PubMed · 296442
The source did not provide an abstract. Follow the original record for more information.
Explore connections, maps & timelines
W M Ringsdorf, E Cheraskin. 1978. The lingual ascorbic acid test.. https://pubmed.ncbi.nlm.nih.gov/296442/
Cite the original work for its findings. Save a collection to share your selection of sources.
KEEP EXPLORING
Diphenylene iodonium (Ph2I), a lipophilic reagent, is an efficient inhibitor of the production of O2- by the activated NADPH oxidase of bovine neutrophils. In a cell-free system of NADPH oxidase activation consisting of neutrophil membranes and cytosol from resting cells, supplemented with guanosine 5'-[gamma-thio]triphosphate, MgCl2 and arachidonic acid, or in membranes isolated from neutrophils activated by 4 beta-phorbol 12-myristate 13-acetate, addition of a reducing agent, e.g. NADPH or sodium dithionite, markedly enhanced inhibition of the NADPH oxidase by Ph2I. The membrane fraction was found to contain the Ph2I-sensitive component(s). In the presence of a concentration of Ph2I sufficient to fully inhibit O2- production (around 10 nmol/mg membrane protein), addition of catalytic amounts of the redox mediator dichloroindophenol (Cl2Ind) resulted in a by-pass of the electron flow to cytochrome c, the rate of which was about half of that determined in non-inhibited oxidase. A marked increase in the efficiency of this by-pass was achieved by addition of sodium deoxycholate. The Cl2-Ind-mediated cytochrome c reduction was negligible in membranes isolated from resting neutrophils. At a higher concentration of Ph2I (100 nmol/mg membrane protein), the Cl2Ind-mediated cytochrome c reductase activity was only half inhibited, which indicated that, in the NADPH oxidase complex, there are at least two Ph2I sensitive components, differing by their sensitivity to the inhibitor. At low concentrations of Ph2I (less than 10 nmol/mg protein), the spectrum of reduced cytochrome b558 in isolated neutrophil membranes was modified, suggesting that the component sensitive to low concentrations of Ph2I is the heme binding component of cytochrome b558. Higher concentrations of Ph2I were found to inhibit the isolated NADPH dehydrogenase component of the oxidase complex. A number of membrane and cytosolic proteins were labeled by [125I]Ph2I. However, the radiolabeling of a membrane-bound 24-kDa protein, which might be the small subunit of cytochrome b558, responded more specifically to the conditions of activation and reduction which are required for inhibition of O2- production by Ph2I. The O2(-)-generating form of xanthine oxidase was also inhibited by Ph2I. Inhibition of xanthine oxidase, a non-heme iron flavoprotein, by Ph2I had a number of features in common with that of the neutrophil NADPH oxidase, namely the requirement of reducing conditions for inhibition of O2- production by Ph2I and the induction of a by-pass of electron flow to cytochrome c by Cl2Ind in the inhibited enzyme, suggesting some similarity in the molecular organization of the two enzymes.
Nitric oxide acts as a widespread signal molecule and represents the endogenous activator of soluble guanylyl cyclase. In endothelial cells and brain tissue, NO is enzymatically formed from L-arginine by Ca2+/calmodulin-regulated NO synthases which require NADPH, tetrahydrobiopterin, and molecular oxygen as cofactors. Here we show that purified brain NO synthase binds to cytochrome c-agarose and exhibits superoxide dismutase-insensitive cytochrome c reductase activity with a Vmax of 10.2 mumol x mg-1 x min-1 and a Km of 34.1 microM. Cytochrome c reduction was largely dependent on Ca2+/calmodulin and cochromatographed with L-citrulline formation during gel filtration. When reconstituted with cytochrome P450, NO synthase induced a moderate Ca(2+)-independent hydroxylation of N-ethylmorphine. NO synthase also reduced the artificial electron acceptors nitro blue tetrazolium and 2,6-dichlorophenolindophenol. Cytochrome c, 2,6-dichlorophenolindophenol, and nitro blue tetrazolium inhibited NO synthase activity determined as formation of L-citrulline from 0.1 mM L-arginine in a concentration-dependent manner with half-maximal effects at 166, 41, and 7.3 microM, respectively. These results suggest that NO synthase may participate in cellular electron transfer processes and that a variety of electron-acceptors may interfere with NO formation due to the broad substrate specificity of the reductase domain of NO synthase.
Quinoids undergo metabolism by a number of flavoenzymes. Reactive species formed during the metabolism of some quinoids might be anticipated to inhibit flavoenzyme activity. Several quinoids have been tested for their ability to inhibit rat liver thioredoxin reductase (TR). The antitumor quinones diaziquone and doxorubicin, and the quinoneimine 2,6-dichloroindophenol, were found to be inhibitors of the reduction of 5,5'-dithiobis-2-nitrobenzoic acid (DTNB) by TR. The inhibition was most marked after incubation of the quinoid with NADPH and the enzyme for 60 min before adding DTNB, with Ki values of 0.5 microM for diaziquone, 0.5 microM for doxorubicin, and 0.07 microM for 2,6-dichloroindophenol. The three quinoids all produced a time-dependent and first order loss of TR activity. There was formation of electron spin resonance-detectable semiquinoid free radicals upon incubation of diaziquone, doxorubicin and 2,6-dichloroindophenol with TR and NADPH under anaerobic conditions. Oxygen radicals formed by redox cycling of the quinoids did not make a major contribution to the inhibition of TR by the quinoids, as shown by the absence of significant reversal of the inhibition by anaerobic incubation conditions and the lack of effect of the oxygen radical scavengers dimethyl sulfoxide, superoxide dismutase and catalase. It was not possible to demonstrate NADPH-dependent covalent binding of radiolabeled diaziquone or doxorubicin to the TR apoprotein. It is possible that the quinoids bind noncovalently to the enzyme apoprotein, or bind to the FAD prosthetic group. The results of the study suggest that some antitumor quinoids are mechanism-based inhibitors of TR showing metabolism- and time-dependent irreversible inhibition of enzyme activity.