Comparative studies on mannosylphosphoryl dolichol and glucosylphosphoryl dolichol synthases.
Factors affecting the synthesis of mannosylphosphoryl dolichol and glucosylphosphoryl dolichol hen oviduct microsomes were compared in order to gain insight into the properties of their respective synthases. A stabilized form of mannosylphosphoryl dolichol synthase, but not glucosylphosphoryl dolichol synthase, was released from microsomes by freezing the membranes after exposure to the detergent CHAPSO. The activation energy for mannosylphosphoryl dolichol synthesis in membranes was 9.4 glucosylphosphoryl dolichol synthesis in membranes had a similar activation energy, 8.1 kcal/mol, but below 18 degrees C the value was 16.7 kcal/mol. Tryptic digestion of sealed microsomes preferentially inactivated mannosylphosphoryl dolichol synthase; however, both synthases were equally inactivated in detergent-permeabilized microsomes. Periodate-oxidized UDP-Glc was used to probe the topological orientation of glucosylphosphoryl dolichol synthase in rat liver microsomes. Sealed microsomes treated with oxidized UDP-Glc were inactive in synthesis of glucosylphosphoryl dolichol. However, when these treated microsomes were permeabilized, glucosylphosphoryl dolichol synthase activity was readily detected. From these studies we conclude that although mannosyl- and glucosylphosphoryl dolichol synthases catalyze chemically similar reactions in the endoplasmic reticulum, they differ in several respects. These differences were interpreted in terms of a topological model in which the active sites of the two enzymes reside on opposite faces of the endoplasmic reticulum, with that of the glucosyl lipid synthase facing the lumen and that of the mannosyl lipid synthase facing the cytosol.
Clarke
B L
BL
Department of Physiology and Biophysics, University of Alabama, Birmingham 35294.
Naylor
C
C
Lennarz
W J
WJ
D000818:Animals; D002645:Chickens; D004721:Endoplasmic Reticulum / Q000201:enzymology; D005260:Female; D005964:Glucosyltransferases / Q000037:antagonists & inhibitors / Q000378:metabolism*; D006602:Hexosyltransferases / Q000378:metabolism*; D066298:In Vitro Techniques; D008364:Mannosyltransferases / Q000037:antagonists & inhibitors / Q000378:metabolism*; D008861:Microsomes / Q000201:enzymology; D008954:Models, Biological; D010057:Oviducts / Q000201:enzymology; D051381:Rats; D013696:Temperature; D014357:Trypsin / Q000494:pharmacology
D005964:Glucosyltransferases; D006602:Hexosyltransferases; D008364:Mannosyltransferases; C033917:UDPglucose dolicholphosphate glucosyltransferase; C022755:dolichyl-phosphate beta-D-mannosyltransferase; D014357:Trypsin