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PubMed1994

Stimulatory effects of lung cytochrome b5 on benzphetamine N-demethylation in a reconstituted system containing lung cytochrome P450LgM2.

Cytochrome b5 was partially purified from sheep lung microsomes in the presence of detergents Emulgen 913 and cholate by three consecutive DEAE-cellulose and Sephadex G-100 gel filtration chromatographies. The specific content of cytochrome b5 was 16.5 nmol/mg protein and purified cytochrome b5 fractions were free of cytochrome P450, NADPH-cytochrome P450 reductase and NADH-cytochrome b5 reductase activities. The influences of increasing concentrations of lung cytochrome b5 on benzphetamine N-demethylation reactions were examined in four different reconstitution systems containing lung cytochrome P450LgM2, lung cytochrome P450 reductase and lipid. In each system concentration of reductase was doubled with respect to former system. In all systems cytochrome b5 stimulated benzphetamine N-demethylase activity especially when cytochrome b5 was present at 0.5:1 molar ratio with respect to cytochrome P450LgM2. Besides, the greatest fold of increase in benzphetamine N-demethylation activity due to addition of cytochrome b5 was observed in System 1 with the lowest concentration of reductase.

Animals↗
PubMed1987

The effect of complex formation upon the reduction rates of cytochrome c and cytochrome c peroxidase compound II.

The effect of complex formation between ferricytochrome c and cytochrome c peroxidase (Ferrocytochrome-c:hydrogen peroxide oxidoreductase, EC 1.11.1.5) on the reduction of cytochrome c by N,N,N',N'-tetramethyl-p-phenylenediamine (TMPD), reduced N-methylphenazonium methosulfate (PMSH), and ascorbate has been determined at low ionic strength (pH 7) and 25 degrees C. Complex formation with the peroxidase enhances the rate of ferricytochrome c reduction by the neutral reductants TMPD and PMSH. Under all experimental conditions investigated, complex formation with cytochrome c peroxidase inhibits the ascorbate reduction of ferricytochrome c. This inhibition is due to the unfavorable electrostatic interactions between the ascorbate dianion and the negatively charged cytochrome c-cytochrome c peroxidase complex. Corrections for the electrostatic term by extrapolating the data to infinite ionic strength suggest that ascorbate can reduce cytochrome c peroxidase-bound cytochrome c faster than free cytochrome c. Reduction of cytochrome c peroxidase Compound II by dicyanobis(1,10-phenanthroline)iron(II) (Fe(phen)2(CN)2) is essentially unaffected by complex formation between the enzyme and ferricytochrome c at low ionic strength (pH 6) and 25 degrees C. However, reduction of Compound II by the negatively changed tetracyano-(1,10-phenanthroline)iron(II) (Fe(phen)(CN)4) is enhanced in the presence of ferricytochrome c. This enhancement is due to the more favorable electrostatic interactions between the reductant and cytochrome c-cytochrome c peroxidase Compound II complex then for Compound II itself. These studies indicate that complex formation between cytochrome c and cytochrome c peroxidase does not sterically block the electron-transfer pathways from these small nonphysiological reductants to the hemes in these two proteins.

Ascorbic Acid↗
PubMed2005

Role of cytochrome b5 in catalysis by cytochrome P450 2B4.

