Search PubMed⌕ Search

Biomedical subjects

I Fridovich

Publications and source records attributed to I Fridovich.

At least 109 records · Page 6Linked to original sources
PubMed1991

Superoxide sensitivity of the Escherichia coli aconitase.

Mutants of Escherichia coli lacking superoxide dismutase (SOD) activity were used to explore the sensitivity of aconitase toward O2 and O2-. The aconitase activity in SOD-free extracts was rapidly lost under aerobic conditions and exogenous SOD afforded a concentration-dependent protection. The rate of the inactivating reaction between O2- and aconitase was estimated to be of the order of 10(9) M-1 s-1. The competitive inhibitors fluorocitrate and tricarballylate provided some protection, and at saturating concentrations, they decreased the rate of the inactivating reaction by 100- and 10-fold, respectively. Aconitase was markedly less sensitive to O2 than it was to O2-. Aerobic growth on succinate involves a greater dependence upon aconitase than does growth on glucose and, as expected, the deleterious consequences of SOD deficiency were more pronounced on succinate than on glucose. Moreover, aconitase activity was lower in extracts of aerobically grown SOD mutants, than it was in the parental strain. We suppose that inactivation of aconitase by O2- involves oxidative attack on the prosthetic iron-sulfur cluster. The extreme sensitivity of aconitase to inactivation by O2- suggests that its inactivation will be an early event in the oxidative stress imposed by hyperoxia, ultraviolet irradiation or redox-cycling agents, such as viologens or quinones.

Aconitate Hydratase↗
PubMed1991

Desferal-Mn(III) in the therapy of diquat-induced cataract in rabbit.

In rabbit lenses subjected to oxidative stress, induced by 1 mM diquat in vitro, there were 7- to 10-fold increases (p less than 0.001) in malondialdehyde, conjugated dienes, and carbonyl dienes, indicating extensive peroxidation of cellular membrane lipids, and approximately a 60% decrease in reduced glutathione. In the presence of 0.1-5 mM Desferal-Mn(III) these changes were diminished by 50-70%. In an experimental group of 12 rabbits having diquat-induced cataract, Desferal-Mn(III) (5% w/v) applied topically as a 50-microliters eye drop four times per day and a single intraperitoneal dose of 64 mg/kg body wt daily for 5 weeks (including pretreatment for 1 week) retarded the progression of lens opacities, whereas, in a control group of 6 rabbits treated with the vehicle (0.15 M NaCl) cataract progressed to an advanced grade. Treatment with Desferal-Mn(III) also significantly diminished production of O2.- and OH. in the lens, aqueous humor, and vitreous humor, and of H2O2 in the aqueous humor and vitreous humor. It also suppressed lipid peroxidation and oxidation of protein-SH of the lens and restored lenticular glutathione and ascorbate to normal levels.

Animals↗
PubMed1991

Effects of overproduction of superoxide dismutase on the toxicity of paraquat toward Escherichia coli.

Gross overproduction of the manganese-containing superoxide dismutase in Escherichia coli, by virtue of a multicopy plasmid bearing the sodA gene, decreases enumeration on paraquat-containing agar plates. This reflects growth inhibition, not lethality, since cells on these plates can be rescued by exclusion of dioxygen. Growth in liquid medium revealed that the control strain adapted to growth in the presence of paraquat more rapidly than did the overproducer. Glucose-6-phosphate dehydrogenase, taken as a representative of the superoxide-inducible soxR regulon, was induced during exposure to paraquat to a much greater extent in the control than in the superoxide dismutase-over-producing strain. These results support the view that overproduction of superoxide dismutase interferes with induction of the soxR regulon and thus prevents a balanced adaptation to the multiple aspects of the toxicity of aerobic paraquat.

Anaerobiosis↗
PubMed1991

Assay of metabolic superoxide production in Escherichia coli.

Superoxide production has been measured in subcellular fractions of SOD-deficient Escherichia coli provided with physiological reductants. Although cytosolic enzyme(s) do generate O2-., the larger portion is produced by autoxidation of components of the respiratory electron-transport chain. At 37 degrees C and with pO2, NADH, and NAD+ levels matching those in vivo, respiring membrane vesicles generate 3 O2-./10,000 electrons transferred. This corresponds to intracellular O2-. production, in glucose-fed cells, of 5 microM/s. The high SOD content of normal cells restricts O2-. accumulation to 2.10(-10) M, with a moderate gradient from the membrane to the center of the cell. SOD-deficient mutants achieve a much higher steady-state content of O2-.. Rates of superoxide-mediated inactivation of certain enzymes are sufficiently rapid that even 10(-10) M O2-. imposes a significant oxidative stress.

