Isolation, Purification, and Properties of Malate Dehydrogenase from Sulfur Bacterium Beggiatoa leptomitiformis

Malate dehydrogenase (E.C. 1.1.1.37) from the bacterium Beggiatoa leptomitiformis was isolated and purified 123-fold using a five-step purification procedure including the enzyme extraction, ammonium sulfate protein fractionation, gel filtration, ion exchange chromatography, and gel chromatography. The enzyme was homogenous according to the electrophoresis data; its activity was 20.43 U/mg protein. This malate dehydrogenase is a homotetramer (Mr = 172 kDa). The catalytic and thermodynamic properties, as well as the analysis of the published data suggest that the tetrameric structure of the enzyme allows it to participate in constructive metabolism supplying the cell with organic acids as a source of carbon.     

N5-(L-1-carboxyethyl)-L-ornithine:NADP+ oxidoreductase from Streptococcus lactis. Purification and partial characterization

N5-(L-1-Carboxyethyl)-L-ornithine:NADP+ oxidoreductase (EC 1.5.1.-) from Streptococcus lactis K1 has been purified 8,000-fold to homogeneity. The NADPH-dependent enzyme mediates the reductive condensation between pyruvic acid and the delta- or epsilon-amino groups of L-ornithine and L-lysine to form N5-(L-1-carboxyethyl)-L-ornithine and N6-(L-1-carboxyethyl)-L-lysine, respectively. The five-step purification procedure involves ion-exchange (DE52 and phosphocellulose P-11), gel filtration (Ultrogel AcA 44), and affinity chromatography (2',5'-ADP-Sepharose 4B). Approximately 100-200 micrograms of purified enzyme of specific activity 40 units/mg were obtained from 60 g of cells, wet weight. Anionic polyacrylamide gel electrophoresis revealed a single enzymatically active protein band, whereas three species (pI 4.8-5.1) were detected by analytical electrofocusing. The purified enzyme is active over a broad pH range of 6.5-9.0 and is stable to heating at 50 degrees C for 10 min. Substrate Km values were determined to be: NADPH, 6.6 microM; pyruvate, 150 microM; ornithine, 3.3 mM; and lysine, 18.2 mM. The oxidoreductase has a relative molecular mass (Mr = 150,000) as estimated by high pressure liquid chromatography exclusion chromatography and by polyacrylamide gradient gel electrophoresis. Conventional gel filtration indicated an Mr = 78,000, and a single protein band of Mr = 38,000 was revealed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The enzyme is composed of identical subunits of Mr = 38,000, which may associate to yield both dimeric and tetrameric forms. Polyclonal antibody to the purified protein inhibited enzyme activity. The amino acid composition of the enzyme is reported, and the sequence of the first 37 amino acids from the NH2 terminus has been determined by stepwise Edman degradation.     

Isolation and characterization of flavin-linked glycerol-3-phosphate dehydrogenase from rabbit skeletal muscle mitochondria and comparison with the enzyme from rabbit brain

Mitochondrial glycerol-3-P dehydrogenase (EC 1.1.99.5) has been purified in 20% yield from both rabbit skeletal muscle and brain using a four step procedure involving osmotic shock, solubilization with Triton X-100, hydrophobic chromatography, gel filtration, and preparative column isoelectrofocusing. The active muscle and brain enzymes were found to be 95% and 80% homogeneous, respectively. Final purification was performed on the denatured subunit. The active enzyme from each of the tissues focused at pH 5.25 +/- 0.12 and each produced similar biphasic thermal inactivation plots at 50 degrees C. Mixtures of the purified brain and muscle enzymes co-migrated in discontinuous electrophoresis gels and each enzyme exhibited a single polypeptide component on sodium dodecyl sulfate (SDS) gels either when run separately or in mixtures. The subunit molecular weight was shown to be 76,000 +/- 3,000 by SDS-gel electrophoresis and gel filtration in 6 M guanidine HCl. One mole of noncovalently bound FAD and 1 mole of iron were measured per Mr = 100,000. The amino acid composition was determined based on the assumption of 70 aspartate residues per subunit to give a Mr = 76,000. The absorption spectrum has a maximum at 416 nm and a shoulder at 450 to 460 nm which is bleached on treatment with sodium dithionite. The maximum at 416 nm is removed by treatment with mersalyl.     

