Coenzyme B12-dependent diol dehydrase: purification, subunit heterogeneity, and reversible association.

A purification procedure for diol dehydrase (dl-1,2-propanediol hydro-lyase, EC 4.2.1.28) of Klebsiella pneumoniae (Aerobacter aerogenes) ATCC 8724 has been developed which gives the highest specific activity for this enzyme obtained so far. The purified enzyme is homogeneous by the criteria of ultracentrifugation (s_20,w = 8.9 S) and disc gel electrophoresis in the presence of substrate. The molecular weight of approximately 230,000 was obtained by gel filtration and ultracentrifugal sedimentation equilibrium. The enzyme is composed of components F and S whose molecular weights were determined to be approximately 26,000 and 200,000, respectively, by gel filtration. The incubation of both components F and S with the substrate leads to complete reassociation of the components. Disc gel electrophoresis in the presence of sodium dodecyl sulfate and terminal amino acid analyses indicate that component S consists of at least four nonidentical subunits. The reversible association and heterogeneity of the subunits were also demonstrated with the crude enzyme by immunoelectrophoresis.     

Purification of ornithine transcarbamylase from rat liver by affinity chromatography with immobilized transition-state analog

Ornithine transcarbamylase (EC 2.1.3.3) was purified to homogeneity from rat liver. The basis of the method is the chromatography of a high-speed supernatant fraction of a homogenized rat liver on an affinity column consisting of the transition-state analog of ornithine transcarbamylase, δ-N-(phosphonacetyl)-l-ornithine, immobilized on epoxy-activated Sepharose 6B through the α-amino group. The enzyme was eluted from the column using a gradient of the substrate, carbamyl phosphate, and further purified by gel filtration. The enzyme elutes with a constant specific activity of 250 to 260 μmol min^?1 mg^?1 at pH 8.5, 37°C, and is free of contaminating proteins on sodium dodecyl sulfate gel electrophoresis. Determination of the molecular weight of the purified enzyme by centrifugation (98,000) and by gel electrophoresis in the presence of sodium dodecyl sulfate (35,300) indicates that the enzyme from rat liver is a trimer. The enzyme exhibits conventional Michaelis-Menten kinetics at pH 7.4 and in this respect differs from the enzyme prepared by other methods.     

Purine nucleoside phosphorylases purified from rat liver and Novikoff hepatoma cells.

Purine nucleoside phosphorylase (PNP) was purified from rat hepatoma cells and normal liver tissue utilizing the techniques of ammonium sulfate fractionation, heat treatment, ion-exchange and molecular exclusion chromatography, and polyacrylamide gel electrophoresis. Homogeneity was established by disc gel electrophoresis in the presence and absence of sodium dodecyl sulfate. Purified rat hepatoma and liver PNPs appeared to be identical with respect to subunit and native molecular weight, substrate specificity, heat stability, kinetics and antigenic identity. A native molecular weight of 84,000 was determined by gel filtration. A subunit molecular weight of 29,000 was determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. A single isoelectric point was observed at pH 5.8, and the pH optimum was 7.5. Inosine, guanosine, xanthosine, and 6-mercaptopurine riboside were substrates for the enzymes. The apparent K_m for both inosine and guanosine was about 1.0 × 10^?4m and for phosphate was 4.2 × 10^?4m. Hepatoma and liver PNP showed complete cross-reactivity using antiserum prepared against the liver enzyme.     

Purification and Some Properties of α-Galactosidase from Tubers of Stachys affinis

An α-galactosidase from tubers of S. affinis was purified about 130 fold by ammonium sulfate fractionation, chromatography on DEAE-cellulose and gel filtration on Sephadex G-75. The purified enzyme showed a single protein band on disc gel electrophoresis. The molecular weight of the enzyme was determined to be approximately 42,000 by gel filtration and 44,000 by SDS disc gel electrophoresis. The optimum reaction pH was 5.2. The enzyme hydrolyzed raffinose more rapidly than planteose. The activation energy of raffinose and planteose by the enzyme was estimated to be 7.89 and 11.4 kcal/mol, respectively. The enzyme activity was inhibited by various galactosides and structural analogs of d-galactose. Besides hydrolytic activity, the enzyme also catalyzed the transfer reaction of d-galactosyl residue from raffinose to methanol.     

