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Biomedical subjects

I Fridovich

Publications and source records attributed to I Fridovich.

At least 145 records · Page 8Linked to original sources
PubMed1988

Reactions of hypochlorite with catalase.

OCl-/HOCl imposed a rapid inactivation of catalase (hydrogen-peroxide: hydrogen-peroxide oxidoreductase, EC 1.11.1.6), some of which was slowly reversible upon subsequent exposure to H2O2. Ethanol accelerated this restoration of activity by H2O2. OCl- caused biphasic changes in the visible absorption spectrum of catalase, which were partially reversed by dithionite. A scheme of reactions involving axial ligation of one or two OCl- to heme iron, followed by heterolytic or homolytic cleavages of the O-Cl bond, is proposed to account for the behavior of the system.

Animals↗
PubMed1988

Does copper (II) ethylenediaminetetraacetate disproportionate superoxide?

W. M. Willingham and J. R. J. Sorenson have recently reported [1988) Biochem. Biophys. Res. Commun. 150, 252-258) that the Cu(II)EDTA complex catalyzes the dismutation of O-2 and thereby call into question several earlier reports. These authors concluded that the widespread use of EDTA to suppress dismutation of O-2 by adventitious Cu(II) and other metals, during assays for superoxide dismutase, is unsound. We have undertaken a clarification of this issue by examining the ability of Cu(II)EDTA to catalyze the dismutation of O-2 and have found this complex to be devoid of this activity at pH 6.0 or at 7.8; whether assayed by the cytochrome c or the nitroblue tetrazolium assays. The probable reasons for the results of Willingham and Sorenson and some peculiarities of the nitroblue tetrazolium assay have been explored.

Cytochrome c Group↗
PubMed1988

An ultrasensitive colorimetric assay for manganese.

An ultrasensitive colorimetric assay for manganese is described. It is based upon the catalysis, by Mn(II), of the photochemical oxidation of o-dianisidine, sensitized by riboflavin. Catalase increases the Mn(II)-catalyzed rate of photosensitized oxidation of dianisidine to the bisazobiphenyl, while superoxide dismutase inhibits the rate. The mechanism appears to involve oxidation of Mn(II) by O2-, followed by oxidation of dianisidine by MnO2+ in equilibrium Mn(III). Cu(II) interferes, but Zn(II), Fe(II), Fe(III), Co(II), and Ni(II) do not. Chelating agents and thiol reductants also interfere. Interference by Cu(II) can be overcome by the addition of cyanide, while interference by organic compounds can be surmounted by wet ashing. This assay provides a linear response to Mn(II) over the range 10-2500 nM. The limit of detection was 5 nM Mn(II).

Catalysis↗
PubMed1988

Inductions of superoxide dismutases in Escherichia coli under anaerobic conditions. Accumulation of an inactive form of the manganese enzyme.

Escherichia coli growing anaerobically respond to NO3- with a 3-fold induction of the iron-containing superoxide dismutase. Mutants lacking nitrate reductase do not show this response. Anaerobically grown cells also contain an inactive form of the manganese-containing superoxide dismutase (MnSOD) which can be activated by addition of Mn(II) salts in the presence of acidic guanidinium chloride, followed by dialysis against neutral buffer. Direct addition of Mn(II) to a neutral solution of the inactive MnSOD does not impart activity. This inactive MnSOD thus behaves as would the apoenzyme or the enzyme bearing a metal other than Mn(II) at its active sites. Terminal electron acceptors, such as NO3- or trimethylamine N-oxide, increase the amount of inactive MnSOD produced by anaerobic E. coli. Paraquat, which is itself ineffective in this regard, markedly augments the effect of these terminal electron acceptors. It appears that flow of electrons to sinks such as NO3- or trimethylamine N-oxide, facilitated by paraquat, is sufficient to elicit biosynthesis of the MnSOD protein and that O2- is not needed for this process. Yet, oxygenation and concomitant O2- production do appear important for the insertion of manganese into the growing MnSOD polypeptide, possibly because O-2 oxidizes Mn(II) to Mn(III), and the latter is the valence state most effective in combining with the apoenzyme.

Anaerobiosis↗
PubMed1988

Stimulation of the activity of horseradish peroxidase by nitrogenous compounds.