Cell Membrane↗
PubMed1991

Superoxide sensitivity of the Escherichia coli 6-phosphogluconate dehydratase.

The activity of 6-phosphogluconate dehydratase was significantly lower in extracts of aerobically grown Escherichia coli deficient in superoxide dismutase (sodAsodB) and in mutants lacking the inducible manganese-containing superoxide dismutase (sodA), exposed to the redox-cycling agent paraquat, than in the parental strain. Growth of these strains on a gluconate minimal medium was also impaired under these conditions. The enzyme was most susceptible to dioxygen in superoxide dismutase (SOD)-free extracts, and exogenous SOD afforded a concentration-dependent protection against inactivation. The amount of SOD necessary for full protection was comparable to the amount normally present in extracts of aerobic E. coli (7-36 units/mg protein), and the rate of reaction of O2- with the dehydratase was estimated to be approximately 2.0 x 10(8) M-1 s-1. The dehydratase was much less sensitive to O2 or H2O2 than to O2-. The virtual substrate, alpha-glycerophosphate, provided partial protection. Iron chelators, thiol-reactive reagents, and oxidants, including nitrite and diamide, inactivated the enzyme. Fluoride ions stabilized the dehydratase and blocked the effect of oxidants. The O2(-)-sensitive target site is proposed to be an iron-sulfur cluster which is readily destroyed by oxidation.

Bacterial Proteins↗
PubMed1991

In vivo competition between iron and manganese for occupancy of the active site region of the manganese-superoxide dismutase of Escherichia coli.

Three forms of the dimeric manganese superoxide dismutase (MnSOD) were isolated from aerobically grown Escherichia coli which contained 2 Mn, 1 Mn and 1 Fe, or 2 Fe, respectively. These are designated Mn2-MnSOD, Mn,Fe-MnSOD, and Fe2-MnSOD. Substitution of iron in place of manganese, eliminated catalytic activity, decreased the isoelectric point, and increased the native electrophoretic anodic mobility, although circular dichroism, high performance liquid chromatography gel exclusion chromatography, and sedimentation equilibrium revealed no gross changes in conformation. Moreover, replacement of iron by manganese restored enzymatic activity. Fe2-MnSOD and the iron-superoxide (FeSOD) of E. coli exhibit distinct optical absorption spectra. These data indicate that the active site environments of E. coli MnSOD and FeSOD must differ. They also indicate that competition between iron and manganese for nascent MnSOD polypeptide chains occurs in vivo, and copurification of these variably substituted MnSODs can explain the substoichiometric manganese contents and the variable specific activities which have been reported for this enzyme.

Amino Acids↗
PubMed1991

Isolation and genetic analysis of a mutation that suppresses the auxotrophies of superoxide dismutase-deficient Escherichia coli K12.

The most striking phenotype associated with superoxide dismutase (SOD) deficiency in Escherichia coli is the inability to grow in aerobic minimal medium, which is due to the sensitivity of several amino acid biosynthetic pathways to superoxide. We have isolated two classes of pseudorevertants that grow on minimal medium at modest rates. Of these, the class that exhibited the faster growth carries mutations at a single locus, denoted ssa, which was mapped to 4 min on the E. coli chromosome. This class constituted the majority of the spontaneous pseudorevertants that were selected by the transfer of independent SOD-deficient cultures in minimal medium from anaerobic to aerobic growth conditions. Pseudoreversion at ssa suppressed requirements for a variety of unrelated amino acid supplements. Further, the SOD-deficient strains were unable to assimilate diaminopimelic acid from the growth medium, whereas the ssa pseudorevertants did so. The viability of these pseudorevertants indicates that superoxide-sensitive biosynthetic enzymes do retain some function in SOD-deficient cells during aerobic growth.

Alleles↗
PubMed1991

The roles of O2-, HO(.), and secondarily derived radicals in oxidation reactions catalyzed by vanadium salts.

