Membrane redox as an essential component of how cells increase in size following cell division

Under investigation is the hypothesis that cell enlargement in both plants and animals is not a passive process but the result of an ECTO-NOX-driven physical membrane displacement. Cell enlargement correlates with ECTO-NOX activity and is stimulated when ECTO-NOX activities are stimulated and inhibited when ECTO-NOX activities are inhibited. Both are blocked by thiol reagents. Additionally, cell enlargement emerges as having an energy requirement. An energy requirement is universal among membrane displacement models and is met at the cell surface through coupling with a plasma membrane-associated AAA-ATPase

Role of membrane redox in aging-related diseases

A number of different ECTO-NOX forms have been described as being connected with aging-related diseases. The constitutive form, CNOX, serves as a terminal oxidase of plasma membrane electron transport and functions in the growth process. tNOX is present in addition to CNOX on the surface of all cancer cells and contributes to the unregulated growth characteristic of cancer cells. An age-related ECTO-NOX, arNOX, generates superoxide and may contribute to age-related generation of reactive oxygen species. ECTO-NOX proteins and prions share properties in common as do amyloid-forming proteins of various neurodegenerative disorders. A better understanding of ECTO-NOX proteins may lead to new therapeutic strategies for these several age-related disorders

The plasma membrane NADH oxidase of HeLa cells has hydroquinone oxidase activity

AbstractThe plasma membrane NADH oxidase activity partially purified from the surface of HeLa cells exhibited hydroquinone oxidase activity. The preparations completely lacked NADH:ubiquinone reductase activity. However, in the absence of NADH, reduced coenzyme Q10 (Q10H2=ubiquinol) was oxidized at a rate of 15±6 nmol min−1 mg protein−1 depending on degree of purification. The apparent Km for Q10H2 oxidation was 33 μM. Activities were inhibited competitively by the cancer cell-specific NADH oxidase inhibitors, capsaicin and the antitumor sulfonylurea N-(4-methylphenylsulfonyl)-N′-(4-chlorophenyl)urea (LY181984). With coenzyme Q0, where the preparations were unable to carry out either NADH:quinone reduction or reduced quinone oxidation, quinol oxidation was observed with an equal mixture of the Q0 and Q0H2 forms. With the mixture, a rate of Q0H2 oxidation of 8–17 nmol min−1 mg protein−1 was observed with an apparent Km of 0.22 mM. The rate of Q10H2 oxidation was not stimulated by addition of equal amounts of Q10 and Q10H2. However, addition of Q0 to the Q10H2 did stimulate. The oxidation of Q10H2 proceeded with what appeared to be a two-electron transfer. The oxidation of Q0H2 may involve Q0, but the mechanism was not clear. The findings suggest the potential participation of the plasma membrane NADH oxidase as a terminal oxidase of plasma membrane electron transport from cytosolic NAD(P)H via naturally occurring hydroquinones to acceptors at the cell surface

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

AbstractNOX 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

STRUCTURAL CHANGES REVEALED BY FOURIER TRANSFORM INFRARED AND CIRCULAR DICHROISM SPECTROSCOPIC ANALYSES UNDERLIE tNOX PERIODIC OSCILLATIONS

A recurring pattern of spectral changes indicative of periodic changes in the proportion of β-structure and α-helix of a recombinant ECTO-NOX fusion protein of tNOX, with a cellulose binding domain peptide, was demonstrated by Fourier transform infrared (FTIR) and circular dichroism (CD) spectroscopic analyses. The pattern of structural changes correlated with oscillatory patterns of enzymatic activities exhibited by the protein previously interpreted as indicative of a clock function. The pattern consisted of a repeating pattern of oscillations with a period length of 21 min with five maxima (two separated by 5 min and 3 separated by 4 to 4.5 min) within each 21 min repeat. Oscillatory patterns were not obvious in comparable FTIR or CD spectra of albumin, ribonuclease or concanavalin A. The period length was constant at 5, 15, 25, 35 and 45° C (temperature compensated) and oscillations occurred independently of substrate presence. Spectra obtained in deuterium oxide yielded a longer period length of 26 min both for oscillations in enzymatic activity and absorbance ratios determined by FTIR. Taken together the findings suggest that the regular patterns of oscillations exhibited by the ECTO-NOX proteins are accompanied by recurrent global changes in the conformation of the protein backbone that directly modulate enzymatic activity

Synchronous Oscillations Intrinsic to Water: Applications to Cellular Time Keeping and Water Treatment

A homodimeric, growth-related and time-keeping hydroquinone oxidase (ENOX1) of the eukaryotic cell surface capable of oxidizing intracellular NADH exhibits properties of the ultradian driver of the biological 24 h circadian clock by exhibiting a complex 2 + 3 set of oscillations of copper salts and appear to derive from periodic variations in the ratio of ortho and para nuclear spins of the paired hydrogen atoms of the elongated octahedral structure of the ENOX1 protein bound copper II hexahydrates. A corollary of these observations is that the ortho/para oscillations must occur in a highly synchronized matter. Our findings suggest that water molecules communicate with each other via very low frequency electromagnetic fields and that these fields also appear to be generated by the energetics of the synchronous ortho to para interconversions of the nuclear spin pairs of the water hydrogens. Further evidence for energy absorbed and emitted by water and correlated with ortho/para oscillations of ortho/para spin pairs of water hydrogens is indicated from the auto-oscillations in water luminescence. The emissions oscillate with period lengths of 18.8 min that agree with our previously found period of oscillation of about 18 min for pure water, reflective of ortho to para spin isomers based on measurements of redox potential. The period length of pure water (increased by about 25% in D2O) and varies depending on the dominant cation present (copper salts in solution are unique in that the period length is exactly 24 min). Synchrony is maintained through generation of and response to LFEMF generated by the ortho-para spin pairs. Changes in redox potential sufficient to catalyze NADH oxidation were used to monitor synchronous water oscillations that appear to extend indefinitely over great distances in contiguous bodies of either still or flowing water. Adjacent out-of-phase water samples contained in thin plastic cuvettes auto-synchronize in a matter of seconds when placed side by side. Potential applications from water treatment along with opportunity related to human health are anticipated to derive from a better understanding of how water synchrony is generated and maintained, and to be aided by methodological advances in measurement and analysis