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I Fridovich

Publications and source records attributed to I Fridovich.

At least 163 records · Page 9Linked to original sources
PubMed1987

Effect of hydrogen peroxide on the iron-containing superoxide dismutase of Escherichia coli.

The iron-containing superoxide dismutase from Escherichia coli is inactivated by H2O2 to a limit of approximately 90%. When corrected for the H2O2-resistant portion, this inactivation was first order with respect to residual activity and exhibited a pseudo-first-order rate constant of 0.066 min-1 at 25 degrees C in 0.24 mM H2O2 at pH 7.8. The superoxide dismutase activity remaining after treatment with H2O2 differed from the activity of the native enzyme with respect to heat stability, inhibition by azide, and inactivation by light in the presence of rose bengal and by N-bromosuccinimide. The native and the H2O2-modified enzymes were indistinguishable by electrophoresis on polyacrylamide gels. Inactivation of the enzyme by H2O2 was accompanied by loss of tryptophan and some loss of iron, but there was no detectable loss of histidine or of other amino acids. H2O2 treatment caused changes in the optical spectrum of the enzyme. Inactivation of the enzyme by H2O2 depends upon the iron at the active site. Thus, the apoenzyme and the manganese-substituted enzyme were unaffected by H2O2. We conclude that reaction of H2O2 with the iron at the active site generates a potent oxidant capable of attacking tryptophan residues. A mechanism is proposed.

Amino Acids↗
PubMed1987

Assaying for superoxide dismutase activity: some large consequences of minor changes in conditions.

Most assays for superoxide dismutase depend upon competition between the enzyme and some indicating scavenger for O-2. We have investigated the effects of experimental variables on assays based upon the use of either ferricytochrome c or nitro blue tetrazolium. Our results should help investigators to avoid the numerous potential pitfalls which necessarily surround these assay methods.

Animals↗
PubMed1987

Mechanism of the cyanide-catalyzed oxidation of alpha-ketoaldehydes and alpha-ketoalcohols.

Cyanide catalyzes the reduction of dioxygen or of ferricytochrome c by dihydroxyacetone phosphate. The rapid initial phase of these reactions, but not the subsequent slow phase, was augmented by incubating the triose phosphate aerobically or anaerobically at pH 9.0 prior to adding the cyanide. The aerobic incubation, which was most effective, was associated with a decline in enediol, whereas the less effective anaerobic incubation was accompanied by an increase in enediol content. This suggested that the alpha-ketoaldehyde product of autoxidation of the enediol, rather than the enediol itself, was responsible for the rapid phase reaction which followed addition of cyanide. This was confirmed by exploring the cyanide-catalyzed oxidation of the alpha-ketoaldehyde, phenylglyoxal. The inhibitory effect of the manganese-containing superoxide dismutase indicated that O2- was a kinetically important intermediate of the rapid phase reaction. A reaction mechanism is proposed which is consistent with the results presented.

Catalase↗
PubMed1987

The oxidation of NADH by tetravalent vanadium.

Vanadyl (V(IV)) salts autoxidize in neutral aqueous solution yielding O2- plus vanadate (V(V)) and these, in turn, cause the oxidation of NADH, by a free radical chain reaction. This oxidation of NADH was inhibited by superoxide dismutase, but not by a scavenger of HO.. When H2O2 was present V(IV) caused rapid oxidation of NADH by a process which was unaffected by superoxide dismutase but was inhibited by a scavenger of HO.. This appeared to be dependent upon reduction of H2O2 to OH- plus HO., by V(IV)), followed by oxidation of NADH by HO.. Since there are reductants, within cells, capable of reducing V(V) to V(IV), these reactions are likely to contribute to the toxicity of vanadate.

Free Radicals↗
PubMed1987

Contrasting fates of the paraquat monocation radical in Escherichia coli B and in Dunaliella salina.

Anaerobic cultures of Escherichia coli exposed to paraquat (PQ2+) accumulated the corresponding monocation radical PQ+., both within the cells and in the suspending medium. The green alga, Dunaliella salina, which is susceptible to a light- and O2-dependent toxicity of PQ2+, was nevertheless unable to cause accumulation of PQ+. when illuminated anaerobically and could, moreover, discharge the ESR signal and the blue color of PQ+. accumulated by E. coli. Spin trapping allowed demonstration of the photoproduction of O2- within D. salina and of the augmentation of that O-2 production by PQ2+. D. salina appears to contain an electron sink and a heat-labile mechanism for transferring electrons from PQ+. to that sink. This mechanism was demonstrable anaerobically but did not prevent PQ+.-mediated O2- production under aerobic conditions.

Anaerobiosis↗
PubMed1987

Effects of paraquat on the green alga Dunaliella salina: protection by the mimic of superoxide dismutase, Desferal-Mn(IV).

