Biomedical subjects

I Fridovich

Publications and source records attributed to I Fridovich.

At least 73 records · Page 4Linked to original sources

PubMed1995

The effect of detergents on the reduction of tetrazolium salts.

Detergents, such as Triton X-100, markedly increase the reduction of tetrazolium salts by xanthine oxidase plus xanthine, or by NADH. This effect of detergent, in the case of the xanthine oxidase catalyzed process, is seen aerobically but not anaerobically. Increasing the rate of accumulation of formazan, whether by increasing the concentration of the tetrazolium salt or by adding detergent, decreased susceptibility to inhibition by superoxide dismutase or by O2. These results are accommodated by a scheme of reactions the essence of which is the univalent reduction of the tetrazolium to an uncharged tetrazoinyl radical which can reduce O2 to O2- or which can partition into the detergent micelles and there dismute to generate the stable formazan.

Detergents↗

PubMed1995

A superoxide dismutase mimic protects sodA sodB Escherichia coli against aerobic heating and stationary-phase death.

Superoxide appears to be a major cause of stationary-phase death and heat kill. In support of this conclusion are the following observations: (a) Stationary-phase death was apparent in the sodA sodB, but not in the superoxide dismutase (SOD)-competent parental strain; (b) Stationary phase death in the sodA sodB strain was dioxygen-dependent; (c) A manganic porphyrin, which catalyzes the dismutation of superoxide, protected the sodA sodB strain against stationary-phase death; (d) Heating the sodA sodB strain to 42 degrees C caused a loss of viability not seen with the SOD-competent parental strain and preventable by the manganic porphyrin. Exposure to aerobic conditions induced antibiotic resistance in the sodA sodB, but not in the parental strain and the manganic porphyrin prevented that induction. This again indicates its ability to substitute for SOD in Escherichia coli.

Aerobiosis↗

PubMed1995

A cationic manganic porphyrin inhibits uptake of paraquat by Escherichia coli.

A manganic porphyrin (MnTMPyP), which catalyzed the dismutation of O2-, facilitated the aerobic growth of a sodA sodB strain of Escherichia coli and protected a superoxide dismutase (SOD)-competent parental strain against paraquat. Surprisingly, the latter effect was more complete than the former and the mimic could block the inductions of fumarase C and of glucose 6-phosphate dehydrogenase by paraquat, even though SOD could not. An explanation for these apparent paradoxes was found in the ability of MnTMPyP to inhibit the uptake of paraquat by E. coli. MnTMPyP was accumulated by E. coli until its intracellular concentration was 20-fold greater than the extracellular concentration. This happened in a glucose plus salts medium, but not from a rich LB medium. MnTMPyP was bound onto cellular macromolecules and was maintained in the reduced state within E. coli. The free form of the reduced MnTMPyP was autoxidizable, but the bound form was not. Consequently, the free form could catalyze the oxidation of ascorbate, while the bound form did not.

Ascorbic Acid↗

PubMed1995

Copper, zinc superoxide dismutase in Escherichia coli: periplasmic localization.

Cu,ZnSOD purified from Escherichia coli has been used to raise antibodies in rabbits. The resultant antiserum was found to recognize a single band on Western blots of SDS-polyacrylamide gel electropherograms, and that single band coincided with the position of the Cu,ZnSOD. Ultrathin sections of fixed E. coli were treated with the antibody followed by protein A bearing 10-nm gold particles. Electron microscopy revealed that Cu,ZnSOD was largely localized in the periplasm in polar bays.

Animals↗

PubMed1995

Superoxide from glucose oxidase or from nitroblue tetrazolium?

Glucose oxidase reduces nitroblue tetrazolium, or ferricytochrome c, faster anaerobically than aerobically. This result is inconsistent with the conclusion that GO2 can reduce O2 to O2- which is then responsible for the reduction of NBT and of cytochrome c. Nevertheless, the aerobic reductions are partially inhibitable by superoxide dismutase. A scheme of reactions is proposed which explains why these electron acceptors cause an O2- production which does not occur in their absence when O2 is the sole electron acceptor.

Aerobiosis↗

PubMed1995

Adaptation to oxidative stress in young, but not in mature or old, Caenorhabditis elegans.

The effect of aging on the ability of Caenorhabditis elegans to adapt to oxidative stress was examined. Oxidative stress was applied with the quinone plumbagin or with hyperoxia, both of which are expected to increase intraorganismal production of O2.- and of H2O2. Young nematodes adapted by increasing their content of superoxide dismutase (SOD) and they survived, whereas older nematodes did not induce superoxide dismutase and suffered loss of viability. It thus appears that, in C. elegans, loss of adaptability to oxidative stress, monitored in terms of induction of SOD, is a hallmark of senescence.

Adaptation, Physiological↗

PubMed1995

Superoxide dismutase protects against aerobic heat shock in Escherichia coli.

