The
enzyme GDPFuc:GM1 alpha 1----2
fucosyltransferase, induced by chemical
carcinogens in precancerous rat liver as well as rat
hepatoma cells, was found previously to be membrane bound, and was inactivated by various
detergents, while the activities of many other
transferases are generally enhanced by
detergents (Holmes, E.H. & Hakomori, S. (1983) J. Biol. Chem. 258, 3706-3717). The effects of
phospholipids and
detergents on rat
hepatoma H35 cells, the conditions of solubilization and subsequent affinity chromatography of the
enzyme, and a possible association of
phospholipids with the
enzyme have been studied with the following major results: The alpha 1----2
fucosyltransferase activity in Golgi membrane was diminished on treatment of membranes with
phospholipase A1 or
phospholipase C. The
enzyme activity was stimulated 7-fold in the presence of
cardiolipin or
phosphatidylglycerol (and 3-fold by
phosphatidylethanolamine) but not other
phospholipids. The stimulatory effect of
phosphatidylglycerol was eliminated when a variety of ionic or non-ionic
detergents were added to the reaction mixture, with the exception of the cationic
detergent G-3634-A, which provided a 10-fold total stimulation in the presence of
phosphatidylglycerol. The kinetic analysis indicated that addition of
phosphatidylglycerol has a negligible effect on apparent Km values but increases the Vmax of the
enzyme 5- to 6-fold. The
enzyme activity was solubilized by the dialyzable
detergent CHAPSO without inhibition of the
enzyme activity, and the solubilized
enzyme in the presence of 0.4%
CHAPSO is partially purified by chromatography on
GDP-hexanolamine-
Sepharose. Removal of
CHAPSO from the affinity purified
enzyme by dialysis resulted in a 66% loss of the original activity, which was restored by addition of
phosphatidylglycerol. Chromatography of the affinity-purified
enzyme with 3H-labeled
phosphatidylglycerol on a Biogel A0.5 column indicated an association of the
enzyme with the
phospholipid that occurred only in the absence of
detergent. These results suggest that
phospholipid has a direct effect on the
enzyme and that the inhibitory effect of
detergents can be ascribable to disturbing interaction between
phospholipids and the
enzyme. A possible role of specific
phospholipids on in vivo
transferase activity for
glycolipids is discussed.