Purification and characterization of a microsomal bile acid beta-glucosidase from human liver

• Published in: The Journal of biological chemistry Vol. 272; no. 17; pp. 11261 - 11267
• Main Authors: Matern, H, Heinemann, H, Legler, G, Matern, S
• Format: Journal Article
• Language: English
• Published: United States 25.04.1997
• Subjects: beta-Glucosidase - antagonists & inhibitors; beta-Glucosidase - immunology; beta-Glucosidase - isolation & purification; beta-Glucosidase - metabolism; Bile Acids and Salts - metabolism; Chenodeoxycholic Acid - analogs & derivatives; Chenodeoxycholic Acid - metabolism; Chromatography, Affinity; Cross Reactions; Detergents - pharmacology; Enzyme Inhibitors - pharmacology; Glucosides - metabolism; Glucosylceramidase - immunology; Humans; Lithocholic Acid - analogs & derivatives; Lithocholic Acid - metabolism; Microsomes, Liver - enzymology; Phospholipids - pharmacology; Substrate Specificity
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1074/jbc.272.17.11261

A human liver microsomal beta-glucosidase has been purified to apparent homogeneity in sodium dodecyl sulfate-polyacrylamide gel electrophoresis where a single protein band of Mr 100,000 was obtained under reducing conditions. The enzyme was enriched about 73, 000-fold over starting microsomal membranes by polyethylene glycol fractionation, anion exchange chromatographies on DEAE-Trisacryl, and Mono Q followed by affinity chromatography on N-(9-carboxynonyl)-1-deoxynojirimycin-AH-Sepharose 4B. The purified enzyme had a pH optimum between 5.0 and 6.4, was activated by divalent metal ions, and required phospholipids for exhibition of activity. The enzyme catalyzed the hydrolysis of 3beta-D-glucosido-lithocholic and 3beta-D-glucosido-chenodeoxycholic acids with high affinity (Km, 1.7 and 6.2 microM, respectively) and of the beta-D-glucoside (Km, 210 microM) and the beta-D-galactoside of 4-methylumbelliferone. The ratio of relative reaction rates for these substrates was about 6:3:11:1. No activity was detectable toward 6beta-D-glucosido-hyodeoxycholic acid, glucocerebroside, and the following glycosides of 4-methylumbelliferone: alpha-D-glucoside, alpha-L-arabinoside, beta-D-fucoside or beta-D-xyloside. Immunoinhibition and immunoprecipitation studies using antibodies prepared against lysosomal glucocerebrosidase showed no cross-reactivity with microsomal beta-glucosidase suggesting that these two enzymes are antigenically unrelated.