A rapid four column purification of 2-deoxy-D-glucoside-2-sulphamate sulphohydrolase from human liver

2-Deoxy-D-glucoside-2-sulphamate sulphohydrolase was extracted from human liver and purified 40 000-fold by a simple four column procedure. The purification was followed using a specific substrate isolated from an acid hydrolysate of heparin, O-(alpha-2-sulphamino-2-deoxy-D-glucopyranosyl)-(1 leads to 3)-L-[6,3H]idonic acid. Only one form of the enzyme was seen on either ion exchange chromatography or isoelectric focussing, with a pI of 6.8. The apparent Mr of the holoenzyme as determined by gel filtration was 190 000 +/- 20 000. Two other larger Mr protein peaks observed on gel filtration appear to be an inactive dimer of the 190 000 dalton peak and a larger aggregate near the exclusion limit of the column. On polyacrylamide disc gel electrophoresis in sodium dodecyl sulphate, with or without prior reduction, each protein peak from the gel filtration column electrophoresed as a single major band with an apparent Mr corresponding to 55 000 +/- 6000.     

Purification and characterization of glycerol-3-phosphate dehydrogenase (flavin-linked) from rat liver mitochondria

We have purified the membrane-intrinsic glycerol-3-phosphate dehydrogenase from both normal and hyperthyroid rat liver mitochondria by extraction with Triton X-100, hydrophobic affinity chromatography, ion exchange chromatography, gel filtration, and FAD-linked Sepharose 4B affinity chromatography. The yields in both cases were over 20%, and purification ranged from 800- to 650-fold in mitochondria from hyperthyroid and normal rats, respectively. The final preparations appeared to be greater than 95% pure by polyacrylamide gel electrophoresis in the presence or absence of sodium dodecyl sulfate. The pure enzyme focused at pH 5.5 and produced a biphasic thermal inactivation plot at 50 degrees C. The holoenzyme was found to have a molecular mass of 250,000 daltons on gel filtration. The subunit molecular mass was found to be 74,000 daltons +/- 3,000 by sodium dodecyl sulfate-gel electrophoresis and high-performance liquid chromatography gel filtration in 0.1% sodium dodecyl sulfate. 1 mol of the holoenzyme preparation contains 1.1 mol of non-heme iron and 0.7-0.9 mol of noncovalently bound FAD. The absorption spectrum has a maximum at 375 nm and a shoulder at 450 nm which is bleached on treatment with sodium dithionite. The enzymatic reaction is competitively inhibited by glyceraldehyde 3-phosphate, dihydroxyacetone phosphate, phosphoenolpyruvate, and phosphoglycolic acid. The apparent Km for DL-alpha-glycerol 3-phosphate and noncovalently bound FAD were found to be 6 mM and 7 microM, respectively.     

Light modulation of maize leaf phosphoenolpyruvate carboxylase

Phosphoenolpyruvate carboxylase (PEPC) was extracted from maize (Zea mays L. cv Golden Cross Bantam T51) leaves harvested in the dark or light and was partially purified by (NH₄)₂SO₄ fractionation and gel filtration to yield preparations that were 80% homogeneous. Malate sensitivity, PEPC activity, and PEPC protein (measured immunochemically) were monitored during purification. As reported previously, PEPC from dark leaves was more sensitive to malate inhibition compared to enzyme extracted from light leaves. Extraction and purification in the presence of malate stabilized the characteristics of the two forms. During gel filtration on Sephacryl S-300, all of the PEPC activity and PEPC protein emerged in a single high molecular weight peak, indicating that no inactive dissociated forms (dimers, monomers) were present. However, there was a slight difference between the light and dark enzymes in elution volume during gel filtration. In addition, specific activity (units at pH 7/milligram PEPC protein) decreased through the peak for both enzyme samples; because the dark enzyme emerged at a slightly higher elution volume, it contained enzyme with a relatively lower specific activity. The variation in specific activity of the dark enzyme corresponded with changes in malate sensitivity. Immunoblotting of samples with different specific activity and malate sensitivity, obtained from gel filtration, revealed only a single polypeptide with a relative molecular mass of 100,000. When the enzyme was extracted and purified in the absence of malate, characteristic differences of the light and dark enzymes were lost, the enzymes eluted at the same volume during gel filtration, and specific activity was constant through the peak. We conclude that maize leaf PEPC exists in situ as a tetramer of a single polypeptide and that subtle conformation changes can affect both enzymic activity and sensitivity to malate inhibition.     

