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Biomedical subjects

I Fridovich

Publications and source records attributed to I Fridovich.

At least 235 records · Page 13Linked to original sources
PubMed1980

Mechanism of the antibiotic action pyocyanine.

Exposure of Escherichia coli growing in a rich medium to pyocyanine resulted in increased intracellular levels of superoxide dismutase and of catalase. When these adaptive enzyme syntheses were prevented by nutritional paucity, the toxic action of pyocyanine was augmented. The antibiotic action of pyocyanine was dependent upon oxygen and was diminished by superoxide dismutase and by catalase, added to the suspending medium. Pyocyanine slightly augmented the respiration of E. coli suspended in a rich medium, but greatly increased the cyanide-resistant respiration. Pyocyanine was able to cause the oxidation of reduced nicotinamide adenine dinucleotide, with O2- production, in the absence of enzymatic catalysis. It is concluded that pyocyanine diverts electron flow and thus increases the production of O2- and H2O2 and that the antibiotic action of this pigment is largely a reflection of the toxicity of these products of oxygen reduction.

Catalase↗
PubMed1980

Superoxide radical and superoxide dismutases: threat and defense.

An enzymic flux of O2- and H2O2 caused strand breaks in the supercoiled covalently closed circular Col El plasmid. Subnanomolar levels of superoxide levels of superoxide dismutase or of catalase prevented this attack on DNA, signifying that both O2- and H2O2 were required. Benzoate, mannitol or histidine, which do not scavenge O2- or H2O2, also protected the DNA suggesting that the proximate attacking species had a reactivity comparable to that of the hydroxyl radical. Replacing EDTA with diethylene triamine pentaacetic acid eliminated this attack. In toto the data suggest a metal-catalyzed interaction between O2- and H2O2 which generates a potent oxidant, possibly OH, which can cause DNA strand scission. The biological implications of the production and the enzymic scavenging of the superoxide radical are discussed.

Animals↗
PubMed1979

Autoinactivation of xanthine oxidase: the role of superoxide radical and hydrogen peroxide.

Xanthine oxidase suffers autoinactivation in the course of catalyzing the oxidation of acetaldehyde. When no special efforts were made to maintain a high pO2 in these reaction mixtures catalase protected the xanthine oxidase, but superoxide dismutase did not. However, when oxygen depletion was slowed or prevented by working at lower concentrations of xanthine oxidase, at lower temperatures or by vigorous agitation under an atmosphere of 100% oxygen, superoxide dismutase or catalase protected markedly when added separately and protected almost completely when added together. This result correlates with the greater production of O2-, relative to H2O2, by xanthine oxidase, at elevated pO2. Since histidine also provided some protection and the high levels of acetaldehyde used would have precluded any significant effect of OH., we conclude that singlet oxygen, or something with similar reactivity, was generated from O2- plus H2O2 and contributed significantly to the observed autoinactivation.

Histidine↗
PubMed1979

Purification and characterization of hydroperoxidase II of Escherichia coli B.

The second heme-containing hydroperoxidase isozyme (HP-II) has been isolated from aerobic cultures of Escherichia coli B. The protein exists as a stable tetramer of subunits of equal size, with a combined molecular weight of 312,000. The heme spectrum of HP-II is unusual, in that it exhibits two absorbance maxima at 407 and 591 nm; the alkaline pyridine hemochromogen spectrum shows maxima at 425, 559, and 609 nm. HP-II differs in several respects from the HP-I isozyme previously reported (Claiborne, A., and Fridovich, I. (1979) J. Biol. Chem. 254, 4245-4252). Thus HP-II is virtually devoid of peroxidatic activity toward o-dianisidine but has a 6-fold higher catalatic activity than HP-I. Antisera to HP-II do not cross-react with HP-I, and analyses of chymotryptic and cyanogen bromide digests suggest differences in primary structure between these two isozymes.

Escherichia coli↗
PubMed1979

Subunit association and side-chain reactivities of bovine erythrocyte superoxide dismutase in denaturing solvents.

The copper- and zinc-containing superoxide dismutase of bovine erythrocytes retains its native molecular weight of 32 000 in 8.0 M urea for at least 72 h at 25 degrees C, as evidenced by sedimentation equilibrium analysis. Subsequent to prolonged exposure to urea, the dimeric enzyme could be dissociated by sodium dodecyl sulfate in the absence of reductants, indicating the absence of unnatural disulfide cross-links. The sulfhydryl group of cysteine-6 was unreactive toward 5,5'-dithiobis(2-nitrobenzoic acid) or bromoacetic acid in both neutral buffer and 8.0 M urea. The histidine residues of the enzyme were resistant to carboxymethylation in neutral buffer and 8.0 M urea. However, when the enzyme was exposed to bromoacetic acid in the presence of 6.0 M guanidinium chloride and 1 mM (ethylenedinitriol)tetraacetic acid (EDTA), both sulfhydryl and histidine alkylation were observed. Guanidinium chloride (6.0 M) increased the reactivity of the sulfhydryl group of cysteine-6 and allowed the oxidative formation of disulfide-bridged dimers. This was prevented by 1 mM EDTA. It follows that 8.0 M urea neither dissociates the native enzyme into subunits nor produces a conformation detectably different than that possessed under native conditions.

