Edgar Pick

A simple colorimetric method for the measurement of hydrogen peroxide produced by cells in culture

A simple, rapid and inexpensive method for the measurement of hydrogen peroxide (H2O2) produced by cells in culture is described. The assay is based on the horseradish peroxidase (HRPO)-mediated oxidation of phenol red by H2O2 which results in the formation of a compound demonstrating increased absorbance at 610 nm. A linear relationship between absorbance at 610 nm and concentration of H2O2 was found in the 1--60 microM (1--60 nmoles/ml) range. Due to the non-toxic character of phenol red and HRPO, the assay permits measurement of H2O2 production and release by macrophages for time intervals of 5--60 min under regular tissue culture conditions. Using this assay, the ability of a number of agents to induce H2O2 release by guinea pig peritoneal macrophages was demonstrated. These agents were: phorbol myristate acetate (PMA), opsonized zymosan, concanavalin A (Con A), wheat germ agglutinin (WGA), N-formyl-methionyl-leucyl-phenylalanine (FMLP) and A23187.

Superoxide anion and hydrogen peroxide production by chemically elicited peritoneal macrophages—Induction by multiple nonphagocytic stimuli☆

Abstract The ability of a number of stimulants to activate an oxidative burst (OB) in oil-elicited guinea pig peritoneal exudate macrophages (MPs) was examined. The parameters of the OB were the generation and extracellular release of Superoxide anions (O2−) and hydrogen peroxide (H2O2). We found that: (1) The cocarcinogen and skin irritant phorbol myristate acetate (PMA) was the most potent OB activator—The weak cocarcinogen 4-O-methyl PMA was a proportionally less effective OB activator; (2) The lectins concanavalin A (Con A) and wheat germ agglutinin (WGA), but not soybean, Lotus, and pokeweed lectins, were also quite effective OB activators—The ability of Con A to stimulate O2− production was abolished by succinylation and could be prevented by the presence of α-methyl-D-mannoside; (3) Other stimulators of an OB in MPs were: N-formyl-methionyl peptides, opsonized zymosan, the Ca2+ ionophore A23187, phospholipase C, NaF, antimacrophage antibody, microtubule-disrupting drugs, and sodium nitroprusside—O2− generation induced by A23187 (but not that stimulated by PMA) was dependent on extracellular Ca2+; (4) The amount of O2− produced per cell was higher at low cell densities; (5) The addition of Superoxide dismutase (SOD) to the medium totally prevented the detection of O2− and augmented twice the amount of H2O2 recovered; (6) Pretreatment of MPs with the SOD inhibitor sodium diethyldithiocarbamate had no effect on the release of O2− but blocked H2O2 release in a dose-dependent manner. These data were interpreted as indicating that the bulk of H2O2 was derived by enzymatic dismutation of O2−; (7) The common mechanism by which such a variety of stimuli provoke an OB in MPs was not elucidated. No evidence was found to suggest a role for a cyclic nucleotide messenger.

Unsaturated fatty acids stimulate NADPH-dependent superoxide production by cell-free system derived from macrophages.

Arachidonic acid (C20:4) and other unsaturated fatty acids are shown to activate superoxide (O2-) production in a cell-free system represented by sonically disrupted guinea pig peritoneal macrophages. The reaction requires a heat-sensitive cellular component and NADPH, is enhanced by flavin adenine dinucleotide (FAD), and is not linked to enzymatic oxidation of the fatty acid. C20:4-elicited O2- formation is dependent on the cooperation between a subcellular component sedimentable at 48,000g (probably containing the O2- -forming enzyme) and a cytosolic factor. This appears to be the first report of O2- generation being elicited in a cell-free system derived from unstimulated cells and supports the idea that unesterified unsaturated fatty acids act as second messengers of O2- formation in intact phagocytes.

