ as a cofactor, which could not be replaced by NADP+. Sulfhydryl reagents inhibited the enzyme activity. The pH optimum was 10.7 for oxidative deamination of L-valine and 9.0 for reductive amination of alpha-ketoisovalerate. The Michaelis constants were 2.5 mM for L-valine and 0.10 mM for NAD+. For reductive amination the Km values were 1.25 mM for alpha-ketoisovalerate, 0.023 mM for NADH, and 18.2 mM for NH4Cl.     

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 characterization of dipeptidyl peptidase IV from Streptococcus salivarius HHT.

Dipeptidyl peptidase IV (DAP IV) was purified from Streptococcus salivarius HHT by anion-exchange chromatography, gel filtration and affinity chromatography after lysis of cell walls with N-acetylmuramidase. DAP IV was purified 114-fold with a yield of 16.6% from total activity of the crude extract. The purified enzyme was shown to be homogeneous by disc gel electrophoresis. The molecular weight of the enzyme was estimated to be about 109,000 by gel filtration and 47,000 by sodium dodecylsulphate SDS-polyacrylamide gel electrophoresis, suggesting that the native enzyme is a dimeric form. The optimum pH for the reaction was 8.7 in Gly-NaOH buffer, and the isoelectric point of the enzyme was pH 4.2. The enzyme hydrolysed specifically N-terminal X-Pro from X-Pro-p-nitroanilides. The enzyme activity was hardly affected by various cations, sulphydryl-blocking reagents and metal chelators. The enzyme activity was markedly inhibited by 1 mM diisopropylfluoride, and the desialysed enzyme was attacked by proteinases.     

Purification, properties and primary structure of alanine dehydrogenase involved in taurine metabolism in the anaerobe Bilophila wadsworthia

Alanine dehydrogenase [L-alanine:NAD+ oxidoreductase (deaminating), EC 1.4.1.4.] catalyses the reversible oxidative deamination of L-alanine to pyruvate and, in the anaerobic bacterium Bilophila wadsworthia RZATAU, it is involved in the degradation of taurine (2-aminoethanesulfonate). The enzyme regenerates the amino-group acceptor pyruvate, which is consumed during the transamination of taurine and liberates ammonia, which is one of the degradation end products. Alanine dehydrogenase seems to be induced during growth with taurine. The enzyme was purified about 24-fold to apparent homogeneity in a three-step purification. SDS-PAGE revealed a single protein band with a molecular mass of 42 kDa. The apparent molecular mass of the native enzyme was 273 kDa, as determined by gel filtration chromatography, suggesting a homo-hexameric structure. The N-terminal amino acid sequence was determined. The pH optimum was pH 9.0 for reductive amination of pyruvate and pH 9.0-11.5 for oxidative deamination of alanine. The apparent Km values for alanine, NAD+, pyruvate, ammonia and NADH were 1.6, 0.15, 1.1, 31 and 0.04 mM, respectively. The alanine dehydrogenase gene was sequenced. The deduced amino acid sequence corresponded to a size of 39.9 kDa and was very similar to that of the alanine dehydrogenase from Bacillus subtilis.     

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.     

Purification and properties of saccharopine dehydrogenase (glutamate forming) in the Saccharomyces cerevisiae lysine biosynthetic pathway.

Saccharopine dehydrogenase (glutamate forming) of the biosynthetic pathway of lysine in Saccharomyces cerevisiae was purified 1,122-fold by using acid precipitation, ammonium sulfate precipitation, DEAE-Sepharose, gel filtration, and Reactive Red-120 agarose chromatography. The enzyme exhibited a native molecular size of 69,000 daltons by gel filtration and consisted of a single 50,000-dalton polypeptide based upon sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The enzyme was readily denatured by exposures to temperatures exceeding 46 degrees C. The pH optimum for the reverse reaction was 9.5. The apparent Kms for L-saccharopine and NAD+ were 2.32 and 0.054 mM, respectively. The enzyme was inhibited by mercuric chloride but not by carbonyl or metal complexing agents.     

