Staphylococcal L-asparaginase: purification and properties of enzymic protein

Staphylococcal L-asparaginase has been purified 400-fold with 40% recovery. The procedure involves ammonium sulphate precipitation and a column chromatography on Sephadex G-200 gel filtration). The enzyme is composed of not identical subunits. protein (pI 4.4) with the approximate molecular weight of 125,000 (estimated by Sephadex G-200 gel filtration). The enzyme is composed of not identical subunits. The polyacrylamide-SDS gel electrophoresis indicated two subunits with molecular weight 18,000 and 22,000.     

Subunits of cytochrome a-type terminal oxidases derived from Thiobacillus novellus and Nitrobacter agilis.

Cytochrome a-type terminal oxidases derived from Thiobacillus novellus and Nitrobacter agilis have been purified to a homogeneous state as judged from their electrophoretic behavior and their subunit structures studied by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. The T. novellus enzyme is composed of two kinds of subunits of 32,000 and 23,000 daltons and its minimum molecular weight is 55,000 on the basis of heme content and amino acid composition. The N. agilis enzyme also has two kinds of subunits of 40,000 and 27,000 daltons and its minimum molecular weight is 66,000 on the basis of heme content and amino acid composition. Therefore, the molecule of each enzyme is composed of two kinds of subunits which resemble the subunits of the eukaryotic cytochrome oxidase biosynthesized in the mitochondrion at least with respect to molecular weight.     

Two Escherichia coli fructose-6-phosphate kinases. Preparative purification, oligomeric structure and immunological studies.

Two isoenzymes of fructose-6-phosphate kinase (ATP: D-fructose-6-phosphate 1-phosphotransferase, EC 2.7.1.11) are present in Escherichia coli K12. One isoenzyme is allosterically inhibited by phosphoenolpyruvate and activated by nucleoside diphosphates, and is a tetramer composed of four subunits of molecular weight 35 000. A simple method for the purification of this enzyme is reported. Equilibrium dialysis indicates that there are four ATP sites and four GDP sites per tetramer. The second isoenzyme is present in low quantity in wild type bacteria. This enzyme is devoid of allosteric properties. A complete method of purification is described. Determination of its molecular weight under native and denaturing conditions indicates that this protein is a dimer composed of two subunits of molecular weight 36 000. Antisera have been produced against both isoenzymes. The antiserum against one isoenzyme does not cross-react with the other. Discrepancies between our results and those of other workers are discussed.     

Isolation and characterization of a manganese-containing superoxide dismutase from rat liver.

A manganese-containing superoxide dismutase has been purified from rat liver and characterized. The enzyme has a molecular weight of 89,000 and is composed of four subunits. One atom of manganese is contained per subunit. The metal content, molecular weight, and amino acid analyses show that the rat enzyme is similar to the manganosuperoxide dismutase isolated from human liver.     

Further Characterization of Glycerol Dehydrogenase from Cellulomonas sp. NT3060

Glycerol dehydrogenase prepared from Cellulomonas sp. NT3060 by an improved purification procedure was characterized. The molecular weight was calculated to be about 390,000 by gel filtration and about 336,000 by the sedimentation equilibrium method. The enzyme was composed of eight identical subunits whose molecular weight was 42,000 ~ 43,000. The NH₂-terminal and COOH-terminal amino acids of the subunit were identified as serine and histidine, respectively. The octameric subunit model of the enzyme was confirmed by electron micrographs, which showed as octad aggregate, composed of two tetragons face to face. Studies on the initial velocity and product inhibition were consistent with an ordered Bi-Bi mechanism in which NAD⁺ is bound first to the enzyme and NADH released last.     

Purification and characterization of catabolic dehydroquinase, an enzyme in the inducible quinic acid catabolic pathway of Neurospora crassa.

Catabolic dehydroquinase which functions in the inducible quinic acid catabolic pathway in Neurospora crassa has been purified 8000-fold. The enzyme was purified by two methods. One used heat denaturation of contaminating proteins; the other used antibody affinity chromatography. The preparations obtained by these two methods were identical by all criteria. The purified enzyme is extremely resistant to thermal denaturation as well as denaturation 0y urea and guanidine hydrochloride at 25 degrees. It is irreversibly inactivated, although not efficiently dissociated, by sodium dodecyl sulfate and guanidine hydrochloride at 55 degrees. At pH 3.0, the enzyme is reversibly dissociated into inactive subunits. At high concentrations catabolic dehydroquinase aggregates into an inactive, high molecular weight complex. The native enzyme, which has a very high specific activity, has a molecular weight of approximately 220,000 and is composed of identical subunits of 8,000 to 12,000 molecular weight each. The native enzyme and the subunit are both asymmetric.     