Inhibition of NADPH Oxidase Activation by 4-(2-Aminoethyl)-benzenesulfonyl Fluoride and Related Compounds

The elicitation of an oxidative burst in phagocytes rests on the assembly of a multicomponental complex (NADPH oxidase) consisting of a membrane-associated flavocytochrome (cytochrome b 559), representing the redox element responsible for the NADPH-dependent reduction of oxygen to superoxide (O·̄2), two cytosolic components (p47 phox , p67 phox ), and the small GTPase Rac (1 or 2). We found that 4-(2-aminoethyl)-benzenesulfonyl fluoride (AEBSF), an irreversible serine protease inhibitor, prevented the elicitation of O·̄2 production in intact macrophages and the amphiphile-dependent activation of NADPH oxidase in a cell-free system, consisting of solubilized membrane or purified cytochrome b 559 combined with total cytosol or a mixture of recombinant p47 phox , p67 phox , and Rac1. AEBSF acted at the activation step and did not interfere with the ensuing electron flow. It did not scavenge oxygen radicals and did not affect assay reagents. Five other serine protease inhibitors (three irreversible and two reversible) were found to lack an inhibitory effect on cell-free activation of NADPH oxidase. A structure-function study of AEBSF analogues demonstrated that the presence of a sulfonyl fluoride group was essential for inhibitory activity and that compounds containing an aminoalkylbenzene moiety were more active than amidinobenzene derivatives. Exposure of the membrane fraction or of purified cytochrome b 559, but not of cytosol or recombinant cytosolic components, to AEBSF, in the presence of a critical concentration of the activating amphiphile lithium dodecyl sulfate, resulted in a marked impairment of their ability to support cell-free NADPH oxidase activation upon complementation with untreated cytosol or cytosolic components. Kinetic analysis of the effect of varying the concentration of each of the three cytosolic components on the inhibitory potency of AEBSF indicated that this was inversely related to the concentrations of p47 phox and, to a lesser degree, p67 phox . AEBSF also prevented the amphiphile-elicited translocation of p47 phox and p67 phox to the membrane. These results are interpreted as indicating that AEBSF interferes with the binding of p47 phox and/or p67 phox to cytochromeb 559, probably by a direct effect on cytochromeb 559.

The Cytosolic Component p47phox Is Not a Sine Qua Non Participant in the Activation of NADPH Oxidase but Is Required for Optimal Superoxide Production

The superoxide (O2)-generating NADPH oxidase of phagocytes is a multicomponent complex consisting of a membrane-associated flavocytochrome (cytochrome b559), bearing the NADPH binding site and two redox centers (FAD and heme) and three cytosolic activating components: p47phox, p67phox, and the small GTPase Rac (1 or 2). The canonical view is that the induction of O2 generation involves the stimulus-dependent assembly of all three cytosolic components with cytochrome b559, a process mimicked in vitro by a cell-free system activated by anionic amphiphiles. We studied the requirement for individual cytosolic components in the activation of NADPH oxidase in a cell-free system consisting of purified and relipidated cytochrome b559, recombinant p47phox, p67phox, and Rac1, and the amphiphile, lithium dodecyl sulfate. We found that pronounced activation of NADPH oxidase can be achieved by exposing cytochrome b559 to p67phox and Rac1, in the total absence of p47phox (turnover = 60 mol O2/s/mol cytochrome b559). However, maximal activation (turnover = 153 mol O2/s/mol cytochrome b559) could only be obtained in the presence of p47phox. O2 production, in the absence of p47phox, was dependent on: high molar ratios of p67phox and Rac1 to cytochrome b559, Rac1 being in the GTP-bound form, cytochrome b559 being saturated with FAD, and an optimal concentration of amphiphile. Single cytosolic components or combinations of two cytosolic components, other than p67phox and Rac1, were incapable of activation. We conclude that p67phox and Rac1 are the only cytosolic components directly involved in the induction of electron transport in cytochrome b559. p47phox appears to facilitate or stabilize the interaction of p67phox and, possibly, Rac1 with cytochrome b559, and is required for optimal generation of O2 under physiological conditions.

Unsaturated fatty acids as second messengers of superoxide generation by macrophages.

