Partial Purification and Characterization of Naegleria fowleriβ‐Glucosidase

SIDDHARTHA DAS, ASISH K. SAHA, THOMAS A. NERAD, A. JULIO MARTINEZ, KAREN L. LAMARCO, ALAKANANDA BASU, GUNTER LEGLER, ROBERT H. GLEW

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Abstract

Naegleria fowleri cells, grown axenically, contain high levels of β‐D‐glucosidase which catalyzes the hydrolysis of 4‐methylumbelliferyl‐β‐D‐glucopyranoside (4MUGlc) (Km, 0.9 mM), octyl‐β‐D‐glucoside (Km, 0.17 mM), and p‐nitrophenyl‐β‐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 β‐glucosidase activity appears in the supernatant fraction. The β‐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 β‐D‐galactosidase activity in the Naegleria extract copurifies with the β‐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 Naegleriaβ‐D‐glucosidase has an apparent molecular weight of 66,000, a Stokes radius of 25 Å, and a sedimentation coefficient of 4.2S. The β‐glucosidase is not inhibited by conduritol β‐epoxide or galactosylsphingosine but is completely inhibited by 1.25 mM bromo conduritol β‐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 β‐glucosidase in the metabolism of the ameba and its possible role in pathogenic mechanisms are discussed.

Original languageEnglish
Pages (from-to)68-74
Number of pages7
JournalThe Journal of Protozoology
Volume34
Issue number1
DOIs
StatePublished - Feb 1987

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    DAS, SIDDHARTHA., SAHA, ASISH. K., NERAD, THOMAS. A., MARTINEZ, A. JULIO., LAMARCO, KAREN. L., BASU, ALAKANANDA., LEGLER, GUNTER., & GLEW, ROBERT. H. (1987). Partial Purification and Characterization of Naegleria fowleriβ‐Glucosidase. The Journal of Protozoology, 34(1), 68-74. https://doi.org/10.1111/j.1550-7408.1987.tb03134.x