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.

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 site-directed mutagenesis analysis of tNOX functional domains

Constitutive NADH oxidase proteins of the mammalian cell surface exhibit two different activities, oxidation of hydroquinones (or NADH) and protein disulfide-thiol interchange which alternate to yield oscillatory patterns with period lengths of 24 min. A drug-responsive tNOX (tumor-associated NADH oxidase) has a period length of about 22 min. The tNOX cDNA has been cloned and expressed. These two proteins are representative of cycling oxidase proteins of the plant and animal cell surface. In this report, we describe a series of eight amino acid replacements in tNOX which, when expressed in Escherichia coli, were analyzed for enzymatic activity, drug response and period length. Replacement sites selected include six cysteines that lie within the processed plasma membrane (34 kDa) form of the protein, and amino acids located in putative drug and adenine nucleotide (NADH) binding domains. The latter, plus two of the cysteine replacements, resulted in a loss of enzymatic activity. The recombinant tNOX with the modified drug binding site retained activity but the activity was no longer drug-responsive. The four remaining cysteine replacements were of interest in that both activity and drug response were retained but the period length for both NADH oxidation and protein disulfide-thiol interchange was increased from 22 min to 36 or 42 min. The findings confirm the correctness of the drug and adenine nucleotide binding motifs within the tNOX protein and imply a potential critical role of cysteine residues in determining the period length.     

A role for copper in biological time-keeping

A family of cell surface and growth related proteins that oxidize both NADH and hydroquinones and carry out protein disulfide-thiol interchange (ECTO-NOX proteins) exhibits unique characteristics. The two activities they catalyze, hydroquinone or NADH oxidation and protein disulfide-thiol interchange, alternate in CNOX (the constitutive ECTO-NOX), to generate a regular period length of 24 min. For NADH or hydroquinone oxidation each period is defined by maxima that recur at intervals of 24 min. Here, we report that bound Cu^II is required to sustain the 24 min oscillation cycle of CNOX. CNOX preparations from plasma membranes of soybean, when unfolded in the presence of the copper chelator bathocuproine and refolded, lose activity. When refolded in the presence of copper, activity is restored. Unexpectedly, however, the released copper is capable of catalyzing NADH (or hydroquinone) oxidation in the absence of protein. Solvated Cu^II as the chloride or other salts alone is capable of catalyzing NADH oxidation and the oxidation rates oscillate with an overall period length of 24 min. With Cu^IICl₂ the pattern consists of five maxima, two of which are separated by an interval of 6 min and three of which are separated by intervals of 4.5 min [6 min + 4 (4.5 min)]. The period length is independent of temperature and pH. The asymmetry of the oscillatory pattern is retained after solvation of the Cu^II salts in D₂O but the overall period length is increased to 30 min. The findings suggest that the bound copper of CNOX and perhaps of ECTO-NOX proteins in general, is essential to maintain the structural changes that underlie the periodic alternations in activity that define the 24 min time-keeping cycle of the protein.     

Periodic NADH oxidase activity associated with an endoplasmic reticulum fraction from pig liver. Response to micromolar concentrations of retinol

An endoplasmic reticulum fraction from pig liver enriched in transitional endoplasmic reticulum vesicles capable of forming 50-60 nm buds in the presence of ATP and retinol was assayed for retinol-responsive oxidation of NADH and cleavage of a dithiodipyridine (DTDP) protein disulfide-thiol interchange substrate. Maxima for the two activities alternated giving rise to a 24 min period. The NADH oxidase activity was inhibited by micromolar and submicromolar concentrations of retinol. Retinol at 0.1 mM stimulated the activity. The inhibition was confined to two activity maxima separated in time by about 5 min. In contrast, with the DTDP substrate, the activity was stimulated by retinol and the stimulations were in the part of the oscillatory pattern where retinol inhibition of NADH oxidation was observed. The findings support an earlier proposed mechanism whereby retinol exerted opposing effects on NADH oxidation and protein disulfide reductions.     