Exposure of a superoxide dismutase-null (sodA sodB) strain of Escherichia coli to aerobic heat stress (45 to 48 degrees C) caused a profound loss of viability, whereas the same heat stress applied anaerobically had a negligible effect. A superoxide dismutase-competent parental strain was resistant to the lethal effect of the aerobic heating. It follows that aerobic heating imposes an oxidative burden of which O2- must be a major component. This effect is not seen at 53 degrees C, presumably because, at this higher temperature, direct thermolability of vital cell components overrides the effect of superoxide radicals.

Aerobiosis↗

PubMed1995

Superoxide radical and superoxide dismutases.

O2- oxidizes the [4Fe-4S] clusters of dehydratases, such as aconitase, causing-inactivation and release of Fe(II), which may then reduce H2O2 to OH- +OH.. SODs inhibit such HO. production by scavengingO2-, but Cu, ZnSODs, by virtue of a nonspecific peroxidase activity, may peroxidize spin trapping agents and thus give the appearance of catalyzing OH. production from H2O2. There is a glycosylated, tetrameric Cu, ZnSOD in the extracellular space that binds to acidic glycosamino-glycans. It minimizes the reaction of O2- with NO. E. coli, and other gram negative microorganisms, contain a periplasmic Cu, ZnSOD that may serve to protect against extracellular O2-. Mn(III) complexes of multidentate macrocyclic nitrogenous ligands catalyze the dismutation of O2- and are being explored as potential pharmaceutical agents. SOD-null mutants have been prepared to reveal the biological effects of O2-. SodA, sodB E. coli exhibit dioxygen-dependent auxotrophies and enhanced mutagenesis, reflecting O2(-)-sensitive biosynthetic pathways and DNA damage. Yeast, lacking either Cu, ZnSOD or MnSOD, are oxygen intolerant, and the double mutant was hypermutable and defective in sporulation and exhibited requirements for methionine and lysine. A Cu, ZnSOD-null Drosophila exhibited a shortened lifespan.

Animals↗

PubMed1994

Superoxide and peroxynitrite inactivate aconitases, but nitric oxide does not.

The Escherichia coli and recombinant human cytosolic aconitases are inactivated by O2-., with a rate constant of approximately 3 x 10(7) M-1 s-1; the corresponding value for the porcine mitochondrial aconitase is approximately 0.8 x 10(7) M-1 s-1. Nitric oxide, which is reported to inactivate aconitase, did not do so at a perceptible rate, while incubation with peroxynitrite led to a rapid loss of aconitase activity. We propose that the reported inactivation of aconitase by nitric oxide in vivo is actually mediated through peroxynitrite, the product of the reaction between O2-. and NO..

Aconitate Hydratase↗

PubMed1994

Escherichia coli expresses a copper- and zinc-containing superoxide dismutase.

A mutant of Escherichia coli, unable to produce manganese- or iron-containing superoxide dismutase (SOD), was found to contain modest levels of an SOD that was judged to be a copper- and zinc-containing SOD on the basis of inhibition by cyanide and inactivation by either H2O2 or diethyldithiocarbamate. Moreover, the diethyldithiocarbamate-inactivated enzyme could be reactivated with Cu(II), and this reconstituted enzyme, like the native enzyme, was unaffected by EDTA and was inhibited by cyanide. This enzyme was, furthermore, selectively released by osmotic shock, in keeping with a periplasmic localization, and it was strongly induced during aerobic growth. This enzyme was also present in the SOD-competent parental strain. Failure to detect it previously can be attributed to its periplasmic localization, thermal lability, sensitivity to pH, and to its relative paucity. It will now be interesting to explore the phenotypic consequences imposed by the absence of this SOD.

Cell Compartmentation↗

PubMed1994

Stable Mn(III) porphyrins mimic superoxide dismutase in vitro and substitute for it in vivo.

Several manganic porphyrins, with substituents on the methine bridge carbons, were prepared and examined for stability, redox behavior, catalysis of the dismutation of superoxide radical (O2-), and the ability to protect a superoxide dismutase (SOD)-null strain of E. coli against dissolved oxygen and a SOD-competent strain against paraquat. All of the compounds tested exhibited reversible redox behavior and were stable to EDTA in both the oxidized and reduced states, and several were able to catalyze the dismutation of O2- with the rate constants of approximately 10(7) M-1 s-1. The marked protective effects of some of these compounds exceeded that which could be anticipated on the basis of such rate constants. The tetrakis (1-methyl-4-pyridyl) compound was reduced enzymatically at the expense of NADPH and nonenzymatically by GSH and was kept in the reduced state within E. coli. Since the rate constant for reoxidation of the reduced form by O2- is 4 x 10(9) M-1 s-1, it appears that this compound acts in vivo as an NADPH/GSH:O2- oxidoreductase rather than as an SOD mimic. Its ability to facilitate aerobic growth of the SOD-null strain can be explained on this basis.