Studies on the characterization of the sodium-potassium transport adenosine triphosphatase. Purification and properties of the enzyme from the electric organ of Electrophorus electricus

An unspecific carboxylesterase was purified 180-fold from acid-precipitated human liver microsomes. The final preparation was homogeneous on disc electrophoresis and polyacrylamide gel electrophoresis in the presence of 6.25 M urea at pH 3.2. A single symmetrical peak was also found on gel filtration and on velocity sedimentation in the analytical ultracentrifuge, whereas slight heterogeneity was observed on isoelectric focusing.The amino acid composition of the purified enzyme is presented. From the results the partial specific volume (0.745 ml × g^?1) and the minimal molecular weight (60,000) could be calculated. Fingerprint maps of tryptic peptides from the carboxymethylated enzyme are shown.The molecular weight as determined by gel filtration, disc electrophoresis, and analytical ultracentrifugation is in the range of 181,000–186,000. For the molecular weight of the subunits a value of 61,500 has been obtained by sodium dodecylsulfate polyacrylamide gel electrophoresis. The equivalent weight of the enzyme has been estimated to be 62,500 from stoichiometry of its reaction with diethyl-p-nitrophenyl-phosphate. Partial cross-linking of the subunits with dimethyl suberimidate and subsequent sodium dodecylsulfate polyacrylamide gel electrophoresis yielded three bands with molecular weights of 60,000, 120,000, and 180,000.From these results it is concluded that human liver esterase is a trimeric protein. It is composed of three subunits of equal size, and there is one active site per subunit.     

Metabolism of trans-Aconitic Acid in Maize : I. PURIFICATION OF TWO MOLECULAR FORMS OF CITRATE DEHYDRASE

Trans-aconitate synthesis via citrate dehydrase was determined in crude extracts of maize (Zea mays L.) coleoptiles. Two molecular forms of this enzyme were purified by substrate-specific elution from DEAE-cellulose, ammonium sulfate precipitation, and gel filtration. Each molecular form migrates as a single band in isoelectric focusing. Gel filtration and sodium dodecyl sulfate electrophoresis provided evidence that one enzyme form is composed of four 80,000-dalton subunits while the other is composed of two 60,000-dalton subunits. There was no evidence of proteolytic conversion of the large to the small molecular weight form when the former was incubated with either the 15,000_g supernatant or with proteases. The data indicate that the two molecular forms of citrate dehydrase are isozymes.     

Purification of the restriction endonuclease PalI.

The restriction endonuclease PalI was purified from Providencia alcalifaciens 1650-fold with a yield of 33%. The purified protein moved as a single band upon polyacrylamide gel electrophoresis. When this was carried out in the presence of sodium dodecyl sulfate, a molecular weight of 31,000 was obtained for PalI. Gel filtration through Sephacryl S200 gave molecular weights ranging from 44,000 to 53,000 when 58 to 1870 ng/ml enzyme were used, respectively. Other properties of the enzyme are described.     

Partial Purification and Characterization of a Bacteriocin Produced by Propionibacterium thoenii

A partially purified bacteriocin produced by Propionibacterium thoenii designated propionicin PLG-1 was found to be active against closely related species and exhibited a broad spectrum of activity against other microorganisms. Propionicin PLG-1 was found to be heat labile, sensitive to several proteolytic enzymes, and stable at pH 3 to 9. Propionicin PLG-1 was isolated from solid medium, partially purified by ammonium sulfate precipitation, and purified further by gel filtration. Gel filtration experiments revealed that bacteriocin PLG-1 was present as two different protein aggregates with apparent molecular weights of more than 150,000 and approximately 10,000. Resolution of these protein aggregates by sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed the presence of a protein common to both with an apparent molecular weight of 10,000.     