Response of the regulatory oscillatory behavior of copperII-containing ECTO-NOX proteins and of CuIICl2 in solution to electromagnetic fields

A family of cell surface and growth-related proteins, designated ECTO-NOX proteins, carry out both copper-dependent NADH and hydroquinone oxidation and protein disulfide-thiol interchange. The two activities they catalyze alternate to generate a regular period of 24 min in length for the constitutive CNOX. Unexpectedly, Cu^II salts alone in solution catalyze NADH (or hydroquinone) oxidation with a similar oscillatory pattern. Both patterns consist of five maxima, two of which at physiological temperatures are separated by an interval of 6 min and three of which are separated by intervals of 4.5 min [6 min + 4 (4.5 min)]. EXAFS and infrared spectroscopic measurements on pure water have shown previously that the ratios of ortho and para isomers of the hydrogen atoms of water occur on a similar time scale and produce regular patterns of unequally spaced oscillations similar to those observed with ECTO-NOX proteins and Cu^IICl₂ solutions. Here, we provide results from Cu^IICl₂ solutions that demonstrate that ECTO-NOX-/Cu^II-catalyzed oscillations in NADH oxidation are phased by exposure to low frequency electromagnetic fields.     

Regular oscillatory behavior of aqueous solutions of CuII salts related to effects on equilibrium dynamics of ortho/para hydrogen spin isomers of water

Cell surface and growth-related NADH oxidases with protein disulfide-thiol interchange activity, ECTO-NOX, exhibit copper-dependent, clock-related, temperature-independent and entrainable patterns of regular oscillations in the rate of oxidation of NAD(P)H as do aqueous solutions of copper salts. Because of time scale similarities, a basis for the oscillatory patterns in nuclear spin orientations of the hydrogen atoms of the copper-associated water was sought. Extended X-ray absorption fine structure (EXAFS) measurements at 9302 eV on pure water were periodic with a ca. 3.5 min peak to peak separation. Decomposition fits revealed 5 unequally spaced maxima similar to those observed previously for Cu(II)Cl(2) to generate a period length of about 18 min. With D(2)O, the period length was proportionately increased by 30% to 24 min. The redox potential of water and of D(2)O also oscillated with 18 and 24 min period lengths, respectively. Measurements in the middle infrared spectral region above a water sample surface revealed apparent oscillations in the two alternative orientations of the nuclear spins (ortho and para) of the hydrogen atoms of the water or D(2)O with 5 unequally spaced maxima and respective period lengths of 18 and 24 min. Thus, the time keeping oscillations of ECTO-NOX proteins appear to reflect the equilibrium dynamics of ortho-para hydrogen atom spin ratios of water where the presence of metal cations such as Cu(II) in solution determine period length.     

Prion proteins and ECTO-NOX proteins exhibit similar oscillating redox activities

Both recombinant full-length mouse prion protein expressed in Escherichia coli and native prion protein (PrPsc) from mouse brain exhibited NADH oxidase and protein disulfide-thiol interchange activities similar to those formerly thought to be properties exclusive to the growth-related, cell surface ECTO-NOX proteins. The two activities exhibited the complex 2+3 pattern of oscillations characteristic of ECTO-NOX proteins where the two activities alternate to generate a period length of 24 min. The oscillations were augmented by copper and diminished by addition of the copper chelator bathocuproene. That the activity might be attributable to a contaminating protein was ruled out by experiments where the purified recombinant prion-containing extracts were resolved by SDS-PAGE and the activity was restricted to a single band corresponding to the predicted Mr of the recombinant prion as verified by Western blot analyses.     

Specificity of coenzyme Q inhibition of an aging-related cell surface NADH oxidase (ECTO-NOX) that generates superoxide

Our laboratories have described a novel class of ectoproteins at the cell surface with both NADH or hydroquinone oxidase (NOX) and protein disulfide-thiol interchange activities (ECTO-NOX proteins). The two activities exhibited by these proteins alternate to generate characteristic patterns of oscillations where the period length is independent of temperature. The period length for the constitutive ECTO-NOX is 24 min. Here we describe a distinctive age-related ECTO-NOX (arNOX) whose activity is blocked by coenzyme Q10. arNOX occurs exclusively in aged cells and tissues. The period length of the oscillations is 26 min. Rather than reducing 1/2 O2 to H2O, electrons are transferred to O2 to form superoxide. Superoxide formation was demonstrated by superoxide dismutase-sensitive reduction of ferricytochrome c and by reduction of a superoxide-specific tetrazolium salt. Quinone inhibition was given by coenzymes Q8, 9 and Q10 but not by Q0, Q2, Q4, Q6 or 7. The arNOX provides a mechanism to propagate reactive oxygen species generated at the cell surface to surrounding cells and circulating lipoproteins of importance to atherogenesis. Inhibition of arNOX by dietary coenzyme Q10 provides a rational basis for dietary coenzyme 10 use to retard aging-related arterial lesions.     

