Macrophage variants in oxygen metabolism.

Whereas phagocytic cells from normal individuals have the capacity to kill ingested bacteria and parasites, those from patients with several uncommon genetic deficiency diseases are known to be defective in bactericidal activity. Studies on neutrophils of these patients have revealed fundamental defects in their ability to reduce molecular oxygen and metabolize it to superoxide anion, hydrogen peroxide, and oxygen radicals. In the present experiments, we describe a clone of a continuous murine macrophage-like cell line, J774.16, that, upon appropriate stimulation, activates the hexose monophosphate shunt, and produces superoxide anion and hydrogen peroxide. With nitroblue tetrazolium to select against cells capable of being stimulated by phorbol myristate acetate to reduce the dye to polymer--formazan--which is toxic fot cells, we have selected for variants that are defective in oxygen metabolism. Four of these subclones have been characterized and found to be lacking in the ability (a) to generate superoxide anion, as measured by cytochrome c reduction; (b) to produce hydrogen peroxide, as measured by the ability to form complex I with cytochrome c peroxidase; and (c) to be stimulated to oxidize glucose via the hexose monophosphate shunt. These variants appear to represent a useful model for studying the molecular basis for macrophage cytocidal activity.

The stimulation of microsomal azoreduction by flavins.

The reduction of the azo dye, amaranth, by rat liver microsomes is inhibited about 90% by carbon monoxide, suggesting that the reaction largely depends on cytochrome P-450. Reducing equivalents for this reaction are supplied by NADPH. This reaction is stimulated by riboflavin, FMN and FAD, as well as by methylviologen. A large fraction of the stimulated reaction is not blocked by CO, indicating that there is a pathway of electron transfer which is independent of cytochrome P-450. Rat liver microsomes can reduce FAD, with reducing equivalents supplied by NADPH. The FADH2 thus produced is quickly oxidized by amaranth so that two FADH2 are oxidized for every amaranth reduced. The same stoichiometry is observed with photochemically prepared FADH2, formed in the absence of microsomes.

A comparison of the intrinsic fluorescence of red kangaroo, horse and sperm whale metmyoglobins.

Several metmyoglobins (red kangaroo, horse and sperm whale), containing different numbers of tyrosines, but with invariant tryptophan residues (Trp-7, Trp-14), exhibit intrinsic fluorescence when studied by steady-state front-face fluorometry. The increasing tyrosine content of these myoglobins correlates with a shift in emission maximum to shorter wavelengths with excitation at 280 nm: red kangaroo (Tyr-146) emission maximum 335 nm; horse (Tyr-103, -146) emission maximum 333 nm; sperm whale (Tyr-103, -146, -151) emission maximum 331 nm. Since 280 nm excites both tyrosine and tryptophan, this strongly suggests that tyrosine emission is not completely quenched but also contributes to this fluorescence emission. Upon titration to pH 12.5, there is a reversible shift of the emission maximum to longer wavelengths with an increase greater than 2-fold in fluorescence intensity. With excitation at 305 nm, a tyrosinate-like emission is detected at a pH greater than 12. These studies show that: (1) metmyoglobins, Class B proteins containing both tyrosine and tryptophan residues, exhibit intrinsic fluorescence; (2) tyrosine residues also contribute to the observed steady-state fluorescence emission when excited by light at 280 nm; (3) the ionization of Tyr-146 is likely coupled to protein unfolding.

A proton nuclear magnetic resonance study of sulfmyoglobin cyanide.

The proton nuclear magnetic resonance spectrum of sulfmyoglobin cyanide was studied at 400 MHz. The position of a methyl-group resonance at low field is consistent with a chlorin-like structure for the prosthetic group. The proton NMR spectrum of the cyanide derivative of the purified prosthetic group which decomposes upon extraction from the protein was found to be the same as that of the cyanide derivative of the prosthetic group extracted from myoglobin and a sample prepared from hemin-Cl.

Resistance of cultured hepatoma cells to copper toxicity. Purification and characterization of the hepatoma metallothionein.

