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.

A monte carlo method for generating side chain structural ensembles.

We report a Monte Carlo side chain entropy (MC-SCE) method that uses a physical energy function inclusive of long-range electrostatics and hydrophobic potential of mean force, coupled with both backbone variations and a backbone dependent side chain rotamer library, to describe protein conformational ensembles. Using the MC-SCE method in conjunction with backbone variability, we can reliably determine the side chain rotamer populations derived from both room temperature and cryogenically cooled X-ray crystallographic structures for CypA and H-Ras and NMR J-coupling constants for CypA, Eglin-C, and the DHFR product binary complexes E:THF and E:FOL. Furthermore, we obtain near perfect discrimination between a protein's native state ensemble and ensembles of misfolded structures for 55 different proteins, thereby generating far more competitive side chain packings for all of these proteins and their misfolded states.

[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.

Heme, iron, and the mitochondrial decay of ageing.

Heme, the major functional form of iron, is synthesized in the mitochondria. Although disturbed heme metabolism causes mitochondrial decay, oxidative stress, and iron accumulation, all of which are hallmarks of ageing, heme has been little studied in nutritional deficiency, in ageing, or age-related disorders such as Alzheimer's disease (AD). Biosynthesis of heme requires Vitamin B(6), riboflavin, biotin, pantothenic acid, and lipoic acid and the minerals zinc, iron, and copper, micronutrients are essential for the production of succinyl-CoA, the precursor for porphyrins, by the TCA (Krebs) cycle. Only a small fraction of the porphyrins synthesized from succinyl-CoA are converted to heme, the rest are excreted out of the body together with the degradation products of heme (e.g. bilirubin). Therefore, the heme biosynthetic pathway causes a net loss of succinyl-CoA from the TCA cycle. The mitochondrial pool of succinyl-CoA may limit heme biosynthesis in deficiencies for micronutrients (e.g. iron or biotin deficiency). Ageing and AD are also associated with hypometabolism, increase in heme oxygenase-1, loss of complex IV, and iron accumulation. Heme is a common denominator for all these changes, suggesting that heme metabolism maybe altered in age-related disorders. Heme can also be a prooxidant: it converts less reactive oxidants to highly reactive free radicals. Free heme has high affinity for different cell structures (protein, membranes, and DNA), triggering site-directed oxidative damage. This review discusses heme metabolism as related to metabolic changes seen in ageing and age-related disorders and highlights the possible role in iron deficiency.

Role of the aryl hydrocarbon receptor in cell cycle regulation.

One of the most puzzling aspects of the biological impact of polycyclic aromatic hydrocarbon compounds is that they elicit an apparently unrelated variety of toxic, teratogenic, and carcinogenic responses in exposed animals and in humans. At the cellular level, these environmental toxicants affect cell cycle regulatory mechanisms and signal transduction pathways in ways that are equally diverse and often contradictory. For example, depending on the particular cell lines studied, exposure to these compounds may lead to cell proliferation, to terminal differentiation, or to apoptosis. These effects are mediated by the aryl hydrocarbon receptor, a ligand-activated transcription factor well known for its regulatory activity on the expression of several phase I detoxification cytochrome P450 genes. Research into the molecular mechanisms of aryl hydrocarbon receptor function has uncovered a novel role for this protein during cell cycle progression. The activated receptor acts as an environmental sensor and cell cycle checkpoint that commits cells exposed to adverse environmental stimuli to arrest before the onset of DNA replication.

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.

Structural analyses of the deduced amino acid sequences of a novel type heme-copper terminal oxidase, cytochrome aco3, from alkalophilic Bacillus YN-2000.

