Characterization and localization of phosphatidylglycerophosphate and phosphatidylserine synthases in Rhodobacter sphaeroides |
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Authors: | Cynthia W. Radcliffe Francis X. Steiner George M. Carman Robert A. Niederman |
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Affiliation: | (1) Department of Biochemistry, Rutgers University, P. O. Box 1059, 08855-1059 Piscataway, NJ, USA;(2) Department of Food Science, Rutgers University, 08903 New Brunswick, NJ, USA;(3) Present address: Biology Department, Hillsdale College, 49242 Hillsdale, MI, USA |
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Abstract: | Catalytic properties and membrane associations of the phosphatidylglycerophosphate (PGP) and phosphatidylserine (PS) synthases of Rhodobacter sphaeroides were examined to further characterize sites of phospholipid biosynthesis. In preparations of cytoplasmic membrane (CM) enriched in these activities, apparent Km values of PGP synthase were 90 M for sn-glycerol-3-phosphate and 60 M for CDP-diacylglycerol; the apparent Km of PS synthase for l-serine was near 165 M. Both enzymes required Triton X-100 with optimal PS synthase activity at a detergent/CDP-diacylglycerol (mol/mol) ratio of 7.5:1.0, while for optimal PGP synthase, a range of 10–50:1.0 was observed. Unlike the enzyme in Escherichia coli and several other Gram-negative bacteria, the PS synthase activity had a specific requirement for magnesium and was tightly associated with membranes rather than ribosomes in crude cell extracts. Sedimentation studies suggested that the PGP synthase ws distributed uniformly over the CM in both chemoheterotrophically and photoheterotrophically grown cells, while the PS synthase was confined mainly to a vesicular CM fraction. Solubilized PGP synthase activity migrated as a single band with a pI value near 5.5 in a chromatofocusing column and 5.8 on isoelectric focusing; in the latter procedure, the pI was shifted to 5.3 in the presence of CDP-diacylglycerol. The PGP synthase activity gave rise to a single polypeptide band in lithium dodecyl sulfatepolyacrylamide gel electrophoresis at 4°C.Abbreviations CM cytoplasmic membrane - ICM intracytoplasmic photosynthetic membrane - OM outer membrane - PGP phosphatidylglycerophosphate - PS phosphatidylserine - TLC thin-layer chromatographySupported in part by a Fellowship Awards from the Charles and Johanna Busch Memorial Fund Award to the Rutgers Bureau of Biological Research |
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Keywords: | Rhodobacter sphaeroides Cytoplasmic membrane Photosynthetic membranes Phosphatidylglycerophosphate synthase Phosphatidylserine synthase |
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