Isolation and Identification of a Protein with Capsaicin-Inhibited NADH Oxidase Activity from Culture Media Conditioned by Growth of HeLa Cells

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 EC50of about 5 nMfor 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 with capsaicin-inhibited NADH activity from total defined culture media conditioned by growth of HeLa cells. The NADH oxidase activity of both the soluble and the plasma membrane-associated form of the activity was inhibited by antisera corresponding to the 33.5-kDa protein. The antisera also immunoprecipitated and reacted on Western blots with both the soluble (33.5 kDa) and plasma membrane (34 kDa)-associated forms of the capsaicin-inhibited activity. The results identify the capsaicin-inhibited NADH oxidase of the conditioned media of HeLa cells as being a ca. 33.5-kDa shed form of the previously reported capsaicin-inhibited NADH oxidase of the HeLa cell plasma membrane.