Purification and properties of a mannosyltransferase solubilized from mitochondrial outer membranes.
The enzyme GDPmannose: dolichyl monophosphate mannosyltransferase has been solubilized and purified from mice liver mitochondrial outer membranes. The purification combines detergent extraction of purified outer membranes using Nonidet P-40, with subsequent ion-exchange chromatography on DEAE-cellulose. At this stage, a 400-fold purification is obtained. The partially purified mannosyltransferase is activated by choline-containing lipids such as phosphatidylcholine, lysophosphatidylcholine and sphingomyelin. The reaction is dependent upon the addition of exogenous dolichyl monophosphate. The sole reaction product has been identified as dolichyl phosphate-mannose. The partially purified mannosyltransferase exhibits a Km of 1.33 microM for GDPmannose. Enzyme activity, eluted from DEAE-cellulose, could be further purified after incorporation into sphingomyelin vesicles containing dolichyl monophosphate followed by a sucrose density gradient centrifugation. The mannosyltransferase activity is completely associated with the liposomes at the top of the gradient. Significant stabilization and purification (approx. 1600-fold) of enzyme activity associated with these liposomes is obtained. Furthermore, the reconstitution of this purified enzyme within specific liposomes provides a good model membrane to investigate the molecular requirement of this mitochondrial mannosyltransferase.
D000818:Animals; D002499:Centrifugation, Density Gradient; D002848:Chromatography, DEAE-Cellulose; D002852:Chromatography, Ion Exchange; D003902:Detergents; D004288:Dolichol Phosphates / Q000494:pharmacology; D006602:Hexosyltransferases / Q000302:isolation & purification*; D008081:Liposomes / Q000302:isolation & purification; D008364:Mannosyltransferases / Q000302:isolation & purification*; D051379:Mice; D008930:Mitochondria, Liver / Q000201:enzymology*; D010743:Phospholipids / Q000494:pharmacology; D012995:Solubility
D003902:Detergents; D004288:Dolichol Phosphates; D008081:Liposomes; D010743:Phospholipids; C028306:dolichol monophosphate; D006602:Hexosyltransferases; D008364:Mannosyltransferases; C022755:dolichyl-phosphate beta-D-mannosyltransferase