Structural and chemical characterization of a homogeneous peptide N-glycosidase from almond

A peptide N-glycosidase that catalyzes the hydrolysis of N-linked oligosaccharide chains from glycopeptides and glycoproteins has been purified to homogeneity from almond emulsin and from almond meal. Purification from almond emulsin using ion-exchange chromatography, gel filtration chromatography, and preparative polyacrylamide gel electrophoresis gave an enzyme which was purified more than 700-fold and with a yield of 63%. An alternative procedure, more suitable for efficient large scale purification, used ion-exchange, affinity, and gel filtration chromatography. When purification began with almond emulsin, the enzyme was purified 1200-fold with a 37% yield, while when purification began with almond powder, the enzyme was purified 9000-fold with a yield of 45%. The homogeneous enzyme is stable at 4 degrees C for several months in 10 mM sodium acetate, pH 5.0, buffer. The peptide N-glycosidase is itself shown to be a glycoprotein consisting of a single polypeptide chain with a molecular weight of 66 800 on sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis. Circular dichroism spectra of the native molecule indicate the presence of a high (approximately 80%) alpha-helix content. The amino acid and carbohydrate contents of the enzyme are presented. When a convenient new assay with a turkey ovomucoid glycopeptide as a substrate is used, the enzyme preparation exhibits a broad pH optimum centered between pH 4 and pH 6. The enzyme is readily inactivated by SDS and guanidine hydrochloride, but it is stable in the presence of moderate concentrations of several other protein denaturants.(ABSTRACT TRUNCATED AT 250 WORDS)     

The covalent structure of hepatic microsomal epoxide hydrolase. II. The complete amino acid sequence

The complete amino acid sequence of hepatic microsomal epoxide hydrolase has been determined. The protein contains 455 amino acid residues in a single polypeptide chain and has Mr = 52,691. Peptides from selective chemical and proteolytic cleavages were isolated by a combination of gel filtration and high performance liquid chromatography and sequenced by automated Edman degradation. Overlapping peptide sequences were used to deduce the complete sequence. This is the first epoxide hydrolase and the third microsomal enzyme for which the complete sequence has been determined.     

Schistosoma mansoni: Purification and characterization of malate dehydrogenases

Isoelectric focusing of a homogenate of Schistosoma mansoni, followed by malate dehydrogenase-specific staining, showed the presence of two major and five minor malate dehydrogenase isoenzymes (EC 1.1.1.37), with isoelectric points ranging from 7.3 to 9.5. The malate dehydrogenase isoenzymes were purified by gel filtration, followed by ion-exchange chromatography on DEAE- and CM-cellulose. The isoenzymes could be differentiated by their susceptibility to substrate inhibition. No differences in the Michaelis-Menten constants for substrate were found. One of the isoenzymes is inhibited by 5′-AMP. Further purification of this particular isoenzyme was achieved by affinity chromatography on 5′-AMP-Sepharose 4B. Analysis after subcellular fractionation indicated a mitochondrial origin for this isoenzyme. The mitochondrial isoenzyme (at a recovery of 80%) was purified 218-fold compared to the crude soluble extract, and contained about 40% of the total malate dehydrogenase activity. The enzyme has a molecular weight of 65,500 and showed absolute specificity for l-malic acid, NAD, and NADH. The final preparation has a specific activity of 451 U/mg protein. Physicochemical studies, including binding constants, substrate inhibition, thermostability, and pH optima, demonstrated differences between the mitochondrial and cytoplasmic enzymes. A role for malate dehydrogenase in Schistosoma mansoni metabolism is discussed.     

