Purification of Phosphatidylinositol Synthetase from Rat Brain by CDP‐Diacylglycerol Affinity Chromatography and Properties of the Purified Enzyme

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Abstract
A purification procedure for rat brain phosphatidylinositol synthetase (PI synthetase; CDP-1,2-diacylsn-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.

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