Glutamate dehydrogenase from Escherichia coli: induction, purification and properties of the enzyme.

When Escherichia coli was grown in a minimum medium with glucose as sole carbon source and a proper level of ammonia, NADP+ specific glutamate dehydrogenase (L-glutamate: NADP+ oxidoreductase (deaminating), ED 1.4.1.4) was induced. The enzyme was solubilized by French press treatment and purified to homogeneity by (NH4)2SO4 fractionation, heat treatment followed by DEAE-cellulose, hydroxylapatite and Bio-Gel chromatography with an overall yield of 30%. The enzyme proved to be heat stable and relatively resistant to protein denaturants. The optimum of enzymic activity for the reductive amination is at pH 8 and at pH 9 for the oxidative deamination. The activity is affected by adenine nucleotides. The molecular weight (about 250 000 for the native form and 46 000 for the inactive subunit) and amino acid composition, suggest strict similarities with the NADP+ enzyme from fungal origin.     

Simultaneous purification of L-malate dehydrogenase and L-lactate dehydrogenase from bovine heart by biomimetic-dye affinity chromatography

Two commercially important enzymes, L-lactate dehydrogenase (LDH) and L-malate dehydrogenase (MDH) were purified simultaneously from bovine heart, on an agarose affinity adsorbent. This adsorbent bears a dye-ligand composed of an anthraquinone chlorotriazine chromophore linked to a biomimetic terminal 4-aminophenyloxanylic acid moiety. The purification protocol exploited the biomimetic affinity adsorbent, in combination with a cross-linked agarose DEAE anion-exchanger. The procedure comprised a preliminary anion-exchange first step, for the separation of the three enzyme activities, mMDH, cMDH and LDH. In the second step, that of affinity chromatography, the unbound mMDH obtained from the first step, was purified by specific elution with NAD⁺/sulphite (22.5-fold purification, 55% step-yield). The procedure afforded mMDH preparation of specific activity approx. 1,300?u/mg (25?°C) at 45% overall yield, free of cytoplasmic MDH, glutamic-oxaloacetic transaminase (GOT) and fumarase. The LDH activity, which, bound to the anion-exchanger during the first step, was recovered from the adsorbent in 200?mM KCl, and finally purified by biomimetic-dye affinity chromatography (NAD⁺/sulphite elution) and a second ion-exchange chromatography step (elution with 200?mM KCl). The LDH preparation exhibited specific activity approx. 500?u/mg at 25?°C (content of impurities: pyruvate kinase and GOT were not detected; MDH, 0.01%).     

Metal-dependent proteinase of the lens. Assay, purification and properties of the bovine enzyme.

1. Two new assay methods were developed for the lens proteinase. In both, the substrate was alpha2-crystallin (a major lens protein); in the first method, the products were detected by reaction with trinitrobenzenesulphonate in the presence of SO32-, whereas in the second method, 3H-labelled substrate was used, and the products were detected as radioactivity soluble in trichloroacetic acid. 2. The neutral proteinase from bovine lens was partially purified by extraction of the lens at pH5.0 and column chromatography on hydroxyapatite and Sepharose 6B gel. 3. The purified enzyme had no detectable activity against haemoglobin, azo-casein or gamma-crystallin under optimum conditions for alpha2-crystallin. 4. The enzyme showed greatest activity and stability at pH7.5. It was reversibly inhibited by EDTA and 1,10-phenanthroline, and activated by Ca2+ and Mg2+. 5. Molecular weights obtained for the enzyme by chromatography on Sepharose 6B were approx. 500,000 in buffer of I = 0.02, and 250,000 at I = 1.02. 6. The properties of the purified lens proteinase are such as to suggest that this enzyme could account for the entire endopeptidase activity of the lens.     

Carbon monoxide dehydrogenase from Methanosarcina barkeri. Disaggregation, purification, and physicochemical properties of the enzyme

Carbon monoxide dehydrogenase from acetate-grown cells of Methanosarcina barkeri exists in a high molecular weight form (approximately 3 X 10(6)) under conditions of high ionic strength but is converted to a much smaller form by dialysis. The enzyme was purified by a procedure which exploits isolation of the aggregated form by gel filtration and subsequent dissociation. Following this, the enzyme was purified to within 92% of homogeneity by chromatography on phenyl-Sepharose and finally on hydroxylapatite. Due to the extreme oxygen lability of the enzyme, the entire procedure was carried out within the anaerobic laboratory at the National Institutes of Health. The enzyme has an alpha 2 beta 2 oligomeric structure composed of subunits with molecular weights of 19,700 and 84,500. The amino acid compositions of the individual subunits were determined. Analysis of the metal content by plasma emission spectroscopy indicated 1.3 +/- 0.3 (n = 4) nickel and 15.6 +/- 5.6 (n = 5) iron per mol of alpha 2 beta 2. The enzyme did not contain significant amounts of cobalt or molybdenum. Ferredoxin, FAD, FMN, 2,3,5-triphenyltetrazolium chloride, methyl viologen, and phenazine methosulfate served as electron acceptors; however, the enzyme failed to reduce NAD+, NADP+, or the 8-hydroxy-5-deazaflavin factor F420. The optimum pH was between 7 and 9. The apparent Km for methyl viologen was 7.1 mM, whereas the value for 2,3,5-triphenyltetrazolium chloride was below 0.5 mM. Strong inhibition was observed by oxygen and cyanide. Inactivation by glyoxaldehyde required enzymatic turnover which suggested that a reactive group was formed, or exposed, on an enzyme intermediate in catalysis. A high degree of thermostability was noted. Carbon monoxide, however, rendered the enzyme more susceptible to temperature inactivation.     

Chemical modification of lysine and arginine residues of bovine heart 2-oxoglutarate dehydrogenase: effect on the enzyme activity and regulation.