Paraquat caused a time-, dose-, and light-dependent bleaching of the halophilic green alga Dunaliella salina. Sublethal levels of paraquat elicited increases in cell content of both superoxide dismutase and catalase, with changes in the pattern of electromorphs of these enzymes. Desferal-Mn(IV), which catalyzes the dismutation of O2- in vitro, protected against the toxic effect of paraquat. Desferal (desferoxamine mesylate) alone was toxic to D. salina, and the salts of Mn(II), Mn(III), and Mn(IV), in the absence of Desferal, did not protect. Desferal-Mn(IV) is green, but its absorbance was 15% or less than the peak absorbances due to the chlorophyll in D. salina under the conditions of exposure; hence, masking of incident light could not have been the basis of the protective effect of the complex. Incubation of the cells with Desferal-Mn(IV), for up to 8 h prior to the addition of paraquat, did not increase its protective action, and brief washing, following 30 min incubation with the complex, eliminated its protective effect. Neither catalase nor superoxide dismutase, added to the medium, provided protection against paraquat. These results support the view that Desferal-Mn(IV) gains entry into D. salina and protects against the lethal effect of paraquat by there catalyzing the dismutation of O2- into H2O2 + O2.

Catalase↗
PubMed1987

Induction of superoxide dismutase in Escherichia coli by heat shock.

Exposure of midlogarithmic-phase cultures of Escherichia coli B to 48 degrees C for 1 hr elicited an induction of the manganese-containing superoxide dismutase (MnSOD), which became more pronounced during 1 hr of recovery at 37 degrees C. This induction required protein biosynthesis, since it was suppressed by chloramphenicol. Induction of MnSOD appeared to be a response to a heat-mediated increase in O2- production because it was dioxygen-dependent and because heating to 48 degrees C doubled the cyanide-resistant fraction of the total respiration.

Enzyme Induction↗
PubMed1986

Stabilization and partial characterization of endothelium-derived relaxing factor from cultured bovine aortic endothelial cells.

The extreme lability of the chemically undefined vasoregulatory mediator, endothelium-derived relaxing factor (EDRF), has been overcome. The activity of EDRF, from cultured bovine aortic endothelial cells stimulated with A23187, was stabilized by acidification. An additional action of EDRF, platelet disaggregation was used as a sensitive and convenient bioassay to monitor purification. EDRF appears to be a hydrophilic molecule, rapidly inactivated under alkaline conditions. However, activity is restored upon reacidification suggesting that this instability results from a readily reversible chemical process. The stabilization and partial purification of EDRF sets the stage for its further biochemical and chemical characterization.

Animals↗
PubMed1986

Phosphate inhibition of the copper- and zinc-containing superoxide dismutase: a reexamination.

Phosphate was reported to be an inhibitor of copper- and zinc-containing superoxide dismutase (SOD) [de Freitas, D.M., & Valentine, J.S. (1984) Biochemistry 23, 2079-2082]. Thus SOD activity, in 50 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) (pH 7.4), was decreased by approximately 50% when the assay was made 10 mM in phosphate, and the ionic strength was adjusted with sodium fluoride. The inhibitory effect of phosphate was attributed to the neutralization of the positive charge on the guanidino residue of Arg-141. We have reexamined the effects of phosphate inhibition of SOD and found that the enzyme has identical activity in phosphate or HEPES buffer when the ionic strength is adjusted with NaBr. The putative inhibitory effect of phosphate appears to have been due to fluoride inhibition of the superoxide generating system of xanthine/xanthine oxidase. We have confirmed this result by using a photochemical generation of O2- in addition to the enzymatic generation of O2-. Chemical modification of the lysine residues to homoarginines does not affect the activity of the enzyme and does not impart a phosphate sensitivity. Chemical modification with phenylglyoxal caused approximately 80% inactivation of the native enzyme and 90% inactivation of the O-methylisourea-modified enzyme. Our results suggest that phosphate does not inhibit the copper- and zinc-containing superoxide dismutase (Cu,Zn-SOD) beyond the expectations of its effect on ionic strength.

Animals↗
PubMed1986

Free-radical chain oxidation of 2-nitropropane initiated and propagated by superoxide.

The superoxide radical O2.-, whether produced by the xanthine/xanthine oxidase reaction or infused as KO2, solubilized by a crown ether in dry dimethyl sulphoxide, initiated a free-radical chain oxidation of anionic 2-nitropropane. Superoxide dismutase, but not catalase, inhibited oxidation of the nitroalkane. Xanthine oxidase suffered a syncatalytic inactivation, during the co-oxidation of 2-nitropropane, which was reversed by dialysis. Cyanide exacerbated this syncatalytic inactivation and rendered it irreversible. The frequently observed oxidations of nitroalkanes by flavoenzymes now need to be re-examined to clarify the extent to which O2.--initiated free-radical chain oxidation contributed to the overall nitroalkane oxidation.

