Protein Disulfide-thiol Interchange Activity Research Articles

Molecular Cloning and Characterization of a Candidate Plant Growth-Related and Time-Keeping Constitutive Cell Surface Hydroquinone (NADH) Oxidase (ENOX1) from <i>Arabidopsis lyrata</i>

ENOX (ECTO-NOX) proteins are proteins of the external surface of the plasma membrane that catalyze oxidation of both NADH and hydroquinones as well as carry out protein disulfidethiol interchange. They exhibit both prion-like and time-keeping (clock) properties. The oxidative and interchange activities alternate to generate a regular period of 24 min in length. Here we report the cloning, expression, and characterization of a plant candidate constitutive ENOX (CNOX or ENOX1) protein from Arabidopsis lyrata. The gene encoding the 335 (165) amino acid protein is found in accession XP-002882467. Functional motifs characteristics of ENOX proteins previously identified by site-directed mutagenesis and present in the candidate ENOX1 protein from plants include adenine nucleotide and copper binding motifs along with essential cysteines. However, the drug binding motif (EEMTE) sequence of human ENOX2 is absent. The activities of the recombinant protein expressed in E. coli were unaffected by capsaicin, EGCg, and other ENOX2-inhibiting substances. Periodic oxidative activity was exhibited both with NAD(P)H and reduced coenzyme Q as substrate. Bound copper was necessary for activity and activity was inhibited by the ENOX1-specific inhibitor simalikalactone D. Addition of melatonin phased the 24-min period such that the next complete period began 24 min after the melatonin addition as appeared to be characteristic of ENOX1 activities in general. Periodic protein disulfide-thiol interchange activity also was demonstrated along with the 2 oxidative plus 3 interchange activity pattern characteristics of the 24-min ENOX1 protein period. Concentrated solutions of the purified plant ENOX1 protein formed insoluble aggregates, devoid of enzymatic activity, resembling amyloid. Activity was restored to aggregate preparations by isoelectric focusing.

Cloning and Characterization of a Candidate Auxin Plant Growth Regulator-Activated Cell Surface Hydroquinone (NADH) Oxidase

ENOX (ECTO-NOX) proteins of the external surface of the plasma membrane catalyze oxidation of both NADH and hydroquinones and protein disulfide-thiol interchange. They exhibit both prion-like and time-keeping (clock) properties. The oxidative and interchange activities alternate to generate a regular period of 24 min in length. Here we report the cloning, expression and characterization of a constitutive plant ENOX protein activated by both natural (Indole-3-acetic acid, IAA) and synthetic (2,4-dichlorophenoxyacetic acid, 2,4-D) auxin plant growth regulators with an optimum of about 1 μM, higher concentrations being less effective. The gene encoding the 213 amino acid protein (ABP20) is found in EMBL accession number U81162. Functional motifs characteristic of ENOX1 proteins, previously identified by site-directed mutagenesis, are present in the candidate auxin-activated ENOX (dNOX, ENOX5), including adenine nucleotide and copper binding motifs along with essential cysteines and a motif having homology with a previously identified auxin-binding motif. Periodicity was exhibited by both the oxidative and protein disulfide-thiol inter-change activities as is characteristic for other ENOX proteins. Activity was blocked by the ENOX2-specific quassinoid inhibitor glaucarubolone and other ENOX2 inhibitors but not by the ENOX1-specific quassinoid inhibitor simalikalactone D. Activity required both auxin and bound copper. The inactive auxin 2,3-D was without effects.

Molecular Cloning and Characterization of a Candidate ENOX Protein of <i>Saccharomyces cerevisiae</i> with a 25 Min Period Insensitive to Simalikalactone D Inhibition and Melatonin

A yeast deletion library was screened based on NADH fluorescence using a 384-well plate assay to identify a yeast isolate lacking a previously identified cell surface oxidase exhibiting an oscillatory pattern with a period length of 25 min and resistant to the ENOX1-specific inhibitor simalikalactone D (YNOX for yeast-specific ENOX = ENOX4). The cDNA was cloned from a yeast over expression library using NADH fluorescence analyzed by Fast Fourier transform and decomposition fits. The objective was to identify and sequence an ENOX homologue in Saccharomyces cerevisiae with a 25 min rather than a 24 min period length (YNOX). The finding identified YDR005C as the yeast ENOX protein with a temperature-independent 25 min period length and insensitive to inhibition by simalikalactone D. The encoded protein was expressed in bacteria and characterized. Gel slices corresponding to 55 kDa and 39 kDa His-tagged proteins exhibited 25 min oscillatory patterns not inhibited by 1 μM simalikalactone D for both NADH oxidation and reduced coenzyme Q10 oxidation as well as a protein disulfide-thiol interchange activity which alternated with the oxidative activities. Activities were phased by low-frequency electromagnetic fields but, in contrast, to yeast ENOX1, not by addition of melatonin. The assay in the presence of D2O shifted the length of the oscillatory period from 25 min to 32 min. The YDR005C deletion mutant cells lacked the ENOX4 clock output present in the wild type yeast.

