Partial purification of a GTP‐insensitive (1 → 3)‐β‐glucan synthase from Phytophthora sojae

FEBS Letters

Ebel, Jürgen¹  Hertkorn, Norbert²  Antelo, Luis¹  Cosio, Eric G.³ 

[1] Botanisches Institut der Universität, Menzinger Strasse 67, D-80638 Munich, Germany;Institut für Ökologische Chemie, GSF-Forschungszentrum für Umwwelt und Gesundheit, Ingolstädter Landstrasse 1, D-85764 Neuherberg, Germany;Seccion Química, Pontificia Universidad Católica del Perú, Apartado Postal 1761, Lima 100, Peru

关键词: Cell wall synthesis;    (1 → 3)-β-Glucan synthase;    Product entrapment;    Phytophthora sojae;    CHAPS;    3-[(3-cholamidopropyl)dimethylammonio]-1-propane sulfonate;    HPTLC;    high performance thin layer chromatography;    Zwittergent 3-12;    N-dodecyl-N;    N-dimethyl-3-ammonio-1-propane sulfonate;   

DOI  :  10.1016/S0014-5793(98)00904-1

学科分类：生物化学/生物物理

来源: John Wiley & Sons Ltd.

【 摘 要 】

A (1 → 3)-β-glucan synthase activity was identified in cell membrane preparations from the oomycete Phytophthora sojae, a soybean pathogen. The activity could be solubilized using the zwitterionic detergent CHAPS at relatively low concentrations (3 mg/ml). High salt concentrations were not effective in removing the activity from the membranes. Detergent solubilization of the enzyme resulted in a six-fold increase of calculated Vmax values (2.5 vs. 0.4 nkat/mg protein) but only minor alteration of the K _m (10.6 vs. 10.7 mM). Analysis of the reaction product of the solubilized enzyme by enzymatic degradation and by 2D NMR spectroscopy confirmed its identity as a linear high molecular weight (1 → 3)-β-glucan. Glucan synthase activity in both membrane and solubilized preparations was not activated by GTP or divalent cations as reported for other fungal or plant glucan synthases, The activity was inhibited, as expected, in a competitive manner by UDP with a K _i of 2.9 mM. Partial purification of the enzyme was achieved by anion exchange chromatography followed by product entrapment. This procedure resulted in the selective enrichment of a protein band with apparent M _r 108 000 in SDS-PAGE which was not visible in any of the steps preceding product entrapment. The glucan pellets from product entrapment contained up to 3% of the initial enzyme activity present in the fraction used for the procedure.

【 授权许可】

Unknown