Cytochrome aco3 from a facultatively alkalophilic bacterium, Bacillus YN-2000, was found to be alkaline- and heat-tolerant. To better understand the structural features of Bacillus YN-2000 cytochrome aco3, the gene encoding this enzyme was cloned and sequenced. Nucleotide sequence analyses of the region neighboring the acoI (subunit I) gene revealed that the acoII (subunit II) and acoIII (subunit III) genes were concomitantly clustered upstream and downstream of the acoI gene, respectively, forming an operon with transcriptional polarity. The deduced amino acid sequence of subunit I was highly similar to that of cytochrome caa3 from thermophilic bacterium Bacillus PS3 in which the heme a3 could be replaced with heme o. Furthermore, a marked paucity of basic amino acid residues was found in the cytochrome c-binding subunit II, which might be a result of the adaptation to a highly alkaline external milieu.

Propofol impairment of mitochondrial respiration in isolated perfused guinea pig hearts determined by reflectance spectroscopy.

OBJECTIVE: To simultaneously determine the effect of propofol on myocardial oxygenation, mitochondrial function, and whole organ function in an isolated heart model, using optical reflectance spectroscopy. DESIGN: Controlled laboratory investigation. SETTING: Research laboratory. SUBJECTS: Twenty adult guinea pigs. INTERVENTIONS: Isolated hearts were perfused alternately with a modified oxygenated Krebs-Henseleit buffer and with buffer containing varied concentrations of propofol. Ninety seconds of ischemia were produced during perfusion with each solution studied. MEASUREMENTS AND MAIN RESULTS: Myoglobin oxygen saturation, cytochrome c and cytochrome a/a3 redox state, and ventricular pressure were continuously measured from isolated guinea pig hearts during a 2-hr period. Myoglobin oxygen saturation increased and both cytochromes became more oxidized in the presence of propofol. During ischemia, myoglobin desaturation and cytochrome reduction were delayed and less complete in the presence of propofol. The mean ischemic time to 50% myoglobin desaturation was, on average, 14.3 secs with buffer perfusion, and increased to 24.5, 27.9, and 41.8 secs, with 50, 100, and 200 microM propofol perfusion, respectively. Ventricular function decreased linearly with increasing propofol concentration. From baseline buffer perfusion, maximal dP/dt per cardiac cycle decreased on average by 30.4%, 40.9%, and 69.4%, with 50, 100, and 200 microM propofol perfusion, respectively. CONCLUSIONS: Propofol impairs either oxygen utilization or inhibits electron flow along the mitochondrial electron transport chain in the guinea pig cardiomyocyte. Propofol also significantly decreases ventricular performance in the isolated perfused heart. These effects are linearly correlated with propofol concentration in the range studied.

Femtosecond resolution of ligand-heme interactions in the high-affinity quinol oxidase bd: A di-heme active site?

Interaction of the two high-spin hemes in the oxygen reduction site of the bd-type quinol oxidase from Escherichia coli has been studied by femtosecond multicolor transient absorption spectroscopy. The previously unidentified Soret band of ferrous heme b(595) was determined to be centered around 440 nm by selective excitation of the fully reduced unliganded or CO-bound cytochrome bd in the alpha-band of heme b(595). The redox state of the b-type hemes strongly affects both the line shape and the kinetics of the absorption changes induced by photodissociation of CO from heme d. In the reduced enzyme, CO photodissociation from heme d perturbs the spectrum of ferrous cytochrome b(595) within a few ps, pointing to a direct interaction between hemes b(595) and d. Whereas in the reduced enzyme no heme d-CO geminate recombination is observed, in the mixed-valence CO-liganded complex with heme b(595) initially oxidized, a significant part of photodissociated CO does not leave the protein and recombines with heme d within a few hundred ps. This caging effect may indicate that ferrous heme b(595) provides a transient binding site for carbon monoxide within one of the routes by which the dissociated ligand leaves the protein. Taken together, the data indicate physical proximity of the hemes d and b(595) and corroborate the possibility of a functional cooperation between the two hemes in the dioxygen-reducing center of cytochrome bd.

Preliminary evidence for the existence of specific functional assemblies between enzymes of the beta-oxidation pathway and the respiratory chain.