Molecular cloning and characterization of a tumor-associated, growth-related, and time-keeping hydroquinone (NADH) oxidase (tNOX) of the HeLa cell surface

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.     

Cell enlargement of plant tissue explants oscillates with a temperature-compensated period length of CA. 24 min

Summary Rate of plant cell enlargement, measured at intervals of 3 min using a sensitive linear transducer, oscillates with a minimum period of about 24 min that parallels the 24-min periodicity observed with the oxidation of NADH by the external plasma membrane NADH oxidase and of single cells measured previously by video-enhanced light microscopy. Also exhibiting 24-min oscillations is the steady-state rate of cell enlargement induced by the addition of the auxin herbicide 2,4-dichlorophenoxyacetic acid (2,4-D) or the natural auxin indole-3-acetic acid (IAA). Immediately following 2,4-D addition, a very complex pattern of oscillations is frequently observed. However, after several hours a dominant 24-min period emerges. The length of the 24 min period is temperature compensated and remains constant at 24 min when measured at 15, 25 or 35°C, despite the fact that the rate of cell enlargement approximately doubles for each 10°C rise over this same range of temperatures.     

Monoclonal antibody to a cancer-specific and drug-responsive hydroquinone (NADH) oxidase from the sera of cancer patients

Monoclonal antibodies were generated in mice to a 34-kDa circulating form of a drug-responsive hydroquinone (NADH) oxidase with a protein disulfide-thiol interchange activity specific to the surface of cancer cells and the sera of cancer patients. Screening used Western blots with purified 34-kDa tNOX from HeLa cells and the sera of cancer patients. Epitopes were sought that inhibited the drug-responsive oxidation of NADH with the sera of cancer patients, but which had no effect on NADH oxidation with the sera of healthy volunteers. Two such antisera were generated. One, designated monoclonal antibody (mAb) 12.1, was characterized extensively. The NADH oxidase activity inhibited by mAb 12.1 also was inhibited by the quinone site inhibitor capsaicin (8-methyl- N-vanillyl-6-noneamide). The inhibition was competitive for the drug-responsive protein disulfide-thiol interchange activity assayed either by restoration of activity to scrambled RNase or by cleavage of a dithiodipyridine substrate, and was uncompetitive for NADH oxidation. Both the mAb 12.1 and the postimmune antisera immunoprecipitated drug-responsive NOX activity and identified the same 34-kDa tNOX protein in the sera of cancer patients that was absent from sera of healthy volunteers, and was utilized as immunogen. Preimmune sera from the same mouse as the postimmune antisera was without effect. Both mouse ascites containing mAb 12.1 and postimmune sera (but not preimmune sera) slowed the growth of human cancer cell lines in culture, but did not affect the growth of non-cancerous cell lines. Immunocytochemical and histochemical findings showed that mAb 12.1 reacted with the surface membranes of human carcinoma cells and tissues.     

Auxin-activated nadh oxidase activity of soybean plasma membranes is distinct from the constitutive plasma membrane NADH oxidase and exhibits prion-like properties

Summary The hormone-stimulated and growth-related cell surface hydroquinone (NADH) oxidase activity of etiolated hypocotyls of soybeans oscillates with a period of about 24 min or 60 times per 24-h day. Plasma membranes of soybean hypocotyls contain two such NADH oxidase activities that have been resolved by purification on concanavalin A columns. One in the apparent molecular weight range of 14–17 kDa is stimulated by the auxin herbicide 2,4-dichlorophenoxyacetic acid (2,4-D). The other is larger and unaffected by 2,4-D. The 2,4-D-stimulated activity absolutely requires 2,4-D for activity and exhibits a period length of about 24 min. Also exhibiting 24-min oscillations is the rate of cell enlargement induced by the addition of 2,4-D or the natural auxin indole-3-acetic acid (IAA). Immediately following 2,4-D or IAA addition, a very complex pattern of oscillations is frequently observed. However, after several hours a dominant 24-min period emerges at the expense of the constitutive activity. A recruitment process analogous to that exhibited by prions is postulated to explain this behavior.     

