Glycosyltransferase activities in liver mitochondria. Phospholipid-dependence of inner membrane mannosyltransferase.
The role of phospholipids in the activity of inner mitochondrial mannosyltransferase was investigated. This enzyme catalyzes the direct transfer from GDP-mannose to lipidic acceptor. Inner mitochondrial membranes from purified mice liver mitochondria are prepared by digitonin treatment. Swelling of mitoplasts leads to the formation of inner membrane vesicles, which are then purified on a discontinuous sucrose gradient. The validity of this fractionation procedure is controlled by measurements of specific enzymatic activities and by electron microscopy. Measurement of mannosyltransferase activity in native inner mitochondrial membranes is unsuccessful, even in the presence of exogenous dolichyl monophosphate. Treatment of inner membranes with specific phospholipid liposomes in the presence of exogenous dolichyl monophosphate is essential in order to measure this enzymatic activity. Addition of phosphatidylcholine, phosphatidylethanolamine, phosphatidylinositol and cardiolipin in the presence of Mg2+ results in a high degree of activation of the mannosyltransferase system. Maximal enzymatic activity is obtained with an approximate 3:7 mass ratio of exogenous phospholipid to inner membrane proteins. These experiments establish that sensitivity to activation by phospholipids is an inherent property of inner membrane mannosyltransferase. Another approach to this problem was to reconstitute an in vivo lipidic environment of the inner membrane. The results of this procedure suggest that the activity of inner mitochondrial mannosyltransferase may be subject to modulation by outer membrane lipidic extract treatment.
D000818:Animals; D002413:Cations, Divalent / Q000378:metabolism; D002458:Cell Fractionation / Q000379:methods; D002499:Centrifugation, Density Gradient; D002855:Chromatography, Thin Layer; D004789:Enzyme Activation; D006602:Hexosyltransferases / Q000378:metabolism*; D007425:Intracellular Membranes / Q000201:enzymology*; D008081:Liposomes / Q000378:metabolism; D008364:Mannosyltransferases / Q000378:metabolism*; D051379:Mice; D008930:Mitochondria, Liver / Q000201:enzymology*; D010743:Phospholipids / Q000502:physiology*
D002413:Cations, Divalent; D008081:Liposomes; D010743:Phospholipids; D006602:Hexosyltransferases; D008364:Mannosyltransferases