PURIFICATION AND CHARACTERIZATION OF A MICROSOMAL BILE-ACID BETA-GLUCOSIDASE FROM HUMAN LIVER

A human liver microsomal beta-glucosidase has been purified to apparen t homogeneity in sodium dodecyl sulfate-polyacrylamide gel electrophor esis where a single protein band of M-r 100,000 was obtained under red ucing conditions, The enzyme was enriched about 73,000 fold over start ing microsomal membranes by polyethylene glycol fractionation, anion e xchange chromatographies on DEAE-Trisacryl, and Mono Q followed by aff inity chromatography on N-(9-carboxynonyl)-1-deoxynojirimycin-AH-Sepha rose 4B. The purified enzyme had a pH optimum between 5.0 and 6.4, was activated by divalent metal ions, and required phospholipids for exhi bition of activity, The enzyme catalyzed the hydrolysis of 3 beta-D-gl ucosido-lithocholic and 3 beta-D-glucosido-chenodeoxycholic acids with high affinity (K-m, 1.7 and 6.2 mu M, respectively) and of the beta-D -glucoside (K-m, 210 mu M) and the beta-D-galactoside of 4-methylumbel liferone. The ratio of relative reaction rates for these substrates wa s about 6:3:11:1, No activity was detectable toward 6 beta-D-glucosido -hyodeoxycholic acid, glucocerebroside, and the following glycosides o f 4-methylumbelliferone: alpha-D-glucoside, alpha-L-arabinoside, beta- D-fucoside or beta-D-xyloside. Immunoinhibition and immunoprecipitatio n studies using antibodies prepared against lysosomal glucocerebrosida se showed no cross-reactivity with microsomal beta-glucosidase suggest ing that these two enzymes are antigenically unrelated.