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  • Title: Phosphatidylinositol synthase from canine pancreas: solubilization by n-octyl glucopyranoside and stabilization by manganese.
    Author: Parries GS, Hokin-Neaverson M.
    Journal: Biochemistry; 1984 Sep 25; 23(20):4785-91. PubMed ID: 6093865.
    Abstract:
    Phosphatidylinositol synthase (CDP-1,2-diacyl-sn-glycerol:myo-inositol 3-phosphatidyltransferase) is active in mammalian pancreas, where it plays a role in the resynthesis of phosphatidylinositol (PI) during agonist-stimulated inositol-phospholipid metabolism. The enzyme was found to be present in relatively high specific activity [30 nmol of PI formed min-1 (mg of protein)-1] in dog pancreas microsomal membranes, and its activity in these membranes was partially characterized. The Km for myo-inositol was 0.76 mM, and the apparent Km for cytidine(5')diphospho-1,2-diacylglycerol (CDP-diacylglycerol) was 18 microM. The apparent Ka values for activation by Mn2+ and Mg2+ were respectively 42 microM and 2.5 mM. The pH optimum was 8.5-9.0. The enzyme was solubilized in stable form and in nearly quantitative yield with 40 mM n-octyl glucopyranoside (OG), with 4-6 mg of OG/mg of microsomal protein. In the presence of solubilizing levels of OG, the enzyme exhibited less than maximal activity, but full activity was restored by dilution of the OG to below its critical micelle concentration of 20-25 mM. The presence of Mn2+ was essential for stabilization of the OG-solubilized enzyme, with half-maximal stabilization at 40 microM Mn2+. The stability of the OG-solubilized enzyme was sufficient to facilitate purification of the enzyme in the presence of this detergent, with 67% of the activity remaining after 3 days at 4 degrees C. The enzyme was partially purified by OG extraction and DEAE-cellulose chromatography, in 98% yield, to a specific activity of 290 nmol of PI formed min-1 (mg of protein)-1.(ABSTRACT TRUNCATED AT 250 WORDS)
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