Difference Spectrum Of Cytochrome P-450 Research Articles

The expression of a catalytically active cholesterol 7 alpha-hydroxylase cytochrome P450 in Escherichia coli.

We have recently cloned a full-length cDNA encoding the rat hepatic cholesterol 7 alpha-hydroxylase cytochrome P450 (P450c7) (Li, Y. C., Wang, D. P., and Chiang, J. Y. L. (1990) J. Biol. Chem. 265, 12012-12019), which catalyzes the rate-limiting reaction of bile acid synthesis in the liver. By using the polymerase chain reaction, we have designed two P450c7 cDNAs. One has the second Met codon deleted and the third Thr codon replaced with an Ala. The other lacks codons for the NH2-terminal hydrophobic sequence of amino acids 2-24 (P450c7 delta 2-24). The cDNAs were separately cloned into the expression vector pKK233-2 and transformed into Escherichia coli. After induction with isopropyl-beta-D-thiogalactopyranoside, bacteria harboring recombinant plasmids expressed a polypeptide which reacted with the antibody against cholesterol 7 alpha-hydroxylase in immunoblots. The slightly modified full-length enzyme was expressed to 0.2% of the total bacterial lysate and was located in the membrane fraction, whereas P450c7 delta 2-24 was expressed at a 10-fold higher level (2%), of which 85% was in the cytosol and the remaining associated with the membranes. We have purified P450c7 delta 2-24 which showed a typical reduced-CO difference spectrum of cytochrome P450 and reconstituted cholesterol 7 alpha-hydroxylase activity in the presence of NADPH-cytochrome P450 reductase. P450c7 delta 2-24 has a similar Km for cholesterol (24.6 microM) but a lower Vmax (0.10 nmol/min) and a lower turnover number (1.93 min-1) as compared with the enzyme isolated from rat liver microsomes. The purified P450c7 delta 2-24 has an unique hydrophilic NH2 terminus and contains monomers and dimers in equal amounts. This is the first report demonstrating that a genetically engineered cytochrome P450 enzyme lacking a typical NH2-terminal hydrophobic sequence is mainly cytosolic and catalytically active.

Oxidant, mixed-function oxidase and peroxisomal responses in channel catfish exposed to a bleached kraft mill effluent

Channel catfish (Ictalarus punctatus) were exposed to 0, 10, 20, and 40% bleached kraft pulp and paper mill effluent (BKME) for up to two weeks. Fish were sacrificed on days 1, 3, 7, and 14 of exposure. Blood, liver, and gill tissues were assayed for biochemical responses associated with oxidative stress (superoxide dismutase, catalase, and glutathione peroxidase activities and methemoglobin, reduced glutathione, and malondialdehyde concentrations), peroxisome proliferation (palmitoyl-CoA oxidase and lauroyl-CoA oxidase activities) and the mixed-function oxidase system (cytochrome P-450 difference spectra and ethoxyresorufin O-deethylase activities). There were significant dose-related increases in hepatic catalase and ethoxyresorufin O-deethylase activities and significant decreases in hepatic reduced glutathione concentrations. There were also significant increases in lauroyl-CoA oxidase and palmitoyl-CoA oxidases activities, both components of the fatty acid beta-oxidation system and located in peroxisomes. No statistically significant changes were seen in the other components studied. The increases in catalase and the fatty-acid oxidase activities suggest that BKME exposure may have resulted in peroxisome proliferation in catfish hepatic tissue.

Drug metabolism components in liver microsomes from Hypostomus punctatus, a brazilian benthic fish (cascudo)

1. The major components of hepatic drug biotransformation system were identified in a Brazilian freshwater benthic fish. 2. Cytochrome P-450 difference spectra were obtained adding 0.02 mM phenazine ethosulphate and 2 mM ascorbate to microsomal suspensions. Basal levels of P-450 were high (0.9 nmol/mg of microsomal protein) and were not induced by 3-MC. 3. Microsomal NADPH-cytochrome C reductase activity was determined in presence of 1.3 × 10 −4M NADPH, 3.3 × 10 −5 M cytochrome C, 1.0 × 10 − M EDTA, 66 μg of microsomal protein per ml in a 0.3 M Tris-HCl buffer, pH8.6. Basal levels of NADPH-cytochrome C were 152.7nmoles/min/mg of microsomal protein.

The effects of nicotine, cotinine and anabasine on rat adrenal 11β-hydroxylase and 21-hydroxylase

The effects of nicotine, cotinine and anabasine on rat adrenal steroidogenesis were examined by spectral and enzymatic techniques. The addition of nicotine, cotinine or anabasine to preparations of rat adrenal mitochondria produced type II cytochrome P-450 difference spectra. The addition of nicotine or anabasine, but not cotinine, to rat adrenal microsomes yielded type II cytochrome P-450 difference spectra. Nicotine and anabasine competitively inhibited rat adrenal mitochondrial 11β-hydroxylase and microsomal 21-hydroxylase. Cotinine competitively inhibited mitochondrial 11β-hydroxylase, but did not inhibit microsomal 21-hydroxylase. The apparent enzymatic inhibition constants for cotinine, nicotine, anabasine and metyrapone inhibition of the mitochondrial 11β-hydroxylase were 32, 96, 120 and 74 μM respectively. These studies suggest that components of cigarette smoke may alter patterns of adrenal steroidogenesis.