The mechanism of resistance to the toxic effects of copper was investigated using a series of copper-resistant hepatoma cell lines maintained in copper-enriched medium. Gel electrophoresis of carboxyamidated cell extracts demonstrated the presence of a pair of low molecular mass cysteine-rich proteins in wild-type and resistant cell lines. These proteins were purified to homogeneity and contained approx. 60% of the total cellular copper. Comparisons of molecular masses, pI values and amino-acid compositions for the purified hepatoma proteins with authentic rat liver metallothionein, as well as cross-reactivity with anti-rat metallothionein antibody, confirmed that the cysteine-rich hepatoma proteins were metallothioneins. The cellular concentration of these hepatoma copper-metallothioneins was proportional to both the level of metal resistance and the amount of copper accumulated by individual cell lines. Further, resistant cells removed from copper-enriched medium for 6-12 months, yet maintaining their level of resistance, showed only a slight decrease in metallothionein concentration. Thus it is proposed that the level of resistance to metal toxicity is mediated by the concentration of copper-metallothionein. It is also suggested that the steady-state level of copper metallothionein is controlled by the degree of metal exposure.

The demonstration of ferrihemochrome intermediates in heinz body formation following the reduction of oxyhemoglobin A by acetylphenylhydrazone.

Interaction of acetylphenylhydrazine with oxyhemoglobin A in a hemolysate or in intact red cells resulted in the formation of ferrihemochromes as shown by a characteristic optical spectrum. The same optical spectrum was observed in a suspension of red cell ghosts containing numerous Heinz bodies. Electron paramagnetic resonance of actylphenylhydrazine-incubated red cells disclosed the presence of previously identified reversible ferrihemochromes, which can be reduced to functional hemoglobin, and irreversible ferrihemochromes, which cannot be reduced to functional hemoglobin. (Ferrihemochromes are defined as low spin forms of ferric hemoglobin having heme ligands endogenous to the protein structure). In contrast, only irreversible ferrihemochromes could be observed in ghosts containing Heinz bodies. In addition both optical and magnetic features of sulfhemoglobin were observed in an acetylphenylhydrazine-treated red cell hemolysate. Similar optical features are produced by the interaction of aromatic nitrogen-containg reductants with purified oxyhemoglobin in the presence of (NH4)2S. This reaction is not effected by the presence of catalase, suggesting that H2O2 is not an intermediate of the reaction. It is concluded that the mechanism of action of acetylphenylhydrazine with oxyhemoglobin is two-fold, ultimate reduction to high spin ferric hemoglobin followed by ferrihemochrome formation. Thus it appears that the pathway of denaturation of hemolytic anemias and thalassemia or induced by chemical reagents, entails a common route involving the formation of ferric hemoglobin by a reductive mechanism, followed by reversible ferrihemochromes, irreversible ferrihemochromes, and ultimately, precipitation.

High resolution EPR studies of the fine structure of heme proteins. Third harmonic detection approach.

When third harmonic detection is applied in EPR studies of the superhyperfine structure of nitrosyl derivatives of a number of human hemoglobin variants, significant resoltuion enhancement is obtained. This has allowed a detailed analysis of the number of superhyperfine lines, their g-values and their splittings, and has led to a more complete understanding of the interaction between the axial ligands of the heme iron. Sudies of the effect of the modulation amplitude on EPR line-shapes revealed that the amplitude required to resolve fine structure in the third harmonic mode is 3-10 times larger than that used to record an undistorted first derivative spectrum. The application of this approach for other systems is discussed, and practical guidelines for its use are given.

Electron spin-echo spectroscopic studies of Escherichia coli fumarate reductase.

Electron spin-echo envelope modulation (ESEEM) spectroscopy was applied to the study of reduced Centre 1 of Escherichia coli fumarate reductase (succinate:(acceptor) oxidoreductase, EC 1.3.99.1). The ESEEM spectrum derived from stimulated (3-pulse) echo envelopes obtained at 8.8 GHz contained lines at 0.9, 2.1, 3.0 and 4.2 MHz in the g = 1.94 region. When studied at 11.4 GHz, these low-frequency components scale with magnetic field in a manner indicating interaction between the unpaired electron spin of the Fe-S cluster and a weakly coupled 14N nucleus. Spectral simulations of these ESEEM data yield nuclear quadrupole interaction parameters indicative of peptide nitrogen. For oxidized protein, the magnetic-field dependence of the linear electric-field effect (LEFE) for Centre 3 was measured, and the results confirm the presence of a [3Fe-4S] cluster in the protein.

