An enhanced in vivo stable isotope labeling by amino acids in cell culture (SILAC) model for quantification of drug metabolism enzymes.

Many of the enzymes involved in xenobiotic metabolism are maintained at a low basal level and are only synthesized in response to activation of upstream sensor/effector proteins. This induction can have implications in a variety of contexts, particularly during the study of the pharmacokinetics, pharmacodynamics, and drug-drug interaction profile of a candidate therapeutic compound. Previously, we combined in vivo SILAC material with a targeted high resolution single ion monitoring (tHR/SIM) LC-MS/MS approach for quantification of 197 peptide pairs, representing 51 drug metabolism enzymes (DME), in mouse liver. However, as important enzymes (for example, cytochromes P450 (Cyp) of the 1a and 2b subfamilies) are maintained at low or undetectable levels in the liver of unstimulated metabolically labeled mice, quantification of these proteins was unreliable. In the present study, we induced DME expression in labeled mice through synchronous ligand-mediated activation of multiple upstream nuclear receptors, thereby enhancing signals for proteins including Cyps 1a, 2a, 2b, 2c, and 3a. With this enhancement, 115 unique, lysine-containing, Cyp-derived peptides were detected in the liver of a single animal, as opposed to 56 in a pooled sample from three uninduced animals. A total of 386 peptide pairs were quantified by tHR/SIM, representing 68 Phase I, 30 Phase II, and eight control proteins. This method was employed to quantify changes in DME expression in the hepatic cytochrome P450 reductase null (HRN) mouse. We observed compensatory induction of several enzymes, including Cyps 2b10, 2c29, 2c37, 2c54, 2c55, 2e1, 3a11, and 3a13, carboxylesterase (Ces) 2a, and glutathione S-transferases (Gst) m2 and m3, along with down-regulation of hydroxysteroid dehydrogenases (Hsd) 11b1 and 17b6. Using DME-enhanced in vivo SILAC material with tHR/SIM, therefore, permits the robust analysis of multiple DME of importance to xenobiotic metabolism, with improved utility for the study of drug pharmacokinetics, pharmacodynamics, and of chemically treated and genetically modified mouse models.

[The state of the cytochrome part of respiratory chain in tumor carrier rats' liver in the conditions of preliminary low-level irradiation].

The effect of low-level irradiation on the structural and functional organization of the cytochrome part of the respiratory chain in tumor carrier rats' liver is studied. The preliminary low-level irradiation leading to the mitochondrial cytochrome a, b and c content reduction at the latent stage of Guerin's carcinoma is shown. At the same time, the maximal reduction of the content of all liver cytochromes is observed at the terminal stages of oncogenesis. The content of cytochome c undergoes the most significant changes in the liver mitochondrial fracture. The possible mechanism of mitochondrial haem-containing cytochromes content reduction may be associated with the disorder of their formation caused by the heam synthesis inhibition found in our study. Simultaneously, the cytochrome oxydase (key enzyme of the cytochrome part) activity inhibition is observed to be caused by preliminary low-level irradiation at the latent growth stage of Guerin's carcinoma. The determined differences between irradiated and non-irradiated tumor carrier groups allow us to come to the conclusion that low-level irradiation has an impact only at the initial stages of the aftereffect. At the following stages, the state of the cytochrome part of the respiratory chain is defined by growth conditions of tumor.

Regulation of apoptosis by BH3 domains in a cell-free system.

