Folylpolyglutamate synthetase from beef liver: purification and some properties.

The polypeptide chain of folylpolyglutamate synthetase from beef liver has been isolated and partially characterized. This polypeptide has an apparent molecular weight of 73,000. Its amino-terminal residue is blocked. Amino acid analysis agrees with the hydrophobic properties and the pI (6.0) of this cytosolic enzyme. Polyclonal antibodies to the denatured enzyme have been prepared.     

Human spleen dihydroorotate dehydrogenase: properties and partial purification

Human spleen dihydroorotate dehydrogenase is associated with the mitochondrial membrane and is linked to the respiratory chain via ubiquinone. The enzyme activity was unaffected by pyridine nucleotides. The product of the reaction, orotate, was a potent inhibitor. However, a range of other naturally occurring pyrimidines or purines had no significant effect on the activity. No evidence for the involvement of a complexed metal ion or for an active sulfhydryl group was obtained. Purification of the enzyme was achieved by preparation of an acetone powder and extraction with Triton X-100, followed by preparative polyacrylamide gel electrophoresis. Activity was observed by the addition of the artificial electron acceptors, ubiquinone 50 or PMS. Purification resulted in alteration of the pH optimum and of other kinetic characteristics. Two molecular-weight species, of molecular weight 88,000 and 98,000, were consistently observed. The properties of the human spleen enzyme were similar in principle to those for the rat liver enzyme. Differences in the mode of linkage to the respiratory chain for the mitochondrially bound enzyme, and in the characteristics of the purified enzyme, were observed.     

Purification and some properties of buffalo spleen cathepsin B

Purification of cathepsin B from buffalo-spleen, a hitherto unstudied system has been achieved by a simple procedure developed by incorporating suitable modifications in the existing methods for isolation of the enzyme from other sources. The purified enzyme has a molecular weight of 25 KDa and its Stokes radius was found to be 2·24 nm. Effects of several reducing agents, urea and thiol-protease inhibitors such as leupeptin and antipain, have been studied and the data unequivocally support the contention that the buffalo-enzyme is similar to cathepsin B from other tissues with respect to these properties.     

Chicken ornithine transcarbamylase: purification and some properties

Ornithine transcarbamylase [EC 2.1.3.3] has been purified from chick kidney to homogeneity. The molecular weight is 110,000 as determined by gel filtration. Sodium dodecylsulfate polyacrylamide gel electrophoresis of the enzyme showed that the enzyme exists as a trimer of identical subunits of 36,000 daltons like other mammalian species ornithine transcarbamylases. In 0.1 M triethanolamine/HCl, the apparent optimum pH of the purified enzyme was 7.5 in the presence of 5 mM ornithine. The curve shifted toward a more alkaline region with a decrease in ornithine concentration. The specific activity of the purified enzyme as 77 units at pH 7.5. The Km for carbamyl phosphate was 0.11 mM and the Km for ornithine was 1.21 mM. With an increase in pH, a decrease in Km values for ornithine and an increase in the extent of inhibition by ornithine were observed. On using antibody against bovine liver ornithine transcarbamylase, the precipitin lines for the chick and bovine enzymes showed a spur pattern. Even when excess amounts of the antibody were added, the chick enzyme did not lose the activity while the bovine enzyme activity was inhibited completely.     

The purification and some properties of pig liver hyaluronidase

Hyaluronidase (hyaluronate 4-glycanohydrolase, EC 3.2.1.35) has been isolated from pig liver and purified 1720-fold with an overall yield of 9.5%. The enzyme was purified using an acid-extraction technique followed by successive chromatography on DEAE-cellulose, two boronate affinity columns and Sephadex G-75. This final preparation, which was essentially homogeneous as determined by gel electrophoresis, was a single subunit enzyme of apparent molecular weight 70 000 with an isoelectric point of 5.0. No contaminant enzymes capable of degrading glycosaminoglycans could be detected in the final preparation. The substrate specificity of the enzyme was the same as for bovine testicular hyaluronidase; however, both the Km and V values were significantly lower for the pig liver enzyme with all of the substrates tested (hyaluronate, chondroitin 4-sulphate, chondroitin 6-sulphate). A full kinetic analysis of the enzyme using hyaluronate as a substrate showed that the activity of pig liver hyaluronidase was uncompetitively activated by either protons or NaCl.     

Biological properties and partial purification of a growth factor from porcine spleen

3T3-Fibroblast growth is enhanced in a dose-dependent way by a factor isolated from porcine spleen after limited proteolysis. The factor is not dialyzable and is stable for 5 min at 100 degrees C and at pH 2 at room temperature. Trypsin or collagenase treatment does not affect its biological activity. The stimulation of cell growth is independent of serum concentration in the culture medium but is accelerated by insulin supplement. The biochemical properties of this factor indicate a novel growth promotor different from other growth factors.     

Affinity purification and properties of cathepsin-E-like acid proteinase from rat spleen.

A unique acid proteinase different from cathepsin D was purified from rat spleen by a method involving precipitation at pH 3.5, affinity chromatography on pepstatin-Sepharose 4B and concanavalin A-Sepharose 4B, chromatography on Sephadex G-100 and DEAE-Sephacel, and isoelectric focusing. A purification of 4200-fold over the homogenate was achieved and the yield was 11%. The purified enzyme appeared to be homogeneous on electrophoresis in polyacrylamide gels. The isoelectric point of the enzyme was determined to be 4.1-4.4. The enzyme hydrolyzed hemoglobin with a pH optimum of about 3.1. The molecular weight of the enzyme was estimated to be about 90000 by gel filtration on Sephadex G-100. In sodium dodecylsulfate polyacrylamide gel electrophoresis, the purified enzyme showed a single protein band corresponding to a molecular weight of about 45000. The hydrolysis of bovine hemoglobin by the enzyme was much higher than that of serum albumin. Various synthetic and natural inhibitors of the enzyme were tested. The enzyme was inhbited by Zn2+, Fe3+, Pb2+, cyanide, p-chloromercuribenzoate, iodoacetic acid and pepstatin, whereas 2-mercaptoethanol, phenylmethyl-sulfonyl fluoride and leupeptin showed no effect.