Partial purification and characterization of Naegleria fowleri beta-glucosidase.
Naegleria fowleri cells, grown axenically, contain high levels of beta-D-glucosidase which catalyzes the hydrolysis of 4-methylumbelliferyl-beta-D-glucopyranoside (4MUGlc) (Km, 0.9 mM), octyl-beta-D-glucoside (Km, 0.17 mM), and p-nitrophenyl-beta-D-glucopyranoside at relative rates of 1.00, 2.88, and 1.16, respectively (substrate concentration, 3.0 mM). When the amebae are subjected to freeze-thawing, sonication, and centrifugation (100,000 g, 1 h), 85% of the beta-glucosidase activity appears in the supernatant fraction. The beta-glucosidase was purified 40-fold (34% yield) using a combination of chromatographic steps involving DE-52 cellulose, concanavalin A-Sepharose, and hydroxylapatite followed by isoelectric focusing. The predominant soluble beta-D-galactosidase activity in the Naegleria extract copurifies with the beta-D-glucosidase; the two activities have the same isoelectric point (pI, 6.9), similar heat stabilities, are both inhibited by lactobionic acid (Ki, 0.40 mM), and exhibit optima at pH 4.5, indicating that they are probably the same enzyme. The Naegleria beta-D-glucosidase has an apparent molecular weight of 66,000, a Stokes radius of 25 A, and a sedimentation coefficient of 4.2S. The beta-glucosidase is not inhibited by conduritol beta-epoxide or galactosylsphingosine but is completely inhibited by 1.25 mM bromo conduritol beta-epoxide. The latter compound, when present in the growth medium, inhibits the growth of the organism and profoundly alters its ultrastructure, the main effect being the apparent inhibition of cytokinesis and the generation of multinucleate cells. The issue of the role of the beta-glucosidase in the metabolism of the ameba and its possible role in pathogenic mechanisms are discussed.
D000656:Amoeba / Q000187:drug effects / Q000201:enzymology* / Q000254:growth & development / Q000648:ultrastructure; D000818:Animals; D002850:Chromatography, Gel; D004187:Disaccharides / Q000494:pharmacology; D005959:Glucosidases / Q000302:isolation & purification*; D006358:Hot Temperature; D006863:Hydrogen-Ion Concentration; D007294:Inositol / Q000031:analogs & derivatives / Q000494:pharmacology; D007525:Isoelectric Focusing; D007700:Kinetics; D008854:Microscopy, Electron; D013379:Substrate Specificity; D001617:beta-Glucosidase / Q000037:antagonists & inhibitors / Q000302:isolation & purification* / Q000378:metabolism
D004187:Disaccharides; D007294:Inositol; C005608:lactobionic acid; D005959:Glucosidases; D001617:beta-Glucosidase; C011423:conduritol epoxide