BACKGROUND: The Bcl-2 family of proteins plays a key role in the regulation of apoptosis. Some family members prevent apoptosis induced by a variety of stimuli, whereas others promote apoptosis. Competitive dimerisation between family members is thought to regulate their function. Homologous domains within individual proteins are necessary for interactions with other family members and for activity, although the specific mechanisms might differ between the pro-apoptotic and anti-apoptotic proteins. RESULTS: Using a cell-free system based on extracts of Xenopus eggs, we have investigated the role of the Bcl-2 homology domain 3 (BH3) from different members of the Bcl-2 family. BH3 domains from the pro-apoptotic proteins Bax and Bak, but not the BH3 domain of the anti-apoptotic protein Bcl-2, induced apoptosis in this system, as determined by the rapid activation of specific apoptotic proteases (caspases) and by DNA fragmentation. The apoptosis-inducing activity of the BH3 domains requires both membrane and cytosolic fractions of cytoplasm, involves the release of cytochrome c from mitochondria and is antagonistic to Bcl-2 function. Short peptides, corresponding to the minimal sequence of BH3 domains required to bind anti-apoptotic Bcl-2 family proteins, also trigger apoptosis in this system. CONCLUSIONS: The BH3 domains of pro-apoptotic proteins are sufficient to trigger cytochrome c release, caspase activation and apoptosis. These results support a model in which pro-apoptotic proteins, such as Bax and Bak, bind to Bcl-2 via their BH3 domains, inactivating the normal ability of Bcl-2 to suppress apoptosis. The ability of synthetic peptides to reproduce the effect of pro-apoptotic BH3 domains suggests that such peptides may provide the basis for engineering reagents to control the initiation of apoptosis.

On the origin of respiration: electron transport proteins from archaea to man.

All aerobic organisms use the exergonic reduction of molecular oxygen to water as primary source of metabolic energy. This reaction is catalyzed by membrane residing terminal heme/Cu-oxidases which belong to a superfamily of widely varying structural complexity between mitochondrial and bacterial members of this family. Over the last few years, considerable information from this and other laboratories accumulated also on archaeal respiratory chains and their terminal oxidases. In the following, the molecular and catalytic properties of the latter are discussed and compared to those from bacteria and eucarya under the aspect of their energy conserving capabilities and their phylogenetic relations. The Rieske iron-sulfur proteins being important functional constituents of energy transducing respiratory complexes are included in this study. A number of essential conclusions can be drawn. (1) Like bacteria, archaea can also contain split respiratory chains with parallel expression of separate terminal oxidases. (2) The functional core of all oxidases is the highly conserved topological motif of subunit I consisting of at least 12 membrane spanning helices with the 6 histidine residues of the heme/Cu-binding centers in identical locations. (3) Some archaeal oxidases are organized in unusual supercomplexes with other cytochromes and Rieske [2Fe2S] proteins. These complexes are likely to function as proton pumps, whereas on a structural basis several subunit I equivalents alone are postulated to be unable to pump protons. (4) The genes of two archaeal Rieske proteins have been cloned from Sulfolobus; phylogenetically they are forming a separate archaeal branch and suggest the existence of an evolutionary ancestor preceding the split into the three urkingdoms. (5) Archaeal oxidase complexes may combine features of electron transport systems occurring exclusively as separate respiratory complexes in bacteria and eucarya. (6) As far back as the deepest branches of the phylogentic tree, terminal oxidases reveal a degree of complexity comparable to that found in higher organisms. (7) Sequence analysis suggests a monophyletic origin of terminal oxidases with an early split into two types found in archaea as well as bacteria. This view implies an origin of terminal oxidases prior to oxygenic photosynthesis in contrast to the widely accepted inverse hypothesis.

Purification and properties of a 'cytochrome a1'-like hemoprotein from a magnetotactic bacterium, Aquaspirillum magnetotacticum.

A novel hemoprotein was purified from a magnetotactic bacterium, Aquaspirillum magnetotacticum MS-1. The protein showed absorption peaks at 437 nm in the oxidized form, and 592, 550 and 450 nm in the reduced form. Although the spectral properties of the hemoprotein were very similar to those of 'cytochrome a1', the hemoprotein contained no molecules of heme a. The protein contained two kinds of hemes; one was extracted with HCl-acetone and the other was covalently bound to the protein. The pyridine ferrohemochrome of the former heme showed absorption peaks at 440, 545 and 585 nm. The chromatographic behavior of the heme on reverse-phase HPLC was different from that of heme a. The pyridine ferrohemochrome of the covalently bound heme showed an alpha peak at 565 nm. On the basis of the iron analysis, the hemoprotein contained one molecule of each of the two kinds of heme in the holoprotein. The protein was composed of two kinds of subunit with molecular weights of 41,000 and 17,000 and showed very little cytochrome c oxidase activity. The amounts of the hemoprotein in the magnetic cells of A. magnetotacticum were larger than those in non-magnetic cells. These results suggest that the 'cytochrome a1'-like hemoprotein is not the terminal oxidase of the bacterium and may be related to the formation of magnetosome in the magnetic cells of A. magnetotacticum.

