Biosynthesis of glucosidase II in suspension-cultured soybean cells.
Since antibody against homogeneous mung bean glucosidase II cross-reacted with a 110-kDa protein from cultured soybean cells and also precipitated this activity from extracts of soybean cells, we used this antibody to examine the biosynthesis, turnover, and cellular localization of glucosidase II in soybean cells. Time course studies of [35S]methionine incorporation into glucosidase II (as well as pulse-chase studies) showed that this enzyme is synthesized as a 110-kDa protein that does 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 extensive modification of the carbohydrate. Furthermore, glucosidase II remained 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 secretion of this protein occurred. Membranes of gently disrupted cells were 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 suggested that glucosidase II did not have a hydrophobic domain and is probably a luminal endoplasmic reticulum protein.
Kaushal
G P
GP
Department of Biochemistry and Molecular Biology, University of Arkansas for Medical Sciences, Little Rock 72205.
Zeng
Y
Y
Elbein
A D
AD
D001345:Autoradiography; D002240:Carbohydrate Sequence; D002478:Cells, Cultured; D002850:Chromatography, Gel; D004591:Electrophoresis, Polyacrylamide Gel; D005959:Glucosidases / Q000037:antagonists & inhibitors; D007212:Indolizines / Q000494:pharmacology; D007700:Kinetics; D008715:Methionine / Q000378:metabolism; D008969:Molecular Sequence Data; D008970:Molecular Weight; D009844:Oligosaccharides / Q000302:isolation & purification / Q000378:metabolism; D011523:Protoplasts / Q000201:enzymology; D013025:Glycine max / Q000201:enzymology*; D013347:Subcellular Fractions / Q000201:enzymology; D013462:Sulfur Radioisotopes; D013997:Time Factors; D000520:alpha-Glucosidases / Q000096:biosynthesis* / Q000302:isolation & purification / Q000378:metabolism; D054836:Indolizidines
D005959:Glucosidases; D007212:Indolizines; D008715:Methionine; D009844:Oligosaccharides; D013462:Sulfur Radioisotopes; D000520:alpha-Glucosidases; C034251:4-nitrophenyl-alpha-glucosidase; C037806:castanospermine; D054836:Indolizidines