A chemical modification of cytochrome-c lysines leading to changes in heme iron ligation.

Although 13 lysines of horse cytochrome c are invariant, and three more are extremely conserved, the modification of their side-chain epsilon-amino groups by beta-thiopropionylation caused important changes in protein properties for only three of them; lysines 72,73 and 79. Optical spectroscopy, electron and nuclear paramagnetic resonance, electron spin echo envelope modulation, and molecular weight studies, as well as the unique features of their reaction with cytochrome-c oxidase, indicate that in the oxidized state the modification of these lysines resulted in equilibria between two different states of iron ligation: the native state, in which the metal is coordinated by the methionine-80 sulfur, and a new state in which this ligand is displaced by the sulfhydryl groups of the elongated side chains. The reduction potentials of the TP Lys-72 and the TP Lys-79 derivatives were 201 and 196 millivolt, respectively, indicating that the equilibria favored the sulfhydryl ligated state by 1.5 and 1.7 kcal/mol, respectively. In the ferric state, the protein modified at lysine 72 remained stable as a monomer, but that modified at lysine 73 dimerized rapidly through disulfide bond formation, while the TP Lys-79 cytochrome c dimerized with a half-time of approx. 3 h, both recovering the native-like iron ligation. By contrast, in the ferrous state the monomeric state and the native ligation were preserved in all cases, indicating that the affinity of the cytochrome-c ferrous iron for the methionine-80 sulfur is particularly strong. The dimerized derivatives lost most, but not all, of the capability of the native protein for electron transfer from ascorbate-TMPD to cytochrome-c oxidase.

Oxidation state dependence of proton exchange near the iron-sulfur centers in ferredoxins and high-potential iron-sulfur proteins.

For both the [2Fe-2S] and the [4Fe-4S] ferredoxins, dialysis against 2H2O prior to single electron reduction leads to the appearance of a deuterium modulation pattern in the electron spin echo decay envelope indicative of deuteron-proton exchange very near the paramagnetic center. In contrast, if the ferredoxin is exposed to 2H2O after its reduction in H2O, far less deuterium exchange near the metal center takes place. Thus, proton exchange with solvent is in part dependent on the redox state of the protein. For high potential iron-sulfur proteins, this type of proton-deuteron exchange near the metal center does not occur unless the protein is partially unfolded in dimethylsulfoxide in 2H2O.

The nature of the copper atoms of cytochrome c oxidase as studied by optical and x-ray absorption edge spectroscopy.

X-ray absorption edge spectroscopy has been used to study the copper of 1--2 mM cytochrome c oxidase in the resting oxidized, mixed-valence, and fully reduced states. A comparison was made of this protein with copper complexes and with natural and artificial copper proteins. Spectra were obtained with synchrotron radiation from the SPEAR storage ring using highly sensitive fluorescence detectors. Temperatures of -80 to -120 degrees C were employed further to improve the stability of the samples and to avoid the possibility of either auto- or photon-induced reduction of the materials, which might have occurred in previous studies. In order to characterize the valence states of the Cu and Fe components, the samples were monitored by infrared and visible spectroscopy before and after irradiation by the X-ray beam. The combination of the optical and X-ray absorption techniques has afforded a deconvolution of the four species of copper in the various states of cytochrome c oxidase and the tentative assignment of Cu alpha, the copper redox coupled to the heme alpha of cytochrome alpha, as a highly covalent type of copper and Cu alpha 3, the copper of cytochrome alpha 3, as a more ionic 'blue' type I copper. The implications of these findings upon the mechanism of action of cytochrome oxidase are briefly outlined.

Role of the divalent metal ion in the NAD:malic enzyme reaction: an ESEEM determination of the ground state conformation of malate in the E:Mn:malate complex.

