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PubMed1978

Definition of cytochrome c binding domains by chemical modification. II. Identification and properties of singly substituted carboxydinitrophenyl cytochromes c at lysines 8, 13, 22, 27, 39, 60, 72, 87, and 99.

Sensitive thin layer peptide mapping is employed to establish the identity and the homogeneity of eight singly substituted 4-carboxy-2,6-dinitrophenyl derivatives of horse cytochrome c. Seven of the components, all of greater than 95% homogeneity, are modified at lysyl residues 13, 72, 87, 8, 27, 39, and 60. The eighth component is a mixture of derivatives at lysines 22 and 99. The positions of the modified residues were confirmed by the amino acid analysis and Edman sequential degradation of the CDNP-peptides. Physiochemical properties characteristic of cytochrome c are unchanged in the chemically modified products examined. These properties, that include the proton NMR spectrum, are sensitive probes of the polypeptide organization surrounding the heme prosthetic group. The lack of any discernable changes indicates that modification of the epsilon-amino groups on the surface of cytochrome c does not perturb the overall structure of the protein. The widespread distribution of the modifications on the surface of the molecule, together with the homogeneity and native conformation of the CDNP-derivatives make them well suited for assessing the effects of changes in the charge topography on the electron transfer activity of cytochrome c.

Binding Sites↗
PubMed1979

Comparative solvent perturbation of horse heart cytochrome c and Rhodospirillum rubrum cytochrome c2.

The extent of exposure of heme to solvent in horse heart cytochrome c and Rhodospirillum rubrum c2 was investigated to determine whether a correlation exists between the properties of these oxidation-reduction proteins and their heme environments. Solvent perturbation absorption difference spectra were measured using ethylene glycol, glycerol, and sucrose at concentrations between 0 and 30%. Cytochrome c appears to exhibit a somewhat greater extent of heme exposure than cytochrome c2 for both the oxidized and reduced states. These results suggest that the lower oxidation-reduction potential of cytochrome c may in part be due to a greater extent of exposure of the heme. The oxidized state of both proteins appears to exhibit a greater exposure than that of the reduced state which is consistent with a more favorable environment for the charge on the ferric heme coordination center.

Animals↗
PubMed1988

A mutant of Neurospora crassa deficient in cytochrome c heme lyase activity cannot import cytochrome c into mitochondria.

The nuclear cyt-2-1 mutant of Neurospora crassa is characterized by a gross deficiency of cytochrome c (Bertrand, H., and Collins, R. A. (1978) Mol. Gen. Genet. 166, 1-13). The mutant produces mRNA that can be translated into apocytochrome c in vitro. Apocytochrome c is also synthesized in vivo in cyt-2-1, but it is rapidly degraded and thus does not accumulate in the cytosol. Mitochondria from wild-type cells bind apocytochrome c made in vitro from either wild-type or cyt-2-1 mRNA and convert it to holocytochrome c. This conversion depends on the addition of heme by cytochrome c heme lyase and is coupled to translocation of cytochrome c into the intermembrane space. Mitochondria from the cyt-2-1 strain are deficient in the ability to bind apocytochrome c. They are also completely devoid of cytochrome c heme lyase activity. These defects explain the inability of the cyt-2-1 mutant to convert apocytochrome c to the holo form and to import it into mitochondria.

Chromosome Deletion↗
PubMed1989

Thermodynamic and spectroscopic characteristics of the cytochrome bc1 complex. Role of quinone in the behavior of cytochrome b562.

Cytochrome b562 does not behave as a single independent thermodynamic component in preparations of purified quinol cytochrome c reductase. This effect is much more pronounced in quinone sufficient preparations; in such preparations, the epr spectrum of the cytochrome is Eh sensitive, with a peak shift from g = 3.42 to 3.48 occurring as the potential is lowered from 100 mV to 0 mV. The peak shift is dependent on the presence of quinone and can be restored to quinone-depleted preparations by supplementation with ubiquinol 2 if phospholipid depletion is not too severe. The results suggest that cytochrome b562 is strongly interacting with the Qc quinone binding site.

