BIOSYNTHESIS OF GLUCOSIDASE-II IN SUSPENSION-CULTURED SOYBEAN CELLS

Since antibody against homogeneous mung bean glucosidase II cross-reac ted with a 110-kDa protein from cultured soybean cells and also precip itated this activity from extracts of soybean cells, we used this anti body to examine the biosynthesis, turnover, and cellular localization of glucosidase II in soybean cells. Time course studies of [S-35]methi onine incorporation into glucosidase II (as well as pulse-chase studie s) showed that this enzyme is synthesized as a 110-kDa protein that do es not change in size from very early labeling times to those as late as 60 h, indicating the absence of a cleavable signal sequence or exte nsive modification of the carbohydrate. Furthermore, glucosidase II re mained susceptible to digestion by endo-beta-N-acetylglucosaminidase H throughout this time period, and the major oligosaccharide structure was a Man9(GlcNAc)2 with small amounts of Glc1Man9(GlcNAc)2. The half- life of the biosynthesized glucosidase II was about 36 h, and no secre tion of this protein occurred. Membranes of gently disrupted cells wer e separated by sucrose-density gradient centrifugation, and fractions were tested for glucosidase II activity as well as for marker enzymes. The bulk of the glucosidase II activity fractionated with endoplasmic reticulum membranes. Detergent solubility studies with Triton X-114 s uggested that glucosidase II did not have a hydrophobic domain and is probably a luminal endoplasmic reticulum protein.