Purification and molecular properties of urate oxidase from Chlamydomonas reinhardtii.
Urate oxidase (urate: oxygen oxidoreductase, EC 1.7.3.3) from the unicellular green alga Chlamydomonas reinhardtii has been purified to electrophoretic and immunological homogeneity by a procedure which includes as main steps ammonium sulfate fractionation, gel filtration, ion exchange and xanthine-agarose affinity chromatography. The native enzyme has a relative molecular mass (Mr) of 124,000 and consists of four identical or similar-sized subunits of Mr 31,000 each. The enzyme has a Stokes's radius of 3.87 nm, a sedimentation coefficient of 6.8 S and an f/f0 of 1.23, and exhibits its maximal absorption at 276 nm. Optimum pH was 8.5 and maximum activity was shown at 40 degrees C, with an activation energy of 53 kJ.mol-1 and a Q10 of 1.96. Absorption spectrum of native reduced enzyme showed two transient maxima at 392 and 570 nm, very similar to those of metal-urate complexes, which disappeared in the presence of cyanide. Inhibition by cyanide and neocuproin, but not by salicylhydroxamic acid, strongly suggests that copper is the metal involved in enzymatic urate oxidation. By using a sensitive photokinetic method for copper determination, a content of 4 mol of copper per mol of enzyme has been found.
Alamillo
J M
JM
Departamento Bioquímica, Biología Molecular y Fisiología, Facultad de Ciencias, Universidad de Córdoba, Spain.
Cárdenas
J
J
Pineda
M
M
D002151:Calorimetry; D002696:Chlamydomonas / Q000201:enzymology*; D002846:Chromatography, Affinity / Q000379:methods; D002850:Chromatography, Gel / Q000379:methods; D002852:Chromatography, Ion Exchange / Q000379:methods; D007700:Kinetics; D046911:Macromolecular Substances; D008970:Molecular Weight; D014503:Urate Oxidase / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism
D046911:Macromolecular Substances; D014503:Urate Oxidase