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At least 19 recordsLinked to original sources

PubMed1991

NADH-Ferricyanide Reductase of Leaf Plasma Membranes : Partial Purification and Immunological Relation to Potato Tuber Microsomal NADH-Ferricyanide Reductase and Spinach Leaf NADH-Nitrate Reductase.

Plasma membranes obtained by two-phase partitioning of microsomal fractions from spinach (Spinacea oleracea L. cv Medania) and sugar beet leaves (Beta vulgaris L.) contained relatively high NADH-ferricyanide reductase and NADH-nitrate reductase (NR; EC 1.6.6.1) activities. Both of these activities were latent. To investigate whether these activities were due to the same enzyme, plasma membrane polypeptides were separated with SDS-PAGE and analyzed with immunoblotting methods. Antibodies raised against microsomal NADH-ferricyanide reductase (tentatively identified as NADH-cytochrome b(5) reductase, EC 1.6.2.2), purified from potato (Solanum tuberosum L. cv Bintje) tuber microsomes, displayed one single band at 43 kilodaltons when reacted with spinach plasma membranes, whereas lgG produced against NR from spinach leaves gave a major band at 110 kilodaltons together with a few fainter bands of lower molecular mass. Immunoblotting analysis using inside-out and right-side-out plasma membrane vesicles strongly indicated that NR was not an integral protein but probably trapped inside the plasma membrane vesicles during homogenization. Proteins from spinach plasma membranes were solubilized with the zwitterionic detergent 3-[(3-cholamidopropyl) dimethylammonio] 1-propane-sulfonate and separated on a Mono Q anion exchange column at pH 5.6 with fast protein liquid chromatography. One major peak of NADH-ferricyanide reductase activity was found after separation. The peak fraction was enriched about 70-fold in this activity compared to the plasma membrane. When the peak fractions were analyzed with SDS-PAGE the NADH-ferricyanide reductase activity strongly correlated with a 43 kilodalton polypeptide which reacted with the antibodies against potato microsomal NADH-ferricyanide reductase. Thus, our data indicate that most, if not all, of the truly membrane-bound NADH-ferricyanide reductase activity of leaf plasma membranes is due to an enzyme very similar to potato tuber microsomal NADH-ferricyanide reductase (NADH-cytochrome b(5) reductase).

Journal Article↗

PubMed1981

Evidence for the extracellular reduction of ferricyanide by rat liver. A trans-plasma membrane redox system.

1. Reduction of ferricyanide by the isolated perfused rat liver and by isolated rat hepatocytes was studied. 2. Ferricyanide was reduced to ferrocyanide by the perfused liver at a linear rate of 0.22mumol/min per g of liver. Ferricyanide was not taken up by the liver and the perfusate concentration of ferricyanide+ferrocyanide remained constant throughout the perfusion. Perfusate samples from livers perfused without ferricyanide did not reduce ferricyanide. 3. Isolated hepatocytes reduced ferricyanide in a biphasic manner. The initial rate of 2.3mumol/min per g of cells proceeded for approx. 3min and derived from low-affinity sites (apparent K(m)>1.3mm). The secondary rate of 0.29mumol/min per g of cells was maintained for the remainder of the incubation and derived from higher affinity sites (apparent K(m)0.13mm). Disruption of the cells resulted in an increase in the low-affinity rate and a decrease in the high-affinity rate. 4. Ferrocyanide was oxidized by isolated hepatocytes but not by perfused liver. The apparent K(m) for ferrocyanide oxidation by hepatocytes was 1.3mm. 5. Oxidized cytochrome c was reduced by isolated hepatocytes in the presence of 1mm-KCN but at a rate less than that of the reduction of ferricyanide. 6. Properties of the ferricyanide-reducing activities of intact hepatocytes and the perfused liver were examined. The low-affinity rate, present only in cell and broken cell preparations, was inhibited by 1mum-rotenone and 0.5mm-ferrocyanide, and stimulated by 0.1mm-KCN. The mitochondrial substrate, succinate, also stimulated this rate. The perfused liver showed only a high-affinity activity for ferricyanide reduction. This activity was also present in liver cells and was unaffected by rotenone, antimycin A, KCN, NaN(3), or p-hydroxymercuribenzoate but was inhibited by 2.6mm-CaCl(2), 2-heptyl-4-hydroxyquinoline-N-oxide and ferrocyanide. Overall, these results are consistent with the occurrence of a trans-plasma membrane redox system of liver that reduces extracellular ferricyanide to ferrocyanide. The reduction process shows properties which are similar to that of the NADH:ferricyanide oxidoreductase found in isolated liver plasma membranes but different from that of mitochondria.

