Topological studies on the enzymes catalyzing the biosynthesis of Glc-P-dolichol and the triglucosyl cap of Glc3Man9GlcNAc2-P-P-dolichol in microsomal vesicles from pig brain: use of the processing glucosidases I/II as latency markers.
In the current model for Glc3Man9GlcNAc2-P-P-Dol assembly, Man5GlcNAc2-P-P-Dol, Man-P-Dol, and Glc-P-Dol are synthesized on the cytoplasmic face of the ER and diffuse transversely to the lumenal leaflet where the synthesis of the lipid-bound precursor oligosaccharide is completed. To establish the topological sites of Glc-P-Dol synthesis and the lipid-mediated glucosyltransfer reactions involved in Glc3Man9GlcNAc2-P-P-Dol synthesis in ER vesicles from pig brain, the trypsin-sensitivity of Glc-P-Dol synthase activity and the Glc-P-Dol:Glc0-2Man9GlcNAc2-P-P-Dol glucosyltransferases (GlcTases) was examined in sealed microsomal vesicles. Since ER vesicles from brain do not contain glucose 6-phosphate (Glc 6-P) phosphatase activity, the latency of the lumenally oriented, processing glucosidase I/II activities was used to assess the intactness of the vesicle preparations. Comparative enzymatic studies with sealed ER vesicles from brain and kidney, a tissue that contains Glc 6-P phosphatase, demonstrate the reliability of using the processing glucosidase activities as latency markers for topological studies with microsomal vesicles from non-gluconeogenic tissues lacking Glc 6-P phosphatase. The results obtained from the trypsin-sensitivity assays with sealed microsomal vesicles from brain are consistent with a topological model in which Glc-P-Dol is synthesized on the cytoplasmic face of the ER, and subsequently utilized by the three Glc-P-Dol-mediated GlcTases after "flip-flopping" to the lumenal monolayer.
Rush
J S
JS
Department of Biochemistry, University of Kentucky College of Medicine, Lexington, KY 40536, USA.
Waechter
C J
CJ
D017485:1-Deoxynojirimycin / Q000494:pharmacology; D000818:Animals; D001665:Binding Sites / Q000502:physiology; D001921:Brain / Q000201:enzymology / Q000378:metabolism*; D002240:Carbohydrate Sequence; D004721:Endoplasmic Reticulum / Q000201:enzymology / Q000378:metabolism; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005964:Glucosyltransferases / Q000378:metabolism; D008861:Microsomes / Q000201:enzymology / Q000378:metabolism*; D008969:Molecular Sequence Data; D017830:Octoxynol / Q000378:metabolism; D009844:Oligosaccharides / Q000096:biosynthesis*; D011103:Polyisoprenyl Phosphate Monosaccharides / Q000378:metabolism*; D013552:Swine; D014357:Trypsin / Q000378:metabolism; D000520:alpha-Glucosidases / Q000378:metabolism*
D004791:Enzyme Inhibitors; D009844:Oligosaccharides; D011103:Polyisoprenyl Phosphate Monosaccharides; D017485:1-Deoxynojirimycin; C002968:dolichol-D-glucosylmonophosphate; D017830:Octoxynol; D005964:Glucosyltransferases; C033917:UDPglucose dolicholphosphate glucosyltransferase; C034251:4-nitrophenyl-alpha-glucosidase; C042059:glucosidase I; D000520:alpha-Glucosidases; D014357:Trypsin