A Rapid Purification of L-Glutamic Acid Decarboxylase from the Brain of the Locust Schistocerca gregaria

L-Glutamic acid decarboxylase (GAD; EC 4.1.1.15) was purified to apparent homogeneity from the brain of the locust Schistocerca gregaria using a combination of chromatofocusing (Mono P) and gel filtration (Superose 12) media. The homogeneity of the enzyme preparation was established by native polyacrylamide gel electrophoresis (PAGE) with silver staining. The molecular weight of the purified enzyme was estimated from native gradient gel electrophoresis and gel filtration chromatography to be 97,000 +/- 4,000 and 93,000 +/- 5,000, respectively. When analysed by sodium dodecyl sulphate-PAGE, the enzyme was found to be composed of two distinct subunits of Mr 51,000 +/- 1,000 and 44,000 +/- 1,500. Tryptic peptide maps of iodinated preparations of these two subunits showed considerable homology, suggesting that the native enzyme is a dimer of closely related subunits. The purified enzyme had a pH optimum of 7.0-7.4 in 100 mM potassium phosphate buffer and an apparent Km for glutamate of 5.0 mM. The enzyme was strongly inhibited by the carbonyl-trapping reagent aminooxyacetic acid with an I50 value of 0.2 microM.     

Purification and properties of carnitine dehydrogenase from Pseudomonas putida

Carnitine dehydrogenase (carnitine:NAD+ oxidoreductase, EC 1.1.1.108) from Pseudomonas putida IFP 206 catalyzes the oxidation of L-carnitine to 3-dehydrocarnitine. The enzyme was purified 72-fold to homogeneity as judged by polyacrylamide gel electrophoresis. The molecular mass of this enzyme is 62 kDa and consists of two identical subunits. The isoelectric point was found to be 4.7. the carnitine dehydrogenase is specific for L-carnitine and NAD+. The optimum pH for enzymatic activity in the oxidation reaction was found to be 9.0 and 7.0 in the reduction reaction. The optimal temperature is 30 degrees C. The Km values for substrates were determined.     

Isolation and properties of cytoplasmic alpha-glycerol 3-phosphate dehydrogenase from the pectoral muscle of the fruit bat,Eidolon helvum

Cytoplasmic alpha-glycerol-3-phosphate dehydrogenase from fruit-bat-breast muscle was purified by ion-exchange and affinity chromatography. The specific activity of the purified enzyme was approximately 120 units/mg of protein. The apparent molecular weight of the native enzyme, as determined by gel filtration on Sephadex G-100 was 59,500 +/- 650 daltons; its subunit size was estimated to be 35,700 +/- 140 by SDS-polyacrylamide gel electrophoresis. The true Michaelis-Menten constants for all substrates at pH 7.5 were 3.9 +/- 0.7 mM, 0.65 +/- 0.05 mM, 0.26 +/- 0.06 mM, and 0.005 +/- 0.0004 mM for L-glycerol-3-phosphate, NAD(+), DHAP, and NADH, respectively. The true Michaelis-Menten constants at pH 10.0 were 2.30 +/- 0.21 mM and 0.20 +/- 0.01 mM for L-glycerol-3-phosphate and NAD(+), respectively. The turnover number, k(cat), of the forward reaction was 1.9 +/- 0.2 x 10(4)s(-1). The treatment of the enzyme with 5,5'-dithiobis-2-nitrobenzoic acid (DTNB) under denaturing conditions indicated that there were a total of eight cysteine residues, while only two of these residues were reactive towards DTNB in the native enzyme. The overall results of the in vitro experiments suggest that alpha-glycerol-3-phosphate dehydrogenase of the fruit bat preferentially catalyses the reduction of dihydroxyacetone phosphate to glycerol-3-phosphate.     

