Galactosyltransferase activities in mitochondrial outer membrane: biosynthesis of dolichylmonophosphate-galactose

Mitochondrial outer membranes were prepared from mouse liver homogenates by swelling purified mitochondria in phosphate buffer and were purified on a discontinuous sucrose gradient. Assays for marker enzymes and controls in electron microscopy confirmed the purity and homogeneity of this subfraction. Mitochondrial outer membranes had significant galactosyltransferase activity when incubated with UDP-[14C]galactose: 14C-labelling was found in products extractable with organic solvents and in a residual precipitate. Addition of exogenous dolichylmonophosphate loaded into phosphatidylcholine liposomes strongly enhanced the incorporation of [14C]galactose into chloroform/methanol (2:1, v/v) -extractable products. Thin-layer chromatography of these 2:1 extracts showed that the increase of [14C]galactose incorporation was attributable to the synthesis of a new galactosylated lipid, 'lipid L'. This 'lipid L' has been purified on silicic acid columns by elution with chloroform/methanol (1:1, v/v). The purified 'lipid L' was labile in acid and released [14C]galactose. It had the same chromatographic behaviour as dolichylmonophosphate-mannose in neutral, acid and alkaline solvent systems. Upon incubation in presence of [3H]dolichylmonophosphate and UDP-[14C]galactose, purified 'lipid L' contained both 3H- and 14C-labelling. 'Lipid L', synthesized by mitochondrial outer membranes, was therefore characterized as dolichylmonophosphate-galactose.