Cytochrome b5 has been shown to stimulate, inhibit or have no effect on catalysis by P450 cytochromes. Its action is known to depend on the isozyme of cytochrome P450, the substrate, and experimental conditions. Cytochrome P450 2B4 (CYP 2B4) has been used in our laboratory as a model isozyme to study the role of cytochrome b5 in cytochrome P450 catalysis using two substrates, methoxyflurane and benzphetamine. One substrate is the volatile anesthetic, methoxyflurane, whose metabolism is consistently markedly stimulated by cytochrome b5. The other is benzphetamine, whose metabolism is minimally modified by cytochrome b5. Determination of the stoichiometry of the metabolism of both substrates showed that the amount of product formed is the net result of the simultaneous stimulatory and inhibitory actions of cytochrome b5 on catalysis. Site-directed mutagenesis studies revealed that both cytochrome b5 and cytochrome P450 reductase interact with cytochrome P450 on its proximal surface on overlapping but non-identical binding sites. Comparison of the rate of reduction of oxyferrous CYP 2B4 and the rate of substrate oxidation by cyt b5 and reductase with stopped-flow spectrophotometric and rapid chemical quench experiments has demonstrated that although cytochrome b5 and reductase reduce oxyferrous CYP 2B4 at the same rate, substrate oxidation proceeds more slowly in the presence of the reductase.

Animals↗
PubMed1978

Effect of specific lysine modification on the reduction of cytochrome c by succinate-cytochrome c reductase.

The reduction of cytochrome c by succinate-cytochrome c reductase was studied at very low cytochrome c concentrations where the reaction between cytochrome c1 and cytochrome c was rate limiting. The rate constant for the reaction was found to be independent of ionic strength up to 0.1 M chloride, and to decrease rapidly at higher ionic strength, suggesting that the interaction between cytochrome c1 and cytochrome c was primarily electrostatic. The reaction rates of cytochrome c derivatives modified at single lysine residues to form trifluoroacetylated or trifluoromethylphenylcarbamylated cytochromes c were studied to determine the role of individual lysines in the reaction. None of the modifications affected the reaction at low ionic strength, but at higher ionic strength the reaction rate was substantially decreased by modification of those lysines surrounding the heme crevice, lysine-8, -13, -27, -72, and -79. Modification of lysine-22, -25, -55, -99, and -100 had no effect on the rate. These results indicate that the binding site on cytochrome c for cytochrome c1 overlaps considerably with that for cytochrome oxidase, suggesting that cytochrome c might undergo some type of rotational diffusion during the electron-transport process.

Animals↗
PubMed1999

Ubiquinone at center N is responsible for triphasic reduction of cytochrome b in the cytochrome bc(1) complex.

We have examined the pre-steady state reduction kinetics of the Saccharomyces cerevisiae cytochrome bc(1) complex by menaquinol in the presence and absence of endogenous ubiquinone to elucidate the mechanism of triphasic cytochrome b reduction. With cytochrome bc(1) complex from wild type yeast, cytochrome b reduction was triphasic, consisting of a rapid partial reduction phase, an apparent partial reoxidation phase, and a slow rereduction phase. Absorbance spectra taken by rapid scanning spectroscopy at 1-ms intervals before, during, and after the apparent reoxidation phase showed that this was caused by a bona fide reoxidation of cytochrome b and not by any negative spectral contribution from cytochrome c(1). With cytochrome bc(1) complex from a yeast mutant that cannot synthesize ubiquinone, cytochrome b reduction by either menaquinol or ubiquinol was rapid and monophasic. Addition of ubiquinone restored triphasic cytochrome b reduction, and the duration of the reoxidation phase increased as the ubiquinone concentration increased. When reduction of the cytochrome bc(1) complex through center P was blocked, cytochrome b reduction through center N was biphasic and was slowed by the addition of exogenous ubiquinone. These results show that ubiquinone residing at center N in the oxidized cytochrome bc(1) complex is responsible for the triphasic reduction of cytochrome b.

Cytochrome b Group↗
PubMed1990

NMR characterization of surface interactions in the cytochrome b5-cytochrome c complex.