Age-Related Surface Oxidases Shed into Body Fluids as Targets to Prevent Skin Aging and Reduce Cardiovascular Risk

Age-related Ecto-Nicotinamide Adenine Dinucleotide Oxidase Disulfide Thiol Exchangers 3 (ENOX3) or age-related NADH oxidases (arNOX) are expressed at the cell surface as five members of the TM-9 superfamily, initially membrane anchored, all functionally similar, with the N-termini exposed at the cell’s exterior. ECTO-NOXes are cell surface proteins with both time-keeping CoQH2 [NAD(P)H] oxidase and protein disulfidethiol interchange activities. They are designated as ECTO-NOX proteins because of their localization on the outer surface of the plasma membrane and to distinguish them from the phox-NOXes of host defense. A ca. 30 kDa N-terminal fragment is cleaved and accumulates in body fluids (serum, saliva, urine, perspiration). arNOXes appear around age 30 and increase steadily thereafter. Reduced quinones, i.e., reduced coenzyme Q, of the plasma membrane are natural substrates. NAD(P)H is oxidized as an artificial substrate. In one phase of the arNOX cycle electrons are transferred to oxygen to generate superoxide. Substrates for the shed forms of arNOX appear to be proteins of body fluids. Circulating lipoproteins and skin matrix proteins emerge as potentially important health-related targets. Through oxidation of collagen, elastin and other proteins of the skin matrix, arNOXes are major contributors to skin aging through tyrosine and thiol oxidation and subsequent cross linking. The main destructive action of arNOX, however, may be to directly oxidize circulating lipoproteins. arNOX in the blood is structured as an integral component of the LDL particle through site-specific binding. As such, arNOXes are implicated as major risk factors for cardiovascular disease due to specific oxidation of LDLs. The superoxide produced and its conversion to hydrogen peroxide would be one part of the potentially destructive properties by contribution to lipid oxidation. Inhibition of arNOX proteins provides a rational basis for anti-aging interventions and their elimination as a major risk factor of atherogenesis.

Essential role of copper in the activity and regular periodicity of a recombinant, tumor-associated, cell surface, growth-related and time-keeping hydroquinone (NADH) oxidase with protein disulfide-thiol interchange activity (ENOX2)

ECTO-NOX proteins are growth-related cell surface proteins that catalyze both hydroquinone or NADH oxidation and protein disulfide interchange and exhibit time-keeping and prion-like properties. A bacterially expressed truncated recombinant 46kDa ENOX2 with full ENOX2 activity bound ca 2 moles copper and 2 moles of zinc per mole of protein. Unfolding of the protein in trifluoroacetic acid in the presence of the copper chelator bathocuproine resulted in reversible loss of both enzymatic activities and of a characteristic pattern in the Amide I to Amide II ratios determined by FTIR with restoration by added copper. The H546-V-H together with His 562 form one copper binding site and H582 represents a second copper site as determined from site-directed mutagenesis. Bound copper emerges as having an essential role in ENOX2 both for enzymatic activity and for the structural changes that underly the periodic alternations in activity that define the time-keeping cycle of the protein.

Cancer Site-Specific Isoforms of ENOX2 (tNOX), A Cancer-Specific Cell Surface Oxidase

Abstract Introduction All neoplastic cells express one or more members of a unique family of tumor-associated cell surface ubiquinone (NADH) oxidase proteins with protein disulfide-thiol interchange activity (ENOX2 or tNOX proteins) that are characteristically blocked by quinone site inhibitors with anti-cancer activity. Methods Analyses using two-dimensional gel electrophoresis with detection on western blots using a pan ENOX2 recombinant antibody revealed unique ENOX2 isoforms or unique combinations of isoforms of differing molecular weights and/or isoelectric points in sera of patients with cancers of different cellular or tissue origins. Results and Discussion Isoform presence provides for broad-range cancer detection. The specific patterns and molecular weights of the isoforms present allows for identification of the cell type and/or tissue of origin of the neoplasm. ENOX2 isoform presence and relative amounts are largely independent of stage but may be proportional to tumor burden to provide indications of response to therapy and disease progression.

