ENZYMATIC SULFATION OF A LARGE MOLECULAR-WEIGHT GLYCOPROTEIN ASSOCIATED WITH PIG BRAIN MEMBRANES

Pig brain membranes catalyze the transfer of [35S]sulfate from 3′-phosphoadenosine 5′-phospho[35S]sulfate into two macromolecular endogenous acceptors. Several operational enzymatic properties of the sulfotransferase activity have been defined. An apparent Km = 0.65 μm for 3′-phosphoadenosine 5′-phosphosulfate has been determined for the pig brain in vitro sulfotransferase system. Direct proof for the absolute requirement of the 3′-phosphate moiety of 3′-phosphoadenosine 5′-phosphosulfate is presented. The nucleotide end product, 3′,5′-ADP, is a potent competitive inhibitor of the pig brain sulfotransferase activity. One of the major products enzymatically labeled during incubation with 3′-phosphoadenosine 5′-phospho[35S]sulfate is a membrane-bound glycoprotein of high molecular weight. The sulfated glycoprotein appears to be an integral membrane glycoprotein, requiring 1% Triton X-100 for extraction. An 35S-labeled oligosaccharide, released by mild base treatment, contains O-sulfate ester groups and at least one N-acetylneuraminic acid residue. The sulfated glycoprotein has an apparent molecular weight of 198,000. Under the same in vitro conditions [35S]sulfate is also incorporated into a membrane-associated 35S-labeled proteoglycan having the properties of heparan sulfate. The 35S-labeled proteoglycan is electrostatically bound to the pig brain membranes, and can be readily extracted with 1 m NaCl. © 1979.