Purification and partial characterization of glycogen synthase kinase-3 from rabbit liver

Summary Glycogen synthase kinase-3 (GSK-3) was purified from rabbit liver to homogeneity by ultracentrifugation, ion-exchange chromatography on DEAE-cellulose, Cellulose phosphate, CM-Sephadex and Fast Protein Liquid Chromatography (FPLC) on Mono-S column. The enzyme was purified approximately 20,000 fold with an approximate 2% recovery. The purified enzyme showed a single band on SDS-polyacrylamide gel electrophoresis. GSK-3 is a monomeric enzyme with a molecular weight of 50,000–52,000 as derived from SDS-polyacrylamide gel electrophoresis and gel filtration. The purified enzyme was indeed a GSK-3 since it phosphorylated three sites, i.e., 3a, 3b, and 3c on liver glycogen synthase. GSK-3 incorporated up to 2.6 mol Pi/mol glycogen synthase subunit with a concomitant inactivation of glycogen synthase activity.     

Subunit Structure of Glucoamylase of Saccharomyces diastaticus

Extracellular glucoamylase produced by a starch-fermenting yeast, Saccharomyces diastaticus 5106-9A, was purified. The enzyme was found to be heterogeneous in molecular weight, ranging from approximately 80K to 66K as estimated by gel filtration, and consisted of two subunits, H and Y. The molecular weight of subunit H was heterogeneous and was determined to be approximately 68K, 59K, and 53K by acrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. The molecular weight of subunit Y was 14K, estimated by the same gel. the molecular weight of the deglycosylated form of subunit H was 41K, suggesting that the heterogeneity of the enzyme was due to glycosyl moieties of subunit H. Subunits H and Y were separated by gel filtration in the presence of sodium dodecyl sulfate. Subunit Y seemed to be hydrophobic, since it was insoluble in an aqueous buffer without detergent.     

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.     

Purification and properties of the allophanate hydrolase from Chlamydomonas reinhardii

Allophanate hydrolase was purified to homogeneity from extracts of Chlamydomonas reinhardii grown phototrophically using urea as sole source of nitrogen. The following sequence of steps comprised the purification procedure: (1) protamine sulfate precipitation; (2) ammonium sulfate fractionation; (3) poly(ethylene glycol) fractionation; (4) batch-wise DEAE-cellulose adsorption; (5) Sepharose 6-B gel filtration; (6) hydroxyapatite chromatography. This procedure yielded an allophanate hydrolase preparation which was homogenous as judged by polyacrylamide gel electrophoresis. The molecular weight, as determined by gradient polyacrylamide electrophoresis and gel filtration, was 110 000 and 100 000, respectively. The pH optimum of this enzyme was approximately 9.0, while the K_m for allophanate was 0.55 mM. Allophanate hydrolase was sensitive to N-ethylmaleimide but was protected from this inhibition by allophanate. Malonic acid, oxaloacetic acid, and acetoacetic acid were inhibitory to allophanate hydrolysis.     

Characteristics of human milk glutathione peroxidase

Human milk glutathione peroxidase (GPx) was purified 4500-fold using acetone precipitation and purification by repetitive ion-exchange and gel filtration chromatography with an overall yield of 34%. Homogeneity was established by gel electrophoresis. Using gel filtration, the molecular weight (mol wt) of the enzyme was estimated to be 92 kdalton (kD). The monomeric molecular weight was estimated to b 23 kD from polyacrylamide gel electrophoresis, indicating that the native enzyme consists of four identical subunits. The molecular weight of each subunit was supported by amino acid analysis. Selenium (Se) content of the purified enzyme was 0.31%, in a stoichiometry of 3.7 g-atoms/mol. Data from these studies reveal that GPx provided approximately 22% of total milk Se, but only 0.025% of the total protein.     

Studies on the "aerobic" acetyl-coenzyme A synthetase of Saccharomyces cerevisiae: purification, crystallization, and physical properties of the enzyme.

A procedure for the purification of a stable acetyl-coenzyme A synthetase (ACS) from aerobic cells of Saccharomyces cerevisiae is presented. The steps include differential centrifugation, solubilization of the bound enzyme from the crude mitochondrial fraction, ammonium sulfate fractionation, crystallization to constant specific activity from ammonium sulfate solutions followed by Bio-Gel A-1.5 m column chromatography. The resulting enzyme preparation is homogeneous as judged by chromatography on Bio-Gel columns, QAE-Sephadex A-50 anion exchange columns, analytical ultracentrifugal studies, and polyacrylamide gel electrophoresis.Sedimentation velocity runs revealed a single symmetric peak with an s_20,w value of 10.6. The molecular weight of the native enzyme, as determined by gel filtration and analytical ultracentrifugation, is 250,000 ± 500. In polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate, the molecular weight of the single polypeptide chain is 83,000 ± 500. The purified enzyme is inhibited by palmityl-coenzyme A with a Hill interaction coefficient, n, of 2.88. These studies indicate that the ACS of aerobic S. cerevisiae is composed of three subunits of identical or nearly identical size.     