A variety of nitrogenous compounds broaden the activity versus pH profile for the peroxidation of dianisidine catalyzed by horseradish peroxidase (HRP), but not by myeloperoxidase, chloroperoxidase, Escherichia coli hydroperoxidase I, methemoglobin, or microperoxidases. The peroxidation of dianisidine catalyzed by cytochrome c peroxidase was affected by the nitrogenous compounds, but to a lesser extent than was the action of HRP. The peroxidations of a variety of phenols by HRP exhibited broad activity versus pH profiles and were unaffected by the nitrogenous compounds. The energy of activation for the peroxidation of dianisidine by HRP was unaffected by changes of pH in the range 6.5-8.5 and was unchanged by the presence of the nitrogenous compounds. The nitrogenous compounds markedly increased Vm for the peroxidation of dianisidine by HRP, but did not change the slope of Lineweaver-Burk plots of kinetic data. These results are accommodated by a mechanism in which nitrogenous compounds hydrogen-bond to the distal histidine of HRP and in so doing raise its pK alpha. Since the acid form of the distal histidine is thought to facilitate peroxidations catalyzed by HRP by hydrogen bonding to the ferryl oxygen of compound II, raising its pK alpha broadens the activity versus pH profile for the peroxidation of anilino substrates, such as dianisidine. We propose that phenolic substrates hydrogen-bond directly to the ferryl oxygen, thus displacing the distal histidine and eliminating the possibility of being influenced by nitrogenous compounds.

Amines↗
PubMed1988

NADPH mediates the inactivation of bovine liver catalase by monochloroamine.

Bovine liver catalase suffered a biphasic inactivation when exposed to NH2Cl. A rapid and irreversible phase of activity loss was followed by a much slower and reversible inactivation. Removal of tightly bound NADPH from the enzyme decreased the extent of the rapid phase; whereas addition of NADPH augmented it. The catalase from Aspergillus niger, which does not contain bound NADPH, was not nearly as sensitive toward NH2Cl as was the liver enzyme and was not sensitized by added NADPH. NADPH is oxidized by NH2Cl, as evidenced by loss of the 340-nm absorption band, but the product of this oxidation was not NADP+. The rapid inactivation of liver catalase by NH2Cl was accompanied by some bleaching of the bands in the visible, while the slow inactivation was coincident with the appearance of a new band at 570 nm. A tentative explanation for these observations is proposed.

Ammonium Chloride↗
PubMed1988

Superoxide is responsible for the vanadate stimulation of NAD(P)H oxidation by biological membranes.

Vandate augments the oxidation of NAD(P)H, but not of NMNH, by rat liver microsomes. Paraquat increases the vanadate effect on NADPH, but not on NADH, oxidation. Substoichiometric levels of NADPH caused the co-oxidation of NADH or NMNH and SOD inhibited in all cases. The ratio of NADH or NMNH co-oxidized per NADPH added allowed estimation of average chain length, which increased as the pH was lowered from 8.0 to 7.1. The initial rate of this co-oxidation of NMNH was a saturating function of the concentration of microsomes, reflecting a decrease in chain length with an increase in number of concomitant reaction chains, and due to increasing radical-radical termination reactions. Mitochondrial outer membranes behaved like the microsomal membranes, but mitochondrial inner membranes catalyzed a rapid oxidation of NADH which could be augmented by vanadate, whose action was enhanced by paraquat and inhibited by antimycin or rotenone. These and related observations support the view that vanadate stimulates NAD(P)H oxidation by biological membranes, not by virtue of interacting with enzymes, but rather by interacting with O-2.

Animals↗
PubMed1987

Controls on the biosynthesis of the manganese-containing superoxide dismutase of Escherichia coli. Effects of thiols.

In vitro synthesis of Escherichia coli manganese-containing superoxide dismutase, directed by the plasmid pDT1-5, has been achieved. The Mn superoxide dismutase polypeptide was identified by electrophoresis on polyacrylamide gels, immunoprecipitation, and the competitive immunoprecipitation effect of pure, active E. coli Mn superoxide dismutase. Dithiothreitol and glutathione, but not cysteine, suppressed in vitro synthesis of Mn superoxide dismutase. The parallel syntheses of beta-lactamase and of another unidentified polypeptide were not suppressed by thiols. In vitro transcription of the E. coli Mn superoxide dismutase gene was similarly suppressed by glutathione, dithiothreitol, and beta-mercaptoethanol; but not by L-cysteine or thioglycolate. Compounds, such as diamide, 1-chloro-2,4-dinitrobenzene, potassium ferricyanide, and methylene blue, which are expected to deplete intracellular glutathione, caused the induction of Mn superoxide dismutase in anaerobic E. coli.

Anaerobiosis↗
PubMed1987

A mimic of superoxide dismutase activity based upon desferrioxamine B and manganese(IV).

MnO2 reacted with desferrioxamine B yielding a green, water-soluble complex, with absorption maxima at 315 and 635 nm whose extinction coefficients were 925 and 60 M-1 cm-1, respectively. Increasing the proportion of ligand to metal increased both color yield and ability to scavenge O2-, with maximal color yield and activity being achieved at a 1:1 ratio. The complex catalyzed the dismutation of O2- and 1 microM was equivalent to 1 unit of superoxide dismutase activity in the xanthine oxidase-cytochrome c assay. The complex thus exhibited approximately 0.1% as much activity as did the manganese-containing superoxide dismutase, on the basis of manganese content. The activity of the complex was not suppressed by bovine serum albumin or by the soluble proteins extracted from Lactobacillus plantarum. In contrast, the activities of Cu(II) complexes of salicylate or Gly-His-Lys were suppressed by these proteins.

