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PubMed1996

The respiratory chain of Helicobacter pylori: identification of cytochromes and the effects of oxygen on cytochrome and menaquinone levels.

The quinone and cytochrome components of the respiratory chain of the microaerophilic bacterium Helicobacter pylori have been investigated. The major isoprenoid quinone was menaquinone-6, with traces of menaquinone-4; no methyl-substituted or unusual menaquinone species were found. Cell yield was highest after growth at 10% (v/v) oxygen and menaquinone levels (per dry cell mass) were maximal at 5-10% (v/v) oxygen. Helicobacter pylori cells and membranes contained b- and c-type cytochromes, but not terminal oxidases of the a- or d-types, as judged by reduced minus oxidised difference spectra. Spectra consistent with the presence of a CO-binding terminal oxidase of the cytochrome b- or o-type were obtained. The soluble fraction from disrupted cells also contained cytochrome c. There were no significant qualitative differences in the cytochrome complements of cells grown at oxygen concentrations in the range 2-15% (v/v) but putative oxidases were highest in cells grown at 5-10% (v/v) oxygen.

Cytochromes↗
PubMed1985

Perinatal development of cytochrome P-450, cytochrome C reductase, aryl hydrocarbon hydroxylase, styrene monooxygenase, and styrene epoxide hydrolase in rabbit liver microsomes and nuclei.

The development of cytochrome P-450, cytochrome C reductase, aryl hydrocarbon hydroxylase, styrene monooxygenase, and styrene epoxide hydrolase was studied in fresh rabbit liver nuclear and microsomal preparations. Enzymatic activities were determined in rabbit fetus at 21 and 30 days of gestation and in rabbits at 1, 5, 11, 22, 44, and 60 days of age. In extrauterine life, nuclear cytochrome P-450, aryl hydrocarbon hydroxylase, and styrene epoxide hydrolase were found to develop later than their microsomal counterparts, whereas nuclear cytochrome C reductase developed earlier than its microsomal counterpart. The behavior of styrene monooxygenase was more complex. The only activities detectable before birth were styrene epoxide hydrolase, styrene monooxygenase, and cytochrome C reductase in both nuclei and microsomes. The microsomes-to-nuclei epoxide hydrolase activity ratio was almost ten times the microsomes-to-nuclei styrene monooxygenase ratio, indicating a dissociation in the development of the two enzymes in rabbits. Nuclear metabolic activity was high at all ages, suggesting possible toxicological relevance.

Animals↗
PubMed1989

A missense mutation in the neutrophil cytochrome b heavy chain in cytochrome-positive X-linked chronic granulomatous disease.

A membrane-bound cytochrome b, a heterodimer formed by a 91-kD glycoprotein and a 22-kD polypeptide, is a critical component of the phagocyte NADPH-oxidase responsible for the generation of superoxide anion. Mutations in the gene for the 91-kD chain of this cytochrome result in the X-linked form of chronic granulomatous disease (CGD), in which phagocytes are unable to produce superoxide. Typically, there is a marked deficiency of the 91-kD subunit and the cytochrome spectrum is absent (X- CGD). In a variant form of CGD with X-linked inheritance, affected males have a normal visible absorbance spectrum of cytochrome b, yet fail to generate superoxide (X+ CGD). The size and abundance of the mRNA for the 91-kD subunit and its encoded protein were examined and appeared normal. To search for a putative mutation in the coding sequence of the 91-kD subunit gene, the corresponding RNA from an affected X+ male was amplified by the polymerase chain reaction and sequenced. A single nucleotide change, a C----A transversion, was identified that predicts a nonconservative Pro----His substitution at residue 415 of the encoded protein. Hybridization of amplified genomic DNA with allele-specific oligonucleotide probes demonstrated the mutation to be specific to affected X+ males and the carrier state. These results strengthen the concept that all X-linked CGD relates to mutations affecting the expression or structure of the 91-kD cytochrome b subunit. The mechanism by which the Pro 415----His mutation renders the oxidase nonfunctional is unknown, but may involve an impaired interaction with other components of the oxidase.