Chemically elicited guinea pig peritoneal exudate macrophages respond by superoxide (O2-) production to a large number of unrelated stimulants. It has been found that 8 out of 10 stimulants also induce arachidonic acid (20:4) liberation and thromboxane synthesis. The elicitation of O2- production by most stimulants was reduced or totally suppressed by three procedures that inhibit the activity of endogenous phospholipases: the use of drug p-bromophenacyl bromide, elevation of the cellular cyclic AMP level, and the removal of extracellular Ca2+. O2- production in response to concanavalin A, wheat germ agglutinin, and fMet-Leu-Phe were exquisitely sensitive to inhibition of phospholipase activity. Exogenously applied 20:4 as well as other unsaturated fatty acids (linolenic, linoleic, and oleic) induced massive and instantaneous O2- production in a dose-dependent manner. Saturated fatty acids (stearic) and methyl esters of unsaturated acids were inactive. Lysophosphoglycerides were also inactive. Incubation of macrophages with inhibitors of cyclooxygenase or lipoxygenase did not prevent the elicitation of O2- production by stimulants or fatty acids. On the contrary, O2- formation was enhanced by indomethacin and indomethacin by itself was capable of evoking O2- generation. Treatment of 20:4 with soybean lipoxygenase did not abolish its capacity to induce O2- production; native and lipoxygenase-treated 20:4 exhibited similar dose-response ratios. Purified 15-hydroxyeicosatetraenoic acid also elicited O2- production by macrophages with a potency comparable to but not exceeding that of 20:4. Equimolar amounts of prostaglandin E2 were inactive. These findings suggest that liberation of unsaturated fatty acid (principally, 20:4) from membrane phospholipids, as a consequence of phospholipase activation, is a necessary step in the elicitation of an oxidative burst in macrophages. O2- generation is stimulated by unesterified 20:4 and, possibly, by certain metabolites of 20:4. It appears that the lipoxygenase pathway may generate metabolites with stimulating capacity while the cyclooxygenase pathway is abortive.

Generation of superoxide by purified and relipidated cytochrome b559 in the absence of cytosolic activators.

Purified cytochrome b559 from guinea pig macrophages was relipidated with several phospholipid mixtures. Relipidated cytochrome b559 was found capable of NADPH‐dependent Superoxide (O2 −) production in the absence of the cytosolic components of the NADPH oxidase complex. The rate of O2 − generation by cytochrome b559 varied with the type of phospholipid utilized for relipidation, was absolutely dependent on exogenous FAD, and was enhanced by a critical concentration of anionic amphiphile. It is demonstrated that exogenous FAD acts by binding to cytochrome b559 . These results provide firm experimental evidence for the proposal that cytochrome b559 comprises the complete electron transporting apparatus of the O2 − forming NADPH oxidase and that the cytosolic components function merely as activators.

Superoxide production by cytochrome b559: Mechanism of cytosol-independent activation

Purified cytochrome b 559 relipidated with either a mixture of phosphatidylcholine and phosphatidic acid or with phosphatidylcholine only exhibits high and low superoxide (O2) producing ability, respectively, in the absence of cytosolic activators [Koshkin, V. and Pick, E. (1993) FEBS Lett. 327, 57‐62]. This system was used as a model for the study of the mechanism of NADPH oxidase activation. It is shown that, depending on the composition of the phospholipid environment, cytochrome b 559 binds FAD with high or low affinity, this being accompanied by changes in flavin absorbance and fluorescence. High affinity binding of FAD to cytochrome b 559 relipidated with phosphatidylcholine combined with phosphatidic acid is associated with an enhanced NADPH‐driven O2 − producing capacity. A kinetic study of O2 − production by cytochrome b 559 reflavinated under stoichiometric FAD binding conditions revealed an FAD/heme ratio of 1:2. A further kinetic study of O2 production by high‐ and low‐activity relipidated and reflavinated eytochrome b 559, at varying substrate concentrations, and the determination of steady‐state difference spectra of such preparations, reduced by NADPH, indicated that O2 − production is activated by facilitation of electron transfer from NADPH to FAD rather than by an enhancement of NADPH binding.Purified cytochrome b559 relipidated with either a mixture of phosphatidylcholine and phosphatidic acid or with phosphatidylcholine only exhibits high and low superoxide (O2-) producing ability, respectively, in the absence of cytosolic activators [Koshkin, V. and Pick, E. (1993) FEBS Lett. 327, 57-62]. This system was used as a model for the study of the mechanism of NADPH oxidase activation. It is shown that, depending on the composition of the phospholipid environment, cytochrome b599 binds FAD with high or low affinity, this being accompanied by changes in flavin absorbance and fluorescence. High affinity binding of FAD to cytochrome b559 relipidated with phosphatidylcholine combined with phosphatidic acid is associated with an enhanced NADPH-driven O2- producing capacity. A kinetic study of O2- production by cytochrome b559 reflavinated under stoichiometric FAD binding conditions revealed an FAD/heme ratio of 1:2. A further kinetic study of O2- production by high- and low-activity relipidated and reflavinated cytochrome b559, at varying substrate concentrations, and the determination of steady-state difference spectra of such preparations, reduced by NADPH, indicated that O2- production is activated by facilitation of electron transfer from NADPH to FAD rather than by an enhancement of NADPH binding.