Animals↗

PubMed1976

Active transport by membrane vesicles from anaerobically grown Escherichia coli energized by electron transfer to ferricyanide and chlorate.

Active transport of amino acids by membrane vesicles from Escherichia coli, grown anaerobically on glucose in the presence of nitrate, can be energized under anaerobic conditions by electron transfer in the nitrate respiration system with formate as electron donor and nitrate as acceptor. A high rate of amino acid transport is also obtained under anaerobic conditions by electron transfer from formate to the nitrate analogue chlorate or to the membrane-impermeable electron acceptor ferricyanide. Electron transfer from formate to nitrate results in the generation of an electrical potential as is indicated by the uptake of the lipophilic cation triphenylmethylphosphonium. Ferricyanide accpets electrons from at least two sites of the nitrate respiration system. One of these sites appears to be nitrate reductase, because cytochrome b, reduced by formate, is completely reoxidized by ferricyanide and glutamate transport energized by formate plus ferricyanide and formate plus nitrate are affected by the same electron transfer inhibitors. A second site of electron transfer to ferricyanide appears to be located prior to nitrate reductase in the nitrate respiration system, since formate is oxidized at a higher rate in the presence of ferricyanide than with nitrate while formate/ferricyanide energizes transport of amino acids at a lower rate than formate/nitrate. Moreover, electron transfer inhibitors block electron transfer from formate to nitrate to a significantly higher extent than from formate to ferricyanide. The effects of irradiation of the membrane vesicles with near ultra-violet light suggest that quinones play an essential role in the electron transfer from formate to nitrate or ferricyanide. Irradiation blocks completely formate-dependent nitrate and ferricyanide reduction and active transport driven by formate/nitrate and formate/ferricyanide, but has hardly any effect on the activity of formate dehydrogenase and on ascorbate/phenazine methosulphate/oxygen-driven transport. Similar effects of ferricyanide have been observed in membrane vesicles from E. coli, grown anaerobically in the presence of fumarate. In these membrane vesicles a high rate of lactose and triphenylmethylphosphonium uptake under anaerobic conditions is obtained by electron transfer from glycerol 1-phosphate to fumarate and also to ferricyanide and evidence has been presented for the involvement of cytochromes in these electron transfers.

Amino Acids↗

PubMed1998

Ascorbate stimulates ferricyanide reduction in HL-60 cells through a mechanism distinct from the NADH-dependent plasma membrane reductase.

The impermeable oxidant ferricyanide is reduced by the plasma membrane redox system of HL-60 cells. The rate of reduction is strongly enhanced by ascorbate or dehydroascorbate. The aim of this study was to determine the mechanism by which ascorbate and dehydroascorbate accelerate ferricyanide reduction in HL-60 cells. Addition of ascorbate or dehydroascorbate to cells in the presence of ferricyanide led to the intracellular accumulation of ascorbate. Control experiments showed that extracellular ascorbate was rapidly converted to dehydroascorbate in the presence of ferricyanide. These data suggest that intracellular ascorbate originates from extracellular dehydroascorbate. Accumulation of ascorbate was prevented by inhibitors of dehydroascorbate transport into the cell. These compounds also strongly inhibited ascorbate-stimulated ferricyanide reduction in HL-60 cells. Thus, it is concluded that the stimulation of ferricyanide reduction is dependent on intracellular accumulation of ascorbate. Changing the alpha-tocopherol content of the cells had no effect on the ascorbate-stimulated ferricyanide reduction, showing that a nonenzymatic redox system utilizing alpha-tocopherol was not involved. p-Chloromercuribenzenesulfonic acid strongly affected ferricyanide reduction in the absence of ascorbate, whereas the stimulated reaction was much less responsive to this compound. Thus, it appears that at least two different membrane redox systems are operative in HL-60 cells, both capable of reducing ferricyanide, but through different mechanisms. The first system is the ferricyanide reductase, which uses NADH as its source for electrons, whereas the novel system proposed in this paper relies on ascorbate.