Base Sequence↗
PubMed1986

Immunocytochemical evidence for the maintenance of cytochrome P-450 isozymes, NADPH cytochrome C reductase, and epoxide hydrolase in pure and mixed primary cultures of adult human hepatocytes.

Specific polyclonal antibodies were used to investigate the distribution of two cytochrome P-450 isozymes (5 and 8), NADPH cytochrome c reductase, and epoxide hydrolase in adult human hepatocytes cultured alone or co-cultured with rat liver epithelial cells. The enzymes were localized by the indirect immunoperoxidase technique following fixation with a paraformaldehyde-glutaraldehyde mixture and membrane permeabilization with saponin. The pattern of distribution of the four enzymes after 24 hr in culture was similar to that found in vivo. Virtually all the hepatocytes exhibited nearly homogeneous positive staining for cytochrome P-450-8, whereas only 60-80% were positive for cytochrome P-450-5. Nearly homogeneous staining was also observed in all hepatocytes for NADPH cytochrome c reductase and epoxide hydrolase. During the first 12 days in pure culture, the intensity of staining, as well as the number of positively stained cells, decreased slightly except for epoxide hydrolase, which did not show any obvious change. In contrast, even after 15 days in co-culture the extent of staining for all the enzymes decreased less than in pure culture. These results indicate that adult human hepatocytes continue to express specific drug-metabolizing enzymes for several days in culture and provide further evidence that those cells are more stable than rodent hepatocytes in primary culture.

Adolescent↗
PubMed1986

Immunolocalization of cholesterol side-chain-cleavage cytochrome P-450 and 17 alpha-hydroxylase cytochrome P-450 in bovine ovarian follicles.

Follicles were collected from cows and processed for electron microscopy and for immunofluorescent staining at the light microscope level. Key regulatory steroidogenic enzymes cholesterol side-chain-cleavage cytochrome P-450 (P-450scc) and 17 alpha-hydroxylase cytochrome P-450 (P-45017 alpha) were immunolocalized using specific IgG fractions raised against these enzymes. In larger follicles in which the theca interna had differentiated, positive staining for cytochromes P-450scc and P-450(17) alpha was observed in the cells of the theca interna. Electron microscopic examination showed that these cells were rich in endoplasmic reticulum, mainly rough, and had moderate numbers of mitochondria with tubular and lamellar cristae. Positive staining was also present in the theca of follicles undergoing atresia. Positive staining for cytochrome P-450(17) alpha was not observed in the membrana granulosa but cytochrome P-450scc was present in the membrana granulosa in some follicles, particularly in the larger antral follicles. By contrast, positive staining for both enzymes was not observed in stroma, surface epithelium or in small preantral follicles in which the theca interna had not differentiated. These results indicate good agreement between the type(s) of steroidogenic enzyme(s) present in tissues and the type(s) of steroid hormone(s) produced. It is concluded that regulation of steroid hormone production involves, at least in part, regulation of the levels of steroidogenic enzymes.

Animals↗
PubMed2006

Direct electrochemical behavior of cytochrome c on DNA-modified glassy carbon electrode and its application to cytochrome c biosensor.

DNA was immobilized on glassy carbon electrodes to fabricate DNA-modified electrodes. The direct electron transfer of horse heart cytochrome c on DNA-modified glassy carbon electrode was achieved. A pair of well-defined redox peaks of cytochrome c appeared at Epc = -0.017 V and Epa = 0.009 V (vs. Ag/AgCl) in 10 mM phosphate buffer solution (pH 7.0) at a scan rate of 50 mV/s. The electron transfer coefficient (alpha) and the standard rate constant of the surface reaction (Ks) of cytochrome c on DNA-modified electrodes could be estimated to be 0.87 and 34.52 s(-1), respectively. The DNA-modified glassy carbon electrode could be applied to detect cytochrome c by means of differential pulse voltammetry (DPV). The cathodic peak current was proportional to the quantity of cytochrome c in the range of 4.0 x 10(-6) M to 1.2 x 10(-5) M. The correlation coefficient is 0.996, and with the detection limit was 1.0 x 10(-6) M (three times the ratio of signal to noise, S/N = 3).