The electron-transferring flavoprotein (ETF) has been detected in two large soluble-protein complexes partially purified from sonicated porcine liver mitochondria. Size-exclusion chromatography and sucrose-density ultracentrifugation suggested molecular masses in the region of 390 to 420 kDa for the two complexes. Activities of medium-chain acyl-CoA dehydrogenase, sarcosine dehydrogenase and ETF:ubiquinone oxidoreductase were also detected. No evidence of oxidative-phosphorylation properties was obtained. Treatment with antimycin A inhibited the activity of both complexes. Pyridine haemochromogens, prepared from the partially purified species, show the presence of cytochrome proteins. The possible composition of these complexes and their relationship to the electron transport chain are discussed.

Heme-copper oxidase family structure of Magnetospirillum magnetotacticum 'cytochrome a1'-like hemoprotein without cytochrome c oxidase activity.

The genes encoding 'cytochrome a1'-like hemoprotein of Magnetospirillum magnetotacticum were identified and sequenced. Three ORFs, mcalI, mcaI and hosA, were included in the sequenced region. The six histidine residues which were predicted to associate with the prosthetic cofactors of heme-copper oxidase superfamily were conserved in the hemoprotein. However, none of the amino acid residues which were proposed to participate in the oxygen-reducing and the coupled proton pumping reactions in cytochrome c oxidase were at all conserved in the hemoprotein.

Involvement of cytochrome a in iron oxidation of a moderately thermophilic iron-oxidizing bacterium, strain TI-1.

The iron-oxidizing activity of a moderately thermophilic iron-oxidizing bacterium, strain TI-1, was located in the plasma membrane. When the strain was grown in Fe2+ (60 mM)-salts medium containing yeast extract (0.03%), the plasma membrane had iron-oxidizing activity of 0.129 mumol O2 uptake/mg/min. Iron oxidase was solubilized from the plasma membrane with 1.0% n-octyl-beta-D-glucopyranoside (OGL) containing 25% (v/v) glycerol (pH 3.0) and purified 37-fold by a SP Sepharose FF column chromatography. Iron oxidase solubilized from the plasma membrane was stable at pH 3.0, but quite unstable in the buffer with the pH above 6.0 or below 1.0. The optimum pH and temperature for iron oxidation were 3.0 and 55 degrees C, respectively. Solubilized enzyme from the membrane showed absorption peaks characteristic of cytochromes a and b. Cyanide and azide, inhibitors of cytochrome c oxidase, completely inhibited iron-oxidizing activity at 100 microM, but antimycin A, 2-n-heptyl-4-hydroxyquinoline-N-oxide (HOQNO) and myxothiazol, inhibitors of electron transport systems involved with cytochrome b, did not inhibit enzyme activity at 10 microM. The absorption spectrum of the most active enzyme fraction from SP Sepharose FF column chromatography (4.76 mumol O2 uptake/mg/min) compared with lower active fractions from the chromatography (0.009 and 2.10 mumol O2 uptake/mg/min) showed a large alpha-peak of cytochrome a at 602 nm and a smaller alpha-peak of cytochrome b at 560 nm. The absorption spectrum of pyridine ferrohemochrome prepared from the most highly purified enzyme showed an alpha-peak characteristic of heme a at 587 nm, but not the alpha-peak characteristic of heme c at 550 nm. The cytochrome a, but not cytochrome b, in the most highly purified enzyme fraction was reduced by the addition of ferrous iron at pH 3.0, indicating that electrons from Fe2+ were transported to cytochrome a, but not cytochrome b. These results strongly suggest that cytochrome a, but not cytochromes b and c, is involved in iron oxidation of strain TI-1.

The second derivative electronic absorption spectrum of cytochrome c oxidase in the Soret region.

