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A hormone-and growth factor-stimulated NADH oxidase of the mammalian plasma membrane, constitutively activated in transformed cells, was inhibited preferentially in HeLa, ovarian carcinoma, mammary adenocarcinoma, and HL-60 cells, all of human origin, by the naturally occurring quinone analog capsaicin (8-methyl-N-vanillyl-6- Previous reports described a growth factor-and hormonestimulated NADH oxidase activity of rat liver plasma membranes (1, 2). Several correlative studies have produced evidence for the involvement of this growth factor-responsive NADH oxidase in the control of cell proliferation (3). The activity in transformed cells and tissues was distinguished from that of liver in that the growth factor and hormone responsiveness was lost in plasma membranes of transformed liver tissues. These studies were done with hyperplastic nodules of liver induced by the liver carcinogen 2-acetylaminofluorene (4) and with transplanted rat hepatomas (5).The NADH oxidase activity of liver plasma membranes is unique among oxidoreductase activities not only in its response to growth factors and hormones but also in its response to inhibitors and activators other than growth factors and hormones (3, 6, 7). To further characterize this unusual NADH oxidase activity, studies were extended to include responses to quinone analogs. The activity has been shown to require quinones (8) but has been little characterized with respect to response to potentially inhibitory quinone analogs such as capsaicin (8-methyl-N-vanillyl-6-noneamide).The NADH oxidase activity of rat liver plasma membrane was largely unaffected by capsaicin, whereas the NADH oxidase activity of HeLa plasma membranes was strongly inhibited. The results indicate a fundamental difference in response to capsaicin between the NADH oxidase activity of normal and transformed cells and tissues that correlates with inhibition of growth and induction of apoptosis in the transformed cells.

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NOX proteins are growth-related cell surface proteins that catalyze both hydroquinone or NADH oxidation and protein disulfide interchange and exhibit prion-like properties. The two enzymatic activities alternate to generate a regular period length of about 24 min. Here we report the expression, cloning, and characterization of a tumor-associated NADH oxidase (tNOX). The cDNA sequence of 1830 bp is located on gene Xq25-26 with an open reading frame encoding 610 amino acids. The activities of the bacterially expressed tNOX oscillate with a period length of 22 min as is characteristic of tNOX activities in situ. The activities are inhibited completely by capsaicin, which represents a defining characteristic of tNOX activity. Functional motifs identified by site-directed mutagenesis within the C-terminal portion of the tNOX protein corresponding to the processed plasma membrane-associated form include quinone (capsaicin), copper and adenine nucleotide binding domains, and two cysteines essential for catalytic activity. Four of the six cysteine to alanine replacements retained enzymatic activity, but the period lengths of the oscillations were increased. A single protein with two alternating enzymatic activities indicative of a time-keeping function is unprecedented in the biochemical literature.

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