A fluorescence assay for alpha-1,2-mannosidases involved in glycoprotein processing reactions.
A highly specific, sensitive, and convenient fluorescence assay for alpha-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-alpha-D-mannopyranosyl-alpha-D-mannopyranoside by the alpha-1,2-mannosidase. The assay was used to determine the substrate specificity of a calcium ion-activated alpha-1,2-mannosidase purified from rabbit liver microsomes. The microsomal mannosidase was specific for hydrolysis of the alpha-1,2 linkage. The mannosyl linkages in alpha-1,3- and alpha-1,6-linked methyl-disaccharides, in methyl-alpha-D-mannopyranoside, and in yeast mannan were hydrolyzed at rates of 2% or less than that noted with the alpha-1,2-linked disaccharide. Mannosidase activity was linear with time and was proportional to enzyme concentration. The Km for the alpha-1,2-linked methyl-disaccharide is 0.5 mM.
Department of Microbiology, University of Alabama, Birmingham 35294.
J S
JS
Schutzbach
D000818:Animals; D006023:Glycoproteins / Q000378:metabolism; D007700:Kinetics; D008361:Mannosidases / Q000032:analysis*; D008862:Microsomes, Liver / Q000201:enzymology; D011817:Rabbits; D013050:Spectrometry, Fluorescence; D043323:alpha-Mannosidase
D006023:Glycoproteins; D008361:Mannosidases; D043323:alpha-Mannosidase