Antisense Experiments Demonstrate an Exon 4 Minus Splice Variant mRNA as the Basis for Expression of tNOX, a Cancer-Specific Cell Surface Protein

A novel hydroquinone and NADH oxidase with protein disulfide-thiol interchange activity (designated ENOX2 or tNOX), associated exclusively with the outer leaflet of the plasma membrane at the surface of cancer cells and in sera of cancer patients, is absent from the surface of noncancer cells and from sera from healthy individuals. Transfection of HeLa (human cervical carcinoma) cells with antisense oligonucleotides and measurement of mRNA levels by real-time quantitative PCR and growth and drug response by in vitro cytotoxicity assays were combined to demonstrate encoding of a cancer-specific and growth-related cell surface protein, tNOX, via an exon 4 minus splice variant. tNOX mRNA levels of HeLa cells were determined following transfection with antisense relative to control cells transfected with Lipofectamine using the cycle threshold method normalized for GAPDH mRNA. Antisense to tNOX exon 4 mRNA blocked generation of full-length tNOX mRNA but not of exon 4 minus mRNA. Antisense to exon 5 mRNA inhibited the production of exon 4 minus mRNA and full-length tNOX mRNA. Scrambled antisense to exon 5 mRNA was without effect. Antisense to exon 5 mRNA decreased the amount of tNOX protein on the surface of cancer cells. As a control, antisense-mediated downregulation of exon 5 minus mRNA of tNOX also was demonstrated as detected using exon 4/exon 6 primers. Exon 5 antisense blocked the cell surface expression of tNOX whereas exon 4 antisense was without effect. In contrast to nontransfected HeLa cells, cells transfected with exon 5 antisense were not inhibited by the green tea catechin, (-)-epigallocatechin-3-gallate. A relationship of tNOX to unregulated growth of cancer cells was provided by data where growth of HeLa cells was inhibited by transfection with the exon 5 antisense oligonucleotides. Growth inhibition was followed by apoptosis in greater than 70% of the transfected cells.

Alternative Splicing as the Basis for Specific Localization of tNOX, a Unique Hydroquinone (NADH) Oxidase, to the Cancer Cell Surface

A novel hydroquinone and NADH oxidase with protein disulfide-thiol interchange activity (designated ENOX2 or tNOX), associated exclusively with the outer leaflet of the plasma membrane at the surface of cancer cells and in sera of cancer patients, is absent from the surface of noncancer cells and from sera of healthy individuals. Full-length tNOX mRNA is present in both normal and tumor cells but appears not to be expressed in either. Our research suggests alternative splicing as the basis for the cancer specificity of tNOX expression at the cell surface. Four splice variants were found. Of these, the exon 4 minus and exon 5 minus forms present in cancer cell lines were absent in noncancer cell lines. In contrast to full-length tNOX cDNA, transfection of COS cells with tNOX exon 4 minus cDNA resulted in overexpression of mature 34 kDa tNOX protein at the plasma membrane. The exon 4 minus form resulted in initiation of translation at a downstream M231 initiation site distinct from that of full-length mRNA. With replacement of M231 by site-directed mutagenesis, no translation of exon 4 minus cDNA or cell surface expression of 34 kDa mature tNOX was observed. The unprocessed molecular mass of 47 kDa of the exon 4 minus cDNA translated from methionine 231 corresponded to that of the principal native tNOX form of the endoplasmic reticulum. Taken together, the molecular basis of cancer-cell-specific expression of 34 kDa tNOX appears to reside in the cancer-specific expression of exon 4 minus splice variant mRNA.

Regular oscillatory behavior of aqueous solutions of Cu II 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 IICl 2 to generate a period length of about 18 min. With D 2O, the period length was proportionately increased by 30% to 24 min. The redox potential of water and of D 2O 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 2O 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.