Ascorbic Acid↗

PubMed1978

Biodegradative threonine dehydratase. Reduction of ferricyanide by an intermediate of the enzyme-catalyzed reaction.

The threonine-dependent reduction of ferricyanide catalyzed by the purified biodegradative threonine dehydratase of Escherichia coli has been studied. The rate of production of 2-oxobutyrate in the presence of ferricyanide was lower than that found in the absence of ferricyanide. The concentrations of threonine required for half-maximal effects for the reduction of ferricyanide and, in the presence of the dye, for 2-oxobutyrate production, were 3 mM and 9mM, respectively. Reduction of ferricyanide was accompanied by evolution of CO2, and even within a very short incubation time with the enzyme, the ratio of ferricyanide reduced over CO2 evolved was approximately 7. Stopping the enzyme activity after a brief exposure to threonine at pH 9.7 resulted in the accumulation of an intermediate (with a half-life of 4 min at 25 degrees C) which formed an adduct with N-ethylmaleimide; the accumulated intermediate, in the absence of N-ethylmaleimide, reduced ferricyanide with concomitant evolution of CO2. We conclude from these results that 2-aminocrotonate is the intermediate which serves as a source of reducing equivalent for ferricyanide, and nonstoichiometric amount of ferricyanide reduction may be attributed to some secondary reactions of ferricyanide with compounds derived from the oxidation product of 2-aminocrotonate.

Escherichia coli↗

PubMed1976

Evidence for multiple sites of ferricyanide reduction in chloroplasts.

Various sites of ferricyanide reduction were studied in spinach chloroplasts. It was found that in the presence of dibromothymoquinone a fraction of ferricyanide reduction was dibromothymoquinone sensitive, implying that ferricyanide can be reduced by photosystem I as well as photosystem II. To separate ferricyanide reduction sites in photosystem II, orthophenanthroline and dichlorophenyl dimethylurea inhibitions were compared at various pHs. It was noted that at low pH ferricyanide reduction was not completely inhibited by orothophenanthroline. At high pH's, however, inhibition of ferricyanide reduction by orthophenanthroline was complete. It was found that varying concentration of orthophenanthroline at a constant pH showed different degrees of inhibition. In the study of ferricyanide reduction by photosystem II various treatments affecting plastocyanin were performed. It was found that Tween-20 or KCN treatments which inactivated plastocyanin did not completely inactivate ferricyanide reduction. These data support the conclusion that ferricyanide accepts electrons both before and after plastoquinone in photosystem II.

Binding Sites↗

PubMed1988

Plasmalemma Redox Chain and H Extrusion: II. Respiratory and Metabolic Changes Associated with Fusicoccin-Induced and with Ferricyanide-Induced H Extrusion.