Biosensing Techniques↗
PubMed1991

Endogenous cytochrome c and cytochrome oxidase in rat and mouse kidney: light microscopic histochemical study.

Activity of cytochrome c oxidase and the level of endogenous cytochrome c were investigated light microscopically in adult rat and mouse kidney by incubating unfixed frozen sections with diaminobenzidine (DAB) in the absence or presence of exogenous cytochrome c. The results suggest that DAB staining intensity mainly reflects the local density of mitochondria and only occasionally visualizes the differences in cytochrome oxidase activity and/or endogenous cytochrome c content. Most intense reaction was observed in proximal and distal tubules both in rat and mouse. Finer differentiation of reactivity in particular nephron segments and interspecies differences between rat and mouse kidney are also described.

3,3'-Diaminobenzidine↗
PubMed1990

Oxidation-reduction potentials of cytochromes in Ascaris muscle mitochondria: high-redox-potential cytochrome b558 in complex II (succinate-ubiquinone reductase).

Oxidation-reduction midpoint potentials (Ems) were determined at pH 7.0 for cytochromes in the anaerobic respiratory chain of Ascaris mitochondria by redox titration techniques. Cytochrome b558, which is associated with complex II that functions as fumarate reductase in the terminal step of the respiratory chain, was shown to have an Em of -34 mV in the isolated complex II and -54 mV in mitochondria. These values are much higher than the value of Ascaris cytochrome b558. In contrast, Ems of cytochromes C + C1 and cytochrome b559.5 were determined in situ to be 235 mV and 78 mV, respectively, which are comparable to those of their mammalian counterparts.

Animals↗
PubMed1989

Molecular basis for resistance to myxothiazol, mucidin (strobilurin A), and stigmatellin. Cytochrome b inhibitors acting at the center o of the mitochondrial ubiquinol-cytochrome c reductase in Saccharomyces cerevisiae.

The respiratory bc1 complex transfers the electrons from ubiquinol to cytochrome c oxidase. Myxothiazol, strobilurin A (mucidin), and stigmatellin are center o inhibitors preventing electron transfer at the ubiquinone redox site Qo, which is located closer to the outer side of the inner mitochondrial membrane. The cytochrome b gene is carried by the organelle DNA. Yeast mutants resistant to myxothiazol and mucidin have been previously isolated and mapped to specific loci of the cytochrome b gene. In the present work, stigmatellin-resistant mutants were isolated and genetically analyzed. The mutated amino acid residues from seven myxothiazol-, four mucidin-, and six stigmatellin-resistant mutants have been identified by sequencing the relevant segments of the resistant cytochrome b gene. A third myxothiazol-resistant locus and the first stigmatellin-resistant locus were identified. The mutated codons were found to be clustered in two regions of the cytochrome b protein which appeared to be responsible for the resistance to Qo site inhibitors. The first region is within the end of the first, the second, and the beginning of the third exon whereas the second region is within exon five and the beginning of the sixth exon.

Amino Acid Sequence↗
PubMed1986

Identification of a human liver cytochrome P-450 homologous to the major isosafrole-inducible cytochrome P-450 in the rat.

