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 vul garis) leaves and uredospores of two different rust fungi (Uromyces phaseol i and Uromyces fabae) has been purified to electrophoretic homogeneity by a procedure which includes xanthine-agarose affinity chromatography as the m ain 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 tetrame rs consisting of identical or similar-sized subunits of 32-33 kDa. They als o exhibited similar optimum pH (around 9.0), showed hyperbolic kinetics wit h K-m values of 15-34 mu M and behaved similarly against a number of putati ve activators/inhibitors, all of them being inhibited only by oxonate and x anthine. 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 polycl onal antibodies from bean leaves and anti-uricase monoclonal antibodies fro m soybean nodules. Cross-reaction was observed between leaf uricase and nod ule monoclonal antibodies and between nodule enzyme and leaf polyclonal ant ibodies. These results confirm the homogeneity of plant uricases and demons trate that fungal obligate parasites have their own uricase, which is simil ar to the plant enzyme in many molecular and kinetic properties but differe nt in DEAE-cellulose binding characteristics and immunological properties. (C) 1999 Elsevier Science Ireland Ltd. All rights reserved.