Phosphatidylglycerophosphate synthease and phosphatidylserine synthase activites in Clostridium perfringens

Abstract

Cytidine 5'-diphospho (CDP)-1,2-diacyl-sn-glycerol (CDPdiacylglycerol):snglycerol-3-phosphate phosphatidyltransferase (EC 2.7.8.5, and CDPdiacylglycerol:L-serine O-phosphatidyltransferase (EC 2.7.8.8, phosphatidylserine synthase) activities were identified in the cell envelope fraction of the gram-positive anaerobe Clostridium perfringens. The association of phosphatidylglycero-P synthase and phosphatidylserine synthase with the cell envelope fraction of cell-free extracts was demonstrated by sucrose density gradient centrifugation, by both activities sedimenting with the 100,000 x g pellet and solubilization of both activities from the 100,000 x g pellet with Triton X-100. The pH optimum for both enzyme activities was 8.0 with tris(hydroxymethyl)aminomethane-hydrochloride buffer. Phosphatidylglycero-P synthase activity was dependent on magnesium ions (100 mM). Phosphatidylserine synthase activity was dependent on manganese (0.1 mM) or magnesium ions (50 mM). Both enzyme activites were dependent on the addition of the nonionic detergent Triton X-100. Maximum phosphatidylglycero-P synthase and phosphatidylserine synthase activities were obtained when the molar ratio of Triton X-100 to CDPdiacylglycerol was 50:1 and 12:1, respectively. The Km for sn-glycero-3-P in the phosphatidylglycero-P synthase reaction was 0.1 mM. The Km for L-serine in the phosphatidylserine synthase reaction was 0.15 mM. Both enzyme activities were 100% stable for at least 20 min at 600C.

Hosphatidylglycerophosphate Synthase From Germinating Soybeans

“…Soybean PGP synthase activity was completely stable at temperatures up to 70°C. The stability of this enzyme to high temperatures is also observed in several bacterial species (Carman and Wieczorek 1980;Larson et al 1976). On the other hand PGP synthase from spinach is labile above 37°C (Marshall and Kates 1972) and the enzyme from yeast is labile above 30°C (Carman and Belunis 1983).…”

Modification of the Agranoff-Suomi Method for the Synthesis of CDP-Diacylglycerol

“…The product phosphatidylinositol was identified by thin-layer chromatography in solvent system 3 (R, = 0.5) as described by Carman and Felder ( 1980). The products phosphatidylserine and phosphatidylglycerophosphate were identified by thin-layer chromatography in solvent system 2 (RF = 0.12 and R, = 0.2, respectively) as described by Carman and Wieczorek (1980).…”

“…Cutler 71) was prepared as described by Carman and Felder (1980) and was used as the source of phosphatidylinositol synthase. The cell envelope fraction of C. perfringens was prepared as described by Carman and Wieczorek (1980) and was used as the source of phosphatidylserine synthase and phosphatidylglycerophosphate synthase.…”

“…Phosphatidylinositol synthase activity was measured as described by Carman and Felder (1980) using 0.5 mM my0-[2-~H] inositol (400 cpm/nmol). Phosphatidylserine synthase and phosphatidylglycerophosphate synthase activities were measured as described by Carman and Wieczorek (1980) The concentration of CDP-diacylglycerol in the assays was 0.2 mM. One unit of enzymatic activity is defined as the amount of enzyme that catalyzes the formation of 1 nmol of product per min under the assay conditions described above.…”

Partial purification and properties of phosphatidylserine synthase from Clostridium perfringens

“…The enzyme has been studied extensively in bacteria and has been purified to apparent homogeneity from two strains of Escherichia coli by means of substrate affinity chromatographic techniques (23,35). The enzyme has never been purified from a gram-positive organism, though it has been identified and studied in cell-free extracts of several Bacillus species (14,22,30) and two Clostridium species (6,8,36). The significance for investigating this particular phospholipid biosynthetic enzyme is twofold.…”