A transplasma membrane redox system in Phycomyces blakesleeanus: properties of a ferricyanide reductase activity regulated by iron level and vitamin K3.

Intact Phycomyces blaskesleeanus mycelia are capable of reducing extracellular ferricyanide and this transmembrane reduction is an enzymatic process, which is enhanced by the presence of 10 mM lactate. It is modulated in response to intracellular iron levels and negatively regulated by iron and copper. It is inhibited by NEM, p CMB, iodoacetate, Zn2+, Cd2+, dicumarol, and capsaicine analog, but not by cloroquine, and activated by Ca2+, Mg2+, Na+, and K+. Ferricyanide reduction was concomitant with proton release into the extracellular medium, both processes being greatly promoted by vitamin K3 following hyperbolic saturation kinetics with regard to ferricyanide concentration. No stoichiometric proton release was observed with regard to ferricyanide reduction in the absence or the presence of vitamin K3. Proton release coupled with ferricyanide reductase activity does not appear to be due to H+-ATPase. The relevance of these findings to the relationship between the two processes is discussed.

Transplasma membrane electron transport in Leishmania donovani promastigotes.

Leishmania donovani promastigotes are capable of reducing certain electron acceptors with redox potential at pH 7 down to -125 mV; outside the plasma membrane promastigotes can reduce ferricyanide. Ferricyanide has been used as an artificial electron acceptor probe for studying the mechanism of transplasma membrane electron transport. Transmembrane ferricyanide reduction by L. donovani promastigotes was not inhibited by such mitochondrial inhibitors as antimycin A or cyanide, but it responded to inhibitors of glycolysis. Transmembrane ferricyanide reduction by Leishmania appears to involve a plasma membrane electron transport chain dissimilar to that of hepatocyte cells. As with other cells, transmembrane electron transport is associated with proton release, which may be involved in internal pH regulation. The Leishmania transmembrane redox system differs from that of mammalian cells in being 4-fold less sensitive to chloroquine and 12-fold more sensitive to niclosamide. Sensitivities to these drugs suggest that transplasma membrane electron transport and associated proton pumping may be targets for the drugs used against leishmaniasis.

Photoactivation of the flavin cofactor in Xenopus laevis (6 - 4) photolyase: observation of a transient tyrosyl radical by time-resolved electron paramagnetic resonance.

The light-induced electron transfer reaction of flavin cofactor photoactivation in Xenopus laevis (6-4) photolyase has been studied by continuous-wave and time-resolved electron paramagnetic resonance spectroscopy. When the photoactivation is initiated from the fully oxidized form of the flavin, a neutral flavin radical is observed as a long-lived paramagnetic intermediate of two consecutive single-electron reductions under participation of redox-active amino acid residues. By time-resolved electron paramagnetic resonance, a spin-polarized transient radical-pair signal was detected that shows remarkable differences to the signals observed in the related cyclobutane pyrimidine dimer photolyase enzyme. In (6-4) photolyase, a neutral tyrosine radical has been identified as the final electron donor, on the basis of the characteristic line width, hyperfine splitting pattern, and resonance magnetic field position of the tyrosine resonances of the transient radical pair.

Evidence for the extracellular reduction of alpha-lipoic acid by Leishmania donovani promastigotes: a transplasma membrane redox system.

Leishmania donovani cells, capable of reducing certain electron acceptors with redox potentials at pH 7.0 down to -290 mV, outside the plasma membrane, can reduce the oxidised form of alpha-lipoic acid. alpha-Lipoic acid has been used as natural electron acceptor probe for studying the mechanism of transplasma membrane electron transport. Transmembrane alpha-lipoic acid reduction by Leishmania was not inhibited by mitochondrial inhibitors as azide, cyanide, rotenone or antimycin A, but responded to hemin, modifiers of sulphhydryl groups and inhibitor of glycolysis. The protonophores carbonyl cyanide chlorophenylhydrazone and 2,4-dinitrophenol showed inhibition of alpha-lipoic acid reduction. This transmembrane redox system differs from that of mammalian cells in respect to its sensitivity of UV irradiation and stimulation by diphenylamine. Thus a naphthoquinone coenzyme appears to be involved in alpha-lipoic acid reduction by Leishmania cells.

Copper- and zinc-containing superoxide dismutase can act as a superoxide reductase and a superoxide oxidase.

The copper- and zinc-containing superoxide dismutase can catalyze the oxidation of ferrocyanide by O(2) as well as the reduction of ferricyanide by O(2). Thus, it can act as a superoxide dismutase (SOD), a superoxide reductase (SOR), and a superoxide oxidase (SOO). The human manganese-containing SOD does not exert SOR or SOO activities with ferrocyanide or ferricyanide as the redox partners. It is possible that some biological reductants can take the place of ferrocyanide and can also interact with human manganese-containing superoxide dismutase, thus making the SOR activity a reality for both SODs. The consequences of this possibility vis à vis H(2)O(2) production, the overproduction of SODs, and the role of copper- and zinc-containing superoxide dismutase mutations in causing familial amyotrophic lateral sclerosis are discussed, as well as the likelihood that the biologically effective SOD mimics, as described to date, actually function as SORs.

[Permeability of 5 methemoglobin formers through red cell membrane].

By measuring the methemoglobin formation, the permeabilities of some cyanide antidotes passing through mouse erythrocyte membrane were studied. K3Fe(CN)6(0.1 mol/L) did not permeate the red cell and no methemoglobin formed. To the red cell suspension, adding PAPP 0.07 mmol/L, an useful cyanide antidote, no methemoglobin was found. On the contrary, PHAPP, the metabolite of PAPP, transported into the cell readily and reacted with hemoglobin to form methemoglobin quickly. DMAP and NaNO2 passed through the red cell membrane easily. With comparable amount of methemoglobin formation, the concentration of NaNO2 was about 200 times as much as that of DMAP. A comparison of the anticyanide potency of DMAP and NaNO2, the permeability rate constant, the half time and activation energy were measured as: 0.217 and 0.0506/min; 3.2 and 13.7 min; 17.1 and 50.2 kJ/mol, respectively. Owing to its ready permeability and formation of methemoglobin, DMAP is a better antidote than NaNO2 against cyanide poisoning.

Cyanide release from sodium nitroprusside during coronary bypass in hypothermia.

Erythrocyte cyanide levels were determined by a sensitive fluorimetric method on four occasions during coronary bypass in hypothermia in 18 consecutive patients treated with sodium nitroprusside (SNP) with an infusion rate less than 1 microgram x kg-1 x min-1. Every second patient received the cyanide antidote thiosulphate simultaneously with the SNP-infusion. At normal body temperature, as well as during hypothermia in cases receiving thiosulphate, the cyanide levels rose slowly but significantly with the infusion rate. Higher erythrocyte cyanide levels in relation to the infusion rates, up to 8.0 mumol/l, were found during hypothermia in two of the cases not receiving thiosulphate. We conclude that SNP is broken down to cyanide even under hypothermia and that low body temperature may impair the conversion of cyanide to thiocyanate, probably by affecting the metabolic pathways providing the sulphur substrate. This effect may add to other factors decreasing sulphur availability in critically ill patients, and simultaneous administration of thiosulphate is therefore recommended to ensure a safe SNP treatment during and after coronary bypass operations.