The electronic absorption spectrum of solubilized beef heart cytochrome c oxidase was analyzed in the 400-500 nm region to identify the origin of doublet features appearing in the second derivative spectrum associated with ferrocytochrome a. This doublet, centered near 22,600 cm(-1), was observed in the direct absorption spectrum of the a(2+)a(3)(3+).HCOO(-) form of the enzyme at cryogenic temperatures. Since evidence for this doublet at room temperature is obtained only on the basis of the second derivative spectrum, a novel mathematical approach was developed to analyze the resolving power of second derivative spectroscopy as a function of parameterization of spectral data. Within the mathematical limits defined for resolving spectral features, it was demonstrated that the integrated intensity of the doublet feature near 450 nm associated with ferrocytochrome a is independent of the ligand and oxidation state of cytochrome a(3). Furthermore, the doublet features, also observed in cytochrome c oxidase from Paracoccus denitrificans, were similarly associated with the heme A component and were correspondingly independent of the ligand and oxidation state of the heme A(3) chromophore. The doublet features are attributed to lifting of the degeneracy of the x and y polarized components of the B state of the heme A chromophore associated with the Soret transition.

MTO1 codes for a mitochondrial protein required for respiration in paromomycin-resistant mutants of Saccharomyces cerevisiae.

Mutations in MTO1 express a respiratory defect only in the context of a mitochondrial genome with a paromomycin-resistance allele. This phenotype is similar to that described previously for mss1 mutants by Decoster, E., Vassal, A., and Faye, G. (1993) J. Mol. Biol. 232, 79-88. We present evidence that Mto1p and Mss1p are mitochondrial proteins and that they form a heterodimer complex. In a paromomycin-resistant background, mss1 and mto1 mutants are inefficient in processing the mitochondrial COX1 transcript for subunit 1 of cytochrome oxidase. The mutants also fail to synthesize subunit 1 and show a pleiotropic absence of cytochromes a, a3, and b. In vivo pulse labeling of an mto1 mutant, however, indicate increased rates of synthesis of other mitochondrial translation products. The respiratory defective phenotype of mto1 and mss1 mutants is not seen in a paromomycin-sensitive genetic background. The visible absorption spectra of such strains indicate a higher ratio of cytochromes b/a and elevated NADH- and succinate-cytochrome c reductase activities. To explain these phenotypic characteristics, we proposed that the Mto1p.Mss1p complex plays a role in optimizing mitochondrial protein synthesis in yeast, possibly by a proofreading mechanism.

From NO to OO: nitric oxide and dioxygen in bacterial respiration.

Nitric oxide reductase (NOR) is a key enzyme in denitrification, reforming the N-N bond (making N2O from two NO molecules) in the nitrogen cycle. It is a cytochrome bc complex which has apparently only two subunits, NorB and NorC. It contains two low-spin cytochromes (c and b), and a high-spin cytochrome b which forms a binuclear center with a non-heme iron. NorC contains the c-type heme and NorB can be predicted to bind the other metal centers. NorB is homologous to the major subunit of the heme/copper cytochrome oxidases, and NOR thus belongs to the superfamily, although it has an Fe/Fe active site rather than an Fe/Cu binuclear center and a different catalytic activity. Current evidence suggests that NOR is not a proton pump, and that the protons consumed in NO reduction are not taken from the cytoplasmic side of the membrane. Therefore, the comparison between structural and functional properties of NOR and cytochrome c- and quinol-oxidizing enzymes which function as proton pumps may help us to understand the mechanism of the latter. This review is a brief summary of the current knowledge on molecular biology, structure, and bioenergetics of NOR as a member of the oxidase superfamily.

Release of cytochrome c and decrease of cytochrome c oxidase in Bax-expressing yeast cells, and prevention of these effects by coexpression of Bcl-xL.

The characteristics of mitochondria of yeast cells expressing the pro-apoptotic gene Bax or coexpressing Bax and the anti-apoptotic gene Bcl-xL have been investigated in whole cells, isolated mitochondria and permeabilized spheroplasts. It is found that Bax-induced growth arrest of yeast cells is related to two defects in the respiratory chain: (i) a decrease in the amount of cytochrome c oxidase, the terminal enzyme of the respiratory chain, and (ii) a dramatic increase in the release of cytochrome c to the cytosol. Other components of the inner mitochondrial membrane (bc1 complex and F0F1-ATPase) are unaffected. Coexpression of Bcl-xL almost fully prevented the effect of Bax. Surprisingly, these results obtained in yeast parallel similar observations reported in mammalian cells.