NADH oxidase activity (NOX) and enlargement of HeLa cells oscillate with two different temperature-compensated period lengths of 22 and 24 minutes corresponding to different NOX forms

Abstract

NOX proteins are cell surface-associated and growth-related hydroquinone (NADH) oxidases with protein disulfide-thiol interchange activity. A defining characteristic of NOX proteins is that the two enzymatic activities alternate to generate a regular period length of about 24 min. HeLa cells exhibit at least two forms of NOX. One is tumor-associated (tNOX) and is inhibited by putative quinone site inhibitors (e.g., capsaicin or the antitumor sulfonylurea, LY181984). Another is constitutive (CNOX) and refractory to inhibition. The periodic alternation of activities and drug sensitivity of the NADH oxidase activity observed with intact HeLa cells was retained in isolated plasma membranes and with the solubilized and partially purified enzyme. At least two activities were present. One had a period length of 24 min and the other had a period length of 22 min. The lengths of both the 22 and the 24 min periods were temperature compensated (approximately the same when measured at 17, 27 or 37 degrees C) whereas the rate of NADH oxidation approximately doubled with each 10 degrees C rise in temperature. The rate of increase in cell area of HeLa cells when measured by video-enhanced light microscopy also exhibited a complex period of oscillations reflective of both 22 and 24 min period lengths. The findings demonstrate the presence of a novel oscillating NOX activity at the surface of cancer cells with a period length of 22 min in addition to the constitutive NOX of non-cancer cells and tissues with a period length of 24 min.

A role for copper in biological time-keeping

“…[1]. The rate of enlargement of plant, animal and bacterial cells also oscillates with a period of 24 min [12][13][14]29,30]. The 24 min periodic behavior is exhibited by pure recombinant tNOX protein and is accompanied by a recurring pattern of amide I/amide II FTIR and CD spectral changes suggestive of a-helix-b-sheet transformations [31].…”

“…The alternation of activities imparts a time-keeping function to ECTO-NOX proteins. The length of the period is temperature independent [12][13][14] and entrainable [15][16][17][18], both features of the biological clock [19,20]. Site directed mutagensis (cysteine to alanine replacements) of the tNOX cDNA has generated recombinant tNOX forms with period lengths of less than and greater than 24 min [21].…”

Molecular Cloning and Characterization of a Tumor-Associated, Growth-Related, and Time-Keeping Hydroquinone (NADH) Oxidase (tNOX) of the HeLa Cell Surface

“…The oxidative and interchange activities of the tNOX protein also alternate but generate a 22 min rather than a 24 min period (9). Inhibition by vanilloids such as capsaicin (3) and by antitumor sulfonylureas (10) distinguishes tNOX from other NADH oxidases.…”

Purification and Characterization of a Doxorubicin-inhibited NADH-quinone (NADH-ferricyanide) Reductase from Rat Liver Plasma Membranes

“…Although physiological roles of the redox system are not yet completely understood, functions in which plasma membrane redox systems have been implicated are proton extrusion and control of internal pH (25), generation of superoxide (26), reduction of ferric irons and iron uptake (27,28), control of cell growth and proliferation (5,29,30), and biological timekeeping (31,32). Comprehensive reviews of the plasma membrane redox system are found elsewhere (33)(34)(35).…”