D-Malic enzyme of Pseudomonas fluorescens

By the enrichment culture technique 14 gram-negative bacteria and two yeast strains were isolated that used D(+)-malic acid as sole carbon source. The bacteria were identified as Pseudomonas putida, Pseudomonas fluorescens, Pseudomonas aeruginosa and Klebsiella aerogenes. In cell-free extracts of P. fluorescens and P. putida the presence of malate dehydrogenase, D-malic enzyme (NAD-dependent) and L-malic enzyme (NADP-dependent) was demonstrated. D-Malic enzyme from P. fluorescens was purified. Stabilization of the enzyme by 50 mM ammonium sulphate an 1 mM EDTA was essential. Preparation of D-malic enzyme that gave one band with disc gel electrophoresis showed a specific activity of 4-5 U/mg. D-Malic enzyme requires divalent cations. The Km values were for malate Km = 0.3 mM and for NAD Km = 0.08 mM. The pH optimum for the reaction was found to be in the range of pH 8.1 to pH 8.8. D-Malic enzyme is partially inhibited by oxaloacetic acid, meso-tartaric acid, D-lactic acid and ATP. Determined by gel filtration and gradient gel electrophoresis, the molecular weight was approximately 175 000.     

Purification and properties of malic dehydrogenase from Trichomonas gallinae

The malic dehydrogenase (MDH², l-malate: NAD oxidoreductase, E.C. 1,1.1.37) of Trichomonas gallinae was purified 215-fold and characterized. The molecular weight was found to be 72,000 and the enzyme protein contained essential cations and sulfhydryl groups. Polyacrylamide gel electrophoresis before and after extensive purification yielded a single band of malic dehydrogenase activity strongly suggesting only one molecular form of the enzyme. Analysis of kinetic data yielded the following K_m values: oxalocetate, 16 μM; malate, 200 μM; NADH 11 μM; and NAD, 70 μM. The enzyme was absolutely specific for l-malic acid, NAD, and NADH. The enzyme exhibited a broad band of heat stability with an optimum of 51 C. The pH optimum in the direction of oxalacetate reduction was 9.0. The pH optima in the reverse direction were 9.0 and 10.5 A role for this enzyme in T. gallinae metabolism is discussed.     

Purification and characterisation of leukotriene A4 hydrolase from rat neutrophils

Leukotriene A4 hydrolase was rapidly and extensively purified from rat neutrophils using anion exchange and gel filtration high-pressure liquid chromatography. The enzyme which converts the allylic epoxide leukotriene A4 to the 5,12-dihydroxyeicosatetraenoic acid leukotriene B4 was localized in the cytosolic fraction and exhibited an optimum activity at pH 7.8 and an apparent Km for leukotriene A4 between 2 X 10(-5) and 3 X 10(-5) M. The purified leukotriene A4 hydrolase was shown to have a molecular weight of 68 000 on sodium dodecylsulfate polyacrylamide gel electrophoresis and of 50 000 by gel filtration. The molecular weight and monomeric native form of this enzyme are unique characteristics which distinguish leukotriene A4 hydrolase from previously purified epoxide hydrolases.     

Microbial production of methyl ketones. Purification and properties of a secondary alcohol dehydrogenase from yeast.

Cell-free extracts derived from yeasts Candida utilis ATCC 26387, Hansenula polymorpha ATCC 26012, Pichia sp. NRRL-Y-11328 Torulopsis sp. strain A1 and Kloeckera sp. strain A2 catalyzed an NAD+-dependent oxidation of secondary alcohols (2-propanol, 2-butanol, 2-pentanol, 2-hexanol) to the corresponding methyl ketones (acetone, 2-butanone, 2-pentanone, 2-hexanone). We have purified a NAD+-specific secondary alcohol dehydrogenase from methanol-grown yeast, Pichia sp. The purified enzyme is homogenous as judged by polyacrylamide gel electrophoresis. The purified enzyme catalyzed the oxidation of secondary alcohols to the corresponding methyl ketones in the presence of NAD+ as an electron acceptor. Primary alcohols were not oxidized by the purified enzyme. The optimum pH for oxidation of secondary alcohols by the purified enzyme is 8.0. The molecular weight of the purified enzyme as determined by gel filtration is 98 000 and subunit size as determined by sodium dodecyl sulfate gel electrophoresis is 48 000. The activity of the purified secondary alcohol dehydrogenase was inhibited by sulfhydryl inhibitors and metal-binding 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 properties of formate dehydrogenase from Moraxella sp. strain C-1

