Properties of the dolichol phosphate: GDPmannose mannosyltransferase of liver microsomes

The reaction of GDP[14C]-mannose with dolichol phosphate (Dol-P) in hepatic microsomes is characterized by an initial brief period of relatively rapid Dol-P-[14C]-mannose synthesis. The time course of this 1--3 min period of rapid synthesis follows approximate first order kinetics. However, the rate of reaction does not decrease to zero as predicted by the kinetics of the initial period of synthesis, but continues instead at a slow, steadily decreasing, rate. Examination of the time course of Dol-P-mannose synthesis for different concentrations of GDP[14C]-mannose revealed that the extrapolated final level of Dol-P-mannose synthesized is increased when the concentration of GDPmannose is raised. These data, plus those derived from studies of the reverse reaction, suggest that the non-linear time course for the synthesis of Dol-P-mannose is due in part to the reaction approaching equilibrium between the forward and reverse reactions. The effects of Mn++ on the time course of the forward and reverse reaction are complex and suggest that the Mn++ complexes of both GDPmannose and GDP are poorer substrates for the enzyme than the free nucleotides. Perturbations of the lipid environment of the microsomal membrane by treatment with phospholipase A, detergent, sonication, or alkaline pH lead to a decrease in the final level of Dol-P-mannose synthesized, but do not affect the time required for half maximal labeling. When the reverse reaction was investigated in phospholipase A-treated microsomes, the final extent of the reaction was also reduced. These data suggest that perturbation of the membrane lipid environment decreases in some undefined way the availability of Dol-P and Dol-P-mannose to enzyme.