The complex formed in solution by native and chemically modified cytochrome c with cytochrome b5 has been studied by 1H and 13C nuclear magnetic resonance spectroscopy (NMR). Contrary to predictions of recent theoretical analysis, 1H NMR spectroscopy indicates that there is no major movement of cytochrome c residue Phe82 on binding to cytochrome b5. The greater resolution provided by 13C NMR spectroscopy permits detection of small perturbations in the environments of cytochrome c residues Ile75 and Ile85 on binding with cytochrome b5, a result that is in agreement with earlier model-building experiments. As individual cytochrome c lysyl residues are resolved in the 1H NMR spectrum of N-acetimidylated cytochrome c, the interaction of this modified protein with cytochrome b5 has been studied to evaluate the number of cytochrome c lysyl residues involved in binding to cytochrome b5. The results of this experiment indicate that at least six lysyl residues are involved, two more than predicted by static model building, which indicates that cytochrome c and cytochrome b5 form two or more structurally similar 1:1 complexes in solution.

Carbon Isotopes↗
PubMed1984

Potentiometric analysis of the cytochromes of an Escherichia coli mutant strain lacking the cytochrome d terminal oxidase complex.

A combination of potentiometric analysis and electrochemically poised low-temperature difference spectroscopy was used to examine a mutant strain of Escherichia coli that was previously shown by immunological criteria to be lacking the cytochrome d terminal oxidase. It was shown that this strain is missing cytochromes d, a1, and b558 and that the cytochrome composition of the mutant is similar to that of the wild-type strain grown under conditions of high aeration. The data indicate that the high-aeration branch of the respiratory chain contains two cytochrome components, b556 (midpoint potential [Em] = +35 mV) and cytochrome o (Em = +165 mV). The latter component binds to CO and apparently has a reduced-minus-oxidized split-alpha band with peaks at 555 and 562 nm. When the wild-type strain was grown under conditions of low aeration, the components of the cytochrome d terminal oxidase complex were observed: cytochrome d (Em = +260 mV), cytochrome a1 (Em = +150 mV) and cytochrome b558 (Em = +180 mV). All cytochromes appeared to undergo simple one-electron oxidation-reduction reactions. In the absence of CO, cytochromes b558 and o have nearly the same Em values. In the presence of CO, the Em of cytochrome o is raised, thus allowing cytochromes b558 and o to be individually quantitated by potentiometric analysis when they are both present.

Cytochrome d Group↗
PubMed1989

[Interaction of cholesterol hydroxylating cytochrome P-450 with cytochrome b5].

Some new relations between cytochrome P-450-dependent monooxygenases were discovered. Cytochrome b5, a representative of "microsomal" monooxygenases, was shown to form a highly specific complex with cytochrome P-450scc, a member of the "ferredoxin" monooxygenase family. This interaction is characterized by a dissociation constant, Kd, of 0.28 microM. The cytochrome P-450scc-cytochrome b5 complex may be cross-linked with water-soluble carbodiimide. Using proteolytic modification of cytochrome b5, it was shown that both hydrophilic and hydrophobic fragments of cytochrome b5 are involved in the interaction with cytochrome P-450scc. Cytochrome b5 immobilized via amino groups is an effective affinity matrix for cytochrome P-450scc purification. The role of some amino acid residues in cytochrome P-450scc interaction with cytochrome b5 was studied. The role and the nature of complexes in cytochrome P-450-dependent monooxygenases as well as interrelationships between "microsomal" and "ferredoxin" monooxygenases are discussed.

Adrenal Cortex↗
PubMed2001

A novel hydrophobic diheme c-type cytochrome. Purification from Corynebacterium glutamicum and analysis of the QcrCBA operon encoding three subunit proteins of a putative cytochrome reductase complex.

