Partial purification of a mannosyltransferase involved in the O-mannosylation of glycoproteins from Saccharomyces cerevisiae.
The mannosyltransferase that catalyses the transfer of mannose from dolichyl-phosphate-mannose (Dol-P-Man) to the hydroxyl group of serine/threonine residues in the acceptor peptide (Tyr-Asn-Pro-Thr-Ser-Val) was partially purified approximately 150-fold from the microsomal membrane fraction of Saccharomyces cerevisiae. The membrane-bound enzyme was solubilized with 0.5% Triton X-100 at a protein:detergent ratio of 2:1, and was then purified by ion-exchange chromatography on DEAE-cellulose, followed by hydroxyapatite column chromatography. The partially purified enzyme had a pH optimum of 7.2 and required Mg2+ at an optimum concentration of 10 mM for activity. The apparent mol. wt of the enzyme, as estimated by gel filtration on Sephacryl S-300, was approximately 125 kDa. The activity of the partially purified enzyme was greatly stimulated by phosphatidylcholine (PC), while other naturally occurring phosphoglycerides had no significant effect. The extent of activation of mannosyltransferase activity was greatly affected by the number of carbons and the degree of saturation/unsaturation of the fatty acid substituents, as well as by their position on the glycerol moiety of the PC molecule. Maximum stimulation of the mannosyltransferase activity was induced by a PC derivative in which both sn-1 and sn-2 positions on the glycerol moiety were occupied by C12:0 fatty acids. In general, mannosyltransferase was found to exhibit greater specificity for the L-alpha-PC derivatives in which the sn-2 position of the glycerol contained a saturated fatty acid.(ABSTRACT TRUNCATED AT 250 WORDS)
Sharma
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Department of Biochemistry, University of Texas Health Science Center, San Antonio 78284.
D'Souza
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C
Elbein
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D000595:Amino Acid Sequence; D004287:Dolichol Monophosphate Mannose / Q000378:metabolism; D007700:Kinetics; D008274:Magnesium / Q000494:pharmacology; D008364:Mannosyltransferases / Q000187:drug effects / Q000302:isolation & purification*; D008969:Molecular Sequence Data; D009842:Oligopeptides / Q000378:metabolism; D010713:Phosphatidylcholines / Q000494:pharmacology; D010743:Phospholipids / Q000494:pharmacology; D012441:Saccharomyces cerevisiae / Q000201:enzymology*; D012694:Serine / Q000378:metabolism; D013379:Substrate Specificity; D013912:Threonine / Q000378:metabolism
D009842:Oligopeptides; D010713:Phosphatidylcholines; D010743:Phospholipids; D013912:Threonine; D012694:Serine; D004287:Dolichol Monophosphate Mannose; D008364:Mannosyltransferases; D008274:Magnesium