CHO cell enlargement oscillates with a temperature-compensated period of 24 min

The rate of increase in cell area of CHO cells when measured at intervals of 1 min using a light microscope equipped with a video measurement system, oscillated with a minimum period of about 24 min. The pattern of oscillations paralleled those of the 24 min period observed with the oxidation of NADH by an external cell surface or plasma membrane NADH oxidase. The increase in cell area was non-linear. Intervals of rapid increase in area alternated with intervals of rapid decrease in area. The length of the 24 min period was temperature-compensated (approximately the same when measured at 14 degrees C, 24 degrees C or 34 degrees C) while the rate of cell enlargement increased with temperature over this same range of temperatures.     

Entrainment in solution of an oscillating NADH oxidase activity from the bovine milk fat globule membrane with a temperature-compensated period length suggestive of an ultradian time-keeping (clock) function

Entrainment in solution of an oscillating activity with a temperature compensated period of 24 min is described for a NADH oxidase (NOX) activity of the bovine milk fat globule membrane, a derivative of the mammary epithelial cell plasma membrane. The period of 24 min remained unchanged at 17°C, 27°C and 37°C whereas the amplitude approximately doubled with each 10°C rise in temperature (Q₁₀≅2). The periodicity was observed with both intact milk fat globule membranes and with detergent-solubilized membranes, demonstrating that the oscillations did not require an association with membranes. The periodicity was not the result of instrument variation or of chemical interactions among reactants in solution. Preparations with different periodicities entrained (autosynchronized) when mixed. Upon mixing, the preparations exhibited two oscillatory patterns but eventually a single pattern representing the mean of the farthest separated maxima of the two preparations analyzed separately emerged. The cell surface NOX protein is the first reported example of an entrainable biochemical entity with a temperature-compensated periodicity potentially capable of functioning as an ultradian or circadian clock driver.     

Human amyloid peptides Abeta1-40 and Abeta1-42 exhibit NADH oxidase activity with copper-induced oscillations and a period length of 24 min

Human amyloid beta peptides Abeta1-40 and Abeta1-42 exhibit NADH oxidase activity with regular oscillations at intervals of ca 6 min. In the presence of copper, the oscillations in Abeta1-40 and Abeta1-42 become more pronounced and now assume a period length of 24 min. In the presence of copper, the oscillations are similar to those observed with NADH oxidase activities of cell surface ECTO-NOX proteins in general including a period length of 24 min. Solutions of copper sulphate in the presence of all the reagents except for the peptides did not exhibit the oscillatory behavior. NOX proteins have been reported previously to have properties of prions and to form amyloid rods of indeterminant length similar to those formed by the 39-43 residue amyloid beta proteins (Abeta). In this report, we demonstrate a second similarity between ECTO-NOX proteins and amyloid beta, that of an oscillating NADH oxidase activity with a period length of 24 min when assayed in the presence of copper.     

Cell Surface NADH Oxidases (ECTO-NOX Proteins) with Roles in Cancer,Cellular Time-keeping,Growth, Aging and Neurodegenerative Diseases

ECTO-NOX (because of their cell surface location) proteins comprise a family of NAD(P)H oxidases of plants and animals that exhibit both oxidative and protein disulfide isomerase-like activities. The two biochemical activities, hydroquinone [NAD(P)H] oxidation and protein disulfide-thiol interchange alternate, a property unprecedented in the biochemical literature. A tumor-associated ECTO-NOX (tNOX) is cancer-specific and drug-responsive. The constitutive ECTO-NOX (CNOX) is ubiquitous and refractory to drugs. The physiological substrate for the oxidative activity appears to be hydroquinones of the plasma membrane such as reduced coenzyme Q₁₀. ECTO-NOX proteins are growth-related and drive cell enlargement. Also indicated are roles in aging and in neurodegenerative diseases. The regular pattern of oscillations appears to be related to α-helix-β-structure transitions and serves biochemical core oscillator of the cellular biological clock. Period length is independent of temperature (temperature compensated) and synchrony is achieved through entrainment.     