Ferricyanide reduction by Elodea densa leaves is associated with a release of protons in the cytoplasm, a fraction of the increase in protons being then extruded by the ATP-driven proton pump (20). The data presented here show that ferricyanide induces a marked increase in O(2) uptake, additive to that induced by fusicoccin plus K(+), and here interpreted as depending on the utilization of ATP by the H(+) pump. Glucose 6-phosphate and malate levels are markedly increased by fusicoccin plus K(+). The simultaneous presence of ferricyanide reduces by about 50% the increase of malate, while it completely suppresses that of glucose 6-phosphate. The ferricyanide-induced decrease of malate is interpreted as due to the acidification of the cytosol associated with ferricyanide reduction, while the more marked decrease of glucose 6-phosphate might depend in part on the pH change and in part on a faster oxidation of this substrate. In fact, ferricyanide reduction is accompanied by a marked decrease of the incorporation into RNA ribose of C-1 as compared with C-2 of [(14)C]glucose. This suggests a stimulation of the release of C-1 as CO(2) at the level of the glucose 6-phosphate oxidation pathway, as expected if NADPH was the electron donor for ferricyanide reduction. These results are interpreted as confirming that the H(+) efflux associated with ferricyanide reduction depends on the activation of the ATP-driven plasmalemma H(+) pump. They also suggest that NADPH is used as an electron donor to some initial component of the plasmalemma redox system.

Journal Article↗

PubMed1985

Transmembrane redox in control of cell growth. Stimulation of HeLa cell growth by ferricyanide and insulin.

The impermeable electron acceptor ferricyanide stimulates the growth of HeLa cells in the absence of serum and increases cell replication with limiting amounts of serum (0.75%). Maximum growth stimulation occurs at low ferricyanide concentration from 0.01 to 0.1 mM. Higher ferricyanide concentrations inhibit growth on serum. Addition of insulin enhances the stimulating effect of ferricyanide. Increase in the transplasmalemma electron transport activity in the presence of insulin is also observed by measuring the rate of ferricyanide reduction by cells. There is a close correlation between insulin stimulation of ferricyanide reduction and insulin induction of cell proliferation and attachment. In addition to ferricyanide, the growth response is observed with other impermeable oxidants, such as indigotetrasulfonate and hexaamine ruthenium III, which are reduced by the transplasma membrane electron transport system. Inactive oxidants such as cytochrome c do not stimulate cell growth. Ferrocyanide does not stimulate growth. We propose that electron flow through the transplasma membrane electron transport system stimulates growth and that insulin acts to increase that flow.

Blood↗

PubMed2000

Ferricyanide reduction by Escherichia coli: kinetics, mechanism, and application to the optimization of recombinant fermentations.

Ferricyanide reduction was studied by flow injection analysis (FIA) and chronoamperometry (CA) using two host strains and one recombinant strain of E. coli. Samples taken from batch cultures of E. coli JM105 and HB101 showed maximal specific ferricyanide reduction rates in the late exponential phase of growth, with values (micromol/min x g) of 24 (FIA) and 17 (CA) for JM105, and 36 (FIA) for HB101, when shake-flask cultures were sampled, and 70 for HB101, when a chemostat was used to control pH and dissolved oxygen concentration throughout the cultivation. Remarkably higher ferricyanide reduction rates were obtained with HB101 cells cultivated continuously at very slow growth rate, when chilled, resuspended cell samples were incubated for 5 min in solutions containing 10 mM succinate or formate. These compounds are substrates for primary, membrane-bound dehydrogenases that transfer electrons via ubiquinone to the cytochrome oxidase complexes. Apparent Michaelis-Menten kinetics were observed with respect to ferricyanide concentration when 10 mM succinate was included in the assay buffer; apparent Km values of 10.1+/-0.6 mM and 14.4+/-1.2 mM ferricyanide were obtained for exponential- and stationary-phase E. coli JM105, respectively. Cyanide inhibition studies show that ferricyanide is reduced mainly by cytochrome o oxidase in exponentially growing cells. The large difference in ferricyanide reduction rates observed in the absence and presence of succinate and formate were used to signal stationary-phase entry 5 h after induction of recombinant human Cu/Zn superoxide dismutase expression in a batch fermentation of E. coli HMS174(DE3)(pET3ahSOD). This new method can be used as an adjunct to the quantitation of medium components for the optimization of recombinant fermentations.

