Purification and properties of intracellular fructosyl transferase fromAureobasidium pullulans

Intracellular frustosyl transferase was purified fromAureobasidium pullulans C-23 by ethanol fractionation, CM-Sephadex chromatography and preparative disc gel electrophoresis. It was shown to be homogeneous on disc polyacrylamide gel electrophoresis, with a molecular size of 190kDa. The pI value of the enzyme was about 3.7. The enzyme has aK _m value of 0.43 mM for sucrose and was optimally active at pH 5.0 and 60°C. The enzyme was stable from pH 2.5 to 12. It was almost completely inhibited by 5mM Hg²⁺ but was not significantly affected by other cations. The transferase was inactivated by treatment with the tryptophan-specific reagentN-bromosuccinimide and the tyrosine-specific reagent, I₂, suggesting that tryptophan and tyrosine residues are probably located at or near the active site of the enzyme.     

Purification and properties of glutathione transferase from Issatchenkia orientalis.

Glutathione transferase (GST) (EC 2.5.1.18) was purified from a cell extract of Issatchenkia orientalis, and two GST isoenzymes were isolated. They had molecular weights of 37,500 and 40,000 and were designated GST Y-1 and GST Y-2, respectively. GST Y-1 and GST Y-2 gave single bands with molecular weights of 22,000 and 23,500, respectively, on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. GST Y-1 and GST Y-2 were immunologically distinguished from each other. GST Y-1 showed specific activity 10.4-times and 6.0-times higher when 1-chloro-2,4-dinitrobenzene and o-dinitrobenzene were used as substrates, respectively, than GST Y-2. GST activity was not detected for either isoenzyme when other substrates such as bromosulfophthalein and trans-4-phenyl-3-buten-2-one were used. GST Y-1 and GST Y-2 had Km values of 0.51 and 0.75 mM for glutathione, respectively, and of 0.16 and 4.01 mM for 1-chloro-2,4-dinitrobenzene. GST Y-1 was significantly inhibited by Cibacron blue 3G-A, and GST Y-2 was significantly inhibited by bromosulfophthalein.     

Purification and properties of ornithine carbamoyl transferase from liver of Squalus acanthias

Citrulline synthesis from ammonia by hepatic mitochondria in elasmobranchs involves intermediate formation of glutamine as the result of the presence of high levels of glutamine synthetase and a unique glutamine- and N-acetyl-glutamate-dependent carbamoyl phosphate synthetase, both of which have properties unique to the function of glutamine-dependent synthesis of urea, which is retained in the tissues of elasmobranchs at high concentrations for the purpose of osmoregulation [P.M. Anderson and C.A. Casey (1984) J. Biol. Chem. 259, 456-462; R.A. Shankar and P.M. Anderson (1985) Arch. Biochem. Biophys. 239, 248-259]. The objective of this study was to determine if ornithine carbamoyl transferase, which catalyzes the last step of mitochondrial citrulline synthesis and which has not been previously isolated from any species of fish, also has properties uniquely related to this function. Ornithine carbamoyl transferase was highly purified from isolated liver mitochondria of Squalus acanthias, a representative elasmobranch. The purified enzyme is a trimer with a subunit molecular weight of 38,000 and a native molecular weight of about 114,000. The effect of pH is significantly influenced by ornithine concentration; optimal activity is at pH 7.8 when ornithine is saturating. The apparent Km values for ornithine and carbamoyl phosphate at pH 7.8 are 0.71 and 0.05 mM, respectively. Ornithine displays considerable substrate inhibition above pH 7.8. The activity is not significantly affected by physiological concentrations of the osmolyte urea or trimethylamine-N-oxide or by a number of other metabolites. The results of kinetic studies are consistent with a steady-state ordered addition of substrates (carbamoyl phosphate binding first) and rapid equilibrium random release of products. Except for an unusually low specific activity, the properties of the purified elasmobranch enzyme are similar to the properties of ornithine carbamoyl transferase from mammalian ureotelic and other species and do not appear to be unique to its role in glutamine-dependent synthesis of urea for the purpose of osmoregulation.     