Purification, crystallization, and molecular properties of aspartase from Pseudomonas fluorescens

Aspartase [L-aspartate ammonia-lyase, EC 4.3.1.1] of Pseudomonas fluorescens was highly purified to homogeneity and crystallized. The purified enzyme sedimented as a monodisperse entity upon ultracentrifugation with a s0(20),w value of 8.6S. Upon polyacrylamide gel electrophoresis (PAGE), the enzyme migrated as a single band. The molecular weight of the native enzyme was 173,000 +/- 3,000, as determined by sedimentation equilibrium analysis, and that of the enzyme subunit was determined to be 50,000 +/- 1,500 by sodium dodecyl sulfate (SDS)-PAGE. Cross-linking experiments using dimethyl suberimidate followed by SDS-PAGE indicated that the native enzyme was composed of four subunits with identical molecular weight. The amino acid composition of the enzyme was determined.     

Purification and subunit structure of nicotinamide adenine dinucleotide specific isocitrate dehydrogenase from Neurospora crassa.

Neurospora crassa nicotinamide adenine dinucleotide specific isocitrate dehydrogenase (EC 1.1.1.41) has been purified to homogeneity by the criteria of disc gel electrophoresis and sedimentation equilibrium. Purification of the enzyme is facilitated by the presence of phenylmethanesulfonyl fluoride and by the use of a ribose-linked adenosine 5'-monophosphate affinity column. The enzyme appears to be composed of nonidentical subunits of molecular weights 42 800 and 38 300 as estimated by polyacrylamide gel electrophoresis in 0.1% sodium dodecyl sulfate. From the intensity of each band and the native molecular weight, it is concluded that the enzyme is composed of either six or eight subunits, three or four of each type, respectively. The availability of pure enzyme will allow clarification of the structure of the enzyme by ligand binding studies.     

A cytosolic FAD-containing enzyme catalyzing cytochrome c reduction in Trypanosoma cruzi. I. Purification and some properties

A cytosolic flavoprotein enzyme for the protozoan, Trypanosoma cruzi, has been purified essentially to homogeneity by DEAE-cellulose and 2',5'-ADP-agarose column chromatography. The native enzyme has a molecular weight of 100,000 +/- 6,000, is composed of two identical subunits of molecular weight 52,000 +/- 1,000, and contains FAD in the ratio of 1 mol of FAD per mol of enzyme subunit. The enzyme is NADPH-dependent and is capable of reducing cytochrome c, ferricyanide, 2,6-dichloroindophenol, and menadione, but not adrenalin. It does not hydroxylate either sodium salicylate or sodium p-hydroxybenzoate, but N-methylaniline and N,N-dimethylaminobenzaldehyde-supported oxidation of NADPH has been demonstrated. Plots of initial velocity against NADPH concentration give hyperbolic curves with Km values of 6.289 X 10(-5) M. The enzyme is clearly different from the microsomal NADPH-cytochrome c reductase in its intracellular distribution, molecular weight, dimeric nature, presence of only FAD, and activity against secondary and tertiary aromatic amines.     

Biosynthesis of bacterial glycogen: purification and structural properties of Rhodospirillum tenue adenosine diphosphate glucose synthetase.

Adenosine diphosphate glucose synthetase from the photosynthetic bacterium Rhodospirillum tenue has been purified greater than 95%. The molecular weight of the enzyme is approximately 215,000, with a subunit molecular weight of about 51,000. The enzyme appears to be composed of four similar if not identical subunits. Although the amino acid composition of the enzyme is similar to that of Escherichia coli and Salmonella typhimurium, no apparent homology has been observed between their N-terminal amino acid sequences. Antisera prepared against the R. tenue enzyme can partially inhibit the activities of adenosine diphosphate glucose synthetases from other photosynthetic bacteria.     

