A new phosphatidylserine spin label with nitroxide stearate attached at the 2 position has been synthesized by the reaction of spin-labeled CDP-diglyceride with L-serine under the catalytic action of phosphatidylserine synthetase. Some structural properties of pure phosphatidylserine (PS) and binary PS-phosphatidylcholine (PC) membranes were studied with the spin label. PS membrane became solidified on lowering solution pH, 50% solidification being attained at pH 3.5. The membrane was also solidified by addition of Ca-2+. The effect of Ba-2+,Sr-2+, and Mg-2+ was smaller than that of Ca-2+. The calcium-induced lateral phase separation in the binary membrane was studied from the side of the calcium-receiving lipid. The results confirmed and extended our previous conclusion drawn with PC spin label. The phase diagram of the binary membrane in the presence of Ca-2+ was determined. Not all PS molecules were aggregated to form the solid patches but some remained dissolved in the fluid PC matrix. The fluid PS fraction was larger for the membranes containing more PC. The membrane with 10% PS still had a significant fraction of solid phase. The rate of calcium-induced aggregation was greatly dependent on the PS content. The aggregation was almost complete within 5 min in the membrane containing 67% PS, while it was still proceeding after several hours in the membrane with 20% PS. The rate-limiting step was suggested to be in the formation of "stable" nuclei consisting of larger aggregates. The possible biological significance of the ionotropic phase separation was discussed whereby a transient density fluctuation was emphasized.
The fractionation, turnover and biosynthesis of the phosphatidylglycerol molecular species of Escherichia coli were studied. Monoacetyldiglycerides derived from phosphatidylglycerol were separated into five subfractions; cis-vaccenyl-palmitoleyl, cis-vaccenyl-cis-vaccenyl, palmitylpalmitoleyl, palmityl-cis-vaccenyl and the disaturated molecular species on a silica gel plate impregnated with silver nitrate. Individual molecular species had different turnover rates. The palmityl-cisvaccenyl species was metabolized faster than the others. Disaturated species were rather stable. Various phosphatidylglycerol molecular species were synthesized in the presence of sn-glycerol 3-phosphate, palmityl-CoA, palmitoleyl-CoA, cis-vaccenyl-CoA and CTP by the E. coli membrane particulate fraction. When only the proportion of palmityl-CoA among the three acyl-CoAs was increased, the molecular species containing the palmityl residue were increased. Similar results were obtained with the other acyl-CoAs. However, a temperature-sensitive incorporation of unsaturated and saturated fatty acids into phosphatidylglycerol molecular species was observed with no change in the proportions of the three acyl-CoAs, completely reflecting the in vivo unsaturated/saturated ratio.We have reported [l-31 that differences in fatty acid composition were found among the major phospholipid classes of Escherichia coli, and that phosphatidylglycerol contained more cis-vaccenic acid than did phosphatidylethanolamine at any temperature. Hence, individual phospholipids appear to consist of different proportions of molecular species, which may control the membrane fluidity and phase separation. In contrast to phosphatidylethanolamine, phosphatidylglycerol plays a dynamic role in bacterial membranes. The rapid turnover of this phospholipid has been reported in E. coli [4-61. However, phosphatidylglycerol is required in the sugar transport process [7,8] and serves as a precursor of oligosaccharide [9]. No relationship has been established between the metabolic significance and variety of molecular species.The present paper deals with the separation, turnover and biosynthesis of the phosphatidylglycerol molecular species of E. coli.
Phosphatidylserine synthetase occurs in a soluble and a ribosome-bound form in Escherichia coli. The soluble enzyme of E . coli B was partially purified by DEAE-Sephadex chromatography.The ribosomal enzyme was obtained by differential centrifugation. Two types of the enzyme had the same K , (150 @ I . ) for L-serine. The soluble enzyme was required for the biosynthesis of phosphatidylethanolamine from phosphatidic acid via CDP-diglyceride in the membrane. The ribosomal enzyme was less effective to the phosphatidylethanolamine formation than the soluble enzyme. By the sucrose-density-gradient centrifugation method the soluble enzyme was proved to be loosely associated with the membrane.Differences in the fatty acid composition among phospholipid species of Escherichia coli demonstrated in this laboratory [I-31 suggest a complicated mechanism for the biosynthesis of phospholipid molecular species. With respect to the polar head group, however, the pathway for the synthesis of phosphatidylethanolamine and phosphatidylglycerol has been well established [4-91. The enzymes responsible for their biosyntheses are localized in the cytoplasmic membrane [lo-121 with the exception of phosphatidylserine synthetase which was described to occur in a soluble and a ribosome-bound forms [13]. I n order to understand the mechanism by which the respective phospholipids are synthesized from the key intermediate, phosphatidic acid, it would be necessary to investigate mutual interaction of those enzymes in the membrane.The present paper deals with the purification of soluble phosphatidylserine synthetase and its participation in the system of the phosphatidylethanolamine biosynthesis in the membrane. MATERIALS AND METHODS Assay of Phosphatidylserine Synthetase and Phosphatidylglycerophosphate SynthetaseThe standard assay contained 33 mM potassium phosphate buffer, pH 7.2, 15 mM MgCl,, 0.11 mM L -[U -14C]serine (7900 counts x min-l x nmol-l), Enzymes. Phosphatidylserine synthetase or CDP-diglyceride : L-serine 0-phosphatidyltransferase (EC 2.7.8.8) ; phosphatidylglycerophosphate synthetase or CDP-diglyceride : sn-glycerol 3-phosphate phosphatidyltransferase (EC 2.7.8.5).
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