The mechanism of electron gating in proton pumping cytochrome c oxidase: the effect of pH and temperature on internal electron transfer.

Electron-transfer reactions following flash photolysis of the mixed-valence cytochrome oxidase-CO complex have been measured at 445, 598 and 830 nm between pH 5.2 and 9.0 in the temperature range of 0-25 degrees C. There is a rapid electron transfer from the cytochrome a3-CuB pair to CuA (time constant: 14200 s-1), which is followed by a slower electron transfer to cytochrome a. Both the rate and the amplitude of the rapid phase are independent of pH, and the rate in the direction from CuA to cytochrome a3-CuB is practically independent of temperature. The second phase depends strongly on pH due to the titration of an acid-base group with pKa = 7.6. The equilibrium at pH 7.4 corresponds to reduction potentials of 225 and 345 mV for cytochrome a and a3, respectively, from which it is concluded that the enzyme is in a different conformation compared to the fully oxidized form. The results have been used to suggest a series of reaction steps in a cycle of the oxidase as a proton pump. Application of the electron-transfer theory to the temperature-dependence data suggests a mechanism for electron gating in the pump. Reduction of both cytochrome a and CuA leads to a conformational change, which changes the structure of cytochrome a3-CuB in such a way that the reorganizational barrier for electron transfer is removed and the driving force is increased.

Rate enhancement of the internal electron transfer in cytochrome c oxidase by the formation of a peroxide complex; its implication on the reaction mechanism of cytochrome c oxidase.

The oxidation of reduced cytochrome c oxidase by hydrogen peroxide was investigated with stopped-flow methods. It was reported by us previously (A.C.F. Gorren, H. Dekker and R. Wever (1986) Biochim. Biophys. Acta 852, 81-92) that at low H2O2 concentrations cytochrome a is oxidised simultaneously with cytochrome a3, but that at higher H2O2 concentrations the oxidation of cytochrome a is slower than that of cytochrome a3. We now report that for high peroxide concentrations (10-45 mM) the oxidation rate of cytochrome a increased linearly with the concentration of H2O2 (k = 700 M-1.S-1). Upon extrapolation to zero H2O2 concentration an intercept with a value of 16 s-1 (at 20 degrees C and pH 7.4) was found. A reaction sequence is described to explain these results; according to this model the rate constant (16 S-1) at zero H2O2 concentration represents the true value of the rate of electron transfer from cytochrome a to cytochrome a3 when the a3-CuB site is oxidised and unligated. However, when a complex of hydrogen peroxide with oxidised cytochrome a3 is formed, this rate is strongly enhanced. The slope (700 M-1.S-1) would then represent the rate of cytochrome a3(3+)-H2O2 complex formation. From experiments in which the pH was varied, we conclude that the reaction of H2O2 with cytochrome a3(2+) is independent of pH, whereas the electron-transfer rate from cytochrome a to cytochrome a3 gradually decreases with increasing pH. From the temperature dependence we could calculate values of 23 kJ.mol-1 and 45 kJ.mol-1 for the activation energies of the oxidations by H2O2 of cytochrome a3(2+) and cytochrome a2+, respectively. The similarity of the values that were obtained for cytochrome a oxidation both with H2O2 and with O2 as the electron acceptor suggests that the reactions share the same mechanism. In 2H2O the reactions studied decreased in rate. For the reaction of 2H2O2 with reduced cytochrome a3 in 2H2O, a small effect was found (15% decrease in rate constant). However, the internal electron-transfer rate from cytochrome a to cytochrome a3 decreased by 50%, Our results suggest that the internal electron transfer is associated with proton translocation.

Cytochrome oxidase content of rat brain during development.

The cytochrome oxidase concentration and content of rat brain during development was measured using a simple new assay for cytochrome a. The cytochrome oxidase concentration increased from 1.2 nmol/g wet wt. of brain at birth to about 5.5 nmol/g in the adult, most of the change occurring between 5 and 25 days after birth.

The distance between cytochromes a and a3 in the azide compound of bovine-heart cytochrome oxidase.