Escherichia coli↗

PubMed1981

Ferricyanide Reduction in Photosystem II of Spinach Chloroplasts.

Ferricyanide can be reduced in Photosystem II of spinach chloroplasts at 2 separate sites, both of which are sensitive to 3-(3,4-dichlorophenyl)-1,1-dimethylurea, but only one of which is sensitive to dibromothymoquinone. Data presented in this paper emphasize ferricyanide site II of Photosystem II, which is sensitive to thiol inhibition and may reflect a cyclic pathway around Photosystem II. Ferricyanide reduction sites 1 and 2 also differ from each other in fractions isolated from discontinuous sucrose gradients, from fragmented chloroplasts, and upon trypsin treatment. Sucrose density gradient centrifugation shows that ferricyanide reduction site 1 activity at pH 6 decreases from 30 to 50% in various isolated fractions, while the dibromothymoquinone-insensitive activity at pH 8 (site 2) is stimulated from 15 to 35%.Fragmentation of chloroplasts also stimulates ferricyanide site II activity, but trypsin treatment destroys ferricyanide reduction site II completely in 6 minutes. Ferricyanide reduction site 1 still retains 50% activity at this point. The meaning of these differences is discussed in terms of the physical location of these two sites on the thylakoid membrane.

Journal Article↗

PubMed1996

Reduction of ferricyanide by thiamine or thiamine pyrophosphate.

The colorimetric assay for the activity of pyruvate dehydrogenase (EC 1.2.4.1) developed by Itokawa is based on a coupled reduction of ferricyanide and the formation of Prussian blue (Brian Research 94, 475-484). In this assay system, we found that the coenzyme, thiamine pyrophosphate, itself reduced ferricyanide independent of both the enzyme and the substrate. Similar effect was also observed with thiamine, but not with thiochrome. The reduction of ferricyanide by thiamine or thiamine pyrophosphate was blocked by trichloroacetic acid. Measurement of the activity of purified pyruvate dehydrogenase based on monitoring the reduction of ferricyanide as described by Schwartz et al (Biochem. Biophys. Res. Commun. 31, 495-500) has been widely used. Our findings clearly disprove the assumption that reduction of ferricyanide is dependent on pyruvate dehydrogenase and invalidate assays for thiamine-dependent dehydrogenases based on the reduction of ferricyanide.

Animals↗

PubMed1982

Transmembrane ferricyanide reduction by cells of the yeast Saccharomyces cerevisiae.

Both respiratory-competent and respiratory-deficient yeast cells reduce external ferricyanide. The reduction is stimulated by ethanol and inhibited by the alcohol dehydrogenase inhibitor, pyrazole. The reduction of ferricyanide is not inhibited by inhibitors of mitochondrial or microsomal ferricyanide reduction. Cells in exponential-phase growth show a much higher rate of ferricyanide reduction. The reduction of ferricyanide is accompanied by increased release of protons by the yeast cells. We propose that the ferricyanide reduction is carried out by a transmembrane NADH dehydrogenase.

Ferricyanides↗

PubMed1985

Effect of ubiquinone extraction on the reaction of the mitochondrial bc1 complex with ferricyanide.

Depletion of endogenous ubiquinone by pentane extraction of mitochondrial membranes lowered succinate-ferricyanide reductase activity, whereas quinone reincorporation restored the enzymatic activity as well as antimycin sensitivity. The oxidant-induced cytochrome b extrareduction, normally found upon ferricyanide pulse in intact mitochondria in the presence of antimycin, was lost in ubiquinone-depleted membranes, even if cytochrome c was added. Readdition of ubiquinone-2 restored the oxidant-induced extrareduction with an apparent half saturation at 1 mol/mol bc1 complex saturating at about 5 mol/mol. These findings demonstrate a requirement for the ubiquinone pool of the cytochrome b extrareduction. Since the initial rates of cytochrome b reoxidation upon ferricyanide addition, in the presence of antimycin, did not saturate by any ferricyanide concentration in ubiquinone-depleted mitochondria, a direct chemical reaction between ferricyanide and reduced cytochrome b was postulated. The fact that such direct reaction is much faster in ubiquinone-depleted mitochondria may explain the lower antimycin sensitivity of the succinate ferricyanide reductase activity after removal of endogenous ubiquinone.

