Moseley Waite

Purification and substrate specificity of rat hepatic lipase

Publisher Summary Hepatic lipase from rat liver is an acylhydrolase that cleaves the fatty acid from the 1(3)-position of a broad spectrum of glycerides. In addition to the hydrolytic reaction, the enzyme can catalyze transacylation reactions in which the free hydroxyl of an acceptor lipid molecule becomes acylated rather than water, which is the acyl acceptor in hydrolysis. Routine assay of hepatic lipase is carried out with 1-oleoyl-[2- 3 H]glycerol ([ 3 H]MO) for the steps in purification and to check enzyme stability. This has the advantage of eliminating the chromatographic separation of products since both hydrolysis and transacylation reactions release [ 3 H]glycerol that partitions into the methanol-water phase during extraction. As the remaining substrate partitions into the chloroform layer, enzyme activity is measured by scintillation counting of an aliquot of the upper phase. The method of enzyme purification involves perfusion of the liver followed by two chromatographic steps. Two important points should be considered, first, the liver should be thoroughly washed by perfusion prior to the addition of heparin to remove as much contaminating protein as possible and second, a minimum of time should be used in the preparation. The adoption of this procedure saves nearly two days in enzyme isolation and increases the specific activity of the final preparation by two- to three-fold. Proteolysis may also occur during preparation that may alter substrate specificity.

On the mechanism of action of enzyme-bound biotin.

Ever since the identification of biotin with the curative factor (vitamin H) for egg white injury (1, 2), investigators have concerned themselves with the mode and mechanism of action of this vitamin. With the use of both microbiological techniques and animal experiments, early investigators recognized various biochemical roles for biotin, especially as a key component in the metabolism of COZ, fatty acids, and various dicarboxylic acids. More specifically, biotin was shown somehow to be required for various carboxylation reactions, including the synthesis of oxaloacetic acid from pyruvate plus COz (3-7). The sparing effect of oleic acid on biotin requirements suggested that the latter may play a role in the synthesis of fatty acids (8-11). Biotin deficiency in animals was also known to cause a reduction in the levels of malic enzyme, citrulline synthetase, and other enzymes (12-19). Melville, Pierce, and Partridge (20) first postulated that biotin may function in carboxylation reactions through the turnover of the carbonyl group of the ureido ring. These workers synthesized ureido-U4-biotin and used it as a growth factor for Lactobacillus arabinosus. Biotin was reisolated from both cells and media and its Cl4 content was determined. Approximately 93 y. of the initial Cl4 was still associated with the biotin; this high recovery seemed to invalidate the aforementioned hypothesis, since if the carbonyl carbon of biotin were the group involved in carboxylation reactions, they would have found little or no radioactivity in biotin. In 1958 Wakil, Titchener, and Gibson (21) and Wakil and Gibson (22) observed that bicarbonate was required for fatty acid synthesis and that one of the enzymes involved in fatty acid synthesis contained large amounts of biotin. Biotin was tightly bound to the protein and was closely associated with the enzymatic activity throughout the purification steps. In addition, Wakil and Gibson (22) demonstrated that avidin, an egg white protein long known for its high binding affinity for biotin (23), inhibited the carboxylation of acetyl-CoA. Treatment of avidin with free biotin, before its interaction with the enzyme, prevented

Phospholipid-Sensitive, Ca2+-Dependent Protein Kinase Activity in Rat Embryo Fibroblasts Transformed by Herpes simplex Virus Type 2

Increased cytosolic phospholipid-sensitive, Ca2+-dependent protein kinase C (PK-C) activity is correlated with the highly tumorigenic potential of rat embryo fibroblasts transformed by herpes simplex virus type 2 (HSV-2). Treatment of the cells with 12-O-tetradecanoylphorbol-13-acetate (TPA) caused a decrease in the cytosolic PK-C with a concomitant increase in PK-C recovered in the membrane fraction. Translocation of the PK-C was dependent upon length of exposure to the phorbol diester. PK-C activity in the cytosolic fraction could be stimulated by TPA without the addition of phosphatidylserine and diacylglycerol. It is tempting to speculate that HSV-2 induction of cellular PK-C activity may be important in phosphorylation of proteins needed for promotion of HSV-2-induced carcinogenesis.