Purification of phosphatidylinositol synthetase from rat brain by CDP-diacylglycerol affinity chromatography and properties of the purified enzyme.
A purification procedure for rat brain phosphatidylinositol synthetase (PI synthetase; CDP-1,2-diacyl-sn-glycerol:myo-inositol 3-phosphatidyltransferase; EC 2.7.8.11) is described. The enzyme was purified 200-250-fold from the homogenate by solubilization with Triton X-100 from microsomal membranes and affinity chromatography on CDP-diacylglycerol-Sepharose. Elution of enzyme activity required the presence of Triton X-100, CDP-diacylglycerol, and either phosphatidylcholine or asolectin. The product that was obtained in 5-10% yield from whole brain and in 70% yield from the microsomal fraction contained three protein bands as determined by sodium dodecylsulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The final preparation contained levels of CDP-diacylglycerol hydrolase and CDP-diacylglycerol: sn-glycero-3-phosphate 3-phosphatidyltransferase activities that were less than 1% of PI synthetase activity. The purified enzyme displayed a pH optimum of 8.5-9.0, required either Mg2+ or Mn2+ and exhibited a Km of 4.6 mM for myo-inositol.
Ghalayini
A
A
Eichberg
J
J
D000818:Animals; D001921:Brain / Q000201:enzymology*; D051607:CDP-Diacylglycerol-Inositol 3-Phosphatidyltransferase; D002846:Chromatography, Affinity / Q000379:methods; D003567:Cytidine Diphosphate Diglycerides / Q000378:metabolism*; D004591:Electrophoresis, Polyacrylamide Gel; D005260:Female; D008565:Membrane Proteins; D008861:Microsomes / Q000201:enzymology; D009702:Nucleoside Diphosphate Sugars / Q000378:metabolism*; D017830:Octoxynol; D010713:Phosphatidylcholines; D010743:Phospholipids; D010770:Phosphotransferases / Q000302:isolation & purification*; D011092:Polyethylene Glycols; D051381:Rats; D011919:Rats, Inbred Strains; D012995:Solubility; D017855:Transferases (Other Substituted Phosphate Groups)
D003567:Cytidine Diphosphate Diglycerides; D008565:Membrane Proteins; D009702:Nucleoside Diphosphate Sugars; D010713:Phosphatidylcholines; D010743:Phospholipids; D011092:Polyethylene Glycols; C024990:asolectin; D017830:Octoxynol; D010770:Phosphotransferases; D017855:Transferases (Other Substituted Phosphate Groups); D051607:CDP-Diacylglycerol-Inositol 3-Phosphatidyltransferase; C496767:Cdipt protein, rat