Purification and properties of the membranal thiol oxidase from porcine kidney.
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 Km was 0.73 mM and the V max was a 4.4 U/mg protein. The reaction catalyzed was 2 RSH + O2----RSSR + H2O2, 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 high-pressure gel permeation column the protein eluted at 70 kDa under dilute conditions but at greater than 200 kDa at high concentrations, indicating that the protein also aggregates into larger multimers. Activity was inhibited by copper chelators, L-(alpha S,5S)-alpha-amino-3-chloro-4,5-dihydro-5-isoxazoleacetic acid (acivicin), H2O2, 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 CuSO4, but not with FeSO4. 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.
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D000818:Animals; D002462:Cell Membrane / Q000201:enzymology; D002845:Chromatography / Q000379:methods; D003300:Copper / Q000032:analysis; D004578:Electron Spin Resonance Spectroscopy; D005978:Glutathione / Q000378:metabolism; D007672:Kidney Cortex / Q000201:enzymology*; D007700:Kinetics; D008970:Molecular Weight; D010084:Oxidation-Reduction; D010088:Oxidoreductases / Q000037:antagonists & inhibitors / Q000302:isolation & purification* / Q000378:metabolism; D013379:Substrate Specificity; D013552:Swine; D013997:Time Factors; D005723:gamma-Glutamyltransferase / Q000302:isolation & purification
D003300:Copper; D010088:Oxidoreductases; C020667:glutathione oxidase; D005723:gamma-Glutamyltransferase; D005978:Glutathione