Deferoxamine↗
PubMed1987

Effects of urea and trimethylamine-N-oxide on enzyme activity and stability.

The interactions of urea, trimethylamine-N-oxide (TMAO), and related solutes on a number of enzymes were examined. Urea inhibited enzymatic activity and accelerated the thermal inactivation of catalase, whereas TMAO activated some enzymes but inhibited others. The effects of urea and of TMAO, whether parallel or in opposition, were exerted independently. Thus, in those cases where TMAO increases enzymatic activity, it did so to the same relative degree, whether or not urea was present. TMAO markedly decreased the rate of thermal inactivation of catalase, indicating that it does favor compact protein structures. The assumption that TMAO factors compaction of protein structure, whereas urea has the contrary effect, does not lead to the expectation that TMAO must always oppose the effect of urea on enzymatic activity, since the most compact form of an enzyme may not always be the most active form.

Alcohol Dehydrogenase↗
PubMed1987

Examination of the role of arginine-143 in the human copper and zinc superoxide dismutase by site-specific mutagenesis.

The active site arginine-143 of human Cu,Zn superoxide dismutase has been replaced by lysine or by isoleucine. The mutant proteins were expressed at high levels in yeast, purified, and the amino acid substitution explored through the use of group specific reagents. The specific activities of these enzymes, measured by the xanthine oxidase/cytochrome c method and by using dry weight determination to establish protein concentration, were: native enzyme, 6570 units/mg; Lys-substituted enzyme, 2840 units/mg, Ile-substituted enzyme, 708 units/mg. The active site arginine thus plays an important, but not an essential, role in the catalytic process.

Amino Acid Sequence↗
PubMed1987

An activity stain for dihydroxy-acid dehydratase.

An activity stain has been devised for the dihydroxy-acid dehydratase. When applied to polyacrylamide gel electropherograms of crude soluble extracts of Escherichia coli, it detected a single electromorph. The intensity of staining increased with the amount of extract protein applied to the gel. Activity staining demonstrated that (a) anaerobically grown cells contain more extractable dehydratase activity than do aerobically grown cells; (b) exposure of E. coli to 4.2 atm O2 caused virtually complete loss of activity; (c) exposure of cells to paraquat or plumbagin in the presence of dioxygen, but not in its absence, caused a massive loss of activity. These data illustrate the utility of this activity stain and demonstrate that the dehydratase is inactivated by O2- generated within cells.

Escherichia coli↗
PubMed1987

The oxidation of NADH by vanadate plus sugars.

Sugars and sugar phosphates enable vanadate to catalyze the oxidation of NADH. Superoxide dismutase inhibits this oxidation. Incubation of sugars with vanadate, prior to addition of NADH, accelerates this oxidation of subsequently added NADH and eliminates the lag phase otherwise noted. Incubation of sugars with vanadate also results in the reduction of vanadate to vanadyl, with appearance of a blue-green color probably associated with a vanadyl-vanadate complex. It appears that sugars reduce vanadate to vanadyl which, in turn, reduces O2 to O2- and that vanadate plus O2- then catalyzes the oxidation of NAD(P)H by a free radical chain reaction. Such oxidation of NAD(P)H may account for several of the biological effects of vanadate.

Carbohydrates↗
PubMed1987

Superoxide radical initiates the autoxidation of dihydroxyacetone.

The aerobic xanthine oxidase reaction causes the cooxidation of dihydroxyacetone in a process which is strongly inhibited by superoxide dismutase but not by catalase, HO X scavengers, or iron-inactivating chelating agents. Several molecules of the sugar can be oxidized per O2- introduced. A free radical chain mechanism, in which O2- acts both as an initiator and as a chain propagator, is proposed. Simple sugars capable of tautomerizing to enediols may now be added to the list of biologically relevant targets for O2-.

Catalase↗
PubMed1987

alpha, beta-Dihydroxyisovalerate dehydratase. A superoxide-sensitive enzyme.

Increasing the intracellular flux of O-2 by incubating aerobic Escherichia coli with paraquat or plumbagin markedly lowered the alpha, beta-dihydroxyisovalerate dehydratase activity detectable in extracts from these cells. This effect was not seen in the absence of dioxygen and was exacerbated by inhibiting protein biosynthesis with chloramphenicol. These effects of paraquat and of plumbagin were both time- and concentration-dependent. Transfer of E. coli from aerobic to anaerobic conditions caused a rebound of the dehydratase activity, in the continued presence of paraquat and of chloramphenicol, indicating the presence of a mechanism for reactivating this enzyme. The instability of the dehydratase activity in cell extracts was exacerbated by selective removal of superoxide dismutase, but not of catalase, by immunoprecipitation. Addition of exogenous superoxide dismutase reversed the effect of immunoprecipitation; whereas catalase or inactive superoxide dismutase were ineffective. We conclude that the dehydratase is inactivated by O-2. This could account for the bacteriostatic effects of dioxygen and of paraquat.

Catalase↗