The electron-spin relaxation rates of the two species of cytochrome a3(3+)-azide found in the azide compound of bovine-heart cytochrome oxidase were measured by progressive microwave saturation at T = 10 K. It has been shown previously that Cyt a3(3+)-azide gives rise to two distinct EPR resonances, depending upon the oxidation state of Cyt a. When Cyt a is ferrous, Cyt a3(3+)-azide has g = 2.88, 2.19 and 1.64; upon oxidation of Cyt a, the a3(3+)-azide g-values become g = 2.77, 2.18, and 1.74 (Goodman, G. (1984) J Biol. Chem. 259, 15094-15099). The relaxation effect of Cyt a on Cyt a3 could be measured as the difference in microwave field saturation parameter H1/2 between the g = 2.77 and g = 2.88 species. For each signal the spin-lattice relaxation time T1 was determined from H1/2 using the transverse relaxation time T2. The value of T2 at 10 K was extrapolated from a plot of line-width vs. temperature at higher temperature. The dipolar contribution to T1 was related to the Cyt a-Cyt a3 spin-spin distance utilizing available information on the relative orientation of Cyt a3-azide and Cyt a (Erecinska, M., Wilson, D.F. and Blasie, J.K. (1979) Biochim. Biophys. Acta 545, 352-364). By taking into account the relaxation parameters for both gx and gz components of the Cyt a3-azide g-tensor, the angle between the gz components of the Cyt a and Cyt a3 g-tensors was determined to be between 0 and 18 degrees, and the Cyt a-Cyt a3 spin-spin distance was found to be 19 +/- 8 A.

Uncoupled and coupled electron transfer reactions.

This paper concerns an NMR study of cytochromes c in an effort to understand the coupling of redox state changes with protein conformation and proton movement. The objective is to find a model which will allow us to understand electron/proton diffusion coupling as seen in cytochrome oxidase and envisaged in one description of energy transduction.

Resonance Raman resolution of a-, b- and c-type cytochromes in membrane vesicles of alkalophilic bacteria.

Resonance Raman spectroscopy has been used to obtain complete spectra of each individual cytochrome type - a, b and c - in the reduced state within membrane vesicle preparations from two species of obligately alkalophilic bacteria: Bacillus alcalophilus and Bacillus firmus RAB. The vibrational spectra, in the range 250-1700 cm-1, were obtained with tunable dye laser excitation in the wavelength range 550-600 nm tuned to resonance with the appropriate reduced alpha band maximum for the cytochrome type of interest. The spectra reveal details which serve to characterize the specific type of cytochrome as well as to confirm the similarity of the heme prosthetic group to previously well-characterized cytochromes of the the a- b- or c-type. Preliminary evidence in support of heterogeneity of b-type, and possibly a-type cytochromes, or of heme-heme interaction within the membrane is presented.

The reduced minus oxidized difference spectra of cytochromes a and a3.

We have re-investigated the reduced minus oxidized difference spectra of the two heme centers of cytochrome oxidase, cytochromes a and a3. In contrast to data obtained in an earlier study (Vanneste, W.H. (1966) Biochemistry 5, 838-848), we find that the spectrum for cytochrome a3 agrees with that found with a 5-coordinate high-spin heme A model compound. Small but significant additional differences are noted for both heme centers.

Oxidation-reduction potentials of respiratory chain components in Thiobacillus A2.

(1) Cells of Thiobacillus A2 grown chemoautotrophically on thiosulfate or heterotrophically on succinate with oxygen contained b-, c-, o-, a- and a3-type cytochromes. The amount of cytochrome per mg of cell protein was much greater in thiosulfate-grown cells and differences in the relative concentrations of cytochromes were observed for the different growth conditions. (2) The half-reduction potentials at pH 7.0 (Em,7.0) and spectral maxima of c-, b-, a- and a3-type cytochromes were similar in cells grown aerobically with thiosulfate or with succinate as the growth substrate. (3) The half-reduction potential of the 'invisible', or high-potential copper, as determined from the potentiometric behavior of the carbon monoxide-reduced cytochrome a3 complex at pH 8.0, was 365 mV. (4) Reducing equivalents from thiosulfate appear to enter the respiratory chain at the cytochrome c level; however, studies in cell-free extracts were limited due to a loss in respiratory activity with thiosulfate as a substrate upon cell disruption.

The oxidation of cytochrome c oxidase by hydrogen peroxide.

