Phosphatidylinositol synthase from Saccharomyces cerevisiae. Reconstitution, characterization, and regulation of activity.
Purified membrane-associated phosphatidylinositol synthase (CDP diacylglycerol:myo-inositol 3-phosphatidyltransferase, EC 2.7.8.11) from Saccharomyces cerevisiae was reconstituted into unilamellar phospholipid vesicles. Reconstitution of the enzyme was performed by removing detergent from an octylglucoside/phospholipid/Triton X-100/enzyme mixed micelle mixture by Sephadex G-50 superfine column chromatography. The average diameter of the vesicles was 40 nm and chymotrypsin treatment of intact vesicles indicated that over 90% of the reconstituted enzyme had its active site facing outward. The enzymological properties and reaction mechanism of reconstituted phosphatidylinositol synthase were determined in the absence of detergent. The reconstituted enzyme was used as a model system to study the regulation of activity. Phosphatidylinositol synthase was constitutive in wild type cells grown in the presence of water-soluble phospholipid precursors as determined by enzyme activity and immunoblotting. Reconstituted enzyme was not effected by water-soluble phospholipid precursors or nucleotides. Maximum activity was found when the enzyme was reconstituted into phosphatidylcholine: phosphatidylethanolamine: phosphatidylinositol: phosphatidylserine vesicles. Phosphatidylserine stimulated reconstituted activity, suggesting that the local phospholipid environment may regulate phosphatidylinositol synthase activity.
D051607:CDP-Diacylglycerol-Inositol 3-Phosphatidyltransferase; D002850:Chromatography, Gel; D004228:Dithionitrobenzoic Acid / Q000494:pharmacology; D006863:Hydrogen-Ion Concentration; D007163:Immunosorbent Techniques; D007294:Inositol / Q000494:pharmacology; D008081:Liposomes; D008274:Magnesium / Q000494:pharmacology; D008345:Manganese / Q000494:pharmacology; D017830:Octoxynol; D010770:Phosphotransferases / Q000378:metabolism*; D011092:Polyethylene Glycols; D012441:Saccharomyces cerevisiae / Q000201:enzymology*; D017855:Transferases (Other Substituted Phosphate Groups)
D008081:Liposomes; D011092:Polyethylene Glycols; D008345:Manganese; D007294:Inositol; D017830:Octoxynol; D004228:Dithionitrobenzoic Acid; D010770:Phosphotransferases; D017855:Transferases (Other Substituted Phosphate Groups); D051607:CDP-Diacylglycerol-Inositol 3-Phosphatidyltransferase; D008274:Magnesium