Activation of dolichyl‐phospho‐mannose synthase by phospholipids

Dolichyl‐phospho‐mannose synthase, or GDPmannose:dolichyl‐phosphate mannosyltransferase (EC 2.4.1.83), was solubilized from rat liver microsomes with 1.0% Nonidet P‐40 and the enzyme was further purified by column chromatography on DEAE‐cellulose in the presence of 0.1 % Nonidet P‐40. The purified enzyme preparation (880‐fold over microsomes) was unstable in the presence of detergent and had no activity in the presence of Nonidet P‐40, Triton X‐100, octyl β‐glucoside, or deoxycholate. Detergent‐free enzyme was active in the presence of phosphatidylethanolamine (PtdEtn) and in the presence of phospholipid mixtures of PtdEtn and phosphatidylcholine (PtdCho) when the molar proportion of PtdCho was 70% or less. The enzyme was inactive in the presence of PtdCho alone. Unsaturated species of PtdEtn have a tendency to destabilize membrane bilayers [Cullis, P. R. & de Kruijff, B. (1978) Biochim. Biophys. Acta 507 , 207–218] and we have shown that dolichol promotes the destabilizing effect of PtdEtn on membranes composed of PtdCho and PtdEtn [Jensen, J. W. & Schutzbach, J. S. (1984) Biochemistry 23 , 1115–1119]. These results suggest that dolichyl‐ P ‐mannose synthase is optimally active in a phospholipid matrix that contains some component phospholipids that prefer non‐bilayer structural organization in isolation. Heat‐inactivation and sedimentation experiments demonstrated that the synthase associated with PtdEtn in the presence of dolichyl‐ P . The PtdEtn‐reconstituted enzyme catalyzed the reversible transfer of mannose from GDP‐mannose to dolichyl‐ P . The K m for GDP‐mannose was found to be 0.69 μM and the apparent K m for dolichyl‐ P was 0.3 μM. GMP, GDP, and GTP inhibited mannosyltransfer 50% at concentrations of 16 μM, 1.3 μM and 3 μM respectively.