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Solubilization of Microsomal-Associated Phosphatidylinositol Synthase from Germinating Soybeans ¹

Department of Food Science, Cook College, New Jersey Agricultural Experiment Station, Rutgers University, New Brunswick, New Jersey 08903

CDP-1,2-diacyl-sn-glycerol (CDP-diacylglycerol):myo-inositol phosphatidyltransferase (EC 2.7.8.11, phosphatidylinositol synthase) catalyzes the final step in the de novo synthesis of phosphatidylinositol in the endoplasmic reticulum fraction of germinating soybeans (Glycine max L. var Cutler 71). A variety of solubilization agents were examined for their ability to release phosphatidylinositol synthase activity from the microsome fraction. The most effective agent to solubilize the enzyme was the nonionic detergent Brij W-1. A 2.1-fold increase in specific activity was achieved using 1% Brij W-1 with 69% activity solubilized.

Maximal solubilization of phosphatidylinositol synthase was completely dependent on Brij W-1 (1%), potassium ions (0.3 M), and manganese ions (0.5 mM). Solubilization of the enzyme was not affected by the protein concentration of microsomes between 3 to 20 milligrams per milliliter. Solubilization was not affected by the pH of solubilization buffer between 6.5 to 8.5. To our knowledge, this is the first phospholipid biosynthetic enzyme solubilized from plant membranes. The Brij W-1-solubilized phosphatidylinositol synthase remained at the top of a glycerol gradient, whereas the membrane-associated enzyme sedimented to the bottom of the gradient. Maximal activity of the Brij W-1-solubilized phosphatidylinositol synthase was dependent on manganese (5 mM) or magnesium (30 mM) ions, and Triton X-100 (3.6 mM) at pH 8.0 with Tris-HCl buffer. The apparent K_m values for CDP-diacylglycerol and myo-inositol for the solubilized enzyme was 0.1 mM and 46 µM, respectively. Solubilized phosphatidylinositol synthase activity was thermally inactivated at temperatures above 30°C.

¹ This work was performed as a part of New Jersey Agricultural Experiment Station Projects 10203 and 10517 supported by the New Jersey Agricultural Experiment Station and by Public Health Service Grant GM-28140 from the National Institutes of Health. This is a paper of the Journal Series, New Jersey Agricultural Experiment Station, Cook College, Rutgers University, New Brunswick, NJ.