Purification and characterization of an NADP+-linked alcohol oxido-reductase which catalyzes the interconversion of gamma-hydroxybutyrate and succinic semialdehyde.

Abstract— An NADP⁺ -linked enzyme, capable of interconverting γ-hydroxybutyrate and succinic semialdehyde, has been isolated from hamster liver and brain. The enzyme which was isolated from liver has been purified 300-fold and exhibits a single band by polyacrylamide gel electrophoresis. The molecular weight of the enzyme is - 31,000 as estimated from gel filtration and 38,000 as estimated from sodium dodccyl sulfate gel electrophoresis. The enzyme is inhibited by amobarbital, diphenylhy-dantoin, 2-propylvalerate, and diethyldithiocarbamate, but not by pyrazole. The enzymes from brain and liver appear to be very similar with regard to their molecular weights and their kinetic constants for γ-hydroxybutyrate and succinic semialdehyde.     

Purification and properties of dipeptidyl peptidase IV from Streptococcus mitis ATCC 9811

Dipeptidyl peptidase IV (EC 3.4.14.—) from Streptococcus mitis ATCC 9811 was purified to a specific activity of 56.2 units/mg protein by a series of column chromatographic techniques. The purified enzyme was apparently homogeneous as judged by disc gel electrophoresis. Gel filtration on a calibrated column indicated an apparent molecular weight of 120,000 for the native enzyme. Gel electrophoresis of the denatured enzyme in the presence of sodium dodecyl sulfate in a constant acrylamide concentration resulted in the appearance of a single component for which a molecular weight of 53,000 was calculated. The purified enzyme has an optimum pH between 6.0 and 8.7 and an isoelectric point of 4.0. The K_m value toward glycylprolyl-p-nitroanilide is about 6.0 × 10^?5m. Substrate specificity studies indicated that the purified enzyme hydrolyzes specifically N-terminal X-proline from X-Pro-p-nitroanilides. Inhibition of this enzyme was achieved with Hg²⁺, Pb²⁺, Zn²⁺, EDTA, and diisopropyl phosphorofluoridate, but not with N-ethyl-maleimide and sulfhydryl inhibitors.     

Spectroscopic properties of spinach catalase

Spinach catalase (hydrogen-peroxide: hydrogen-peroxide oxidoreductase, EC 1.11.1.6) has been purified to homogeneity. The purified enzyme has a specific activity of 25 000 units per mg protein. The presence of 2-mercaptoethanol and phenylmethylsulfonyl fluoride (PMSF) were required for high yields of the enzyme. The molecular weight of the enzyme was estimated to be 125 000 by gel filtration. Subunit analysis by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate revealed a single peptide with M_r 55 000. The enzyme, which exhibits optical absorbance maxima at 279, 403, 542, 592 and 723 nm and shoulders at 290, 500 and 630 nm, contains 2 mol iron per mol protein. One of the two irons can be attributed to protoheme, while the other iron appears to be present in a novel heme. The oxidized catalase exhibited two sets of high-spin, ferriheme EPR signals.     

Purification and properties of two forms of hepatic phenylalanine:pyruvate transaminase from glucagon treated rats.

