Partial Purification and Characterization of Naegleria fowleriβ‐Glucosidase1

Publisher Website
Google Scholar
Abstract
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 amoebae 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 .ANG., and a sedimentation coefficient of 4.2S. The .beta.-glucosidase is not inhibited by conduritol .beta.-exposure 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 it''s 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 amoeba and its possible role in pathogenic mechanisms are discussed.

This publication has 28 references indexed in Scilit: