Characterization of beta-glucosidase as a peripheral enzyme of lysosomal membranes from mouse liver and purification.
Lysosomal membrane fractions were prepared from lysosomes of mouse liver by freeze-thawing in a hypotonic buffer: 54% of beta-glucosidase [EC 3.2.1.45] in lysosomes was associated with the membrane fractions, whereas 96% of beta-glucuronidase [EC 3.2.1.31] was recovered in the soluble fractions of lysosomes. beta-glucosidase was solubilized by pH 9.5 treatment or by Triton treatment of membranes. The enzyme solubilized with alkali and concentrated with (NH4)2SO4 was rapidly inactivated in a solution of pH 9.5, but could be protected against inactivation by acidic detergent. Gel filtration analysis indicated that beta-glucosidase was in an aggregated form at neutral pH and could be disaggregated by alkali and detergents. The enzyme dissociated with detergents also showed a higher activity than the alkali-treated enzyme. These results suggested that beta-glucosidase is a peripheral enzyme bound to acidic lipids in membranes. beta-Glucosidase was purified to apparent homogeneity by (NH4)2SO4 fractionation and chromatographies with Sephacryl S-300, hydroxylapatite and cation exchangers in the presence of detergents. The catalytic activity of the purified enzyme was maximally stimulated by phosphatidylserine and heat-stable protein in the presence of a low concentration of Triton X-100. The stimulation was mainly due to an increase in Vmax.
D000818:Animals; D002458:Cell Fractionation; D002850:Chromatography, Gel; D005959:Glucosidases / Q000032:analysis*; D007425:Intracellular Membranes / Q000201:enzymology; D007700:Kinetics; D008099:Liver / Q000201:enzymology*; D008247:Lysosomes / Q000201:enzymology*; D051379:Mice; D008970:Molecular Weight; D010743:Phospholipids / Q000032:analysis; D012995:Solubility; D013379:Substrate Specificity; D013997:Time Factors; D001617:beta-Glucosidase / Q000032:analysis* / Q000302:isolation & purification
D010743:Phospholipids; D005959:Glucosidases; D001617:beta-Glucosidase