Partial purification and properties of phosphatidylserine synthase from Clostridium perfringens

Cousminer, J. Jeffrey; Fischl, Anthony S.; Carman, George M.

doi:10.1128/jb.151.3.1372-1379.1982

Cited by 20 publications

(16 citation statements)

References 36 publications

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“…The main purification step was affinity chromatography on CDP-DAG-Sepharose. This affinity resin has been used in the purification of several enzymes [17,22,23], including Ptdlns synthase from S. cerevisiae [11] and rat brain [16]. Enzymes have been reported to bind extremely strongly to the CDP-DAG affinity resin [11,22].…”

Section: Discussionmentioning

confidence: 99%

Purification and characterization of phosphatidylinositol synthase from human placenta

Antonsson¹

1994

Biochemical Journal

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Phosphatidylinositol synthase (CDP-1,2-diacyl-sn-glycerol:myoinositol 3-phosphatidyltransferase, EC 2.7.8.11) was purified from the microsomal fraction of human placenta. The Triton X-100-extracted enzyme was purified 8300-fold over the microsomal fraction by affinity chromatography on CDP-diacylglycerol-Sepharose followed by ion-exchange chromatography on Mono Q. The purified enzyme had a molecular mass of 24,000 Da on SDS/PAGE. The enzyme had a pH optimum at 9.0, required Mn2+ or Mg2+, and was inhibited by Ca2+ and Zn2+. The Km for myo-inositol was determined to be 0.28 mM. Optimal activity was obtained at 0.2-0.4 mM CDP-diacylglycerol; higher concentrations of the lipid substrate inhibited the enzyme reaction. The enzyme was inhibited by nucleoside di- and tri-phosphates, Pi and PPi. CDP competitively inhibited the enzyme reaction with a Kis of 4 mM. The optimal temperature for the PtdIns synthase reaction was 50 degrees C.

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Section: Discussionmentioning

confidence: 99%

Purification and characterization of phosphatidylinositol synthase from human placenta

Antonsson¹

1994

Biochemical Journal

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“…The CDPdiacylglycerol-Sepharose affinity resin (20) has been a valuable tool for the purification of CDPdiacylglycerol-dependent enzymes in the presence of the nonionic detergent Triton X-100. These enzymes include phosphatidylglycerophosphate synthase from B. licheniformis (20) and E. coli (17), phosphatidylserine synthase from C. perfringens (11) The only other phospholipid biosynthetic enzyme that has been purified from S. cerevisiae is the mitochondrial-associated CTP:phosphatidic acid cytidylyltransferase (3). This enzyme was purified about 100-fold over mitochondrial membranes and 500-fold over the cell extract.…”

Section: Discussionmentioning

confidence: 99%

Phosphatidylinositol biosynthesis in Saccharomyces cerevisiae: purification and properties of microsome-associated phosphatidylinositol synthase