Cytochrome c Group↗

PubMed1994

Characterization of Mn(III) complexes of linear and cyclic desferrioxamines as mimics of superoxide dismutase activity.

The green complex of Mn(III) with desferrioxamine B has been prepared by air oxidation of Mn(II) in the presence of the ligand or by reaction of Mn(OH)3, or of MnO2, with the ligand. The resultant complex was purified by ion-exchange chromatography and shown by electrospray ionization mass spectrometry to be a 1:1 complex of Mn(III) with desferrioxamine B. A similar complex was prepared from the macrocyclic Tris hydroxamate desferrioxamine E. Mn(III) desferrioxamine B was not stable to equimolar EDTA while the Mn(III) desferrioxamine E was stable to a fivefold molar excess of EDTA. Chelex-100 decomposed the desferrioxamine B complex, but not the desferrioxamine E complex. These complexes prevented the reduction of cytochrome c by O2- but were less active than free Mn(II). The second-order rate constants for reaction with O2- at 25 degrees C and at pH 7.5 were 3.0 x 10(6), 1.0 x 10(6), and 9.5 x 10(4) for Mn(II), Mn(III) desferrioxamine B, and Mn(III) desferrioxamine E, respectively.

Catalysis↗

PubMed1994

NADPH: ferredoxin oxidoreductase acts as a paraquat diaphorase and is a member of the soxRS regulon.

Soluble extracts of Escherichia coli contain four NADPH:paraquat diaphorases that were separable by anion-exchange HPLC over Mono Q. One of these was induced when the cells were exposed to paraquat. This was the case in a soxRS-competent strain but not in a soxRS-null strain, while a soxRS-constitutive strain overexpressed this diaphorase without the stimulus of exposure to paraquat. This NADPH:paraquat diaphorase could use cytochrome c or nitroblue tetrazolium as an electron acceptor, whereas O2 was a relatively poor acceptor. This diaphorase was identified as the NADPH:ferredoxin reductase. A role for reduced ferredoxin and flavodoxin in the adaptive soxRS response to oxidative stress and in the regulation of the redox status of soxR is discussed.

Amino Acid Sequence↗

PubMed1994

Paraquat diaphorases in Escherichia coli.

Extracts of E. coli contain at least three easily separable NAD(P)H:paraquat diaphorases. One of these is identified as thioredoxin reductase, which accounts for most of the PQ++ diaphorase in a thioredoxin reductase overproducer but is only 25% of this activity in a wild type. NADP+, but not NAD+, inhibited the diaphorase activity of thioredoxin reductase. All of the soluble PQ++ diaphorases of E. coli are stable during fractionation by HPLC and none depend upon the cooperative action of components separable by this technique. GSSG reductase is inhibited by PQ++ and is not, to any significant degree, a contributor to the diaphorase activity of E. coli.

Chromatography, High Pressure Liquid↗

PubMed1994

The role of O2.- in the production of HO.: in vitro and in vivo.

In vitro O2.- reduces Fe(III) to Fe(II), which, in turn, reduces the H2O2, yielding Fe(II)O or HO.. In vivo O2.- increases the supply of free iron by oxidatively attacking the [4Fe-4S] clusters of dehydratases such that they release Fe(II), which can then reduce H2O2. In vivo, O2.- also increases the production of H2O2 by acting as an oxidant toward the dehydratases and toward other cellular reductants.

Aconitate Hydratase↗

PubMed1994

Free radicals in cutaneous biology.

During the past 25 years, the field of free radical biology has germinated, sprouted, and flowered. Free radicals derived from molecular oxygen, formerly of interest only to radiation chemists, are now known to play multiple roles in living systems. We will here consider the generalities of this field with some special focus on skin as a site of oxygen radical production and as a target upon which the damaging propensities of these radicals are exerted.

Free Radicals↗

PubMed1993

Excess substrate inhibition of xanthine oxidase: a reexamination.

Xanthine oxidase has long been considered to be subject to inhibition by excess substrate. It is now shown that, although such inhibition can be seen in Tris or N,N-bis(2-hydroxyethyl)glycine buffers, earlier reports in which phosphate, pyrophosphate, or Veronal buffers were used were probably the result of a spectrophotometric artifact imposed by stray light in the incident beam.

Animals↗

PubMed1993

Effects of paraquat on Escherichia coli: sensitivity to small changes in pH of the medium--a cautionary note.

Uric acid appears to protect Escherichia coli against the growth-inhibiting effect of paraquat, but this is actually due to acidification of the medium and does not occur when the pH of the medium is readjusted to neutrality. Any compound which lowers the pH of the medium will thus diminish the effect of paraquat on E. coli, whether that effect is inhibition of growth or adaptive induction of members of the soxRS regulon.

Culture Media↗