The chemical carcinogen-induced enzyme, GDP-fucose: GM1 alpha 1----2 fucosyltransferase in rat liver and hepatoma: modulation by and association with phospholipids.

Abstract

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.