Isolation and characterization of a tumor-associated NADH oxidase (tNOX) from the HeLa cell surface

Cell-surface-located, drug-responsive and tumor-associated NADH oxidase (tN OX) proteins were purified and characterized from HeLa cells. The proteins isolated exhibited NADH oxidase activity inhibited by capsaicin and were re sistant to heating and to protease digestion. The activity was purified 200 - to 500-fold to provide apparently homogeneous gel bands for N-terminal se quencing using three different protocols, AU three protocols involved heat (50 degreesC) and proteinase K treatment. Recovery of the total NADH oxidas e activity was 86% and inhibition by capsaicin was 60 to 80%, After 450-fol d purification, a 52-kDa component was obtained as a single gel band that r etained the capsaicin-inhibitied NADH oxidase activity. Amino acid composit ion and partial amino acid sequences were obtained, The partial amino acid sequences were used to generate peptide antisera, Both the peptide antisera and polyclonal antisera to the 52-kDa component immunoprecipitated capsaic in-inhibited NADH oxidase activity and reacted with 52-, 34-, and 17-kDa co mponents on Western blots from different steps of the purification. The tNO X protein exhibited immunological cross-reactivity and amino acid sequence identity with tNOX cloned from a HeLa cDNA library using a monoclonal antib ody to tNOX from sera of cancer patients. The results provide a direct sequ ence link between tNOX of the HeLa cell surface and the cloned tNOX represe ntative of patient sera. The tNOX form from the surface of HeLa cells yield ed N-terminal sequence consistent with a coidentity of the cell surface and serum forms of the two activities. (C) 2001 Academic Press.