Evidence for the biosynthesis of mannosylphosphoryldolichol and N-acetylglucosaminylpyrophosphoryldolichol by an axolemma-enriched membrane preparation from bovine white matter.

Abstract

AbstractAn axolemma‐enriched membrane fraction prepared by an improved procedure from bovine white matter catalyzes the enzymatic transfer of [14C]mannose and N‐acetyl[14C]glucosamine from their nucleotide derivatives into a mannolipid and an N‐acetylglucosaminyl lipid in the presence of exogenous dolichyl monophosphate. The labeled glycolipid products have the chemical and chromatographic characteristics of mannosylphosphoryldolichol and N‐acetylglucosaminylpyrophosphoryldolichol. The initial rates of synthesis of the glycolipids by the axolemma‐enriched membrane fraction have been compared with the initial rates of glycolipid formation catalyzed by a microsomal preparation and myelin in the presence or absence of dolichyl monophosphate. Essentially no glycolipid synthesis was observed when either GDP‐[14C]mannose or UDP‐N‐acetyl[14C]glucosamine were incubated with myelin in the presence or absence of exogenous dolichyl monophosphate. A comparison of the initial rates of synthesis of the glycolipids using endogenous acceptor lipid revealed that the rate of formation of mannolipid was 7 times faster for the microsomal membranes than the axolemma‐enriched membranes. In the presence of an amount of dolichyl monophosphate approaching saturation the initial rate of glycolipid synthesis was markedly enhanced for both membrane preparations. However, due to a more dramatic enhancement in the axolemma‐enriched membranes the initial rate of mannolipid synthesis was only approx. 2.5 times greater in the microsomal membranes. A similar observation was made when the initial rates of N‐acetylglucosaminyl lipid synthesis were compared for axolemma‐enriched and microsomal preparations in the presence and absence of exogenous dolichyl monophosphate. These studies indicate that the axolemma‐enriched membranes have a relatively lower content of dolichyl monophosphate than the microsomal membranes although the difference in the amount of mannosyltransferase is only two to three‐fold lower.The presence of a sugar nucleotide pyrophosphatase activity capable of degrading GDP‐mannose and UDP‐N‐acetylglucosamine has also been demonstrated in the axolemma‐enriched membrane fraction.