Malic enzyme from archaebacterium Sulfolobus solfataricus. Purification, structure, and kinetic properties

An NADP-preferring malic enzyme ((S)-malate:NADP oxidoreductase (oxalacetate-decarboxylating) EC 1.1.1.40) with a specific activity of 36.6 units per mg of protein at 60 degrees C and an isoelectric point of 5.1 was purified to homogeneity from the thermoacidophilic archaebacterium Sulfolobus solfataricus, strain MT-4. The purification procedure employed ion exchange chromatography, ammonium sulfate fractionation, affinity chromatography, and gel filtration. Molecular weight determinations demonstrated that the enzyme was a dimer of Mr 105,000 +/- 2,000 with apparently identical Mr 49,000 +/- 1,500 subunits. Amino acid composition of S. solfataricus enzyme was determined and found to be significantly higher in tryptophan content than the malic enzyme from Escherichia coli. In addition to the NAD(P)-dependent oxidative decarboxylation of L-malate, S. solfataricus malic enzyme was able to catalyze the decarboxylation of oxalacetate. The enzyme absolutely required divalent metal cations and it displayed maximal activity at 85 degrees C and pH 8.0 with a turnover number of 376 s-1. The enzyme showed classical saturation kinetics and no sigmoidicity was detected at different pH values and temperatures. At 60 degrees C and in the presence of 0.1 mM MnCl2, the Michaelis constants for malate, NADP, and NAD were 18, 3, and 250 microM, respectively. The S. solfataricus malic enzyme was shown to be very thermostable.     

Purification and characterization of a rat brain aldehyde dehydrogenase able to metabolize gamma-aminobutyraldehyde to gamma-aminobutyric acid.

An enzyme which catalyses dehydrogenation of gamma-aminobutyraldehyde (ABAL) to gamma-aminobutyric acid (GABA) was purified to homogeneity from rat brain tissues by using DEAE-cellulose and affinity chromatography on 5'-AMP-Sepharose, phosphocellulose and Blue Agarose, followed by gel filtration. Such an enzyme was first purified from mammalian brain tissues, and was identified as an isoenzyme of aldehyde dehydrogenase. It has an Mr of 210,000 determined by polyacrylamide-gradient-gel electrophoresis, and appeared to be composed of subunits of Mr 50,000. The close similarity of substrate specificity toward acetaldehyde, propionaldehyde and glycolaldehyde between the enzyme and other aldehyde dehydrogenases previously reported was observed. But substrate specificity of the enzyme toward ABAL was higher than those of aldehyde dehydrogenases from human liver (E1 and E2), and was lower than those of ABAL dehydrogenases from human liver (E3), Escherichia coli and Pseudomonas species. The Mr and relative amino acid composition of the enzyme are also similar to those of E1 and E2. The existence of this enzyme in mammalian brain seems to be related to a glutamate decarboxylase-independent pathway (alternative pathway) for GABA synthesis from putrescine.     

NADP-dependent malate dehydrogenase from bovine adrenal cortex cytoplasm. Isolation and properties

The NADP-dependent decarboxylating malate dehydrogenase was isolated from the cytoplasmic fraction of bovine adrenal cortex and purified 3530-fold by 3-fold ammonium sulfate fractionation, ion-exchange chromatography on DEAE-Toyopearl 650 M and DEAE-Sephadex A-50 with subsequent two-fold gel filtration through Toyopearl HW-55. The specific activity of homogeneous enzyme preparations was equal to 60 U/mg protein with a 30% yield. The enzyme molecular weight as determined by gel filtration on Sephadex G-20 was 155000. Upon polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate malate dehydrogenase dissociated into two subunits with Mr 77000. The Arrhenius plot for the reaction rate showed a break at 30 degrees C. The values of activation energy and temperature coefficient above and below the breakpoint were equal to 45049 and 147188 J X mol-1; 1.68 and 2.63, respectively. Within the temperature range of 26-40 degrees C, malate dehydrogenase exhibited hyperbolic kinetics with respect to the substrate. At 30 degrees C, Km for malate was equal to 250 microM, whereas at 40 degrees C it was 130 microM. The curve for the dependence of the initial reaction velocity versus NADP concentration was S-shaped. The Hill coefficient was 1.4, which testifies to positive cooperativity of NADP interaction with malate dehydrogenase.     