Purification and properties of phosphoserine aminotransferase from bovine liver

L-Phosphoserine aminotransferase was purified from bovine liver to apparent homogeneity as judged by nondenaturing and sodium dodecyl sulfate-polyacrylamide gel electrophoresis, analytical ultracentrifugation, and immunochemical analysis. The purification procedure described involves the specific elution of the enzyme from Cibacron blue-agarose by micromolar concentrations of its substrate, phosphohydroxypyruvate. The purified enzyme had a specific activity of approximately 13 mumol of phosphohydroxypyruvate formed min-1 mg-1 of protein at 38 degrees C. Determinations of the native molecular weight and the subunit molecular weight indicated that the phosphoserine aminotransferase from bovine liver was a dimer composed of two subunits with identical molecular weights of 43,000.     

Purification and properties of cis-toluene dihydrodiol dehydrogenase from Pseudomonas putida.

The purification of (+)-cis-1(S),2(R)-dihydroxy-3-methylcyclohexa-3,5-diene dehydrogenase from cells of Pseudomonas putida grown with toluene as the sole source of carbon and energy is reported. The molecular weight of the enzyme is 104,000 at pH 9.7. The enzyme is composed of four apparently identical subunits with molecular weights of 27,000. The enzyme is specific for nicotinamide adenine dinucleotide and oxidizes a number of cis-dihydrodiols. Both enantiomers of a racemic mixture of cis-1,2-dihydroxyl-1,2-dihydronaphthalene dihydrodiol are oxidized by the enzyme. No enzymatic activity is observed with trans-1,2-dihydroxyl-1,2-dihydronaphthalene dihydrodiol.     

Purification and characterization of thylakoid-bound Mn-superoxide dismutase in spinach chloroplasts

Thylakoid-bound superoxide dismutase (SOD; EC 1.15.1.1) was solubilized by Triton X-100 from spinach and purified to a homogeneous state. The molecular weight of thylakoid-bound SOD was 52000; the enzyme was composed of two equal subunits. Its activity was not sensitive to cyanide and hydrogen peroxide, and the isolated SOD contained Mn, but neither Fe nor Cu. Thus, the thylakoid-bound SOD is a Mn-containing enzyme. The subunit molecular weight of thylakoid Mn-SOD is the highest among Mn-SODs isolated so far, a fact which might reflect its binding to the membranes.     

Purification and properties of an NADP-specific 6-phosphogluconate dehydrogenase from Streptococcus faecalis.

A procedure is described for the purification of 6-phosphogluconate dehydrogenase (6-phospho-D-gluconate:NADP oxidoreductase (decarboxylating) EC 1.1.1.44) from cell extracts of Streptococcus gaecalis. A 180-fold purification was achieved with an over-all yield of about 12% and an average specific activity of 14. The enzyme was homogeneous as determined by polyacrylamide gel electrophoresis, immunoelectrophoresis, and sedimentation equilibrium, studies. Its weight average molecular weight, as measured by sedimentation equilibrium, was 108,000 +/- 3,600. Other methods employed for molecular weight determinations gave values that ranged between 106,000 and 115,000. An analysis of the enzyme by sodium dodecyl sulfate polyacrylamide gel electrophoresis showed it to be a dimer composed of subunits having equal molecular weight. The amino acid composition of the streptococcal enzyme is reported. The apparent Km values for NADP and 6-phosphogluconate were calculated from kinetic data and found to be 0.015 mM and 0.024 mM, respectively. Kinetic studies also indicated that the binding of one substrate did not affect the apparent affinity of the enzyme for the other substrate.     

Subunit structure of biodegradative threonine deaminase.

The molecule weight of the biodegradative threonine deaminase from Escherichia coli was determined to be approximately 147,000 by sedimentation equilibrium ultracentrifugation. Similar experiments using 5 M guanidinium chloride gave a value of 39,000 for the molecular weight of the enzyme subunit. On sodium dodecyl sulfate-gel electrophoresis the enzyme also dissociated into a single subunit with an estimated molecular weight of 38,000. The NH2 terminus of the enzyme was determined to be methionine by the dinitrophenylation procedure. Quantitative analysis revealed that 3.6 mol of methionine were detected per 147,000 g of enzyme. The selective tritium labeling method established alanine as the COOH-terminal residue. The sequence of residues at the NH2 terminus, determined using an automated sequence analyzer, was: (formula: see text). The fact that a single amino acid was released at each degradation step in the above experiment strongly suggests that the subunits in the enzyme contain the same amino acid sequence. Therefore, the native enzyme with a molecular weight of 147,000 appears to be composed of four identical polypeptide subunits.     