Electrophoresis of a Corynebacterium glutamicum membrane preparation in the presence of sodium dodecyl sulfate, followed by staining for peroxidase activity (heme staining), showed only one band at about 28 kDa. This 28 kDa protein was purified from C. glutamicum membranes by chromatography in the presence of decylglucoside using DEAE-Toyopearl and hydroxylapatite columns, as the sole c-type cytochrome in the bacterium. The cytochrome showed an alpha band at 551 nm, and its E(m, 7) was about 210 mV. A QcrCAB operon encoding the subunits of a putative quinol cytochrome c reductase was found 3'-downstream of ctaE encoding subunit III of cytochrome aa(3) in the C. glutamicum genome. The deduced amino acid sequence of qcrC, composed of 283 amino acid residues, contained two heme C-binding motifs and was in agreement with partial peptide sequences obtained from the 28 kDa protein after V8 protease digestion. We propose to name this protein cytochrome cc. The presence of cytochrome cc is a common feature of high G+C content Gram-positive bacteria, since we could confirm this protein by electrophoresis; homologous QcrCAB operons are also known in Mycobacterium and Streptomyces. QcrA and qcrB of C. glutamicum encode the Rieske Fe-S protein and cytochrome b, respectively, although these proteins were not co-purified with cytochrome cc. The phylogenetic tree of cytochromes b and b(6) show that C. glutamicum cytochrome b, along with those of other bacteria in the high G+C group, is rather different from the Bacillus counterparts, but highly similar to the Deinococci and Thermus cytochromes. This indicates that there is a fourth group of bacteria in addition to the three clades: proteobacterial cytochrome b, cyanobacterial b(6) and green sulfur-low G+C Gram-positive bacteria.

Amino Acid Sequence↗
PubMed2001

Modification of inhibitor binding sites in the cytochrome bf complex by directed mutagenesis of cytochrome b(6) in Synechococcus sp. PCC 7002.

The cytochrome bf complex, which links electron transfer from photosystem II to photosystem I in oxygenic photosynthesis, has not been amenable to site-directed mutagenesis in cyanobacteria. Using the cyanobacterium Synechococcus sp. PCC 7002, we have successfully modified the cytochrome b(6) subunit of the cytochrome bf complex. Single amino acid substitutions in cytochrome b(6) at the positions D148, A154, and S159 revealed altered binding of the quinol-oxidation inhibitors 2,5-dibromo-3-methyl-6-isopropyl-p-benzoquinone (DBMIB), myxothiazol, and stigmatellin. Cytochrome bf and mitochondrial-type cytochrome bc(1) complexes are closely related in structure and function but exhibit quite different inhibitor specificities. Cytochrome bf complexes are insensitive to myxothiazol and sensitive to DBMIB, whereas cytochrome bc(1) complexes are sensitive to myxothiazol and relatively insensitive to DBMIB. Measurements of flash-induced and steady-state electron transfer rates through the cytochrome bf complex revealed increased resistance to DBMIB in the mutants A154G and S159A, increased resistance to stigmatellin in A154G, and created sensitivity to myxothiazol in the mutant D148G. Therefore these mutations made the cytochrome bf complex more like the cytochrome bc(1) complex. This work demonstrates that cyanobacteria can be used as effective models to investigate structure-function relationships in the cytochrome bf complex.

Alleles↗
PubMed1981

Rapid reduction of cytochrome c1 in the presence of antimycin and its implication for the mechanism of electron transfer in the cytochrome b-c1 segment of the mitochondrial respiratory chain.

Antimycin, a specific and highly potent inhibitor of electron transfer in the cytochrome b-c1 segment of the mitochondrial respiratory chain, does not inhibit reduction of cytochrome c1 by succinate in isolated succinate-cytochrome c reductase complex under conditions where the respiratory chain complex undergoes one oxidation-reduction turnover. If a slight molar excess of cytochrome c is added to the isolated reductase complex in the presence of antimycin, there is rapid reduction of one equivalent of c type cytochrome by succinate, after which reduction of the remaining c type cytochrome is inhibited. Antimycin fully inhibits succinate-cytochrome c reductase activity of isolated succinate-cytochrome c reductase complex in which the b-c1 complex undergoes multiple turnovers in a catalytic fashion. In addition, when antimycin is added to isolated reductase complex in the presence of cytochrome c plus cytochrome c oxidase, the inhibitor causes a "crossover" in the steady state level of reduction of the cytochromes b and c1 comparable to this classical effect in mitochondria. On the basis of these results, it is suggested that linear schemes of electron transfer are not adequate to account for the site of antimycin inhibition and the mechanism of electron transfer in the cytochrome b-c1 segment of the respiratory chain. The effects of antimycin are consistent with cyclic electron transfer mechanisms such as the protonmotive Q cycle.