Chemical modification of arginine and lysine residues of bovine heart 2-oxoglutarate dehydrogenase with phenylglyoxal and pyridoxal 5'-phosphate inactivated the enzyme, indicating the importance of these residues for the catalysis. Inactivation caused by pyridoxal 5'-phosphate was prevented in the presence of thiamine pyrophosphate and Mg2+ allowing the assumption that lysine residues participate in binding of the cofactor.     

Mitochondrial malate dehydrogenase. Crystallographic properties of the pig heart enzyme

Crystals of the mitochondrial form of malate dehydrogenase from pig heart have been prepared using polyethylene glycol and ammonium sulfate. The monoclinic crystalline form obtained from polyethylene glycol is suitable for X-ray analysis and contains two molecules of the dimeric malate dehydrogenase, molecular weight 74,000, in the asymmetric unit. A new crystalline form of a related enzyme, β-hydroxyacyl coenzyme A dehydrogenase has been prepared and characterized. Heavy-atom compounds causing isomorphous changes in the X-ray intensities of the mitochondrial malate dehydrogenase have been identified.     

Stabilization and purification of the secondary metabolism specific enzyme, m-hydroxybenzylalcohol dehydrogenase

m-Hydroxybenzylalcohol dehydrogenase (EC 1.1.1.97), a secondary metabolism associated protein from stationary phase cultures of Penicillium urticae, was stabilized in crude extracts prior to purification. Stabilization studies resulted in the formulation of an optimal cell breakage and purification buffer. This buffer increased the enzyme's in vitro half-life at 30 degrees C from 14 to over 800 min which greatly aided purification and enhanced yields. Purification was achieved by salt fractionation, size-exclusion chromatography, affinity chromatography, and ion-exchange chromatography. The 1200-fold purified protein gave only one major band by sodium dodecyl sulphate - polyacrylamide gel electrophoresis.     

Purification and properties of pyruvate dehydrogenase phosphatase from bovine heart and kidney

Pyruvate dehydrogenase phosphatase was purified to apparent homogeneity from bovine heart and kidney mitochondria. The phosphatase has a sedimentation coefficient (S20,w) of about 7.4 S and a molecular weight (Mr) of about 150 000 as determined by sedimentation equilibrium and by gel-permeation chromatography. The phosphatase consists of two subunits with molecular weights of about 97 000 and 50 000 as estimated by sodium dodecyl sulfate--polyacrylamide gel electrophoresis. Phosphatase activity resides in the Mr 50 000 subunit, which is sensitive to proteolysis. The phosphatase contains approximately 1 mol of flavin adenine dinucleotide (FAD) per mol of protein of Mr 150 000. FAD is apparently associated with the Mr 97 000 subunit. The function of this subunit remains to be established. The phosphatase binds 1 mol of Ca2+ per mol of enzyme of Mr 150 000 at pH 7.0, with a dissociation constant (Kd) of about 35 microM as determined by flow dialysis. Use of ethylene glycol bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetate (EGTA) at pH 7.6 in conjunction with flow dialysis gave a Kd value for Ca2+ of about 8 microM. In the presence of both the phosphatase and the dihydrolipoyl transacetylase (E2) core of the pyruvate dehydrogenase complex, two equivalent and apparently non-interacting CA2+-binding sites were detected per unit of Mr 150 000, with a Kd value of about 24 microM in the absence and about 5 microM in the presence of EGTA. In the presence of 0.2 M KCl, which inhibits phosphatase activity about 95%, the phosphatase exhibited only one Ca2+-binding site, even in the presence of E2. The phosphatase apparently possesses an "intrinsic" Ca2+-binding site, and a second Ca2+-binding site is produced in the presence of E2. The second site is apparently altered by increasing the ionic strength. It is proposed that the second site may be at the interface between the phosphatase and E2, with Ca2+ acting as a bridging ligand for specific attachment of the phosphatase to E2.     

Rabbit liver alcohol dehydrogenase: purification and properties

Alcohol dehydrogenase [EC 1.1.1.1] was purified to homogeneity from rabbit liver by water extraction, DEAE-cellulose treatment, affinity chromatography on 5'-AMP-Sepharose and gel filtration on Sephadex G-150 using dithiothreitol as a stabilizer. The purified enzyme has an estimated molecular weight of 72,000 and consists of two subunits with a molecular weight of about 36,000 each. The enzyme contains 4 g-atoms of zinc and 18 sulfhydryl groups per mol of protein and exhibits maximal activity at pH 10.8, with a second maximum at pH 7.5. The apparent Km values for ethanol and NAD+ are 0.45 mM and 53.19 microM, respectively, at pH 10.8 and 3.33 mM and 6.94 microM, respectively, at pH 7.5. The enzyme oxidizes ethanol most readily among the aliphatic alcohols studied and has very low substrate specificity for methanol. Among steroid alcohols, 5 beta-androstan-3 beta-ol-17-one serves as a substrate for the enzyme. Pyrazole and 4-methylpyrazole (which are well known alcohol dehydrogenase inhibitors), sulfhydryl reagents, heavy metal ions and metal-chelating agents inactivate the enzyme.     

Letter: L-3-hydroxyacyl coenzyme A dehydrogenase: crystallographic properties of the pig heart enzyme

Crystals of l-3-hydroxyacyl coenzyme A dehydrogenase from pig heart that are suitable for X-ray analysis have been prepared and characterized. The enzyme is a mitochondrial protein important in the beta oxidation of fatty acids. It is of special interest because it requires coenzyme A-related molecules for its catalytic activity. For one of the crystalline forms of the enzyme, the X-ray data tend to confirm chemical observations that the molecule is a dimer of identical subunits.