The conformation of L-malate bound at the active site of Ascaris suum malic enzyme has been investigated by electron spin echo envelope modulation spectroscopy. Dipolar interactions between Mn2+ bound to the enzyme active site and deuterium specifically placed at the 2-position, the 3R-position, and the 3S-position of L-malate were observed. The intensities of these interactions are related to the distance between each deuterium and Mn2+. Several models of possible Mn-malate complexes were constructed using molecular graphics techniques, and conformational searches were conducted to identify conformers of malate that meet the distance criteria defined by the spectroscopic measurements. These searches suggest that L-malate binds to the enzyme active site in the trans conformation, which would be expected to be the most stable conformer in solution, not in the gauche conformer, which would be more similar to the conformation required for oxidative decarboxylation of oxalacetate formed from L-malate at the active site of the enzyme.

Cloning, expression, and spectroscopic characterization of Cucumis sativus stellacyanin in its nonglycosylated form.

The cDNA encoding the 182 amino acid long precursor stellacyanin from Cucumis sativus was isolated and characterized. The protein precursor consists of four sequence domains: I, a 23 amino acid hydrophobic N-terminal signal peptide with features characteristic of secretory proteins; II, a 109 amino acid copper-binding domain; III, a 26 amino acid hydroxyproline- and serine-rich peptide characteristic of motifs found in the extension family, extracellular structural glycoproteins found in plant cell walls; and IV, a 22 amino acid hydrophobic extension. Maturation of the protein involves posttranslational processing of domains I and IV. The copper-binding domain (domain II), which shares high sequence identity with other stellacyanins, has been expressed without its carbohydrate attachment sites, refolded from the Escherichia coli inclusion bodies, purified, and characterized by electronic absorption, EPR, ESEEM, and RR spectroscopy. Its spectroscopic properties are nearly identical to those of stellacyanin from the Japanese lacquer tree Rhus vernicifera, the most extensively studied and best characterized stellacyanin, indicating that this domain folds correctly, even in the absence of its carbohydrate moiety. The presence of a hydroxyproline- and serine-rich domain III suggests that stellacyanin may have a function other than that of a diffusible electron transfer protein, conceivably participating in redox reactions localized at the plant cell wall, which are known to occur in response to wounding or infection of the plant.

Inverse correlation between resistance towards copper and towards the redox-cycling compound paraquat: a study in copper-tolerant hepatocytes in tissue culture.

The essential mediatory role of copper or iron in the manifestation of paraquat toxicity has been demonstrated (Kohen and Chevion (1985) Free Rad. Res. Commun. 1, 79-88; Korbashi, P. et al. (1986) J. Biol. Chem. 261, 12472-12476). Several liver cell lines, characterized by their resistance to copper, were challenged with paraquat and their cross-resistance to paraquat and copper was studied. Cell growth and survival data showed that copper-resistant cells, containing elevated copper, are more sensitive towards paraquat than wild type cells. Copper-deprived resistant cells did not have this sensitivity. Paraquat was also shown to cause a marked degradation of cellular glutathione in all cell lines. Albeit the fact that the basal glutathione levels are higher in copper-resistant than in wild type cells, there is more paraquat-induced degradation of cellular glutathione (GSH + GSSG) in resistant cells. It is suggested that in copper-resistant cells which contain elevated levels of copper, paraquat-induced cellular injury is potentiated even where glutathione levels are elevated. Additionally, in vitro experiments are presented that support the in vivo findings demonstrating a role for copper in glutathione degradation.

A pulsed EPR study of redox-dependent hyperfine interactions for the nickel centre of Desulfovibrio gigas hydrogenase.