Animals↗
PubMed1994

Electron microscopy of cytochrome c oxidase crystals. Monomer-dimer relationship and cytochrome c binding site.

Cytochrome c oxidase was isolated from beef heart mitochondria by detergent extraction yielding two different crystal forms. Extraction with Triton detergents produced vesicular crystals with two-dimensional crystalline arrays of cytochrome c oxidase dimers while extraction with sodium deoxycholate produced crystalline sheets of cytochrome c oxidase monomers. The structures of both crystal forms were determined in two-dimensional projection along an axis normal to the plane of the membrane by cryoelectron microscopy of crystals embedded in vitreous ice (frozen-hydrated). The projection structures of unstained frozen hydrated monomers and of dimers are similar to the structures of the crystals in negative stain. The molecular outline of dimers can be approximated by a parallelogram 44 A by 82 A with an included angle of 80 degrees. Monomers are less regular consisting of two large domains with a smaller domain at one end and a total length of approximately 82 A. Comparison of the two structures reveals the orientation of cytochrome c oxidase monomers within dimers, an orientation which is different from earlier models of monomer-monomer interaction, and suggests a very close interaction between monomers when they associate to form dimers. The crystalline sheets of cytochrome c oxidase monomers bind tightly the small peripheral membrane protein substrate, cytochrome c, and this binding accentuates a tendency of these crystals to stack upon one another. Images of crystals of the cytochrome c oxidase/cytochrome c complex were analyzed by crosscorrelation analysis versus the monomer crystal image. Two types of two-layer crystals have been identified. Both types have one layer rotated by 180 degrees with respect to the other, but they differ in the shifts of origin along crystal axes of the two layers. Difference images formed by subtracting simulated multilayered crystal images (which have no bound cytochrome c) from the complex crystals (cytochrome c oxidase plus cytochrome c) contain one positive difference peak for each cytochrome oxidase monomer within a unit cell. Comparison of the difference peak loci among the different crystal forms is interpreted based upon a consensus cytochrome c binding site in the single layer cytochrome oxidase monomer crystal image.

Animals↗
PubMed1989

Catalytic activity of cytochrome oxidase and cytochrome c in apolar solvents containing phospholipids and low amounts of water.

Cytochrome c and cytochrome oxidase, in bovine heart submitochondrial particles and in their purified forms, were transferred to a ternary system that contained phospholipids (10 mg/ml toluene), the apolar solvent toluene, and water at concentrations of 13-15 microliters (high water) and 3 microliters (low water) per milliliter of toluene. When the enzymes were transferred back to an all water system, they exhibited full catalytic capacity. In the low water ternary system, cytochrome c could be reduced by ascorbate introduced via inverted micelles. Also in this system, cytochrome oxidase was reduced by ascorbate and cytochrome c but its oxidation was highly impaired. Data on the kinetics of reduction by ascorbate of cytochrome c and cytochrome oxidase under these conditions are presented. Cytochrome oxidase reduced in the organic solvent by ascorbate failed to form a complex with CO, but formed a complex with cyanide introduced via inverted micelles. The oxidized and the ascorbate-reduced cytochrome oxidase-cyanide complex exhibited a trough at 415 nm and a peak at 433 nm. The extent and rate of formation of the cyanide complex were higher with the reduced form of cytochrome oxidase. To achieve protein-protein interactions (cytochrome c-cytochrome oxidase) in the ternary system, it was necessary to extract the two proteins together. There was no functional interaction when they were extracted separately and mixed. In the high water ternary system reduced cytochrome oxidase was not detected, and it oxidized ascorbate at a higher rate than in the low water system; however, this rate was several orders of magnitude lower than in aqueous media.

