Formation of alpha-1,2-mannosyl-mannose by an enzyme preparation from rabbit liver

Abstract

Rabbit liver microsomes catalyzed the formation of 2-O-alpha-D-mannosyl-D-mannose when incubated with GDP-mannose and free D-mannose. Formation of the disaccharide required the addition of divalent metal ions and was inhibited by EDTA and guanosine nucleotides. An enzyme present in the microsomal preparation transferred mannose from GDP-mannose to an endogenous lipid and to exogenous dolichol-P resulting in the formation of a product tentatively identified as dolichol-P-mannose. Formation of the mannosyl-lipid was also metal ion-dependent and was inhibited by EDTA and guanosine nucleotides. A second enzyme in the preparation catalyzed mannosyl transfer from dolichol-P-mannose to free mannose resulting in the formation of 2-O-alpha-D-mannosyl-D-mannose. Transfer from the mannosyl-lipid required the addition of Triton X-100 but was metal ion-independent. The time course of the transfer of mannose from GDP-mannose to the endogenous lipid and to the exogenous saccharide acceptor suggested that the mannosyl-lipid was an intermediate in the formation of the mannobiose. Other evidence, such as similar requirements for divalent metals, inhibition by guanosine nucleotides, and acceptor specificity, also supported the involvement of a mannosyl-lipid as an intermediate in the formation of alpha-1,2-mannosyl-mannose linkages by rabbit liver microsomes.