Highly acidic proteins from human brain: purification and properties of Glu-50 protein

Three extremely acidic proteins were isolated from human brain and purified to apparent homogeneity. One of them, Glu-50 protein, contained much glutamic acid (about 50% of the total amino acids). Its purification involved ammonium sulfate fractionation, DEAE-Sephadex A-50 chromatography, and gel filtration on Sephadex G-100 and G-75. Its molecular weight was determined to be 11,000 by SDS polyacrylamide gel electrophoresis and 34,000-36,000 by gel filtration on Sephadex G-75, suggesting that it consists of three identical polypeptide chains. Its isoelectric point was pH 3.9. Its N-terminal amino acid sequence was NH2-Asp-Glu-Pro-Pro-Asp-Glu and its C-terminal amino acid was Lys. It contained no detectable carbohydrate.     

Purification and characterization of guanosine diphospho-D-mannose dehydrogenase. A key enzyme in the biosynthesis of alginate by Pseudomonas aeruginosa

Alginate-producing Pseudomonas aeruginosa are usually associated with the cystic fibrosis lung environment and contribute to the high mortality rates observed among these patients. The present paper describes the purification and enzymatic properties of guanosine diphospho-D-mannose dehydrogenase (EC 1.1.1.132), a key enzyme in alginate biosynthesis by mucoid P. aeruginosa. The enzyme was overproduced using a plasmid vector containing algD (the gene encoding this enzyme) under control of the tac promoter. It was purified from cell-free lysates by lowering the pH to 5.0, heating the extract to 57.5 degrees C for 10 min, and discarding the protein pellet. The enzyme was selectively precipitated from the supernatant fraction with 45% acetone, resuspended in a 100 mM triethanolamine acetate buffer, pH 7.6, and ultimately purified by Bio-Sil TSK-400 gel filtration chromatography. The subunit molecular weight (Mr 48,000) as well as the N-terminal amino acid sequence corresponded to those predicted from the DNA sequence of algD. The native protein migrated as a hexamer of 290,000 molecular weight upon Bio-Gel A-1.5m gel filtration chromatography. Kinetic analysis demonstrated an apparent Km of 14.9 microM for the substrate GDP-D-mannose and 185 microM for the cofactor NAD+. GDP-D-mannuronic acid was identified as the enzyme reaction product. Several compounds (including GMP, ATP, GDP-D-glucose, and maltose) were found to inhibit enzymatic activity. GMP, the most potent of these inhibitors, exhibited competitive inhibition with an apparent Ki of 22.7 microM. Enzyme activity was also sensitive to the sulfhydryl group modifying agents iodoacetamide and p-hydroxymercuribenzoate. The addition of excess dithiothreitol restored enzyme activity, suggesting a possible involvement of cysteine residues in enzymatic activity.     

Purification and properties of aldehyde dehydrogenase from Saccharomyces cerevisiae.

A procedure for the purification of aldehyde dehydrogenase from bakers' yeast (Saccharomyces cerevisiae) is reported. Treatment with acid, heat and organic solvents was avoided and chromatographic and filtration techniques in the presence of phenylmethylsulfonylfluoride were mainly used. An affinity chromatography step using the reactive dye Cibacron blue F3G-A, which was covalently bound to Sepharose 4B, was found to be essential. The enzyme was bound to and then released from the dye. The purified enzyme was shown to be homogeneous by gel filtration, disc electrophoresis and SDS electrophoresis. The molecular weight of the purified enzyme determined by gel filtration was 170,000, which agreed with that of the enzyme in the crude extract. The enzyme was composed of subunits of a molecular weight of 57,000. The specific activity of the enzyme was 20 units per mg of protein under the standard assay conditions. The substrate specificity, the relative maximal velocity, the michaelis constants, the pH optimum, the stability and the activation energy of the enzyme are reported.     