Alkanes↗
PubMed1986

Biological effects of the superoxide radical.

Can the superoxide radical exert deleterious effects independent of participating with H2O2 in the production of the hydroxyl radical? Examination of the superoxide-related literature reveals data suggesting an affirmative answer to this question.

Animals↗
PubMed1986

The vanadate-stimulated oxidation of NAD(P)H by biomembranes is a superoxide-initiated free radical chain reaction.

Rat liver microsomes catalyze a vanadate-stimulated oxidation of NAD(P)H, which is augmented by paraquat and suppressed by superoxide dismutase, but not by catalase. NADPH oxidation was a linear function of the concentration of microsomes in the absence of vanadate, but was a saturating function in the presence of vanadate. Microsomes did not catalyze a vanadate-stimulated oxidation of reduced nicotinamide mononucleotide (NMNH), but gained this ability when NADPH was also present. When the concentration of NMNH was much greater than that of NADPH a minimal average chain length could be calculated from 1/2 the ratio of NMNH oxidized per NADPH added. The term chain length, as used here, signifies the number of molecules of NMNH oxidized per initiating event. Chain length could be increased by increasing [vanadate] and [NMNH] and by decreasing pH. Chain lengths in excess of 30 could easily be achieved. The Km for NADPH, arrived at from saturation of its ability to trigger NMNH oxidation by microsomes in the presence of vanadate, was 1.5 microM. Microsomes or the outer mitochondrial membrane was able to catalyze the vanadate-stimulated oxidation of NADH or NADPH but only the oxidation of NADPH was accelerated by paraquat. The inner mitochondrial membrane was able to cause the vanadate-stimulated oxidation of NAD(P)H and in this case paraquat stimulated the oxidation of both pyridine coenzymes. Our results indicate that vanadate stimulation of NAD(P)H oxidation by biomembranes is a consequence of vanadate stimulation of NAD(P)H or NMNH oxidation by O-2, rather than being due to the existence of vanadate-stimulated NAD(P)H oxidases or dehydrogenases.

Animals↗
PubMed1986

The effects of paraquat on Escherichia coli: distinction between bacteriostasis and lethality.

Paraquat exerted a progressively more pronounced bacteriostatic effect on Escherichia coli as its concentration was raised in the range 0-1.0 microM. In contrast, concentrations of 100 microM or greater were required before significant lethality could be observed. This bacteriostatic effect of paraquat could be eliminated by supplementation of the glucose-plus-salts medium with either yeast extract or a casein hydrolysate. This protection was seen whether the supplement was added a few minutes prior to or following the addition of paraquat and was thus not due to the inhibition of active uptake of paraquat by the cells. The lethal effect of high levels of paraquat was not influenced by supplementation of the medium with yeast extract. It follows that the bacteriostatic and lethal effects of paraquat involve attack upon distinct targets within the cell.

Cell Division↗
PubMed1986

A demonstration that O2- is a crucial intermediate in the high quantum yield luminescence of luminol.

The chemiluminescence of luminol, due to its reaction with alkaline H2O2, is inhibited by superoxide dismutase or by hydroxyl radical scavengers. Hematin markedly enhances this H2O2-induced luminescence of luminol and lessens, but does not eliminate, the sensitivity towards these inhibitors. Reaction mechanisms are proposed to account for these results. Since luminol luminescence depends upon a reaction between the luminol radical and O2-, and since the luminol radical can reduce dioxygen to O2-, superoxide dismutase-inhibitable luminol luminescence cannot be reliably used as a detector of O2- production.

Hemin↗
PubMed1986

Effects of salts on the lethality of paraquat.

Escherichia coli suffered 95 to 100% lethality when exposed to 1.0 mM paraquat for 30 min at 37 degrees C in aerobic nutrient broth medium but did not lose viability when the exposure was done in Vogel Bonner or tryptic soy yeast extract medium. Paraquat was, however, bacteriostatic in all of these media. Salts, added to the nutrient broth medium, protected against the lethality of paraquat, whereas sucrose did not. Salts of divalent cations were much more effective than salts of monovalent cations. Paraquat increases cyanide-resistant respiration by E. coli; salts added before, but not after, the paraquat diminished this effect. 2,4-Dinitrophenol similarly decreased the cyanide-resistant respiration when added before, but not after, the paraquat. The lethality imposed by paraquat correlated with the rate of cyanide-resistant respiration whether this respiration was modulated by varying salt concentration at a fixed concentration of paraquat or by varying paraquat concentration at a fixed concentration of salt. We conclude that salts or 2,4-dinitrophenol interferes with the active uptake of paraquat by E. coli and thus prevents its lethal effect. The salt concentrations found in a number of commonly used microbiological media are sufficient to exert this effect.

2,4-Dinitrophenol↗