ZBTB34, a novel human BTB/POZ zinc finger protein, is a potential transcriptional repressor

Our work has identified a cancer-specific, cell surface and growth-related quinol oxidase with both NADH oxidase and protein disulfide-thiol interchange activities, a member of the ECTO-NOX protein family designated tNOX. We provide evidence for tNOX as an alternative drug target to COX-2 to explain the anticancer activity of COX inhibitors. Non-steroidal anti-inflammatory drugs (NSAIDS), piroxicam, aspirin, ibuprofen, naproxen and celecoxib all specifically inhibited tNOX activity of HeLa (human cervical carcinoma) and BT-20 (human mammary carcinoma) cells (IC₅₀ in the nanomolar range) without effect on ECTO-NOX activities of non-cancer MCF-10A mammary epithelial cells. With cancer cells, rofecoxib was less effective and two NSAIDS selective for COX-1 were without effect in inhibiting NOX activity. The IC₅₀ for inhibition of tNOX activity of HeLa cells and the IC₅₀ for inhibition of growth of HeLa cells in culture were closely correlated. The findings provide evidence for a new drug target to account for anticancer effects of NSAIDS that occur independent of COX-2.     

Soybean cell enlargement oscillates with a temperature-compensated period length of CA. 24 min

Summary Rate of enlargement of epidermal cells from soybean, when measured at intervals of 1 min using a light microscope equipped with a video measurement system, oscillated with a period length of about 24 min. This oscillation parallels the 24-min periodicity observed for the oxidation of NADH by the external plasma membrane NADH oxidase. The increase in length was not only non-linear but intervals of rapid increase in area alternated with intervals of rapid decrease in area. The length of the period was temperature compensated, and was approximately the same when measured at 14, 24 and 34°C even though the rate of cell enlargement varied over this same range of temperatures. These observations represent the first demonstration of an oscillatory growth behavior correlated with a biochemical activity where the period length of both is independent of temperature (temperature compensated) as is the hallmark of clock-related biological phenomena.     

Cell surface NADH oxidase activity of brine shrimp oscillates with a period of 25 min and is entrained by light

Plants have a surface NADH oxidase that measures time by oscillating with a 24-min period. The period is synchronized by light. With plants, a new maximum is observed exactly 12 min after the beginning of the light exposure. These experiments were to determine if animals exhibited a cell surface NADH oxidase having a similar periodicity and to answer the question, does the periodicity in animals respond to light? Using brine shrimp as a model, the findings show that plants and animals exhibit similar oscillating NADH oxidase activity and that the periodicity in this invertebrate animal does respond to light. Brine shrimp were grown for two to three days and transferred to darkness for 45 min. After return to light for one min, NADH was added and measurements of NADH oxidation were recorded over 50 min. The brine shrimp exhibited a cell surface NADH oxidase that oscillated with a period of 25 min. After being subjected to light, the brine shrimp showed a new maximum in NADH oxidation between 12 to 13 min after the beginning of the light exposure and again at 37 min and at 25 min intervals thereafter. The findings demonstrate that the periodic oscillations in NADH oxidation of brine shrimp are light entrainable.     

Entrainment in solution of an oscillating NADH oxidase activity from the bovine milk fat globule membrane with a temperature-compensated period length suggestive of an ultradian time-keeping (clock) function

Entrainment in solution of an oscillating activity with a temperature compensated period of 24 min is described for a NADH oxidase (NOX) activity of the bovine milk fat globule membrane, a derivative of the mammary epithelial cell plasma membrane. The period of 24 min remained unchanged at 17 degrees C, 27 degrees C and 37 degrees C whereas the amplitude approximately doubled with each 10 degree C rise in temperature (Q(10)congruent with 2). The periodicity was observed with both intact milk fat globule membranes and with detergent-solubilized membranes, demonstrating that the oscillations did not require an association with membranes. The periodicity was not the result of instrument variation or of chemical interactions among reactants in solution. Preparations with different periodicities entrained (autosynchronized) when mixed. Upon mixing, the preparations exhibited two oscillatory patterns but eventually a single pattern representing the mean of the farthest separated maxima of the two preparations analyzed separately emerged. The cell surface NOX protein is the first reported example of an entrainable biochemical entity with a temperature-compensated periodicity potentially capable of functioning as an ultradian or circadian clock driver.