NAD+-dependent formate dehydrogenase was screened in various bacterial strains. Facultative methanol-utilizing bacteria isolated from soil samples, acclimated to a medium containing methanol and formate at pH 9.5, were classified as members of the genus Moraxella. From a crude extract of Moraxella sp. strain C-1, formate dehydrogenase was purified to homogeneity, as judged by disc gel electrophoresis. The enzyme has an isoelectric point of 3.9 and a molecular weight of approximately 98,000. The enzyme is composed of two identical subunits with molecular weights of about 48,000. The apparent Km values for sodium formate and NAD+ were calculated to be 13 mM and 0.068 mM, respectively.     

Purification and properties of N-acetylneuraminate lyase from Escherichia coli

N-Acetylneuraminate lyase [N-acetylneuraminic acid aldolase EC 4.1.3.3] from Escherichia coli was purified by protamine sulfate treatment, fractionation with ammonium sulfate, column chromatography on DEAE-Sephacel, gel filtration on Ultrogel AcA 44, and preparative polyacrylamide gel electrophoresis. The purified enzyme preparation was homogeneous on analytical polyacrylamide gel electrophoresis, and was free from contaminating enzymes including NADH oxidase and NADH dehydrogenase. The enzyme catalyzed the cleavage of N-acetylneuraminic acid to N-acetylmannosamine and pyruvate in a reversible reaction. Both cleavage and synthesis of N-acetylneuraminic acid had the same pH optimum around 7.7. The enzyme was stable between pH 6.0 to 9.0, and was thermostable up to 60 degrees C. The thermal stability increased up to 75 degrees C in the presence of pyruvate. No metal ion was required for the enzyme activity, but heavy metal ions such as Ag+ and Hg2+ were potent inhibitors. Oxidizing agents such as N-bromosuccinimide, iodine, and hydrogen peroxide, and SH-inhibitors such as p-chloromercuribenzoic acid and mercuric chloride were also potent inhibitors. The Km values for N-acetylneuraminic acid and N-glycolylneuraminic acid were 3.6 mM and 4.3 mM, respectively. Pyruvate inhibited the cleavage reaction competitively; Ki was calculated to be 1.0 mM. In the condensation reaction, N-acetylglucosamine, N-acetylgalactosamine, glucosamine, and galactosamine could not replace N-acetylmannosamine as substrate, and phosphoenolpyruvate, lactate, beta-hydroxypyruvate, and other pyruvate derivatives could not replace pyruvate as substrate. The molecular weight of the native enzyme was estimated to be 98,000 by gel filtration methods. After denaturation in sodium dodecyl sulfate or in 6 M guanidine-HCl, the molecular weight was reduced to 33,000, indicating the existence of 3 identical subunits. The enzyme could be used for the enzymatic determination of sialic acid; reaction conditions were devised for determining the bound form of sialic acid by coupling neuraminidase from Arthrobacter ureafaciens, lactate dehydrogenase, and NADH.     

Beta-glucuronidase of rat preputial gland. Crystallization, properties, carbohydrate composition, and subunits.

Rat preputial gland beta-glucuronidase [ED 3.2.1.31] was purified by ammonium sulfate precipitation, ethanol fractionation, gel filtration on Sephadex G-200 and crystallization. The purified enzyme appeared homogeneous on electrophoresis in polyacrylamide gel, and on analytical ultracentrifugation and had a molecular weight of approximately 320,000, and a sedimentation coefficient of 12S. SDS polyacrylamide gel electrophoresis indicated that the enzyme consisted of subunits with molecular weight of 79,000, so the native enzyme appeared to be a tetramer. The Km with p-nitrophenyl beta-D-glucosiduronic acid as substrate was about 0.53 mM. The enzyme had a single pH optimum at 4.5. The enzyme had a very low content of sulphur-containing amino acid and contained 5.7 per cent carbohydrate, consisting of mannose, glucose, fucose, galactose, and glucosamine in a ratio of 44;9;6;2;41. Sialic acid was not detected in the crystallized enzyme.