The rat 3-methylcholanthrene-inducible family of liver cytochromes P-450 contains two proteins (P-450c and P-450d) that are immunochemically related, possess 68% total sequence homology, and are induced by a number of toxic or carcinogenic compounds. To determine whether equivalent isozymes of hepatic cytochrome P-450 are expressed in humans, as they are in several mammalian species, we performed immunoblot analyses on microsomes prepared from 14 human liver specimens and found that each one contained a 52.5-kDa protein (termed HLd) that reacted with antibodies specific for rat P-450d. In addition, one specimen contained a 54-kDa protein (termed HLc) that reacted with antibodies specific for rat P-450c. HLd was purified through the use of immunoaffinity chromatography and was found to be 56% homologous to rat P-450d and 61% homologous to the equivalent isozyme in the rabbit (P-450 LM4) through their first 18 NH2-terminal amino acids. Finally, levels of immunoreactive HLd varied more than 10-fold among these patients but were unrelated to the patients' drug treatments, smoking habits, or amount of immunoreactive HLp, a human liver cytochrome P-450 related to the glucocorticoid-inducible family of rat cytochromes P-450. We conclude that, in man, there is a cytochrome P-450 family composed of two isozymes (HLc and HLd) that are immunochemically and structurally related to the 3-methylcholanthrene-inducible family observed in several other species.

Adult↗
PubMed1985

Demonstration in multiple species of inducible hepatic cytochromes P-450 and their mRNAs related to the glucocorticoid-inducible cytochrome P-450 of the rat.

We have recently demonstrated that P-450p, a form of rat liver cytochrome P-450 inducible by steroids such as dexamethasone and pregnenolone-16 alpha-carbonitrile, by the macrolide antibiotic triacetyloleandomycin, and by phenobarbital, is immunochemically related to and shares 73% NH2-terminal amino acid sequence homology with rabbit cytochrome LM3c. Extending this interspecies comparison we now report that liver microsomes prepared from the rabbit, hamster, gerbil, and mouse contain inducible cytochromes P-450 that resemble P-450p in: (a) converting triacetyloleandomycin to a metabolite that forms a distinct spectral complex with cytochrome P-450 heme, (b) catalyzing the demethylation of erythromycin, and (c) reacting on immunoblots with antibodies directed against P-450p or LM3c. These three characteristics changed in parallel within treatment groups of a given species receiving different inducers of cytochrome P-450. However, there were striking qualitative and quantitative interspecies differences in the responses to inducers. For example, rifampicin was the most efficacious inducer of LM3c in the rabbit and yet was not at all an inducer of P-450p in the rat whereas pregnenolone-16 alpha-carbonitrile, an inducer in the rat, failed to induce LM3c in the rabbit. Immunoblot analysis of these microsomes revealed in each species except the rabbit a single immunochemically related protein. A second immunoreactive protein was present in microsomes from male and female and rifampicin- and dexamethasone-treated female rabbits. Two cloned cDNAs, which hybridized to a species of liver mRNA directing the synthesis of P-450p in a cell-free translation system, were used to probe Northern blots of liver RNAs. These revealed a single band of hybridizable mRNA in each species (except RNA from the rabbit which gave no signal even under conditions of reduced stringency) that was induced in qualitative proportions to that of the accumulated immunoreactive protein. We conclude that P-450p appears to be conserved in evolution and is represented in each of the species tested by one or more immunochemically related proteins which exhibit similar catalytic activities to those of P-450p.

Animals↗
PubMed1982

Biogenesis of mitochondrial ubiquinol:cytochrome c reductase (cytochrome bc1 complex). Precursor proteins and their transfer into mitochondria.

The precursor proteins to the subunits of ubiquinol:cytochrome c reductase (cytochrome bc1 complex) of Neurospora crassa were synthesized in a reticulocyte lysate. These precursors were immunoprecipitated with antibodies prepared against the individual subunits and compared to the mature subunits immunoprecipitated or isolated from mitochondria. Most subunits were synthesized as precursors with larger apparent molecular weights (subunits I, 51,500 versus 50,000; subunit II, 47,500 versus 45,000; subunit IV (cytochrome c1), 38,000 versus 31,000; subunit V (Fe-S protein), 28,000 versus 25,000; subunit VII, 12,000 versus 11,500; subunit VIII, 11,600 versus 11,200). Subunit VI (14,000) was synthesized with the same apparent molecular weight. The post-translational transfer of subunits I, IV, V, and VII was studied in an in vitro system employing reticulocyte lysate and isolated mitochondria. The transfer and proteolytic processing of these precursors was found to be dependent on the mitochondrial membrane potential. In the transfer of cytochrome c1, the proteolytic processing appears to take place in two separate steps via an intermediate both in vivo and in vitro. In vivo, the intermediate form accumulated when cells were kept at 8 degrees C and was chased into mature cytochrome c1 at 25 degrees C. Both processing steps were energy-dependent.