Purification and partial characterization of bovine heart phosphorylase kinase

Bovine heart phosphorylase kinase has been isolated by a procedure involving precipitation with polyethylene glycol, DEAE-Sephacel chromatography and calmodulin-Sepharose affinity chromatography. The isolated enzyme had a specific activity of 8.3 IU/mg of protein at pH 8.2 at 30 degrees C in the presence of 1% glycogen. The native enzyme had a sedimentation coefficient of 23 S and the Mr of the alpha', beta, gamma, and delta subunits, were 140,000, 130,000, 46,000, and 18,000, respectively. Activation of the phosphorylase kinase by the catalytic subunit of bovine heart cAMP-dependent protein kinase increases the pH 6.8/8.2 activity ratio from 0.01 to 0.32-0.38. Glycogen (1%) decreased the Km of the activated phosphorylase kinase at pH 6.8 for phosphorylase b from 5.5 to 1.25 mg/ml. Trypsin treatment increased the pH 6.8 activity but decreased the pH 8.2 activity. During this process the alpha' subunit was converted to a Mr 110,000 polypeptide and the enzyme activity was converted essentially to a 5.9 S species having an apparent Mr of 100,000 as determined by gel filtration. On extended trypsin treatment only one major polypeptide corresponding to the beta subunit remained. The same polypeptide was present in the active fractions following gel filtration of the trypsinized kinase.     

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.     

Studies on photosystem I. I. Relationship of plastocyanin, cytochrome f and P700

DEAE-Sephadex column chromatography now has been used for the final step in purification of d-amino acid oxidase apoenzyme. A specific enzymatic activity of 35–37 units/mg has been obtained for the pure holoenzyme. The purity has been established by disc and SDS gel electrophoreses and by sedimentation equilibrium. The molecular weight per enzyme monomer has been found to be 38,000 ± 1000. Each enzyme monomer binds one FAD and one benzoate with dissociation constants at 23 °C and pH 8.5 of 5.35 × 10^?7m and 1.96 × 10^?6m, respectively. The holoenzyme is more negatively charged than the apoenzyme at alkaline pH. The amino acid composition and some other physicochemical properties of the oxidase are reported.     

Purification and properties of microsomal UDP-glucuronosyltransferase from rat liver.

p-Nitrophenol conjugating activity associated with liver microsomal UDP-glucuronosyltransferase (EC 2.4.1.17) was purified 150- to 200-fold from cell-free homogenates. The purification scheme included solubilization with the nonionic detergent Lubrol WX, anion exchange chromatography at pH 6.0 and 7.5, and affinity chromatography with UDP-hexanolamine Sepharose 4B. The enzyme purified as a phospholipid-protein complex and was shown to consist of a single polypeptide chain of molecular weight 59,000 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Amino acid analysis indicated approximately 531 mol of amino acids/59,000 g of enzyme and a molar ratio of nonpolar to polar residues of 1.08. During fractionation, the enzyme displayed instability with such steps as gel filtration, dialysis, or ultrafiltration of dilute samples; however, upon adsorption to ion exchange resins or storage in concentrated form, the enzyme was reasonably stable. The active lipoprotein complex showed both size and charge heterogeneity as judged by gel filtration and electrofocusing. Three forms of the enzyme resolved by isoelectric focusing had isoelectric points which averaged pH 6.68, 6.56, and 6.31. Polypeptide compositions of these electrophoretically distinct phospholipid protein complexes were indistinguishable on the basis of sodium dodecyl sulfate-polyacryl-amide gel electrophoresis, suggesting that the charge heterogeneity may be the result of differences in the phospholipid content of the lipoprotein complex.     

The affinity purification and characterization of a dehydrogenase from Aspergillus parasiticus involved in aflatoxin B1 biosynthesis.

A two step scheme has been developed for the purification of a dehydrogenase from mycelia of 84 hours old Aspergillus parasiticus (1-11-105 Wh 1), which catalyzes the conversion of norsolorinic acid (NA) to averantin (AVN). The dehydrogenase was purified from cell-free extracts using reactive green 19-agarose and norsolorinic acid-agarose affinity chromatography. The latter affinity matrix was synthesised by attaching norsolorinic acid to omega-aminohexylagarose. The purified protein was shown to be homogenous on non-denaturing polyacrylamide gel electrophoresis. A final purification of 215-fold was achieved. Results of gel filtration chromatography indicated the approximate molecular mass of the native protein to be 140,000 daltons. The isoelectric point of the protein was about 5.5 as determined by chromatofocusing. The reaction catalyzed by the dehydrogenase was optimum at pH 8.5 and between 25 degrees to 35 degrees C. The Km of the enzyme for NA and NADPH was determined to be 3.45 microM and 103 microM respectively.