JBC, Vol. 252, Issue 15, 5419-5423, Aug, 1977
CDP-diglyceride:inositol transferase from rat liver. Purification and properties
T. Takenawa and K. Egawa
CDP-diglyceride:inositol transferase, which catalyzes the final step of the
de novo synthesis of phosphatidylinositol, was solubilized by sodium
cholate from microsomes prepared from rat liver and purified by ammonium
sulfate fractionation, sucrose density gradient centrifugation, and
DEAE-cellulose column chromatography. Addition of phospholipid during the
purification and the assay procedures prevented irreversible loss of the
enzyme activity to some extent. The resulting preparation was nearly
homogeneous as judged by polyacrylamide gel electrophoresis. The recovery
of the purified enzyme from the microsomal fraction was 3 to 3.3% with
respect to activity and 0.12% with respect to amount of protein. The
molecular weight of the enzyme was estimated by sodium dodecyl
sulfate-polyacrylamide gel electrophoresis to be 60,000. The purified
enzyme required exogenous phospholipds for its activity. Various
phospholipid classes activated the enzyme rather nonspecifically. The Km
for myo-inositol was 2.5 X 10(-3) M and that for CDP-diglyceride was 1.7 X
10(-4) M. The pH optimum was 8.6. The enzyme required Mm2+ or Mg2+ for
activity. The optimal concentration of Mn2+ for activation was 0.5 mM,
while the activity in the presence of Mg2+ increased up to 20 mM. The
enzyme was inhibited by thiol-reactive reagents. There was a competition
for inositol by inosose-2 but not by scyllitol.
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