Abstract
Membranes from Penicillium charlesii were separated into 6 fractions by sucrose density gradient ultracentrifugation. The least dense fraction (ρ = 1.1 g cm^−3^) contained GDP‐mannose: glycopeptide mannosyltransferases that transferred [^14^C] mannose onto mannopyranosyl‐(seryl/threoyl)‐polypeptide and phosphogalactomannan regions of peptidophosphogalactomannan. Approximately 90% of the [^14^C] mannose incorporated was isolated as mannobiose following treatment of peptidophosphogalactomannan with 0.5 N NaOH. The remainder was located in phosphogalactomannan. About 10% of the membrane‐bound mannosyltransferase activity was solubilized with 1% Triton X‐100. The soluble mannosyltransferase activity was purified by affinity chromatography on peptidophosphogalactomannan‐Sepharose 4B and ammonium sulfate fractionation. Mannose incorporation was shown to be a function of the concentration of added acceptor. No incorporation occurred in the absence of added acceptor or when MgCl~2~ was substituted for MnCl~2~. Peptidophosphogalactomannan, phosphogalactomannan, phosphomannan, and mannan, each obtained by appropriate treatment of peptidophosphogalactomannan from P. charlesii, served as mannosyl acceptors. In contrast, α‐mannosidase treated peptidophosphogalactomannan did not serve an acceptor of mannosyl residues. Up to 70% of the mannose from GDP‐mannose was transferred to added acceptor. Treatment of [^14^C] mannosyl‐labeled peptidophosphogalactomannan with 0.5 N NaOH released 90% of the [^14^C] mannose as phosphogalactomannan and the remainder was released as mannobiose. [^14^C] Mannose‐labeled phosphogalactomannan was subjected to acetolysis. Mannobiose was the major [^14^C]‐labeled product isolated. Significant quantities of [^14^C] mannose were isolated also. These results show that soluble mannosyltransferase catalyzes the formation of (1–6)‐linked mannosyl residues as well as the transfer of a mannosyl residue to a (1–6)‐linked mannosyl residue in the phosphogalactomannan. The specificity of the enzyme is shown by its inability to catalyze mannosyl transfer to α‐mannosidase treated peptidophosphogalactomannan, or to incorporate more than 2 mannosyl residues onto the phosphogalactomannan region. Presumably the second mannosyl residue is attached by a (1–2) linkage as the mannan contains only (1–6)‐ and (1–2)‐linked mannosyl residues (Gander et al: J Biol Chem 249:2063, 1974). No evidence was obtained for the participation of a lipid‐linked mannosyl‐containing intermediate in this system.