The nickel centre of hydrogenase from Desulfovibrio gigas was studied by electron spin echo envelope modulation (ESEEM) spectroscopy in the oxidized, unready (Ni-A) and H2-reduced active (Ni-C) states, both in H2O and 2H2O solutions. Fourier transforms of the 3-pulse ESEEM, taken at 8.7 GHz, for Ni-A and Ni-C in H2O contained similar peaks with narrow linewidths at frequencies of 0.4, 1.2 and 1.6 MHz, and a broader peak centered at 4.5 MHz. At 11.6 GHz, the low frequency components showed small field-dependent shifts, while the high frequency component was shifted to 5.1 MHz. These results are consistent with the presence of 14N, possibly from imidazole, coupled to the nickel centre. In 2H2O, Ni-A was shown to be inaccessible for exchange with solvent deuterons. In contrast, Ni-C was accessible to solvent exchange, with a deuterium population being in close proximity to the metal ion. Thus, the nickel environment of the active protein is different from that in the oxidized or unready state. On illumination of Ni-C, although EPR changes are seen, 14N coupling remains, and for the 2H2O sample, deuterium coupling is also retained.

The generation of an organic free radical in substrate-reduced pig kidney diamine oxidase-cyanide.

When the cyanide complex of the copper protein, pig kidney diamine oxidase, is reduced anaerobically by cadaverine (1,5-diaminopentane), the broad, 480 nm, absorption band characteristic of the resting enzyme is bleached and a new absorption spectrum with features at 457, 429, 403 (shoulder), 360 (shoulder) and 332 nm appears. Concomitantly, the EPR spectrum of the enzyme Cu(II)-CN complex decreases in intensity and a new signal is observed that is attributable to an organic free radical. The g values and hyperfine splittings are similar to those previously assigned to a free radical observed when the cyanide complex of lentil seedling diamine oxidase is reacted with the substrate p-dimethylaminomethylbenzylamine [(1984) FEBS Lett. 176, 378-380]. The optical absorption and EPR spectra of the organic radical observed in both proteins are consistent with the same semiquinone-type structure, as expected if pyrroloquinolinequinone (PQQ) is the bound cofactor found in both enzymes.

Induction of hepatic microsomal drug metabolism by azo compounds: a structure-activity relationship.

The structure-activity relationship of 40 azo compounds in their ability to induce cytochrome P-448 and associated monooxygenase activities, as well as UDP-glucuronyltransferase (UDPGT) activity, was investigated. Regardless of their structure, hydrophilic azo dyes and lipophilic azobenzene derivatives were not able to induce these enzyme activities. Only those lipophilic azo dyes with 1-azo-2-naphthol or 1-azo-2-naphthylamine moieties were able to induce cytochrome P-448 and related monooxygenase activities, as well as UDPGT activity. The extent of induction is comparable to or greater than that caused by 3-methylcholanthrene (3-MC). It is suggested that those azo dyes capable of inducing P-450 type cytochromes can form coplanar structures with three fused, 6-membered rings through intramolecular hydrogen bonding. These structures are analogous to polycyclic aromatic hydrocarbons that can also induce.

The effect of Sudan III on drug metabolizing enzymes.

We have examined the induction of drug metabolizing enzymes in rat liver microsomes by azo dye, 1-(p-phenylazophenylazo)-2-naphthol (Sudan III). Marked increases were observed in the levels of cytochrome P-448 as well as in p-nitroanisole O-demethylase (p-NAD), amaranth (AR) and neoprontosil reductases (NPR) and 7-ethoxycoumarin O-deethylase (ECD) activities. On the other hand, aminopyrene N-demethylase activity was not significantly increased. Further, induced ECD activity was inhibited 90% by a specific antibody against cytochrome P-448 while the inhibition observed with an antibody against cytochrome P-450 was less than 25%. Simultaneous administration of Sudan III and 3-methylcholanthene (3-MC) induced cytochrome P-448 up to a level brought about by either Sudan III or 3-MC treatment alone. In contrast, Sudan III did not induce cytochrome P-448 in the 3-MC insensitive DBA/2 mouse. Solubilized microsomes from Sudan III-treated rats showed an identical sodium dodecyl sulfate polyacrylamide gel electrophoretic (SDS-PAGE) pattern with those from 3-MC-treated animals. It is concluded that the cytochrome P-448 induced in liver by Sudan III is very similar to that induced by 3-MC. Sudan III also induced UDP-glucuronyltransferase activity towards 1-naphthol and estradiol. It did not induce NADPH-cytochrome c reductase, nor any of the enzymes which constitute the microsomal electron transport chain except for cytochrome P-448.