Animals↗
PubMed1979

Paraquat and Escherichia coli. Mechanism of production of extracellular superoxide radical.

Paraquat mediates a superoxide dismutase-inhibitable reduction of cytochrome c by suspensions of Escherichia coli B. Glucose was most effective in providing electrons for this cytochrome c reduction, but other nutrients could serve in this capacity, provided the cells were preconditioned by growth on these nutrients. Paraquat reduction depended upon a NADPH:paraquat diaphorase, present in the cytosol. Reduced paraquat could diffuse across the cell envelope and react with dioxygen, in the suspending medium, thus generating O2- in that compartment. Most of the paraquat reduced in the cell, under the conditions used, reoxidized in situ and most of the O2- production was thus intracellular. The partitioning of reduced paraquat between intracellular and extracellular compartments, prior to reaction with dioxygen, depended upon intracellular pO2 and any strategy which raised intracellular pO2 decreased the efflux of reduced paraquat and thus decreased extracellular O2- production. Extracellular O2- and H2O2 did contribute to cell damage in proportion to the amount produced. O2- appeared to be unable to cross the cell envelope in either direction and the only O2- which was effective in raising the rate of biosynthesis of the manganese-superoxide dismutase, was that generated within the cell.

Cyanides↗
PubMed1979

Chemical and enzymatic intermediates in the peroxidation of o-dianisidine by horseradish peroxidase. 2. Evidence for a substrate radical--enzyme complex and its reaction with nucleophiles.

Changes in the optical absorption spectrum of horseradish peroxidase, during the oxidation of o-dianisidine at pH 7.5, reveal an intermediate distinct from the previously described compounds I and II. The rate of decay of this new complex appeared to be rate limiting for the catalytic cycle, in this pH range, since imidazole, which augments the catalytic reaction, also enhanced the rate of decay of this complex. Nitrogenous compounds reportedly unable to ligate to hemes, such as 2-methylimidazole and benzimidazole, were nevertheless capable of augmenting the HRP-catalyzed rate of oxidation of o-dianisidine. The activity of nitrogenous compounds, in this regard, appeared to be a function of their nucleophilicity and was sensitive to steric factors but relatively free of a deuterium solvent isotope effect. The data presented in this and in the preceding paper [Claiborne, A., & Fridovich, I. (1979) Biochemistry 18 (preceding paper in this issue)] lead to the suggestion that the nucleophile-responsive intermediate is an enzyme--dianisidine radical complex and that abstraction of the second electron from the bound radical is facilitated by binding of nitrogenous nucleophiles.

Benzidines↗
PubMed1979

Purification of the o-dianisidine peroxidase from Escherichia coli B. Physicochemical characterization and analysis of its dual catalatic and peroxidatic activities.

Extracts of aerobically grown Escherichia coli B exhibit both catalase and dianisidine peroxidase activities. Polyacrylamide gel electrophoresis demonstrates two distinct catalases which have been designated hydroperoxidases I and II (HP-I and HP-II) in order of increasing anodic mobility. HP-I has been purified to essential homogeneity and found to be composed of four subunits of equal size. Its molecular weight is 337,000, and it contains two molecules of protoheme IX per tetramer. Its amino acid composition is unusual, for so large a protein, in lacking half-cystine. HP-I is a very efficient catalase with an activity optimum at pH 7.5, a Km for H2O2 of 3.9 mM, and a turnover number of 9.8 x 10(5) per min. It is also a broad specificity peroxidase capable of acting upon dianisidine, guaiacol, p-phenylenediamine, and pyrogallol. Dianisidine acted as a powerful reversible inhibitor of the catalatic activity of HP-I and as a suicide substrate when HP-I functioned in its peroxidatic mode.

Amino Acids↗
PubMed1978

The biology of oxygen radicals.

The reactive superoxide radical, O2-, formerly of concern only to radiation chemists and radiobiologists, is now understood to be a normal product of the biological reduction of molecular oxygen. An unusual family of enzymes, the superoxide dismutases, protect against the deleterious actions of this radical by catalyzing its dismutation to hydrogen peroxide plus oxygen.

Animals↗
PubMed1978

Permeation of the erythrocyte stroma by superoxide radical.

Superoxide anion, generated by xanthine oxidase within vesicles formed from washed erythrocyte ghosts, crosses the vesicle membrane to reduce cytochrome c in the medium (Lynch, R. E., and Fridovich, I. (1978) J. Biol. Chem, 253, 1838-1845). To determine whether O2- could travel through the membrane in the "channel" for the exchange of stable anions, the effects of two specific inhibitors of anion exchange, 4-acetamido-4'-isothiocyano-2,2' disulfonic acid stilbene (SITS) and 4,4'-diisothiocyano-2,2' disulfonic acid stilbene (DIDS), on the escape of O2- from vesicles were studied. The reduction of external cytochrome c, caused by O2- produced by the enzymic turnover of internal xanthine oxidase, was 85 to 90% inhibited by SITS and DIDS. If SITS impeded the egress of O2- from vesicles, it should enhance the internal effects of O2- and antagonize the inhibition of these effects by external superoxide dismutase. External superoxide dismutase inhibited the lysis of vesicles containing xanthine oxidase. SITS (60 micron) partially reversed this inhibition. It appears that O2- can cross the membrane of the erythrocyte in the anion channel.

Cytochrome c Group↗