The reaction of H2O2 with mixed-valence and fully reduced cytochrome c oxidase was investigated by photolysis of fully reduced and mixed-valence carboxy-cytochrome c oxidase in the presence of H2O2 under anaerobic conditions. The results showed that H2O2 reacted rapidly (k = (2.5-3.1) X 10(4) M-1 X s-1) with both enzyme species. With the mixed-valence enzyme, the fully oxidised enzyme was reformed. On the time-scale of our experiments, no spectroscopically detectable intermediate was observed. This demonstrates that mixed-valence cytochrome c oxidase is able to use H2O2 as a two-electron acceptor, suggesting that cytochrome c oxidase may under suitable conditions act as a peroxidase. Upon reaction of H2O2 with the fully reduced enzyme, cytochrome a was oxidised before cytochrome a3. From this observation it was possible to estimate that the rate of electron transfer from cytochrome a to a3 is about 0.5-5 s-1.

Cytochrome a3 deficiency in human achondroplasia.

Mitochondria prepared from tissue culture cells (skin fibroblasts) from normal subjects and subjects with homozygous achondroplasia were studied to determine the concentrations of cytochromes a and a3 in the preparations. Cytochrome a3 was markedly decreased (80%) in the achondroplastic preparations with cytochrome a present in normal amounts. Determination of total heme a (as the pyridine hemochromogen) in the normal and achondroplastic preparations demonstrated that the observed decrease in concentration of cytochrome a3 in the achondroplastic preparations was due to an absence of cytochrome a3 and not to a change in its absorbancy (extinction coefficient). The decreased concentrations of cytochrome a3 in the achondroplastic cells may decrease the reactivity or affinity of the mitochondrial oxidative systems for oxygen and result in the phenotypic expression of the disease.

A comparison of three preparations of cytochrome c oxidase. Optical absorbance spectra, EPR spectra and reaction towards ligands.

Three preparations of cytochrome c oxidase, the preparation as traditionally prepared in our laboratory as described by Van Buuren (1992; PhD Thesis, University of Amsterdam), a preparation according to Volpe and Caughey (Biochem. Biophys. Res. Commun. 61 (1974) 502-509) and a preparation of 'fast' cytochrome c oxidase (Brandt, U., Schägger, H. and Von Jagow, G. (1989) Eur. J. Biochem. 182, 705-711), are compared in their reaction with cyanide and carbon monoxide. The reaction with cyanide is nearly as fast for the Van Buuren preparation as for the 'fast' preparation, but much slower for the Volpe-Caughey preparation. Mixed-valence cytochrome c oxidase (cytochrome a3 and CuB reduced with carbon monoxide bound and cytochrome a and CuA oxidized) is prepared by anaerobic incubation with carbon monoxide. With the Van Buuren preparation complete formation of the species takes 4 h, whereas with the Volpe-Caughey preparation it takes 20 h. Longer incubation under CO results in partial reduction of cytochrome a and CuA. With the 'fast' preparation mixed-valence cytochrome c oxidase is formed after more than one day of incubation with CO, but it is stable for at least 3 days. The presence of oxidized cytochrome c did enhance the reactivity towards cyanide and towards carbon monoxide in cytochrome c oxidase of all three preparations. Furthermore, optical and EPR spectra of the preparations of cytochrome c oxidase are compared. The Volpe-Caughey preparation has an intense g' = 12 EPR-signal, the Van Buuren preparation has hardly any g' = 12 signal and the 'fast' preparation has no g' = 12 signal. In the 'fast' preparation the low-spin heme signal is shifted (from g = 3.00 to g = 2.97). The absorbance spectra of the three preparations in the Soret region are similar with a maximum at 424 nm. Only the 'fast' preparation as isolated was completely oxidized, whereas the other preparations were partially reduced. It was concluded that differences in the reaction of cytochrome c oxidase with ligands are determined by the internal or external ligand bound to the cytochrome a3-CuB couple.

A specific uncoupler-binding protein in Tetrahymena pyriformis and Paracoccus denitrificans.

