Purification and characterization of a microsomal bile acid beta-glucosidase from human liver.
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.
Matern
H
H
Department of Internal Medicine III, Aachen University of Technology, D-52074 Aachen, Federal Republic of Germany.
Heinemann
H
H
Legler
G
G
Matern
S
S
D001647:Bile Acids and Salts / Q000378:metabolism*; D002635:Chenodeoxycholic Acid / Q000031:analogs & derivatives / Q000378:metabolism; D002846:Chromatography, Affinity; D003429:Cross Reactions; D003902:Detergents / Q000494:pharmacology; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005960:Glucosides / Q000378:metabolism*; D005962:Glucosylceramidase / Q000276:immunology; D006801:Humans; D008095:Lithocholic Acid / Q000031:analogs & derivatives / Q000378:metabolism; D008862:Microsomes, Liver / Q000201:enzymology*; D010743:Phospholipids / Q000494:pharmacology; D013379:Substrate Specificity; D001617:beta-Glucosidase / Q000037:antagonists & inhibitors / Q000276:immunology / Q000302:isolation & purification* / Q000378:metabolism
C105603:3-glucosido-lithocholic acid; D001647:Bile Acids and Salts; D003902:Detergents; D004791:Enzyme Inhibitors; D005960:Glucosides; D010743:Phospholipids; D002635:Chenodeoxycholic Acid; C105602:3-glucosido-chenodeoxycholic acid; D008095:Lithocholic Acid; D001617:beta-Glucosidase; D005962:Glucosylceramidase