Isolation and characterization of uricase from bean leaves and its comparison with uredospore enzymes

Uricase (urate: oxygen oxidoreductase; EC 1.7.3.3) from bean (Phaseolus vulgaris) leaves and uredospores of two different rust fungi (Uromyces phaseoli and Uromyces fabae) has been purified to electrophoretic homogeneity by a procedure which includes xanthine-agarose affinity chromatography as the main step. Pure preparations had similar specific activities (2-6 U mg(-1)) with turnover numbers from 250 to 750 min(-1), and all enzymes were tetramers consisting of identical or similar-sized subunits of 32-33 kDa. They also exhibited similar optimum pH (around 9.0), showed hyperbolic kinetics with K-m values of 15-34 mu M and behaved similarly against a number of putative activators/inhibitors, all of them being inhibited only by oxonate and xanthine. However, leaf and uredospore uricases differed in the strength of binding to DEAE-cellulose since leaf uricase did not bind to the exchanger and that from U. fabae bound stronger than that of CT phaseoli. Uredospore uricases showed complete antigenic independence against anti-uricase polyclonal antibodies from bean leaves and anti-uricase monoclonal antibodies from soybean nodules. Cross-reaction was observed between leaf uricase and nodule monoclonal antibodies and between nodule enzyme and leaf polyclonal antibodies. These results confirm the homogeneity of plant uricases and demonstrate that fungal obligate parasites have their own uricase, which is similar to the plant enzyme in many molecular and kinetic properties but different in DEAE-cellulose binding characteristics and immunological properties. (C) 1999 Elsevier Science Ireland Ltd. All rights reserved.