Purification and properties of the membranal thiol oxidase from porcine kidney

Abstract

A thiol oxidase was purified from porcine kidney cortex by chromatography of detergent-solubilized plasma membranes on cysteinylsuccinamidopropyl-glass beads, hydroxyapatite, and Sephacryl S-200. The oxidase was purified 2600-fold; 28% recovery of activity was obtained. With glutathione as substrate, the apparent K m was 0.73 m m and the V max was a 4.4 U/mg protein. The reaction catalyzed was 2 RSH + O 2 → RSSR + H 2O 2, and superoxide production was not detected during the reaction. Other low molecular weight thiols, including cysteine, dithiothreitol, N-acetylcysteine, and cysteamine, were substrates for the oxidase; 2-mercaptoethanol, reductively denatured ribonuclease A, and chymotrypsinogen A were not substrates. Analysis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed one band corresponding to 70 kDa; gel filtration on a Sephacryl column produced a single elution of activity with a protein corresponding to 120 kDa, indicating that the functional form is a dimer. On a highpressure gel permeation column the protein eluted at 70 kDa under dilute conditions but at >200 kDa at high concentrations, indicating that the protein also aggregates into larger multimers. Activity was inhibited by copper chelators, l-(α S,5 S)-α-amino-3-chloro-4, 5-dihydro-5-isoxazoleacetic acid (acivicin), H 2O 2, and N-ethylmaleimide, suggesting the presence of copper and a sulfhydryl group at the active site. Following treatment with metal chelators, enzyme activity was reconstituted with CuSO 4, but not with FeSO 4. The purified enzyme contained 1 mol copper per subunit which was undetectable by electron paramagnetic resonance, suggesting that the copper is in a binuclear complex.