Cation-sensitive neutral endopeptidase: isolation and specificity of the bovine pituitary enzyme

A highly purified preparation of a cation-sensitive neutral endopeptidase was obtained from bovine pituitaries. The enzyme constitutes almost 0.1% of the protein in bovine pituitary homogenates. Polyacrylamide gel electrophoresis of the enzyme showed a single protein band, and in gel filtration experiments on calibrated Sepharose 6B columns the enzyme eluted slightly ahead of thyroglobulin, suggesting an apparent molecular weight of about 700,000. Polyacrylamide gel electrophoresis in SDS-containing buffers indicated the presence of three major components with molecular weights ranging from about 24,000 to 28,000. The enzyme hydrolyzes bonds between hydrophobic and small neutral amino acids in both model synthetic substrates and biologically active peptides such as substance P, LH-RH, and bradykinin. Peptide bonds in which the carbonyl group is contributed by a glutamyl or arginyl residue are also hydrolyzed, especially if they are preceded in the sequence by hydrophobic amino acids. Leupeptin exclusively inhibited enzymatic activity toward the arginine-containing substrates. This observation, together with the high molecular weight and broad specificity of the enzyme, raised the possibility that the isolated enzyme represents a proteolytic complex composed of units with distinctly different activities. Preliminary attempts to dissociate the enzyme into catalytic units of lower molecular weight were not successful and led to loss of activity.     

Purification and characterization of NADP+-specific glutamate dehydrogenase fromEscherichia coli

The NADP⁺-specific glutamate dehydrogenase fromEscherichia coli has been purified to electrophoretic homogeneity. The enzyme was purified 40-fold and has a specific activity of 23. Glutamate dehydrogenase fromE. coli is a hexameric enzyme with a native molecular weight of 275 KDa composed of monomers each with a molecular weight of 44.5 KDa. In nondenaturing isoelectric focusing gels, the purified enzyme is resolved into six catalytically active species, each with a molecular weight of 275 KDa and with isoelectric points ranging between pH 5.3 and 5.7. The K_m values for substrates and coenzymes have been determined, and the effect of several divalent ions on catalytic activity has been investigated.     

Novel phenylalanine dehydrogenases from Sporosarcina ureae and Bacillus sphaericus. Purification and characterization

NAD+-dependent phenylalanine dehydrogenases were purified 1,500- and 1,600-fold, and crystallized from Sporosarcina ureae SCRC-R04 and Bacillus sphaericus SCRC-R79a, respectively. The purified enzymes were homogeneous as judged by disc gel electrophoresis. The enzyme from S. ureae has a molecular weight of 305,000, while that of B. sphaericus has a molecular weight of 340,000. Each is probably composed of eight subunits identical in molecular weight. The S. ureae enzyme showed a high substrate specificity in the oxidative deamination reaction acting on L-phenylalanine, while that of B. sphaericus acted on L-phenylalanine and L-tyrosine. The enzymes had lower substrate specificities in the reductive amination reaction acting on alpha-keto acids. The Sporosarcina enzyme acted on phenylpyruvate, alpha-ketocaproate, alpha-keto-gamma-methylthiobutyrate and rho-hydroxyphenylpyruvate. The Bacillus enzyme acted on rho-hydroxyphenylpyruvate, phenylpyruvate, and alpha-keto-gamma-methylthiobutyrate. The enzyme from B. sphaericus catalyzes The enzyme from B. sphaericus catalyzes the transfer of pro-S (B) hydrogen from NADH.     

扬子鳄红细胞超氧物歧化酶的纯化及其性质研究

扬子鳄红细胞超氧物歧化酶的纯化及其性质研究周衍茂（安徽教育学院生物系,合肥２３００６１）尹路明（中国科学技术大学生物系,合肥２３００２６）关键词超氧物歧化酶;纯化;扬子鳄;红细胞超氧物歧化酶（的田广泛存在于各类生物组织中,是生物体内超氧自由基有效的清...     

PaeExo IX: a unique deoxyribonuclease from Pseudomonas aeruginosa active in the presence of EDTA.

A new deoxyribonuclease, PaeExo IX, has been purified to electrophoretic homogeneity from extracts of Pseudomonas aeruginosa strain PAO. This enzyme, which is active in the presence of EDTA, is equally efficient in hydrolyzing native and heart-denatured DNA to acid-s-luble products. The enzyme is partially or totally inhibited by the presence of several divalent cations. The active protein has a molecular weight of 1.6 +/- 0.1 x 10(5) and is composed of two nonidentical polypeptides with molecular weights of 78,000 and 69,000.