Topographical orientation in microsomal vesicles of the N-acetylglucosaminyltransferases which catalyze the biosynthesis of N-acetylglucosaminylpyrophosphoryldolichol and N-acetylglucosaminyl-N-acetylglucosaminylpyrophosphoryldolichol.
The orientation of the N-acetylglucosaminyltransferases which catalyze the biosynthesis of N-acetylglucosaminylpyrophosphoryldolichol and N-acetylglucosaminyl-N-acetylglucosaminylpyrophosphoryldolichol was examined using microsomes isolated from livers of the embryonic chick. Dolichol phosphate liposomes were used as the exogenous substrate, thus avoiding the use of detergents. The formation of both of these intermediates of the dolichol pathway was almost completely inhibited after trypsinization of the microsomes under conditions in which microsomal integrity was maintained as evaluated by determining the latency of mannose-6-phosphatase. Using dolichol [32P]phosphate liposomes, it was shown that trypsinization did not interfere with the binding of liposomes to the microsome, suggesting that the effect of trypsinization was on the GlcNAc-transferases. Although dolichol phosphate liposomes associate with the microsomes during incubation, it was possible by sucrose density centrifugation to separate a population of liposomes which were not bound in this manner. GlcNAc lipids isolated from the free liposomes were shown to contain both the mono-GlcNAc and (GlcNAc)2 derivatives in proportions similar to those obtained from the microsomes. The possibility of transfer of the GlcNAc lipids from endogenous sources on the microsome to the liposome was examined and shown to play little role in these processes. This study strongly suggests a nonluminal, cytoplasmic orientation of the N-acetylglucosaminyltransferases concerned with the biosynthesis of the first two intermediates of the dolichol pathway.
Kean
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Department of Surgery, Case Western Reserve University, Cleveland, Ohio 44106.
D000818:Animals; D002384:Catalysis; D002642:Chick Embryo; D002854:Chromatography, Paper; D005964:Glucosyltransferases / Q000378:metabolism*; D008081:Liposomes / Q000378:metabolism; D008862:Microsomes, Liver / Q000201:enzymology / Q000378:metabolism*; D017351:N-Acetylglucosaminyltransferases; D011103:Polyisoprenyl Phosphate Monosaccharides / Q000378:metabolism*; D011104:Polyisoprenyl Phosphate Oligosaccharides / Q000378:metabolism*; D014357:Trypsin
D008081:Liposomes; D011103:Polyisoprenyl Phosphate Monosaccharides; D011104:Polyisoprenyl Phosphate Oligosaccharides; C018439:N-acetylglucosaminylpyrophosphoryldolichol; C016418:dolichyl-diphosphate-di-N-acetylchitobiose; D005964:Glucosyltransferases; D017351:N-Acetylglucosaminyltransferases; C037649:N-acetyllactosaminide beta-1,6-N-acetylglucosaminyltransferase; D014357:Trypsin