Fischl¹,

Carman²

1983

J Bacteriol

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136

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The membrane-associated phospholipid biosynthetic enzyme phosphatidylinositol synthase (cytidine 5'-diphospho-1,2-diacyl-sn-glycerol:myo-inositol 3-phosphatidyltransferase, EC 2.7.8.11) was purified 1,000-fold from the microsomal fraction of Saccharomyces cerevisiae. The purification procedure included Triton X-100 solubilization of the microsomal membranes, CDPdiacylglycerol-Sepharose (Larson et al., Biochemistry 15:974-979, 1976) affinity chromatography, and chromatofocusing. The procedure resulted in the isolation of a nearly homogeneous protein preparation with an apparent minimum subunit molecular weight of 34,000, as determined by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. Phosphatidylinositol synthase was dependent on manganese and Triton X-100 for maximum activity. The pH optimum was 8.0. Thioreactive agents inhibited enzyme activity. The energy of activation was found to be 35 kcal/mol (146,540 J/mol). The enzyme was reasonably stable at temperatures of up to 60°C.Phosphatidylinositol (PI) has been shown to regulation and the kinetics of PI synthase in have considerable importance in the growth of well-defined systems have been hampered by Saccharomyces cerevisiae. When inositol-re-difficulty in purifying this enzyme from yeast quiring strains of S. cerevisiae are deprived of membrane preparations. PI synthase has been inositol, the cells undergo drastic changes in the solubilized with either the nonionic detergent metabolism of lipids, carbohydrates, proteins, Renex 690 or Triton X-100 in reasonably high and nucleic acids, ultimately resulting in a loss yields with good stability (8). However, further of cell viability (1,15). The requirement for purification of the enzyme by chromatography inositol arises because membranes do not func-with hydroxyapatite, DEAE-cellulose, carboxytion correctly if they are deficient in inositol-methyl cellulose, or phosphocellulose was uncontaining lipids (23). Besides being the third successful (8). A major problem in the purificamajor phospholipid component of yeast mem-tion of some membrane-associated enzymes is branes (1), PI serves as a precursor in the that after solubilization, they tend to bind large synthesis of other inositol-containing lipids, in-quantities of detergents. This binding masks the cluding di-and triphosphoinositides (21) and enzyme hydrodynamic and electrostatic properseveral sphingolipids (2). PI may also play a ties, the manifestation of which is essential for direct role in the synthesis of yeast glycans (15). purification by classical techniques (16). AffinityThe enzyme responsible for the biosynthesis chromatography has proven useful for the purifiof PI is PI synthase (CDP-1,2-diacyl-sn-glycerol: cation of some membrane-associated enzymes myo-inositol 3-phosphatidyltransferase, EC solubilized with nonionic detergents. The pres-2.7.8.11). PI synthase catalyzes the formation of ence of the nonionic detergents Used in solubiliz-PI and CMP from CDPdiacylglycerol and myo-ing these enzymes does not interfere wi...

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“…In contrast, the phosphatidylserine synthases of gram-positive bacilli and the yeast Saccharomyces cerevisiae are associated with membranes (5,9,23,24). Among these, Bacillus licheniformis phosphatidylserine synthase has been purified to near homogeneity and characterized in some detail: it requires Mn2' and assumes a sequential bi-bi reaction mechanism, in contrast to the ping-pong mechanism exhibited by the E. coli counterpart (10).…”

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confidence: 99%

Cloning, sequencing, and expression in Escherichia coli of the Bacillus subtilis gene for phosphatidylserine synthase

et al. 1994

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The Bacillus subtilis pss gene encoding phosphatidylserine synthase was cloned by its complementation of the temperature sensitivity of an Escherichia coli pssA1 mutant. Nucleotide sequencing of the clone indicated that the pss gene encodes a polypeptide of 177 amino acid residues (deduced molecular weight of 19,613). This value agreed with the molecular weight of approximately 18,000 observed for the maxicell product. The B. subtilis phosphatidylserine synthase showed 35% amino acid sequence homology to the yeast Saccharomyces cerevisiae phosphatidylserine synthase and had a region with a high degree of local homology to the conserved segments in some phospholipid synthases and amino alcohol phosphotransferases of E. coli and S. cerevisiae, whereas no homology was found with that of the E. coli counterpart. A hydropathy analysis revealed that the B. subtilis synthase is very hydrophobic, in contrast to the hydrophilic E. coli counterpart, consisting of several strongly hydrophobic segments that would span the membrane. A manganese-dependent phosphatidylserine synthase activity, a characteristic of the B. subtilis enzyme, was found exclusively in the membrane fraction of E. coli (pssA1) cells harboring a B. subtilis pss plasmid. Overproduction of the B. subtilis synthase in E. coli cells by a lac promoter system resulted in an unusual increase of phosphatidylethanolamine (up to 93% of the total phospholipids), in contrast to gratuitous overproduction of the E. coli counterpart. This finding suggested that the unusual cytoplasmic localization of the E. coli phosphatidylserine synthase plays a role in the regulation of the phospholipid polar headgroup composition in this organism.

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Partial purification and properties of phosphatidylserine synthase from Clostridium perfringens

Cited by 20 publications

References 36 publications

Purification and characterization of phosphatidylinositol synthase from human placenta

Purification and characterization of phosphatidylinositol synthase from human placenta

Phosphatidylinositol biosynthesis in Saccharomyces cerevisiae: purification and properties of microsome-associated phosphatidylinositol synthase

Cloning, sequencing, and expression in Escherichia coli of the Bacillus subtilis gene for phosphatidylserine synthase

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