Isolation and identification of a protein with capsaicin-inhibited NADH oxidase activity from culture media conditioned by growth of HeLa cells

Archives of Biochemistry and Biophysics
F E WilkinsonD J Morré

Abstract

A ca. 33.5-kDa protein has been identified as a soluble NADH oxidase activity of culture media conditioned by growth of HeLa cells. The protein appears to be derived from a 34-kDa protein of the HeLa plasma membrane. Both proteins are characterized by an ability to oxidize NADH in the absence of exogenous electron acceptors. The activity is inhibited by 8-methyl-N-vanillyl-6-noneamide (capsaicin). The soluble and the plasma membrane forms of the activity exhibit a similar EC50 of about 5 nM for inhibition of the activity by capsaicin. The activity was purified from culture media conditioned by growth of HeLa cells using DEAE ion exchange chromatography, G-200 size exclusion chromatography, and preparative SDS-PAGE. Purification was monitored on the basis of the capsaicin-inhibited oxidation of NADH, including the final electrophoretic purification. Activity was restored following SDS-PAGE by reduction with dithiothreitol or reduced glutathione in the presence of NADH followed by the addition of 0.03% hydrogen peroxide and preincubation in the presence of NADH for 5-15 min. For affinity purification, the vanillylamine portion of capsaicin was linked to agarose. The agarose-linked vanillylamine bound a ca. 33.5-kDa protein band w...Continue Reading

Citations

Mar 7, 2001·Antioxidants & Redox Signaling·P J Chueh
Sep 4, 2010·Antioxidants & Redox Signaling·Domenico Del PrincipeMaria Valeria Catani
Sep 29, 1998·Archives of Biochemistry and Biophysics·A del Castillo-OlivaresD J Morré

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