Galactosylsphingosine inhibition of the broad-specificity cytosolic beta-glucosidase of human liver.
Glucosylsphingosine is a potent inhibitor of lysosomal glucocerebrosidase and the broad-specificity, cytosolic beta-glucosidase of human liver. In the present study, it was demonstrated that the broad-specificity beta-glucosidase was also inhibited by galactosylsphingosine. The inhibition was observed when the enzyme was assayed for beta-glucosidase, beta-galactosidase, beta-xylosidase, and alpha-arabinosidase activities. Inhibition was of the mixed-type and the degree of inhibition depended on the substrate. For example, galactosylsphingosine was a more potent inhibitor of beta-glucosidase activity (I0.5 = 0.3 mM) than beta-xylosidase activity (I0.5 = 1.2 mM). In addition, the observation that the broad-specificity, cytosolic beta-glucosidase was inhibited by hydrophobic glycosphingolipids prompted the definition of a revised purification procedure which took advantage of hydrophobic affinity chromatography. This revised purification scheme employed Octyl-Sepharose and yielded the largest (68,000 Da) and most active (470,000 nmol h-1 mg protein-1) beta-glucosidase preparation yet described. The beta-glucosidase preparation contained 19% serine and 17% glycine, while 24% of the total amino acids were hydrophobic. The results of this study document the presence of a sphingolipid binding site on the broad-specificity beta-glucosidase. The implications of galactosylsphingosine inhibition of cytosolic beta-glucosidase and the possible role of the enzyme in glycosphingolipid metabolism are discussed.
D000596:Amino Acids / Q000032:analysis; D001665:Binding Sites; D055598:Chemical Phenomena; D002621:Chemistry; D002845:Chromatography / Q000379:methods; D003600:Cytosol / Q000201:enzymology; D005959:Glucosidases / Q000037:antagonists & inhibitors*; D006801:Humans; D007700:Kinetics; D008099:Liver / Q000201:enzymology*; D011609:Psychosine / Q000494:pharmacology*; D013110:Sphingosine / Q000031:analogs & derivatives*; D013379:Substrate Specificity; D001617:beta-Glucosidase / Q000037:antagonists & inhibitors* / Q000302:isolation & purification
D000596:Amino Acids; D011609:Psychosine; D005959:Glucosidases; D001617:beta-Glucosidase; D013110:Sphingosine