V(IV) decomposed H2O2, with evolution of O2, in a free radical chain process involving O2- and HO(.). When V(IV) was limiting, the presence of V(V) augmented O2 evolution because it allowed production of additional V(IV) from the reduction of V(V) by O2-. Gradual addition of V(IV) increased the yield of O2 evolved, per V(IV) added, to greater than 1--a clear indication of a free radical chain reaction. Reductants such as ethanol, Hepes, and NADH imposed a phase of O2 consumption because of HO.-initiated oxidation reactions. The radical produced from the reaction of HO. with ethanol was unable to directly oxidize NADH, whereas that produced from Hepes was able to do so. Ethanol consequently inhibited the oxidation of NADH by anaerobic V(IV) + H2O2, whereas Hepes did not. These results, and others reported herein, are explained on the basis of a coherent set of reactions. Data already in the literature are also clarified on the basis of these reactions.

Free Radical Scavengers↗
PubMed1991

Quinolinate synthetase: the oxygen-sensitive site of de novo NAD(P)+ biosynthesis.

The ability of niacin to relieve the growth-inhibiting effect of hyperoxia on Escherichia coli can be attributed to the dioxygen sensitivity of quinolinate synthetase. The activity of this enzyme within E. coli was diminished by exposure of the cells to 4.2 atm O2, while the activity in extracts was rapidly decreased by 0.2 atm O2. Neither catalase nor superoxide dismutase afforded detectable protection against the inactivating effect of O2, indicating that H2O2 and O2- were not significant intermediates in this process. Nevertheless, H2O2 at 1.0 mM did inactivate quinolinate synthetase, even under anaerobic conditions and in the absence of catalatic activity which might have generated O2. Addition of paraquat to aerobic cultures of E. coli caused an inactivation of quinolinate synthetase, which may be explained in terms of an increase in the production of H2O2. The O2-dependent inactivation of quinolinate synthetase in extracts was gradually reversed during anaerobic incubation and this reactivation was blocked by alpha, alpha'-dipyridyl or by 1,10-phenanthroline. The sequence of the quinolinate synthetase "A" protein contains a--cys-w-x-cys-y-z-cys--sequence, which is characteristic of (Fe-S)4-containing proteins. This sequence, together with the effect of the Fe(II)-chelating agents, suggests that the O2-sensitive site of quinolinate synthetase is an iron-sulfur cluster which is essential for the dehydration reaction catalyzed by the A protein.

2,2'-Dipyridyl↗
PubMed1991

Superoxide production by respiring membranes of Escherichia coli.

O2- production by homogenates and isolated membranes of E. coli has been examined. Approximately one-fourth of the O2- generated by extracts in the presence of NAD (P) H is attributable to the membranes. The autoxidizable membrane component is a member of the respiratory chain, since O2- production is NADH-specific, amplified by cyanide, and absent from membranes lacking the respiratory NADH dehydrogenase. Other respiratory substrates (succinate, 1-phosphoglycerol, D-lactate, and L-lactate) supported O2-production at efficiencies between 3 and 30 O2- released per 10,000 electrons transferred, under conditions of substrate saturation. Membranes from quinoneless mutants quantitatively retain the ability to evolve O2-, indicating that the dehydrogenases are the sites of O2- production. Relative O2- production was greater at low substrate concentrations, probably reflecting the facilitation of unpairing of electrons that may occur when enzymes with multiple redox centers are only partially reduced. Respiration rate, cell volume, rates of membraneous and cytosolic O2- production, and SOD levels were used to calculate a steady-state concentration of O2- between 10(-10) and 10(-9) M in well-fed, aerobic, SOD-proficient cells.

Bacterial Proteins↗
PubMed1991

Anaerobic inductions of active forms of superoxide dismutases in Escherichia coli.

