Abstract
A highly specific, sensitive, and conveient fluorescence assay for α-1,2-mannosidases involved in glycoprotein processing reactions is described. The assay utilizes a coupled enzyme system to determine the amount of free mannose liberated from the disaccharide O-methyl-2- O-α- d-mannopyranosyl-α- d-mannopyranoside by the α-1,2-mannosidase. The assay was used to determine the substrate specificity of a calcium ion-activated α-1,2-mannosidase purified from rabbit liver microsomes. The microsomal mannosidase was specific for hydrolysis of the α-1,2 linkage. The mannosyl linkages in α-1,3- and α-1,6-linked methyl-disacchardes, in methyl-α- d-mannopyranoside, and in yeast mannan were hydrolyzed at rates of 2% or less than that noted with the α-1,2-linked disaccharide. Mannosidase activity was linear with time and was proportional to enzyme concentration. The K m for the α-1,2-linked methyl-disaccharide is 0.5 m m.
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