Animals↗
PubMed1978

Cytochrome c-cytochrome oxidase interaction at subzero temperatures.

Cytochrome oxidase forms two distinctive compounds with oxygen at --105 and --90 degrees C, one appears to be oxycytochrome oxidase (Compound A) and the other peroxycytochrome oxidase (Compound B). The functional role of compound B in the oxidation of cytochrome c has been examined in a variety of mitochondrial preparations. The rate and the extent of the reaction have been found to be dependent upon the presence of a fluid phase in the vicinity of the site of the reaction of cytochrome c and cytochrome oxidase. The kinetics of cytochrome c oxidation and of the slowly reacting component of cytochrome oxidase are found to be linked to one another even in cytochrome c depleted preparations, but under appropriate conditions, especially low temperatures, the oxidation of cytochrome c precedes that of this component of cytochrome oxidase. Based upon the identification of the slowly reacting components of cytochrome oxidase with cytochrome c, various mechanisms are considered which allow cytochrome c to be oxidized without the intervention of cytochrome a at very low temperatures, and tunneling seems an appropriate mechanism.

Animals↗
PubMed1982

The reaction of cytochrome aa3 with (porphyrin) cytochrome c as studied by pulse radiolysis.

(1) Using the pulse-radiolysis and stopped-flow techniques, the reactions of iron-free (porphyrin) cytochrome c and native cytochrome c with cytochrome aa3 were investigated. The porphyrin cytochrome c anion radical (generated by reduction of porphyrin cytochrome c by the hydrated electron) can transfer its electron to cytochrome aa3. The bimolecular rate constant for this reaction is 2 x 10(7) M-1 . s-1 (5 mM potassium phosphate, 0.5% Tween 20, pH 7.0, 20 degrees C). (2) The ionic strength dependence of the cytochrome c-cytochrome aa3 interaction was measured in the ionic strength range between 40 and 120 mM. At ionic strengths below 30 mM, a cytochrome c-cytochrome aa3 complex is formed in which cytochrome c is no longer reducible by the hydrated electron. A method is described by which the contributions of electrostatic forces to the reaction rate can be determined. (3) Using the stopped-flow technique, the effect of the dielectric constant (epsilon) of the reaction medium on the reaction of cytochrome C with cytochrome aa3 was investigated. With increasing epsilon the second-order rate constant decreased.

Animals↗
PubMed1996

Biochemical studies of the c-type cytochromes of the sulfate reducer Desulfovibrio africanus. Characterization of two tetraheme cytochromes c3 with different specificity.

Three c-type cytochromes were isolated and characterized from the sulfate reducer Desulfovibrio africanus. A basic tetraheme cytochrome c3 of molecular mass 16 kDa was previously described and we have extended its characterization. Two other c3-type cytochromes, not previously observed, have also been characterized. These include an acidic tetraheme cytochrome c3 of molecular mass 15 kDa and an octaheme dimeric cytochrome c3 with a native size of 35 kDa. This is the first report of the presence of two distinct tetraheme cytochromes c3 in a Desulfovibrio species. The physico-chemical properties of the three cytochromes, including optical properties, iron content, cysteine and histidine content, N-terminal amino sequence and redox properties, are characteristic of cytochrome c3 family. The acidic tetraheme cytochrome c3 exhibited similar midpoint potential values for all four hemes (Em1 = -210 mV; Em2 = -240 mV; Em3 = -260 mV; Em4 = -270 mV), whereas in the basic tetraheme cytochrome c3 one heme had a much more positive potential than the others (Em1 = -90 mV; Em2 = -260 mV; Em3 = -280 mV; Em4 = -290 mV). The acidic tetraheme cytochrome c3 exhibited unique properties including amino-acid composition and poor reactivity towards hydrogenase. However, it is readily reduced by this enzyme in the presence of the basic cytochrome c3. The weak reactivity of the acidic tetraheme cytochrome c3 towards hydrogenase has been correlated with its low content of basic residues.