Binding Sites↗
PubMed1989

Primary structures of cytochrome P-450 isozyme 5 from rabbit and rat and regulation of species-dependent expression and induction in lung and liver: identification of cytochrome P-450 gene subfamily IVB.

The primary structure of rabbit cytochrome P-450 isozyme 5 has been derived from the nucleotide sequence of cloned cDNA. Identical sequences were obtained for cDNAs constructed with mRNA from four different sources, lung and liver of untreated rabbits and liver from rabbits treated once or four times with phenobarbital. Isozyme 5 shows significant sequence identity only with rabbit P-450p2 (54%) and rat P-450LA omega (53%), which places it in a previously unrecognized cytochrome P-450 gene subfamily (IVB). A cDNA library was also constructed from rat pulmonary mRNA and screened with cDNA encoding rabbit isozyme 5. The amino acid sequence derived from a positive clone was compared with that of rabbit isozyme 5 and found to be 87% identical, significantly greater than observed between other similar forms of cytochrome P-450 from rabbit and rat. Alignment of the primary structures of rabbit isozyme 5 (506 residues), rat isozyme 5 (511 residues), rabbit P-450p2, and rat P-450LA omega shows 43% structural identity and a common 16-residue peptide near position 300 that is unique to these forms of cytochrome P-450. Analysis of mRNA from lung and liver of rabbit, rat, guinea pig, and hamster indicates that species and tissue differences in the expression and induction of isozyme 5 are likely regulated at the level of transcription. These differences fall into one of the following three groups: first, expression in lung and liver and induction in liver by phenobarbital (rabbit); second, expression in lung and liver but no hepatic induction (hamster); and third, expression in lung and little or no expression in liver regardless of treatment (rat and guinea pig).

Amino Acid Sequence↗
PubMed1988

Cytochrome c: ion binding and redox properties. Studies on ferri and ferro forms of horse, bovine, and tuna cytochrome c.

The ion binding properties of horse, bovine, and tuna cytochrome c (both oxidized and reduced) have been measured using a combination of ultrafiltration, neutron activation, and ion chromatography. The ions investigated were chloride, phosphate, and Tris-cacodylate. Ion chromatography and neutron activation analysis techniques were employed to determine the concentration of free anions. Binding constants are obtained from modified Scatchard plots (in the range of 10-2000 M-1). The redox potentials for cytochrome c at different ionic strengths, pH 7.0, have been determined. In this paper we report the ionic strength and ion binding effects on the redox properties of horse, bovine, and tuna cytochrome c. Potential versus ionic strength dependence for horse, bovine, and tuna cytochrome c from the experimental data were compared with a theoretical model.

Animals↗
PubMed1979

Mutations in putative intervening sequences of the mitochondrial cytochrome b gene of yeast produce abnormal cytochrome b polypeptides.

The apoprotein of yeast cytochrome b is translated on mitochondrial ribosomes and coded for by a split gene which is located in the COB-BOX region on mitochondrial DNA. With the aid of an antibody against cytochrome b, we identified the cytochrome b-cross-reacting polypeptides of respiration-deficient mutants mapping either in coding or intervening sequences of the cytochrome b gene. Most mutations in the coding regions caused the accumulation of a single apocytochrome b fragment whose apparent molecular weight (12,000 to 26,600) depended on the map position of the mutation. In contrast, mutations in putative intervening sequences often led to multiple new polypeptides immunologically related to apocytochrome b. Some of these abnormal polypeptides were considerably larger than wild type apocytochrome b. This suggests that mutations in intervening sequences can thus generate aberrant polypeptide products.

Cytochromes↗
PubMed1985

Chromosomal assignments of genes coding for components of the mixed-function oxidase system in mice. Genetic localization of the cytochrome P-450PCN and P-450PB gene families and the nadph-cytochrome P-450 oxidoreductase and epoxide hydratase genes.

