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Glycoprotein biosynthesis: The localization of polypeptidyl: N-acetylgalactosaminyl, collagen: Glucosyl, and glycoprotein: Galactosyl transferases in HeLa cell membrane fractions

1968; Elsevier BV; Volume: 128; Issue: 2 Linguagem: Inglês

10.1016/0003-9861(68)90045-3

1096-0384

Arpi Hagopian, H.Bruce Bosmann, E.H. Eylar,

Carbohydrate Chemistry and Synthesis

Membranes from HeLa cells were purified by centrifugation in a discontinuous sucrose gradient. The distribution and properties of three glycoprotein:glycosyl transferases within these membranes was determined. A multienzyme group of glycosyl transferases, which is involved in the assembly of the carbohydrate units of membrane grycoproteins, was found almost entirely in the fraction representing the smooth internal membranes. Two of the enzymes of this group include a polypeptidyl:N-acetylgalactosaminyl transferase that catalyzes the synthesis of a proteincarbohydrate linkage and a glycoprotein:galactosyl transferase that attaches galactose to N-acetylglucosamine residues during the assembly of the carbohydrate units of the glycoprotein. The N-acetylgalactosaminyl transferase, for example, was purified 45–50-fold, representing 50–60% of the total cellular activity, during the course of purification of the smooth internal membranes. Each of these enzymes is strongly bound to the membranous structure, but can be solubilized with the nonionic detergent, Triton X-100. In contrast to this multienzyme group of the smooth membrane fraction, a collagen:glucosyl transferase, responsible for attachment of the terminal glucose residue to the carbohydrate portion of the secreted protein, collagen, was located exclusively in the plasma membrane. The glucosyl transferase was purified over 140-fold, representing 55% of the total activity of the cell, during the course of purification of the plasma membranes. The specific location of this enzyme suggests it to be a useful marker for the plasma membrane. On the basis of these data, it was suggested that the attachment of carbohydrate to proteins (extracellular) may be a necessary prelude to their secretion. The collagen:glucosyl transferase also differed from the transferases of the smooth membranes in its binding relationship to the membranous structures; it expresses full activity in the membrane-bound state, whereas the transferases require solubilization with Triton X-100 for maximum activity. With regard to the mechanism of glycoprotein biosynthesis, this work supports the concept of a “one enzyme-one linkage” concept in which each monosaccharide is added individually by a highly specific transferase to the growing carbohydrate chain. The intracellular site for assembly of the carbohydrate units of membrane glycoproteins appear to be the smooth internal membranes rather than the microsomes.