Two forms of phenylalanine:pyruvate transaminase (EC 2.6.1. aminotransferases, the exact EC number has not been assigned) termed A and B were obtained from the liver supernatant fraction of glucagon-treated rats by DEAE-Sephadex A-50 column chromatography. Each of the two forms was further purified by hydroxylapatite, Sephadex G-100 chromatography, and preparative gel electrophoresis. Both the A and B forms have been purified to homogeneity as judged by analytical and sodium dodecyl sulfate polyacrylamide gel electrophoresis. Moreover, histidine was found to be a competitive inhibitor of phenylalanine with both purified proteins. These findings conclusively support the view that phenylalanine:pyruvate transaminase and histidine:pyruvate transaminase reactions are catalyzed by the same protein. The overall purification was 710-fold for the A form and 1200-fold for the B form. The apparent molecular weight for both A and B are 74,000 ±6000 as determined by gel filtration. Sodium dodecyl sulfate gel electrophoresis revealed that the A form has two identical subunits of molecular weight 42,000, whereas the B form has two nonidentical subunits of molecular weight 42,000 and 44,000. The amino acid composition for the A and B forms of the enzyme are different. The major differences are in glycine, alanine and leucine. The isoelectric point for A was 7.8 and for B was 7.3. However, the A and B forms of the enzyme are of immunological identity. The substrate specificity determined for both the A and B form was phenylalanine >asparagine >alanine >leucine >histidine. The K_m for phenylalanine was 7.70 mm for the A form, 6.00 mm for the B form. For histidine, the K_m was 13.70 mm for the A form, 12.50 mm for the B form.     

Malonyl-CoA decarboxylase from Mycobacterium tuberculosis and Pseudomonas fluorescens.

Malonyl-CoA decarboxylase was partially purified (nearly 1000-fold) from Mycobacterium tuberculosis H37Ra by ammonium sulfate precipitation, gel filtration with Sepharose 6B, and chromatography on DEAE Sephacel, carboxymethyl-Sephadex, and NADP-agarose. Polyacrylamide gel electrophoresis showed a major band (60–70%), which contained the enzymatic activity, and a minor band which had no decarboxylase activity. The molecular weight of the enzyme was 44,000, and the PI and pH optimum were 6.7 and 5.5, respectively. The enzyme showed a typical Michaelis-Menten substrate saturation, with an apparent K_m and V of 0.2 mm and 3.85 μmol/min/mg, respectively. It catalyzed decarboxylation of methylmalonyl-CoA only at 5% of the rate observed with malonyl-CoA, whereas malonic acid and succinyl-CoA were not decarboxylated. Antibodies prepared against malonyl-CoA decarboxylase from the uropygial glands of goose and rat liver mitochondria did not inhibit the bacterial enzyme. Avidin did not inhibit the enzyme suggesting that biotin was not involved in the reaction. Thiol-directed reagents inhibited the enzyme as did CoA, acetyl-CoA, propionyl-CoA, methylmalonyl-CoA, and succinyl-CoA. Malonyl-CoA decarboxylase was also partially purified from malonate-grown Pseudomonas fluorescens. The molecular weight of this enzyme was 56,000 and the pH optimum and apparent K_m were 5.5 and 1 mm, respectively. Unlike the mycobacterial enzyme, this enzyme was insensitive to p-hydroxymercuribenzoate, acetyl-CoA, and propionyl-CoA, and it was less sensitive to inhibition by succinyl-CoA and CoA than the mycobacterial enzyme. The size and properties of the two bacterial enzymes suggest that these are quite unlike the mammalian and avian enzymes and that they constitute a different class of malonyl-CoA decarboxylases.     

Rat liver alcohol dehydrogenase. Purification and properties

Alcohol dehydrogenase (EC 1.1.1.1) from the rat liver supernatant fraction has been purified 200-fold and partially characterized. The isolation procedure involved ammonium sulphate fractionation, DEAE-Sephadex chromatography and gel filtration. The purified enzyme behaved as a homogeneous preparation as evaluated by cellulose acetate and polyacrylamide-gel disc electrophoresis. Sulphoethyl-Sephadex chromatography and immunoelectrophoresis with rabbit antiserum indicated the presence of a minor component. Rat liver alcohol dehydrogenase appears to contain 4mol of zinc/mol, has an estimated molecular weight of 65000 and consists of two subunits of similar molecular weight. Heavy-metal ions, thiol-blocking reagents, urea at concentrations below 8m, low pH (5.5) and chelating agents deactivate the enzyme but do not dissociate it into subunits. Deactivated enzyme could not be reactivated. The enzyme is strictly specific for NAD(+) and has a broad specificity for alcohols, which are bound at a hydrophobic site. Inhibition occurred with the enzyme equilibrated with Zn(2+) at concentrations above 0.1mm.