Purification and properties of rat brain hexokinase

Rat brain hexokinase has been purified to homogeneity as judged by disc-gel electrophoresis, isoelectric focusing, and analytical ultracentrifugation. More than 50% of the initial activity could be obtained in homogeneous form (sp act, 60 units/mg protein) by a simple procedure consisting essentially of two steps: relatively specific solubilization of the enzyme from the mitochondrial membrane by glucose-6-P, followed by DEAE-cellulose column chromatography. The molecular weight is approximately 98,000; this same molecular weight was observed when the denatured enzyme was examined by the SDS-polyacrylamide electrophoretic technique, strongly suggesting that the enzyme consists of a single polypeptide chain. In accord with this view, a single N-terminal amino acid, glycine, has been recovered in 80% yield based on a molecular weight of 98,000. The amino acid composition of the rat brain hexokinase has been determined and found to be very similar to that previously reported for the bovine brain enzyme (Schwartz, G. P., and Basford, R. E. (1967) Biochemistry6, 1070, suggesting extensive sequence homology. A notable feature of the brain hexokinases is a relatively low aromatic amino acid content, as judged by the amino acid composition and the relatively low molar extinction coefficient.     

Purification and properties of rat stomach kallikrein

Kallikrein (EC 3.4.21.8) was purified from rat stomach by column chromatography on p-aminobenzamidine-Sepharose, DEAE-Sephadex A-50 and Sephadex G-150 and by isoelectric focusing, measuring its activities to hydrolyse L-prolyl-L-phenylalanyl-L-arginine-4-methyl-coumaryl-7-amide and to release kinin from heat-treated rat plasma. the purified stomach kallikrein showed a single band on polyacrylamide gel electrophoresis at pH 7.0. Its molecular weight was calculated to be 29 000 by gel-filtration on a column of Sephadex G-50. The kallikrein was stable between pH 6-11 and hydrolyzed L-prolyl-L-phenylalanyl-L-arginine-4-methyl-coumaryl-7-amide optimally at pH 11.0. The L-prolyl-L-phenylalanyl-L-arginine-4-methyl-coumaryl-7-amide hydrolyzing activity of rat stomach kallikrein was inhibited by diisopropyl fluorophosphate and Trasylol, but not by trypsin inhibitors from soybean, lima bean and ovomucoid. These properties of rat stomach kallikrein are different from those of partially purified rat plasma kallikrein, but similar to those of glandular kallikreins from other species. From these results, it was concluded that kallikrein is present in rat stomach and that it can be classified as a glandular kallikrein.     

Purification and properties of 4-hydroxybutyrate coenzyme A transferase from Clostridium aminobutyricum.

A new coenzyme A (CoA)-transferase from the anaerobe Clostridium aminobutyricum catalyzing the formation of 4-hydroxybutyryl-CoA from 4-hydroxybutyrate and acetyl-CoA is described. The enzyme was purified to homogeneity by standard techniques, including fast protein liquid chromatography under aerobic conditions. Its molecular mass was determined to be 110 kDa, and that of the only subunit was determined to be 54 kDa, indicating a homodimeric structure. Besides acetate and acetyl-CoA, the following substrates were detected (in order of decreasing kcat/Km): 4-hydroxybutyryl-CoA, butyryl-CoA and propionyl-CoA, vinyl-acetyl-CoA (3-butenoyl-CoA), and 5-hydroxyvaleryl-CoA. In an indirect assay the corresponding acids were also found to be substrates; however, DL-lactate, DL-2-hydroxybutyrate, DL-3-hydroxybutyrate, crotonate, and various dicarboxylates were not.     