Animals↗

PubMed1986

Decrease of NADH in yeast cells by external ferricyanide reduction.

Ferricyanide reduction catalyzed by vitamin K-3 was accompanied by the decrease in intracellular (NAD(P)H concentration of yeast cells, and the rate of ferricyanide reduction depended on intracellular concentration of NADH rather than NADPH. The addition of glucose to the cell suspensions enhanced both ferricyanide reduction and intracellular NADH concentration. The catalytic action of vitamin K-3 on ferricyanide reduction was observed in the presence of NADH and plasma membrane preparations. As the toxic action of vitamin K-3 on cell growth of yeast was enhanced by addition of ferricyanide, ferricyanide reduction catalyzed by vitamin K-3 may inhibit cell growth by decreasing intracellular NADH concentration.

Ferricyanides↗

PubMed1986

Primary donor recovery kinetics in reaction centers from Rhodopseudomonas viridis. The influence of ferricyanide as a rapid oxidant of the acceptor quinones.

In reaction centers from Rhodopseudomonas viridis that contain a single quinone, the decay of the photo-oxidized primary donor, P+, was found to be biphasic when the bound, donor cytochromes were chemically oxidized by ferricyanide. The ratio of the two phases was dependent on pH with an apparent pK of 7.6. A fast phase, which dominated at high pH (t1/2 = 1 ms at pH 9.5), corresponded to the expected charge recombination of P+ and the primary acceptor QA-. A much slower phase dominated at low pH and was shown to arise from a slow reduction of P+ by ferrocyanide in reaction centers where QA- has been rapidly oxidized by ferricyanide. The rate of QA- oxidation was linear with respect to ferricyanide activity and was strongly pH-dependent. The second-order rate constant, corrected for the activity coefficient of ferricyanide, approached a maximum of 2 X 10(8) M-1 X s-1 at low pH, but decreased steadily as the pH was raised above a pK of 5.8, indicating that a protonated state of the reaction center was involved. The slow reduction of P+ by ferrocyanide was also second-order, with a maximum rate constant at low pH of 8 X 10(5) M-1 X s-1 corrected for the activity coefficient of ferrocyanide. This rate also decreased at higher pH, with a pK of 7.4, indicating that ferrocyanide also was most reactive with a protonated form of the reaction center. The oxidation of QA- by ferricyanide was unaffected by the presence of o-phenanthroline, implying that access to QA- was not via the QB-binding site. In reaction centers supplemented with ubiquinone, oxidation of reduced secondary quinone, QB-, by ferricyanide was observed but was substantially slower than that for QA-. It is suggested that Q-B may be oxidized via QA so that the rate is modulated by the equilibrium constant for QA-QB in equilibrium with QAQB-.

Ferricyanides↗

PubMed2002

Intracellular flavonoids as electron donors for extracellular ferricyanide reduction in human erythrocytes.