Filter-hybridization studies show that major phenobarbital and pregnenolone-16alpha-carbonitrile-inducible cytochrome P-450 mRNAs in rats were encoded by members of separate, distinct gene families. These gene families are genetically divergent from each and show no cross-hybridization, even under low-stringency conditions. Furthermore, sequences contained in the P-450PB and P-450PCN gene families map to separate chromosomes of the mouse genome. Using mouse X Chinese hamster somatic cell hybrids (EBS cell lines), all distinguishable P-450PCN sequences were found to map to chromosome 6, whereas all P-450PB sequences were located on chromosome 7. Our data support the proposition that the region of the Coh locus on chromosome 7 is the site of the cytochrome P-450PB gene family. The presence of gene families for the cytochromes P-450 occurs in many mammalian species and is likely an important part of the mechanism by which the mixed-function oxidase system is capable of recognizing and metabolizing such a wide array of endogenous and foreign compounds. Conversely, NADPH-cytochrome P-450 oxidoreductase appears to be encoded in many vertebrate species by a single gene and is located on chromosome 6 of the mouse. Corroboratory data are presented to show that the Eph-1 locus on chromosome 1 is the site of at least one microsomal epoxide hydratase gene.

Animals↗
PubMed1984

Induction of cytochrome P-450 by glucocorticoids in rat liver. I. Evidence that glucocorticoids and pregnenolone 16 alpha-carbonitrile regulate de novo synthesis of a common form of cytochrome P-450 in cultures of adult rat hepatocytes and in the liver in vivo.

We administered a series of steroid hormones to primary nonproliferating cultures of adult rat hepatocytes and found that dexamethasone and other glucocorticoids but not sex steroid hormones, mineralocorticoids, or derivatives of pregnenolone other than pregnenolone 16 alpha-carbonitrile (PCN) stimulated de novo synthesis of an immunoreactive protein, indistinguishable from the form of cytochrome P-450 (P450PCN) induced by PCN in rat liver. No difference were discerned among purified liver cytochromes from rats treated with dexamethasone, PCN or dexamethasone plus PCN, among proteolytic digests of these proteins, or among the immunoprecipitated cytochromes prepared from cultured hepatocytes treated with these steroids as judged by electrophoresis on polyacrylamide gels containing sodium dodecyl sulfate followed by immunoblot analysis. Of the steroids tested, dexamethasone proved to be the most efficacious inducer increasing the rate of synthesis of P450PCN from 0.05% of total cellular protein synthesis in incubated control cultures (measured as incorporation of [3H]leucine into immunoprecipitable P450PCN) to as much as 9.4% in cultures incubated for 5 days in medium containing dexamethasone (10(-5) M). As with traditional glucocorticoid-responsive liver functions, induction of immunoreactive P450PCN was dependent on the concentration of dexamethasone (10(-8) to 10(-5) M) and was promptly reversed upon withdrawal of the steroid. However, during the 24-h interval between 24 to 48 h of culture age the hepatocytes were refractory to either induction or de-induction of immunoreactive P450PCN even though continuous exposure of the cells to dexamethasone (including this interval) was mandatory for maximal induction of P450PCN at 120 h in culture. Unlike cultured rat hepatocytes, HTC hepatoma cultures failed to exhibit dexamethasone-responsive expression of immunoreactive P450PCN. We conclude that glucocorticoids and PCN constitute a specific "class" of synthetic and endogenous inducers of a single form of cytochrome P-450.

Animals↗
PubMedDate not supplied

[Pyridoxalphosphate-modified derivatives of cytochrome c. Mono- and disubstituted derivatives: characteristics and effect on electron transport in cytochrome c-depleted mitochondria].