The uncoupler of mitochondrial oxidative phosphorylation, 2-nitro-4-azido-carbonylcyanide phenylhydrazone (N3CCP) which is capable of photoaffinity labeling has been used to examine the effect of uncouplers on the energy conserving membrane of Paracoccus denitrificans and Tetrahymena pyriformis. The N3CCP uncouples respiration in P. denitrificans and T. pyriformis cells with U1/2 values of 1.05 microM and 0.24 microM, respectively. Binding studies show the presence of 0.65 +/- 0.05 high affinity sites per cytochrome alpha with Kd of 0.5 +/- 0.1 microM in P. denitrificans membranes and 1.4 +/- 0.2 sites per cytochrome alpha 2 with a Kd of 0.4 +/- 0.1 microM in T. pyriformis membranes. Irradiation of [3H]-N3CCP bound to the membranes leads to a covalent linking of the radioactive uncoupler to a peptide of 10--15 kdaltons as analyzed by SDS-polyacrylamide gel electrophoresis. It is concluded that these two microbial systems contain a specific high affinity uncoupler binding site very similar to that of mammalian mitochondria (Katre, N.V. and Wilson, D.F. (1978) Arch. Biochem. Biophys. 191, 647--656).

Effect of dysprosium on the spin-lattice relaxation time of cytochrome c and cytochrome a.

The progressive power saturation of the electron paramagnetic resonance of horse heart cytochrome c and solubilized bovine heart cytochrome oxidase has been monitored at low temperature in the presence of the relaxing agent, dysprosium. The saturation of the EPR signal of cytochrome c is relieved even at 6 K. With increasing temperature the effect is enhanced as the relaxation time of the dysprosium becomes shorter; however, the intrinsic spin-lattice relaxation time, T1, for cytochrome c decreases even more rapidly with increasing temperature. T1 for cytochrome c can be described by an intrinsic component, a component which is proportional to the concentration of dysprosium and a third component due to local binding which is independent of dysprosium concentration. The cytochrome a component of cytochrome oxidase is also affected by dysprosium. In the presence of cytochrome oxidase, T1 for cytochrome c is almost unaffected by dysprosium, indicating that access to the cytochrome c heme is blocked by the binding of c to oxidase. Based on the concentration-dependent effect of dysprosium on the lifetime of cytochrome c, it is possible to make distance estimates from the EPR active center to Dy3+. Dysprosium is therefore useful for determining the spatial relationships among paramagnetic enzyme components in a quantitative way.

A comparison of the respiratory chain in particles from Paracoccus denitrificans and bovine heart mitochondria by EPR spectroscopy.

A study is presented on the EPR characteristics of the paramagnetic groups in the respiratory chain present in membrane particles of Paracoccus denitrificans, the respiratory system of which is very similar to that in submitochondrial particles from beef heart. All paramagnetic prosthetic groups of the mitochondrial system are also found in the bacterial plasma membrane. Their properties suggest that the respiratory groups are embedded in very similar protein environments in the two systems.

Unusual cytochrome a592 with low PO2 affinity correlates as putative oxygen sensor with rat carotid body chemoreceptor discharge.

Light-absorption spectra and afferent chemoreceptor discharge were simultaneously recorded on superfused rat carotid bodies (CBs) under the influence of cytochrome a3-CuB ligands (O2, CN-, CO) in order to identify the primary mitochondrial cytochrome c oxidase (CCO) oxygen sensor. Spectra could be described on the basis of weighted light-absorption spectra of cytochrome b558 of the NAD(P)H oxidase and mitochondrial cytochromes b and c, CCO, cytochrome a3, and an unusual cytochrome a peaking at 592 nm. Discharge signals were deconvoluted into phasic and tonic activity for comparing different CB responses. The spectral weight of cytochrome a592 decreased significantly starting at high PO2 (100 mm Hg) and low sodium cyanide (CN-, 10 mM) accompanied by increasing phasic peak discharge. Combined CO-hypoxia or CO-CN- application inhibited photolysis of CO-stimulated chemoreceptor discharge, revealing photometrically cytochrome a592 as central in oxygen sensing. Control spectra in tissue from sympathetic and nodose ganglia did not show any cytochrome a592 contribution. According to these results, cytochrome a592 is assumed as a unique component of CB CCO, revealing in contrast to other cytochromes an apparent low PO2 and high CN- affinity, probably due to a shortcut of electron flow within CCO between CuA and cytochrome a3-CuB.