Uricase from leaves: its purification and characterization from three different higher plants

Abstract.

Uricase (urate: oxygen oxidoreductase, EC␣1.7.3.3) from leaves of chickpea (Cicer arietimum L.), broad bean (Vicia faba major L.), and wheat (Triticum aestivum L.) has been purified to electrophoretic homogeneity by a procedure which includes xanthine-agarose affinity chromatography as the main step. Purification factors of 74 000–83 000 and recoveries of 80–90% were achieved. Purified preparations had specific activities between 600 and 800 nkat · mg protein⁻¹ (turnover numbers between 4400 and 6400 min⁻¹). The three plant uricases were found by sodium dodecyl sulfate-polyacrylamide gel electrophoresis to be tetramers of similar molecular mass (120–130 kDa) and to have identical or similar-sized subunits (32–34 kDa). They also had a similar optimum pH (9–9.5) and showed a hyperbolic kinetics with K _m values from 9–24 μM. All of them showed similar responses to putative activators/inhibitors. Oxonate, xanthine and, to a lesser extent, neocuproin inhibited uricase activity, whereas allantoin, ammonium, citrulline and glutamine did not. The three leaf uricases lacked catalase activity and were not activated by cadaverine. None of the three plant enzymes cross-reacted with anti-uricase monoclonal antibodies from soybean nodules or anti-uricase polyclonal antibodies from Chlamydomonas reinhardtii or rat liver. These results are consistent with the view that uricase in plants is probably a unique enzyme which is expressed at very low level in leaves.