Cytochrome c is modified by covalent binding of pyridoxal phosphate (PLP) to lysine residues. One di-substituted [(PLP)2--C] and two mono-substituted derivatives [(PLP)--c and (PLP)''--c] were obtained and precisely purified. The peak at 695 nm and CD-spectra in 190--600 nm region show that all derivatives have native conformation. The differential UV-spectra of the derivatives against native protein show that in (PLP)2--c there is a contact dipole-dipole interaction between PLP chromophores. It is calculated that the N-atoms of the two PLP-substituted lysines must be at a distance less than or equal to 12 A. Analysing our and literature data, one may suppose that Lys-13 and Lys-87 are the most probable candidates for modification with PLP. (PLP)---c and (PLP)''--c behave differently during ion-exchange chromatography and when added to cytochrom c-depleted mitochondria. (PLP)''--c restores electron transfer at higher concentrations than (PLP)'--c. Both they restore fully succinate and ascorbate oxidation but at considerably higher concentrations than the native protein, i. e. modification of any one of the reactive towards PLP lysines descreases but does not exclude the interaction with its reductase and oxidase. The effective equilibrium constants of binding of modified derivatives to cytochrome c-depleted mitochondria are lower than the constant for native protein. Together with decrease in binding activity, Hill coefficients increase. From our results it may be supposed that probably the binding sites of cytochrome c for its reductase and oxidase partially overlap.

Animals↗
PubMed1980

Phosphatidylcholine vesicle reconstituted cytochrome P-450scc. Role of the membrane in control of activity and spin state of the cytochrome.

Cytochrome P-450scc from bovine adrenal cortex mitochondria was purified and reconstituted into phosphatidylcholine vesicles which varied in both cholesterol content and in the fatty acyl composition of the phospholipid. Under conditions of optimal ionic strength, pH, and excess adrenodoxin and adrenodoxin reductase, it was found that at a constant cholesterol: phospholipid ratio, the membrane composition had large effects on the rate of hemoprotein-catalyzed side chain cleavage of cholesterol. Rate effects were due to phospholipid-induced changes in the enzyme's Km for cholesterol, and not due to Vmax effects. Binding of cholesterol to cytochrome P-450 could also be monitored optically by measuring the fraction of enzyme in the high spin form. Dissociation constants determined in this manner for cholesterol binding in phospholipid of differing fatty acyl composition showed an excellent inverse correlation with the rates of pregnenolone formation in the same lipids (at constant cholesterol concentration) (see Fig. 6); thus, phospholipid exerts its rate effects by modulating the binding of cholesterol to the cytochrome. The membrane-mediated effects on spin state and activity mimic closely the effects seen in mitochondria isolated from adrenocorticotropic hormone-treated versus control adrenal cells. This behavior suggests to us that acute steroidogenic action of adrenocorticotropic hormone may be mediated through changes in the composition of the inner mitochondrial membrane in which cytochrome P-45scc is embedded.

Adrenal Cortex↗
PubMed1983

Transcriptional regulation of rat liver epoxide hydratase, NADPH-Cytochrome P-450 oxidoreductase, and cytochrome P-450b genes by phenobarbital.

The relative rates of transcription of epoxide hydratase, NADPH-cytochrome P-450 oxidoreductase, and cytochrome P-450b genes were determined in purified hepatocyte nuclei isolated at different times after phenobarbital administration. The rates of transcription of the epoxide hydratase and oxidoreductase genes were increased 4- and 9-fold, respectively, above control levels 1 h after administration of the drug. Transcription rates for the oxidoreductase gene slowly decreased to control values at 24 h while transcription of the epoxide hydratase gene rapidly declined to approximately 2-fold above control values at 2.5 h and remained at this level up to 24 h after administration. Interestingly, transcription of the cytochrome P-450b gene followed a markedly different induction time course than the epoxide hydratase and oxidoreductase genes. Specifically, cytochrome P-450b gene transcription increased 23-fold above control values 6 to 8 h after phenobarbital administration and then slowly declined to 14-fold at 24 h. The increased levels of intranuclear pre-mRNA and cytoplasmic mRNA for each enzyme correlated well with transcriptional activity. In contrast to these results, the rate of transcription of the serum albumin gene was not affected, and levels of albumin mRNA actually decreased after administration of phenobarbital. The rapid increase in the rates of transcription and the appearance of elevated levels of nuclear pre-mRNAs and cytoplasmic mRNAs unequivocally demonstrates that phenobarbital elevates levels of these drug-metabolizing enzymes primarily by augmenting the rate of transcription of their respective genes.