Reduction of extracellular ferricyanide [Fe(CN)(6)](-3) to ferrocyanide by intact cells reflects the activity of a trans-plasma membrane oxidoreductase that, in human red blood cells, utilizes ascorbic acid as an electron donor. We herein report that the flavonoids quercetin and myricetin, while inhibiting dehydroascorbic acid uptake-and thus the erythrocyte ascorbic acid content-effectively stimulate the extracellular reduction of ferricyanide. Other flavonoids such as rutin, acacetin, apigenin, and genistein do not show the same effect. The notion that quercetin or myricetin may serve as an intracellular donor for a trans-plasma membrane oxidoreductase is supported by the following lines of evidence: (i) they afford direct reduction of ferricyanide; (ii) extracellular reduction of ferricyanide was not mediated by direct effects of the flavonoids released by the cells and was abolished by the sulphydryl reagent parachloromercuribenzenesulfonic acid (pCMBS); (iii) the intracellular concentrations of quercetin or myricetin well correlate with increases in ferricyanide reduction; (iv) the intracellular concentration of the flavonoids dramatically declines after ferricyanide exposure. Taken together, the results presented in this study demonstrate that myricetin and quercetin, which accumulate in large amounts in red blood cells, act as intracellular substrates of a pCMBS-sensitive trans-plasma membrane oxidoreductase. This may represent a novel mechanism whereby these flavonoids exert beneficial effects under oxidative stress conditions.

4-Chloromercuribenzenesulfonate↗

PubMed1991

Oxidation of molybdopterin in sulfite oxidase by ferricyanide. Effect on electron transfer activities.

The attenuation of the sulfite:cytochrome c activity of sulfite oxidase upon treatment with ferricyanide was demonstrated to be the result of oxidation of the pterin ring of the molybdenum cofactor in the enzyme. Oxidation of molybdopterin (MPT) was detected in several ways. Ferricyanide treatment not only abolished the ability of sulfite oxidase to serve as a source of MPT to reconstitute the aponitrate reductase in extracts of the Neurospora crassa mutant nit-1 but also eliminated the ability of sulfite oxidase to reduce dichlorobenzenoneindophenol after anaerobic denaturation. Additionally, the absorption spectrum of anaerobically denatured ferricyanide-treated molybdenum fragment of rat liver sulfite oxidase was typical of fully oxidized pterins. Ferricyanide treatment had no effect on the protein of sulfite oxidase or on the sulfhydryl-containing side chain of MPT. Quantitation of the ferricyanide reaction showed that 2 mol of ferricyanide were reduced per mol of MPT oxidized, yielding a fully oxidized pterin. These results corroborate the previously reported conclusion that the native state of reduction of MPT in sulfite oxidase is at the dihydro level (Gardlik, S., and Rajagopalan, K.V. (1990) J. Biol. Chem. 265, 13047-13054). As a result of oxidation of the pterin ring, the affinity of MPT for molybdenum is decreased, leading to eventual loss of molybdenum. Because the loss of molybdenum is slow, a population of sulfite oxidase molecules can exist in which molybdenum is complexed to oxidized MPT. These molecules retain sulfite:O2 activity, a function apparently dependent solely on the molybdenum-thiolate complex, yet have greatly decreased sulfite:cytochrome c activity, a function requiring heme as well as the molybdenum center of holoenzyme. These observations suggest that the pterin ring of MPT participates in enzyme function, possibly in electron transfer, directly in catalysis, or by controlling the oxidation/reduction potential of molybdenum.

Animals↗

PubMed2001

Evaluation of hexose monophosphate shunt activity in isolated murine lens by monitoring the potential of the ferricyanide--ferrocyanide system.

A method for evaluation of the activity of the hexose monophosphate shunt (HMS) in isolated lens is presented. The measurement of HMS activity is based on continuous monitoring of the potential of the ferricyanide--ferrocyanide system (where ferricyanide is an artificial electron acceptor) in the presence of a lens. The rate of reduction of ferricyanide increased after the addition of methylene blue (MB) or saponin. The ferricyanide reduction rate did not correlate with GSH content in the contralateral lenses of the same mouse in the absence of MB or saponin. Correlations between the ferricyanide reduction rate and GSH content in the lens were 0.67 (beta = 0.93) in the presence of MB and 0.82 (beta = 0.95) in the presence of saponin. We think that the measured curves of ferricyanide reduction are representative of: 1) normal level of HMS activity (in the absence of methylene blue and saponin); 2) maximal HMS activity (in the presence of methylene blue); 3) the intracellular concentration of reducing equivalents (in the presence of saponin).

Animals↗