Animals↗
PubMed1984

Cytochrome b558 from (bovine) granulocytes. Partial purification from Triton X-114 extracts and properties of the isolated cytochrome.

A membrane-associated b-type cytochrome (a proposed component in the neutrophil microbicidal superoxide generating system) has been partially purified from nonactivated beef granulocytes to a specific heme content of 20 nmol of heme/mg of protein, a value about 10-fold higher than those previously reported. The hemoprotein was solubilized at low temperature (4 degrees C) from mixed granule (30,000 X g) cell fractions using Triton X-114 detergent. Warming the extract to 25 degrees C allowed separation into detergent and aqueous phases; cytochrome b558 partitioned exclusively into the detergent phase, allowing separation from other visible-absorbing species (e.g. myeloperoxidase) and indicated an intrinsic membrane localization (Bordier, C. (1981) J. Biol. Chem. 256, 1604-1607). The partitioned cytochrome was chromatographed on hydroxylapatite and a hydrophobic affinity matrix, allowing a 185-fold (heme content) purification from the granule extract. The cytochrome preparation revealed three equal-staining protein bands by sodium dodecyl sulfate-urea polyacrylamide gel electrophoresis; apparent molecular weights were 14,000, 12,000, and 11,000. The question of heterogeneity of the preparation versus subunit structure is not resolved at present. The hemoprotein binds carbon monoxide, consistent with a proposed role as a terminal oxidase, and has an unusually negative oxidation-reduction potential (-225 mV) similar to that observed in granulocyte membranes. The preparation is devoid of NAD(P)H-diaphorase and cytochrome c reductase activities.

Animals↗
PubMed1982

One-electron photoreduction of bacterial cytochrome P-450 by ultraviolet light. II. A study using laser flash photolysis of the dynamics of the reduction of bacterial cytochrome P-450 in the presence of carbon monoxide. The role of the protein conformation.

The intramolecular photoreduction of the bacterial cytochrome P-450 demonstrated by the formation of the complex of reduced cytochrome m with camphor and CO has been studied by photolysis with a laser flash at 265 nm. This photoreduction of the heme follows the photoionization of a proximal aromatic amino acid residue and especially tryptophan in the nanosecond time scale as evidenced by the N2O effect on the reduction yield. However most of the hydrated electrons released in the solution do not react with the ferric porphyrin. In agreement with steady state studies, the quantum yield for formation of the complex of reduced cytochrome m with camphor and CO is proportional to the concentration of the high-spin species. The low-spin ferric porphyrin is thus not reducible by photoejected electrons. It is also shown that the growth kinetics of the transient absorbance at 446 nm characterizing the complex of reduced cytochrome m with camphor and CO correspond to a structural change affecting the protein and not to the formation of the complex of CO with the camphor-bound reduced cytochrome in which occurs on a shorter time scale in the present experimental conditions.

Carbon Monoxide↗
PubMed1996

Interaction of ferric complexes with NADH-cytochrome b5 reductase and cytochrome b5: lipid peroxidation, H2O2 generation, and ferric reduction.