Purification and properties of an acyl CoA transferase from Ascaris suum muscle mitochondria

An acyl CoA transferase has been purified to electrophoretic homogeneity from the soluble compartment of Ascaris suum muscle mitochondria. From SDS-PAGE, isoelectric focusing and molecular exclusion chromatography, homogeneity was confirmed and the enzyme appears to be composed of two similar or identical subunits of apparent mol. wts of 50,000 resulting in an apparent mol. wt of 100,000 for the holoenzyme. The apparent isoelectric point was 5.6 +/- 0.1 by both chromatofocusing columns and slab gel isoelectric focusing. The transferase was relatively specific for the short, straight-chain acyl CoA donors as well as the CoA acceptors, being active on acetyl CoA, propionyl CoA, butyryl CoA, valeryl CoA and hexanoyl CoA as donors to acetate and propionate. Neither succinyl CoA nor succinate were appreciably active as CoA donor or acceptor, respectively. This enzyme cannot serve physiologically to activate succinate for decarboxylation to propionate, but may serve to ensure a supply of propionyl CoA which appears to be required in catalytic amounts for the decarboxylation of succinate.     

Purification and properties of 4-hydroxybutyrate coenzyme A transferase from Clostridium aminobutyricum.

A new coenzyme A (CoA)-transferase from the anaerobe Clostridium aminobutyricum catalyzing the formation of 4-hydroxybutyryl-CoA from 4-hydroxybutyrate and acetyl-CoA is described. The enzyme was purified to homogeneity by standard techniques, including fast protein liquid chromatography under aerobic conditions. Its molecular mass was determined to be 110 kDa, and that of the only subunit was determined to be 54 kDa, indicating a homodimeric structure. Besides acetate and acetyl-CoA, the following substrates were detected (in order of decreasing kcat/Km): 4-hydroxybutyryl-CoA, butyryl-CoA and propionyl-CoA, vinyl-acetyl-CoA (3-butenoyl-CoA), and 5-hydroxyvaleryl-CoA. In an indirect assay the corresponding acids were also found to be substrates; however, DL-lactate, DL-2-hydroxybutyrate, DL-3-hydroxybutyrate, crotonate, and various dicarboxylates were not.     

Adrenocorticotropin-dependent regulation of glutathione transferase subunit 4 in cultured rat adrenal cells.

After hypophysectomy, the level of glutathione transferase subunit 4 increases in the adrenal, as well as in the liver, as do those of several other forms of glutathione transferase. This increase in subunit 4 can subsequently be down-regulated by administration of adrenocorticotropin. The present investigation demonstrates that also in primary cultures of female rat adrenal cells an increase in the level of glutathione transferase subunit 4 (as shown by immunoblotting) occurs in the absence of adrenocorticotropin. When adrenocorticotropin or dibutyryladenosine 3',5'-phosphate was administered to these cells, a down-regulation of this enzyme level was observed, in agreement with the in vivo situation. This down-regulation was not affected by aminoglutethimide, an inhibitor of the cholesterol-side-chain-cleavage enzyme (cytochrome P-450scc) which is the rate-limiting step in the biosynthesis of steroids. Hence adrenal steroid production is not involved in the down-regulation of glutathione transferase subunit 4 by adrenocorticotropin.     

Isolation, properties and tissue distribution of rat glutathione transferase E

Uptake of Li+ induced by the addition of proline to a cell suspension of Escherichia coli was detected using an Li+-selective electrode. This Li+ uptake was inhibited by L-azetidine 2-carboxylic acid, a competitive inhibitor of the proline transport system. Thus, direct evidence for Li+-proline cotransport via the proline transport system was obtained. Kinetic parameters of the Li+ uptake were determined.     

Purification and properties of arginase of rat kidney

l-Arginase from rat kidney was partially purified and some properties were compared with those of l-arginase of rat liver. The kidney enzyme was firmly bound to the mitochondrial fraction and after solubilization required arginine or an unknown factor in tissue extracts for stabilization after dialysis. The two enzymes differed also in stability with respect to acetone treatment, heating or freezing. In further contrast with liver arginase, arginase from kidney was not adsorbed to CM-cellulose at pH7.5 and its activity was not increased by incubation with Mn(2+). Other differences were seen in relative specificities for substrates, ratio of hydrolysis rates with high and low concentrations of arginine and effects of certain inhibitors. Antisera prepared to pure liver arginase did not cross-react with partially purified kidney arginase.