Mouse embryonic fibroblast cells from transgenic mice overexpressing tNOX exhibit an altered growth and drug response phenotype

Mouse embryonic fibroblast (MEF) cells prepared from transgenic mice overexpressing a cancer-specific and growth-related cell surface NADH oxidase with protein disulfide-thiol interchange activity grew at rates approximately twice those of wild-type embryonic fibroblast cells. Growth of transgenic MEF cells overexpressing tNOX was inhibited by low concentrations of the green tea catechin (-)-epigallocatechin-3-gallate (EGCg) or the synthetic isoflavene phenoxodiol. Both are putative tNOX-targeted inhibitors with anti-cancer activity. With both EGCg and phenoxodiol, growth inhibition was followed after about 48 h by apoptosis. Growth of wild-type mouse fibroblast cells from the same strain was unaffected by EGCg and phenoxodiol and neither compound induced apoptosis even at concentrations 100-1,000-fold higher than those that resulted in apoptotic death in the transgenic MEF cells. The findings validate earlier reports of evidence for tNOX presence as contributing to unregulated growth of cancer cells as well as the previous identification of the tNOX protein as the molecular target for the anti-cancer activities attributed to both EGCg and phenoxodiol. The expression of tNOX emerges as both necessary and sufficient to account for the cancer cell-specific growth inhibitions by both EGCg and phenoxodiol.

ECTO-NOX Target for the Anticancer Isoflavene Phenoxodiol

Phenoxodiol, a synthetic isoflavene with clinical efficacy in the management of ovarian and other forms of human cancer, blocked the activity of a cancer-specific and growth-related cell surface ECTO-NOX protein with both oxidative (hydroquinone) and protein disulfide-thiol interchange activity designated tNOX. Purified recombinant tNOX bound phenoxodiol with high affinity (Kd of 50 nM). The tNOX protein appeared to be both necessary and sufficient for the cancer-specific cytotoxicity of phenoxodiol. Growth inhibition of fibroblasts from embryos of mice expressing a tNOX transgene, but not from wild-type mice, was inhibited by phenoxodiol followed by apoptosis. Both the oxidative and protein disulfide-thiol interchange activities that alternate to generate the complex set of oscillations with a period length of 22 min (24 min for the constitutive counterpart CNOX) that characterize ECTO-NOX proteins respond to phenoxodiol. Oxidation of NADH or reduced coenzyme Q10 was rapidly blocked by phenoxodiol. In contrast, the protein disulfidethiol interchange activity measured either by the restoration of activity to scrambled and inactive RNase or from the cleavage of dithiodipyridine (EC50 of 50 nM) was inhibited progressively over an interval of 60 min that spanned three cycles of activity. Inhibition of the latter paralleled the inhibition of cell enlargement and the consequent inability of inhibited cells to initiate traverse of the cell cycle. Activities of constitutive ECTO-NOX (CNOX) forms of either cancer or noncancer cells were unaffected by phenoxodiol to help explain how the cytotoxic effects of phenoxodiol may be restricted to cancer cells.

TNOX, an alternative target to COX-2 to explain the anticancer activities of non-steroidal anti-inflammatory drugs (NSAIDS)

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 (IC(50) 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 IC(50) for inhibition of tNOX activity of HeLa cells and the IC(50) 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.

Anticancer activity of grape and grape skin extracts alone and combined with green tea infusions

Grapes and grape extracts were compared for inhibition of a growth-related and cancer-specific form of cell surface NADH oxidase with protein disulfide-thiol interchange activity designated tNOX from human cervical carcinoma (HeLa) cells and growth of HeLa and mouse mammary 4T1 cells in culture and transplanted tumors in mice. Grapes and grape extracts of several varieties had activity. With an extracted grape preparation provided by the California Table Grape Commission, an active fraction was eluted with methanol from a Diaion HP-20 column after removal of inactive water-soluble materials. Grape skins were a much more potent source than either grape pulp, juice or seeds. Ethanol extracts of the ground freeze-dried pomace was an excellent source. The grape extracts interacted, often synergistically, with decaffeinated green tea extracts both in the inhibition of tNOX activity and in the inhibition of cancer cell growth. Intratumoral injections of a 25:1 mixture of a green tea extract plus ground freeze-dried pomace was nearly as effective as standard synergistic green tea– Capsicum mixtures in inhibiting growth of 4T1 mammary tumors in situ in mice.