Amino Acid Sequence↗
PubMed1987

Purification and characterization of two constitutive cytochromes P-450 (F-1 and F-2) from adult female rats: identification of P-450F-1 as the phenobarbital-inducible cytochrome P-450 in male rat liver.

Two hepatic microsomal cytochromes P-450, P-450F-1 and P-450F-2 were purified to electrophoretic homogeneity from untreated adult female rats by high-performance liquid chromatography (HPLC) with anion-exchange, cation-exchange, and hydroxyapatite columns. Cytochromes P-450F-1 and P-450F-2 were not adsorbed with the anion-exchange column, but were retained on a cation-exchange column and were separated poorly. These forms separated on hydroxyapatite HPLC. The molecular weights of cytochromes P-450F-1 and P-450F-2 were 50,000 and 49,000, respectively. The absolute spectrum of the oxidized forms indicated that they had the low-spin state of heme, and the CO-reduced spectral maxima of cytochromes P-450F-1 and P-450F-2 were at 450 and 448 nm, respectively. Both forms catalyzed the N-demethylation of benzphetamine and had low catalytic activity for 7-ethoxycoumarin. Cytochrome P-450F-1 had low 2 alpha-hydroxylation activity toward testosterone. Cytochrome P-450F-2 had low 15 alpha-hydroxylation activity. On the basis of these results and those of NH2-terminal sequence analysis, cytochrome P-450F-2 seemed to be the typical female-specific cytochrome P-450. The NH2-terminal sequence of cytochrome P-450F-1 was identical to that of cytochrome P-450PB-2 purified from hepatic microsomes of male rats treated with phenobarbital. Cytochromes P-450F-1 and P-450PB-2 had identical chromatographic properties, minimum molecular weight, spectral properties, and peptide maps. Furthermore, the antibody to phenobarbital-inducible cytochrome P-450PB-2 gave a single immunoprecipitin band with cytochrome P-450F-1 by Ouchterlony double-diffusion analysis.

Amino Acid Sequence↗
PubMed1982

Cytochrome P-450 induction by clofibrate. Purification and properties of a hepatic cytochrome P-450 relatively specific for the 12- and 11-hydroxylation of dodecanoic acid (lauric acid).

Hypolipidaemic drugs induce peroxisomal proliferation in the liver and many induce the formation of the hepatic endoplasmic reticulum in general and the formation of cytochrome P-450 in particular. We have induced the formation of rat liver microsomal cytochrome P-450 by the administration of the hypolipidaemic drug clofibrate, isolated the endoplasmic reticulum, solubilized the cytochrome P-450 from these membranes and subdivided the cytochrome P-450 into four fractions by the use of hydrophobic, anionic, cationic and adsorption chromatography. One of these fractions (cytochrome P-450 fraction 1) was highly purified to a specific content of 17nmol of cytochrome P-450/mg of protein and the protein was active in a reconstituted enzyme system towards the 12- and 11-hydroxylation of the fatty acid, dodecanoic (lauric) acid, with preferential activity towards the 12-hydroxy metabolite. This reconstituted activity was absolutely dependent on NADPH, NADPH-cytochrome P-450 reductase and cytochrome P-450, indicating the role of the mixed-function oxidase system in the metabolism of lauric acid. Another fraction of the haemoprotein (cytochrome P-450 fraction 2) preferentially formed 11-hydroxylauric acid, whereas a third fraction (cytochrome P-450 fraction 3) exhibited only trace laurate oxidase activity and was similar to the phenobarbitone form of the haemoprotein in that these last two cytochromes rapidly turned-over the drug benzphetamine. The molecular weights and spectral properties of these cytochrome P-450 fractions are reported, along with the phenobarbitone-induced form of the enzyme and the nature of the cytochrome(s) induced by clofibrate pretreatment are discussed in the terms of possible haemoprotein heterogeneity.

Animals↗