Animals↗
PubMed1983

Contributions of cytoplasmic free and membrane-bound ribosomes to the synthesis of mitochondrial cytochrome P-450(SCC) and P-450(11 beta) and microsomal cytochrome P-450(C-21) in bovine adrenal cortex.

Rabbit antibodies against cytochrome P-450 (SCC), P-450 (11 beta), and P-450 (C-21) from bovine adrenal cortex were prepared, and it was confirmed that these three cytochrome P-450 species are immunologically distinct from one another. Cytoplasmic sites of synthesis of P-450 (SCC), P-450 (11 beta), and P-450 (C-21) in bovine adrenal cortex were determined by examining the presence of their nascent peptides on isolated free and bound ribosomes. Nascent peptides were released in vitro from ribosomes by [3H]puromycin in a high salt buffer in the presence of a detergent, and the nascent peptides of P-450 (SCC), P-450 (11 beta), and P-450 (C-21) were isolated by immunoprecipitation. The nascent peptides of these three cytochrome P-450 species were found in both free and bound ribosomal fractions, suggesting that they share common sites of synthesis in the cytoplasm. However, the nascent peptides of mitochondrial P-450 (SCC) and P-450 (11 beta) were more concentrated in the free ribosomal fraction, whereas those of microsomal P-450 (C-21) were more abundant in the bound ribosomal fraction. The nascent peptides of the three cytochrome P-450 species were released from the membrane-bound ribosomes of rough microsomes into the cytoplasmic surface of microsomal vesicles by puromycin treatment.

Adrenal Cortex↗
PubMed1983

Effects of polyene cholesterol-binding antibiotics on the binding and removal of cholesterol from bovine adrenocortical cytochrome P-450SCC: difference in properties of the membrane-bound and membrane-free cytochrome.

The polyene cholesterol-binding antibiotics filipin and amphotericin B inhibited the binding of cholesterol to steroid-free, soluble adrenocortical cytochrome P-450SCC. In mitochondria-bound cytochrome P-450SCC, cholesterol was not extracted by filipin, whereas filipin was able to react with cholesterol bound to purified soluble cytochrome P-450SCC. Based on these results we conclude that the cholesterol-binding site of membrane-bound cytochrome P-450SCC is masked by membrane structures.

Adrenal Cortex↗
PubMed1981

Isolation of the membrane-binding peptide of NADPH-cytochrome P-450 reductase. Characterization of the peptide and its role in the interaction of reductase with cytochrome P-450.

A peptide identified as the membrane-associated segment of NADPH-cytochrome P-450 reductase has been generated by steapsin protease treatment of vesicle-incorporated reductase and isolated by preparative gel electrophoresis. This peptide remains associated with vesicles when steapsin protease digests of vesicle-incorporated reductase were fractionated by Sepharose 4B chromatography, confirming its identity as the membrane-binding peptide. The molecular weight of the membrane-binding peptide was 6400 as determined by gel filtration in 8 M guanidine hydrochloride, and its amino acid content was not especially hydrophobic. The activity of reconstituted hydroxylation systems consisting of reductase, cytochrome P-446, and dilauroyl phosphatidylcholine was not inhibited by large molar excesses of purified membrane-binding peptide. Moreover, when purified reductase and cytochrome P-446 were added to liposomes which contained the membrane-binding peptide, it was determined that mixed function oxidase activity was reconstituted as effectively as when vesicles without the membrane-binding peptides were used. Similar results were obtained with reductase, cytochrome P-450, and detergent-solubilized liposomes (with or without the membrane-binding peptide). Thus, the membrane-binding peptide does not appear to interact with either of these two forms of the hemoprotein in a site-specific manner to prevent reconstitution of hydroxylation activity.

7-Alkoxycoumarin O-Dealkylase↗
PubMed1982

Monoclonal antibodies to rabbit liver cytochrome P-450LM2 and cytochrome P-450LM4.