Sera from cancer patients contain two oscillating ECTO-NOX activities with different period lengths

ECTO-NOX protein’s are cell surface-associated and growth-related hydroquinone oxidases with both protein disulfide-thiol interchange activity and the capacity to oxidize NAD(P)H. The activities of these ECTO-NOX proteins are not steady state but fluctuate to create a repeating pattern of oscillations. Two forms of ECTO-NOX activities have been distinguished. The constitutive ECTO-NOX (CNOX), is hormone responsive and refractory to quinone-site inhibitors. A tumor-associated NOX (tNOX) is unregulated, refractory to hormones and growth factors and responds to quinone-site inhibitors. CNOX proteins are widely distributed and exhibit oscillations in enzymatic activity with a period length of 24 min. tNOX proteins are cancer specific and exhibit oscillations with a period length of about 22 min. Our findings now demonstrate the presence of the novel oscillating tNOX activity in sera of patients with cancer whereas the constitutive NOX of non-cancer cells is present in sera of both cancer patients and healthy volunteers. We conclude that ECTO-NOX proteins in sera exhibit oscillatory characteristics similar to those of ECTO-NOX forms of the cell surface.

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.

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.

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°C) whereas the rate of NADH oxidation approximately doubled with each 10°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.

Is the cancer protective effect correlated with growth inhibitions by green tea (−)-epigallocatechin gallate mediated through an antioxidant mechanism?

The preferential inhibition by (−)-epigallocatechin gallate (EGCg) of growth of cancer cells (e.g. HeLa) in culture correlates with the ability of EGCg to inhibit a growth-related, cell surface hydroquinone oxidase with protein disulfide-thiol interchange activity (tNOX) measured as an NADH oxidase and specifically associated with tumorigenically-transformed cells and tissues. tNOX is reduced or absent from the surface of non-cancer cells. Various oxidizing conditions known to render other antioxidants such as thiols, ascorbate and vitamin E ineffective did not reduce the effectiveness of EGCg in inhibiting either the tNOX activity or the growth of HeLa cells. Only after Fenton reaction with iron catalysis in the presence of hydrogen peroxide was the effectiveness of the EGCg reduced. We conclude that it is unlikely that the anticancer action of green tea EGCg on the tNOX protein is mediated through antioxidant properties of EGCg.

Chemical hormesis in cell growth: a molecular target at the cell surface

A multifunctional ubiquinol (NADH) oxidase with protein disulfide-thiol interchange activity of the cell surface, abbreviated as NOX, is described as a molecular target for chemical hormesis of cell growth. The activity of the NOX correlates with rate of cell enlargement, which helps to determine how rapidly cells will divide. When NOX activity is inhibited, cells fail to enlarge following division and the result is a population of small cells unable to reach the minimum size required for them to divide again. In plants, cells fail to enlarge when NOX activity is inhibited. When NOX activity is stimulated or constitutively activated, as in cancer, cells enlarge more rapidly and the rate of cell division also is enhanced. Both cell growth and NOX activity are sometimes stimulated by low concentrations of normally inhibitory molecules. These properties define chemical hormesis, making the NOX molecule a molecular target to explain hormetic growth responses and to utilize hormetic principles to increase, for example, crop yields with plants. The NOX activity at the cell surface oscillates with a temperature-compensated 24-min ultradian (<24 h) periodicity. The indicated function of the NOX protein as a time-keeping mechanism adds to its potential importance as a molecular target for chemical hormesis.

Use of dipyridyl-dithio substrates to measure directly the protein disulfide-thiol interchange activity of the auxin stimulated NADH: protein disulfide reductase (NADH oxidase) of soybean plasma membranes.

Dipyridyl-dithio substrates were cleaved by isolated vesicles of plasma membranes prepared from etiolated hypocotyls of soybean. The cleavage was stimulated by auxins at physiological concentrations. The substrates utilized were principally 2,2'-dithiodipyridine (DTP) and 6,6'-dithiodinicotinic acid (DTNA). The DTP generated 2 moles of 2-pyridinethione whereas the 6,6'-dithiodinicotinic acid generated 2 moles of 6-nicotinylthionine. Both products absorbed at 340 nm. The auxin herbicide, 2,4-dichlorophenoxyacetic acid (2,4-D) stimulated the activity approximately 2-fold to a maximum at about 10 microM. Concentrations of 2,4-D greater than 100 microM inhibited the activity. Indole-3-acetic acid stimulated the activity as well. The growth-inactive auxin, 2,3-dichlorophenoxyacetic acid (2,3-D), was without effect. DTNA cleavage correlated with oxidation of NADH and reduction of protein disulfide bonds reported earlier in terms of location at the external plasma membrane surface, absolute specific activity, pH dependence and auxin specificity. The dipyridyl-dithio substrates provide, for the first time, a direct measure of the disulfide-thiol interchange activity of the protein previously measured only indirectly as an auxin-dependent ability of isolated plasma membrane vesicles to restore activity to scrambled and inactive RNase.