Glycoprotein biosynthesis: The solubilization of glycosyl transferases from membranes of hela cells and bovine submaxillary glycoproteins

Abstract

Two glycosyl transferases, the polypeptidyl: N-acetylgalactosaminyl and glycoprotein: galactosyl transferase, which participate in the assembly of the carbohydrate units of glycoproteins, were purified from both HeLa cells and submaxillary glands. In the case of HeLa cells these enzymes are intimately associated with the smooth internal membranes where they form part of the multienzyme group of transferases synthesizing membrane glycoproteins; in submaxillary tissues, however, these enzymes are involved in synthesis of secreted submaxillary glycoproteins and are strongly bound to membranes, possibly plasma membranes, which sediment to the 35–45% sucrose interface. In preparing the respective membranes, the glycosyl transferases were purified from 10–40-fold over the cellular homogenate. It was found that from 80–95% of the total enzyme activities are masked in the isolated membranes; disruption of the membranous structure with nonionic detergents or with phospholipase A liberated the transferases. The largest increase in activity was obtained with the polyoxyethylene octylphenol type. The linear alcohol polyoxy-ethylene types were slightly less effective; less than 50% of the maximum activity was obtained. These results, together with the effect of phospholipase A, emphasize the significance of nonpolar forces in membrane substructure, particularly with regard to the interaction of the transferases with their membranous environment. By contrast, negatively charged detergents, such as sodium deoxycholate and sodium dodecyl sulfate, led to strong inhibition of enzymes. A positively charged detergent, cetylpyridinium bromide, also produced strong inhibition. It was concluded that the influence of detergents on the activity of membrane-bound transferases appears to depend on two factors, solubilization and inhibition.