Monoclonal antibodies were prepared from hybridoma clones isolated by the fusion of myeloma cells and spleen cells derived from mice immunized with either purified rabbit liver microsomal cytochrome P-450LM2 or cytochrome P-450LM4. Seven hybridoma clones produced three kinds of monoclonal antibodies to P-450LM2. The first class bound, precipitated, and inhibited the enzyme activity of P-450LM2 for both benzo[a]pyrene hydroxylation and 7-ethoxycoumarin deethylation. The other two classes either bound and precipitated or only bound the enzyme. These monoclonal antibodies to P-450LM2 showed a precipitin reaction and inhibition of enzyme activity that was specific for cytochrome P-450LM2. Thus, they did not react with or inhibit the enzyme activity of the other isozyme cytochrome P-450LM4, Fraction 1 or Fraction 7. All of the monoclonal antibodies formed against P-450LM2 were mouse immunoglobulin (Ig) subclass IgG1. The most effective monoclonal antibody strongly inhibited the formation of oxygenated metabolites of benzo[a]pyrene at various positions as well as the deethylation of 7-ethoxycoumarin. Four hybridomas were isolated which produced monoclonal antibodies to P-450LM4. One of the four was of the IgM class and three were of the IgG1 type. The four monoclonal antibodies bound to P-450LM4 but did not precipitate the enzyme, and did not bind to P-450LM2. The monoclonal antibody P-450LM4 complexes interacted with protein A, and the enzyme activity for benzo[a]pyrene hydroxylation could be removed by centrifugation. The high specificity and monoclonality of these antibodies suggest their potential usefulness for studying the genetics, regulation, and roles of the different isozymes of P-450LM in drug and carcinogen metabolism.

7-Alkoxycoumarin O-Dealkylase↗
PubMed1981

The rabbit pulmonary monooxygenase system. partial structural characterization of the cytochrome P-450 components and comparison to the hepatic cytochrome P-450.

Two forms of rabbit pulmonary cytochrome P-450, P-450I and P-450II, are distinguished by unique peptides observed upon electrophoresis of the products of limited proteolysis (with papain or chymotrypsin) in the absence or presence of sodium dodecyl sulfate. In contrast, the peptides from the proteolytic digestions of pulmonary cytochrome P-450I are indistinguishable from those of the major form of hepatic cytochrome P-450 induced by phenobarbital (P-450PB). Electrophoresis of the fragments of P-450, and P-450II produced by treatment with cyanogen bromide also shows different peptides, whereas the peptides produced from P-450I and P-450PB are the same. The amino acid composition and P-450II is different from that of P-450I or P-450PB. P-450II is distinguished further from the other two cytochromes on the basis of amino acid composition and subunit molecular weight (either as calculated from the amino acid composition or as estimated from sodium dodecyl sulfate polyacrylamide gel electrophoresis). The sequence of the first five residues at the NH2-terminal segment of P-450I and P-450PB are identical, while that of P-450II is different.

Amino Acids↗
PubMed1994

Isolation of plant cytochrome P-450 and NADPH: cytochrome P-450 reductase from tulip bulbs (Tulipa fosteriana L.) oxidizing xenobiotics.

Cytochrome P-450 and NADPH: cytochrome P-450 reductase were solubilized by detergents from microsomal fraction of tulip bulbs Tulipa fosteriana L. and purified to electrophoretic homogeneity. The purification was achieved by anion-exchange column chromatography, hydroxyapatite-column chromatography and affinity chromatography. The two enzyme showed relative molecular weights of about 54,200 and 77,600 for cytochrome P-450 and NADPH: cytochrome P-450 reductase, respectively. The purified enzymes were characterized by their absorption spectra and by kinetic characteristics. The interaction with endogeneous as well as exogenous substrates was studied by differential spectroscopy. Both enzymes in the presence of dilauroyl phosphatidylcholine and NADPH were able to oxidize xenobiotics (N-nitroso-N-methylaniline and N-nitroso-N-dimethylamine) in the reconstitution experiments.

Cytochrome P-450 Enzyme System↗