Assembly of the phagocyte NADPH oxidase complex : chimeric constructs derived from the cytosolic components as tools for exploring structure-function relationships

Phagocytes generate superoxide (O2.−) by an enzyme complex known as reduced nicotinamide adenine dinucleotide phosphate (NADPH) oxidase. Its catalytic component, responsible for the NADPH‐driven reduction of oxygen to O2.−, is flavocytochrome b559, located in the membrane and consisting of gp91phox and p22phox subunits. NADPH oxidase activation is initiated by the translocation to the membrane of the cytosolic components p47phox, p67phox, and the GTPase Rac. Cytochrome b559 is converted to an active form by the interaction of gp91phox with p67phox, leading to a conformational change in gp91phox and the induction of electron flow. We designed a new family of NADPH oxidase activators, represented by chimeras comprising various segments of p67phox and Rac1. The prototype chimera p67phox (1–212)‐Rac1 (1–192) is a potent activator in a cell‐free system, also containing membrane p47phox and an anionic amphiphile. Chimeras behave like bona fide GTPases and can be prenylated, and prenylated (p67phox‐Rac1) chimeras activate the oxidase in the absence of p47phox and amphiphile. Experiments involving truncations, mutagenesis, and supplementation with Rac1 demonstrated that the presence of intrachimeric bonds between the p67phox and Rac1 moieties is an absolute requirement for the ability to activate the oxidase. The presence or absence of intrachimeric bonds has a major impact on the conformation of the chimeras, as demonstrated by fluorescence resonance energy transfer, small angle X‐ray scattering, and gel filtration. Based on this, a “propagated wave” model of NADPH oxidase activation is proposed in which a conformational change initiated in Rac is propagated to p67phox and from p67phox to gp91phox.

Electron transfer in the superoxide-generating NADPH oxidase complex reconstituted in vitro

The superoxide (O2-)-generating NADPH oxidase of phagocytic cells is composed of a membrane-bound flavocytochrome (cytochrome b-559) and three cytosolic components, p47-phox, p67-phox, and the small GTPase rac-1 (or 2). Cytochrome b-559 bears the NADPH binding site and the redox centers (FAD and heme). Electron flow through the redox centers, from NADPH to oxygen, is activated consequent to the assembly of the three cytosolic components with cytochrome b-559. We studied the kinetics of electron flow through the redox centers of NADPH oxidase in a cell-free system, consisting of purified relipidated and reflavinated cytochrome b-559 and recombinant cytosolic components, activated by the anionic amphiphile, lithium dodecyl sulphate. The NADPH oxidase complex assembled in vitro exhibited: (a) a high steady-state electron flow (165 electrons/heme/s); (b) low stationary levels of FAD and heme reduction (about 10%), and (c) a high rate constant of heme oxidation by oxygen (1720 s-1). Surprisingly, the kinetic properties of NADPH oxidase assembled in a semi-recombinant cell-free system, lacking p47-phox (found to generate significant amounts of O2-), were similar to those of the complete system, as shown by a steady-state electron flow of 83 electrons/heme/s, low stationary levels of FAD and heme reduction (10%), and a rate constant of heme oxidation by oxygen of 1455 s-1. The kinetic features of NADPH oxidase assembled in vitro from purified and recombinant components differ considerably from those of solubilized enzyme preparations derived from intact stimulated phagocytes. The fast operation of the cell-free system is best explained by the activation-related facilitation of electron flow at both the FAD-->heme and the heme-->oxygen steps.