URICASE FROM LEAVES - ITS PURIFICATION AND CHARACTERIZATION FROM 3 DIFFERENT HIGHER-PLANTS

Uricase (urate: oxygen oxidoreductase, EC 1.7.3.3) from leaves of chic kpea (Cicer arietimum L.), broad bean (Vicia faba major L.), and wheat (Triticum aestivum L.) has been purified to electrophoretic homogenei ty by a procedure which includes xan-, thine-agarose affinity chromato graphy as the main step. Purification factors of 74 000-83 000 and rec overies of 80-90% were achieved. Purified preparations had specific ac tivities between 600 and 800 nkat . mg protein(-1) (turnover numbers b etween 4400 and 6400 min(-1)). The three plant uricases were found by sodium dodecyl sulfate-polyacrylamide gel electrophoresis to be tetram ers of similar molecular mass (120-130 kDa) and to have identical or s imilar-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 mu M. All of them showed similar responses to putative activators/inhibit ors. Oxonate, xanthine and, to a lesser extent, neocuproin inhibited u ricase activity, whereas allantoin, ammonium, citrulline and glutamine did not. The three leaf uricases lacked catalase activity and were no t activated by cadaverine. None of the three plant enzymes cross-react ed with anti-uricase monoclonal antibodies from soybean nodules or ant i-uricase polyclonal antibodies from Chlamydomonas reinhardtii or rat liver. These results are consistent with the view that uricase in plan ts is probably a unique enzyme which is expressed at very low level in leaves.