Partial purification and properties of phosphatidylserine synthase from Clostridium perfringens

J. J. Cousminer, A. S. Fischl, G. M. Carman

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10 Scopus citations

Abstract

The membrane-associated phospholipid biosynthetic enzyme cytidine 5'-diphospho-1,2-diacyl-sn-glycerol: L-serine O-phosphatidyltransferase (phosphatidylserine synthase; EC 2.7.8.8) was partially purified 337-fold from a cell-free extract of the gram-positive pathogenic anaerobe C. perfringens (ATCC 3624). The purification procedure included extraction from the cell envelope with the nonionic detergent Triton X-100, followed by affinity chromatography on cytidine 5'-diphosphate-diacylglycerol-Sepharose. When the partially purified enzyme was subjected to polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate, two major bands were evident with apparent minimum molecular weights of 39,000 and 31,000. Activity of phosphatidylserine synthase was dependent on the addition of manganese ions (3 mM) and Triton X-100 (2.7 mM) for maximum activity. The rate of catalysis was maximal at 40°C (with rapid thermal inactivation above this temperature), and the pH optimum was 8.5. The apparent K(m) values for cytidine 5'diphosphate-diacetylglycerol and L-serine were 0.24 and 0.26 mM, respectively. The synthetic (forward) reaction was favored, as indicated by an equilibrium constant of 82, and the energy of activation was found to be 18 kcal/mol (75,362 J/mol).

Original languageEnglish (US)
Pages (from-to)1372-1379
Number of pages8
JournalJournal of bacteriology
Volume151
Issue number3
StatePublished - 1982

All Science Journal Classification (ASJC) codes

  • Molecular Biology
  • Microbiology

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