Escherichia coli growing anaerobically respond to NO3- with a approximately 3-fold induction of active FeSOD and a approximately 5.5-fold induction of an inactive, but activatable form of MnSOD (pro-MnSOD). Paraquat, which mediates anaerobic electron flow to NO3-, increased the induction of pro-MnSOD to approximately 2.5-fold. Strains with defects in the SOD genes or which lacked nitrate reductase activity failed to accumulate active or pro-forms of SODs in response to NO3- +/- PQ++. Diamide caused anaerobic induction of active MnSOD and this effect was also observed in a glutathione-negative strain. These inductions required de novo synthesis of protein, even when cell content of pro-MnSOD had been elevated by exposure to NO3- +/- PQ++ prior to addition of diamide. These results indicate that oxidation of a cell component increases biosynthesis of the SOD gene product and this postulated oxidation can be caused by terminal electron acceptors, such as dioxygen or NO3-. In addition, it appears that insertion of the correct metal can be rate-limiting, leading to competition by other metals and to the accumulation of inactive, incorrectly substituted pro-forms. Metal insertion may be dependent upon the valence of the metal, which may be influenced, in turn, by the redox status of the cells. Diamide and redox active agents such as ferricyanide may thus allow anaerobic production of active MnSOD by favoring the production of a complexed form of Mn(III) which can compete favorably with other metal cations for the active site of nascent MnSOD.

Anaerobiosis↗
PubMed1991

Alcohols protect Escherichia coli against cold shock.

Alcohols protect Escherichia coli against cold shock, and the concentration of alcohol which provided optimal protection declined with increasing hydrophobicity of the alcohol. The rate of loss of viability after the chilling transition was decreased by n-octanol, even when it was added after that chilling transition. Cold-shocked cells exhibited a sensitivity toward dioxygen, seen as greater enumeration on anaerobic, rather than on aerobic, trypticase-yeast extract agar plates, and addition of catalase or antioxidants, such as alpha-tocopherol or probucol, to the agar plates did not lessen this dioxygen sensitivity. Respiratory capacity was diminished by cold shock, and cyanide-sensitive respiration was more affected than was cyanide-resistant respiration. Discharging the proton gradient, with the uncoupler carbonyl cyanide trifluoromethoxy-phenylhydrazone, did not change sensitivity to cold shock. There was no evidence for minimal medium recovery after cold shock. The data presented, as well as that already in the literature, are explained on the basis of membrane damage caused by patches of ordering transitions in one membrane leaflet, unmatched by comparable transitions in the mating leaflet.

1-Octanol↗
PubMed1990

Anaerobic biosynthesis of the manganese-containing superoxide dismutase in Escherichia coli. Effects of diazenedicarboxylic acid bis(N,N'-dimethylamide) (diamide).

Anaerobically grown Escherichia coli accumulate active manganese-containing superoxide dismutase (MnSOD) upon exposure to diamide. This induction requires de novo biosynthesis of MnSOD. Catalase, glutathione disulfide reductase, and glucose-6-phosphate dehydrogenase were also induced by diamide in anaerobic E. coli. A GSH-negative strain of E. coli did not produce MnSOD under anaerobic conditions and was as responsive to diamide as was the wild type strain. Diamide which had been prereduced, by incubation with GSH, was ineffective. NO3- plus paraquat, which elicits increased anaerobic biosynthesis of the MnSOD polypeptide, but not of active MnSOD, synergized with diamide in the induction of active MnSOD. A similar increase in the ability of diamide to cause anaerobic biosynthesis of active MnSOD was seen when the production of the MnSOD polypeptide was increased by isopropyl-beta-D-thiogalactopyranoside, in a strain bearing the MnSOD gene under the control of the tac promoter. These results are explained in terms of a dual action of diamide, i.e. at both the transcriptional and the maturational levels of biosynthesis of MnSOD. Oxidative inactivation of an Fe(II)-containing repressor and oxidative facilitation of insertion of manganese, in place of iron, into the nascent MnSOD polypeptide, are the postulated bases of this dual action.

Anaerobiosis↗
PubMed1990

Superoxide dismutases enhance the rate of autoxidation of 3-hydroxyanthranilic acid.

The autoxidation of 3-hydroxyanthranilate to cinnabarinate at 37 degrees C and at pH 7.4 is hastened by superoxide dismutase (SOD). The Cu,Zn-containing enzyme from bovine erythrocytes and the Mn-containing enzyme from Escherichia coli were equally effective in this regard; whereas the H2O2-inactivated Cu,Zn enzyme was ineffective. Catalase appears to augment the effect of superoxide dismutase, because it prevents the bleaching of cinnabarinate by H2O2. It follows that O2-, which is a product of the autoxidation, slows the net autoxidation by engaging in back reactions and that SOD increases the rate of autoxidation by removal of O2- and thus by prevention of these back reactions.

3-Hydroxyanthranilic Acid↗