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

A CNOX-like protein disulfide-thiol interchange activity of the cell surface of mouse sperm

Intact frozen mouse sperm were analyzed for the presence of ECTO-NOX-like protein disulfide-thiol interchange activity. Activity was determined both from the cleavage of a dithiodipyridine substrate and from the restoration of activity to scrambled and inactive ribonuclease. An activity was found using both methods of activity determination. The activity was resistant to inhibition by both capsaicin and bacitracin. The activity, which oscillated in the characteristic manner of ECTO-NOX proteins, was characterized by a pattern of five maxima and an overall period length of 24 min. Three of the five maxima were separated by an interval of 6 min and the remaining maxima were separated by intervals of 4.5 min to generate the repeating pattern with a period length of 24 min. The activity pattern was unusual in that all five of the maxima within the 24 min repeat were of approximately equal specific activity. Normally, for somatic cells, the first two maxima are involved in NADH oxidation and less pronounced in terms of protein disulfide-thiol interchange than are the remaining three

NADH oxidase activity of HeLa plasma membranes inhibited by the antitumor sulfonylurea N-(4-methylphenylsulfonyl)-N′-(4-chlorophenyl)urea (LY 181984) at an external site

AbstractNADH oxidase activity from HeLa plasma membranes was inhibited by the antitumor sulfonylurea N-(4-methylphenylsulfonyl)-N′(4-chlorophenyl)urea (LY181984). With sealed right side-out vesicles, the drug inhibited half maximally at about 30 nM and the inhibition was nearly complete. A closely related but growth-inactive sulfonylurea, N-(4-methylphenylsulfonyl)-N′-(phenyl)urea (LY181985), did not inhibit the activity. With plasma membranes first solubilized with 2% Triton X-100, activity also was inhibited by LY181984 and not by LY181985 but the maximum inhibition at 10 μM LY181984 was only 50%. When sealed right side-out plasma membrane vesicles were frozen and thawed repeatedly to evert some of the vesicles into an inside-out configuration, the NADH oxidase activity again was only about 50% inhibited by 1 μM LY181984. In such preparations, the right side-out vesicles exhibited an electrophoretic mobility greater than that of the inside-out vesicles. Sidedness was confirmed by measurements of ATPase latency and binding of immunogold-labeled concanavalin A. When the two vesicle populations were resolved by preparative free-flow electrophoresis, the active antitumor sulfonylurea LY181984 inhibited only the NADH oxidase activity of the right side-out vesicles. These findings suggested two NADH sites or activity isoforms for the plasma membrane NADH oxidase. One activity, inhibited by LY181984, appeared to be accessible to external NADH only with sealed right side-out vesicles. The other, not inhibited by LY181984, was accessible to NADH only with inside-out vesicles or after membrane disruption by Triton X-100. The findings demonstrate that the NADH oxidation site inhibited as a result of binding the active antitumor sulfonylurea LY181984 is at the external cell surface. Plasma membrane vesicles from HeLa cells are able to oxidize NADH supplied to either membrane surface but only with inside-out vesicles is NADH oxidation sensitive to inhibition by the antitumor sulfonylurea

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