Sulfation of Glycosphingolipids and Related Carbohydrates by Brain Preparations from Young Rats

Abstract

Abstract Preparations from the brain of young rats contain enzymes that catalyze the transfer of sulfate-35S from 3'-phosphoadenosine 5'-phosphosulfate-35S to galactose-containing glycosphingolipids and to galactose and water-soluble galactosides. These enzymes were found in all of the subcellular particles and in the high speed supernatant (cytosol). Kinetic experiments, quantitative differences in subcellular distribution, and fractionation with ammonium sulfate of solubilized microsomes showed that one enzyme catalyzes the transfer to glycosphingolipids and a different one to the water-soluble compounds. The relative affinity of 3'-phosphoadenosine 5'-phosphosulfate for the glycosphingolipid sulfotransferase was 4 times higher than the affinity of the same substrate for the sulfotransferase for the water-soluble galactosides. The apparent Km for galactosylceramide was found to be between 3.3 and 8.5 x 10-5 m; for lactosylceramide it was 5.58 x 10-5 m; for galactosylsphingosine it was 4.55 x 10-5 m. Under the same conditions as tested for the sphingolipids the apparent Km for lactose was 1.2 x 10-2 m, and for galactose was 1.12 x 10-1 m. The fastest rates of sulfation for the lipids were with lactosylceramide and galactosylsphingosine; for the water-soluble acceptors the maximal velocity was with p-nitrophenyl-β-galactoside. A pH optimum of 6.8 to 7.0 was found for both enzymes. The rate of release of sulfate under acid conditions and the chromatographic behavior of enzymatically synthesized galactosylceramide and lactosylceramide were compared with those of the natural sulfatides; the results showed that the sulfate attached enzymatically was in position C3 of the galactose moiety. The position of attachment of sulfate to the water-soluble acceptors was not identified. Methods for the determination of the enzymes and for preparations of various glycosphingolipids are given.