NADH is reactive in interacting with iron and liver microsomes to catalyze the formation of reactive oxygen species. NADH-dependent microsomal electron transfer involves the enzymes NADH-cytochrome b5 reductase and cytochrome b5. Experiments were carried out to evaluate the ability of reconstituted systems containing purified reductase in the absence or presence of b5 to reduce several ferric complexes, to generate H2O2, and to catalyze lipid peroxidation. The reductase directly reduced ferric-EDTA; addition of b5 inhibited this reduction probably due to competition for the reductase. Cytochrome b5 was required for reduction of low (5 microM) and high (50 microM) concentrations of ferric-histidine and ferric-ammonium sulfate and low concentrations of ferric-ATP. The reductase could interact directly with high (50 microM) concentrations of ferric-ATP. Peroxidation of phospholipids extracted from liver microsomes by the reductase required b5. Molar ratios of b5 to reductase approximating those found in liver microsomes (e.g., 10) were effective in catalyzing lipid peroxidation and ferric reduction. The role of b5 in catalyzing lipid peroxidation appears to involve reduction of the ferric catalyst to help form an initiation complex and degradation of lipid hydroperoxides by the hemeprotein to catalyze propagation of the peroxidation cycle. In contrast to results with microsomes, lipid peroxidation by the complete reconstituted system was sensitive to super-oxide dismutase; this sensitivity was decreased if the reconstituted system was dialyzed overnight to form vesicular preparations, indicating that accessibility of enzymes to sites of peroxidation was important. High rates of H2O2 formation were observed in the presence of ferric-EDTA plus reductase; rates of H2O2 formation with the other ferric complexes were low even in the presence of b5. These results indicate that the ability of NADH reductase and cytochrome b5 to interact with various ferric complexes depends on the nature of the chelating agent used to complex the iron and on the concentration of the iron.

Adenosine Triphosphate↗
PubMed1995

Experimental and theoretical analysis of the interaction between cytochrome c and cytochrome b5.

Experimental and theoretical investigation of the interaction of cytochrome c and cytochrome b5 performed over nearly twenty years has produced considerable insight into the manner in which these proteins recognize and bind to each other. The results of these studies and the experimental and theoretical strategies that have been developed to achieve these results have significant implications for understanding the behavior of similar complexes formed by more complex and less-well characterized electron transfer proteins. The current review provides a comprehensive summary and critical evaluation of the literature on which the current status of our understanding of the interaction of cytochrome c and cytochrome b5 is based. The general issues related to the study of electron transfer complexes of this type are discussed and some new directions for future investigation of such systems are considered.

Animals↗
PubMed1985

A monoclonal antibody raised to rat liver cytochrome P-448 (form C) which recognises an epitope common to many other forms of cytochrome P-450.

A murine monoclonal antibody has been raised against a partially purified preparation of hepatic cytochrome P-448 (form c) from beta-naphthoflavone-treated rats. The monoclonal origin of the antibody was established by limiting dilution culture and isoelectricfocusing. The antibody has been designated 3/4/2. It reacts with apparently homogeneous cytochrome P-448 from rat liver in solid phase assay. It also cross reacts with a number of other cytochromes P-450, from rat and rabbit. In addition, a positive reaction was obtained with microsomal fractions from a variety of species, including man. None of the species tested was negative. The antibody does not react appreciably with purified haemoproteins other than cytochromes P-450. Antibody 3/4/2 is not inhibitory, either in reconstituted systems or with intact microsomal fraction. However, evidence was obtained that the antibody does cause some perturbation of the tertiary structure of the apoprotein at or near the haem.

Animals↗
PubMed1985

The oxygen reaction of the cytochrome d-terminated respiratory chain of Escherichia coli at sub-zero temperatures. Kinetic resolution by EPR spectroscopy of two high-spin cytochromes.

The oxygen reaction of the fully reduced respiratory chain in membranes from oxygen-limited Escherichia coli was studied at sub-zero temperatures using EPR spectroscopy. Laser photolysis of CO-liganded cytochrome oxidase d precedes oxidation of at least 2 kinetically separable high-spin cytochromes. At -120 to -100 degrees C, a rhombic signal appears, attributable to cytochrome d, followed at above -100 degrees C, by appearance of a second, axial signal near g = 6, here assigned to cytochrome(s) b, and changes in the redox state of iron-sulphur clusters. The data kinetically resolve the 2 high-spin signals attributed to the oxidase complex and suggest schemes for electron flow to oxygen.

Cell Membrane↗