Yeast dolichyl-phosphomannose synthase: reconstitution of enzyme activity with phospholipids.
The activity of purified recombinant yeast dolichyl-phosphomannose synthase (EC 2.4.1.83) was assessed following reconstitution of the enzyme with phospholipids. The yeast synthase, similar to the mammalian enzyme, was active when reconstituted with phosphatidylethanolamine dispersions but had little (less than 5%) activity in stable phosphatidylcholine bilayers. The enzyme was activated by adding increasing amounts of diacylglycerol to phospholipid bilayers, suggesting that activity of the yeast enzyme was dependent on lipid phase properties rather than specific phospholipids. The synthase could also be reconstituted as an active enzyme in bilayers prepared with a commercial crude lipid preparation containing 40% phosphatidylcholine, but at a rate 10% of that occurring in phosphatidylethanolamine. Vesicles composed of the 40% phosphatidylcholine lipid mixture, dolichyl phosphate, and enzyme were leaky in the presence of divalent cations, and dolichyl-phosphomannose synthase did not appear to catalyze the translocation of dolichyl phosphomannose across membranes at a catalytically significant rate under the assay conditions employed.
Schutzbach
J S
JS
Department of Microbiology, University of Alabama, Birmingham 35294.
Zimmerman
J W
JW
D001692:Biological Transport; D003902:Detergents; D004075:Diglycerides / Q000494:pharmacology; D004287:Dolichol Monophosphate Mannose / Q000378:metabolism*; D004288:Dolichol Phosphates / Q000378:metabolism; D004926:Escherichia coli; D005656:Fungal Proteins / Q000378:metabolism*; D008051:Lipid Bilayers; D008358:Mannose / Q000378:metabolism; D008364:Mannosyltransferases / Q000378:metabolism*; D010539:Permeability; D010713:Phosphatidylcholines; D010714:Phosphatidylethanolamines; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000201:enzymology*
D003902:Detergents; D004075:Diglycerides; D004288:Dolichol Phosphates; D005656:Fungal Proteins; D008051:Lipid Bilayers; D010713:Phosphatidylcholines; D010714:Phosphatidylethanolamines; D011993:Recombinant Fusion Proteins; D004287:Dolichol Monophosphate Mannose; D008364:Mannosyltransferases; C022755:dolichyl-phosphate beta-D-mannosyltransferase; D008358:Mannose