Interaction of the substrate analogue of cytochrome P-450 and mixed function oxidases

The interaction of a spin labeled compound carrying an alkylating group 4-(3-iodo-2-oxopropylidene)-2,2,3,5,5-pentamethylimidozolydene-l-oxyl (RJ) and capable of binding covalently to mixed function oxidases (MFO) was studied. Measurements of the difference spectrum of cytochrome P-450 demonstrated that RJ induces spectral changes characteristic of type I substrates ( λ max = 403 nm; λ min = 418nm). The spectral binding constant ( K s ) was 66 μM as determined from the difference spectrum. RJ inhibited the microsomal oxidation of substrates of cytochrome P-450 (aniline, aminopyrine and benzo [ a]pyrene). This inhibition was shown not to be associated with the conversion of cytochrome P-450 to cytochrome P-420, or with the suppression of the activities of NADPH-cytochrome c reductase and NADPH-cytochrome P-450 reductase. Thus, evidence was obtained for the possible interaction of RJ with cytochrome P-450. RJ, injected to rats (5 mg/100 g body wt, i.p.), inhibited the hydroxylation of benzo[a] pyrene, a type I substrate, (21%) and aniline, a type II substrate, (40%) in the microsomes from their livers. The presence of a paramagnetic center in RJ made it possible to study its interaction with microsomes. The electron paramagnetic resonance (EPR) spectrum of RJ was recorded in the rat liver microsomal fraction after in vivo administration of RJ. In rats treated with RJ (5 mg/100 g), hexobarbital sleeping time was prolonged 1.5-fold. Alkylating analogs of substrates of cytochrome P-450 are suggested as agents for structural studies of the active center of cytochrome P-450 and the development of efficient inhibitors of reactions catalyzed by this enzyme.

Hepatic cytochrome P-450-dependent monooxygenase systems of the trout, frog and snake—III. Induction

1. 1. 3-Methylcholanthrene administration increased levels of cytochrome P-450, benzo[a]pyrene hydroxylase activity and p- nitrophenetole O- deethylase activity in hepatic microsomes from the brown trout ( Salmo trutta), leopard frog ( Rana pipiens) and the garter snake ( Thamnophis). The level of aminopyrine N- demethylase activity was increased in trout microsomes, but not in those from the frog, snake or rat. 2. 2. The shift in the Soret maximum of the reduced carbon monoxide difference spectrum of cytochrome P-450 from 450 to 448 nm, which is observed when 3-methylcholanthrene is administered to rats, was not seen in microsomes from the trout, frog or snake. 3. 3. Benzo[a]pyrene hydroxylase activity induced by 3-methylcholanthrene in the trout, frog and snake was inhibited by relatively low concentrations of SKF 525-A, but that induced in the rat was not. α-Naphthoflabone inhibited 3-methylcholanthrene-induced benzo[a]pyrene hydroxylase activity in the trout, frog and rat, but not in the snake. 4. 4. 3-Methylcholanthrene induced an increase in the 455/430 nm peak height ratio of the reduced ethylisocyanide spectrum of microsomes in the rat and trout, but not in the frog or snake. 5. 5. These observations (items 1–4 of the abstract) show that different species of cytochrome P-450 are induced by 3-methylcholanthrene in the four vertebrates. 6. 6. Phenobarbital did not alter the components or the activities of hepatic monooxygenase systems in the trout, frog or snake, even though it was shown to accumulate in the livers of these vertebrates as readily as it does in the liver of the rat.

Oxidation-reduction states of pyridine nucleotide and cytochrome P-450 during mixed-function oxidation in perfused rat liver.

The spectral changes of cytochrome P-450 associated with mixed-function oxidation of hexobarbital and aminopyrine were investigated in perfused rat liver, using reflectance spectrophotometry. Simultaneously, the oxidation-reduction state of pyridine nucleotide(s) and oxygen uptake were measured from the same liver. Difference spectra were observed after infusion of hexobarbital or aminopyrine. Spectra obtained from livers of fasted, sodium phenobarbital-treated rats were similar to those of cytochrome P-450 . substrate complex (type I); the spectrum from fed, sodium phenobarbital-treated rats was a mixture of substrate . cytochrome P-450 complex and reduced (an oxygenated) cytochrome P-450. In fed, sodium phenobarbital-treated rats, the steady state level of reduced (an oxygenated) cytochrome P-450 was dependent on the concentration and the kind of substrates added, and the level correlated well with mixed-function oxidase activity. In the absence of exogenous substrates, the degree of the formation of reduced (an oxygenated) cytochrome P-450 was small. These results indicate that mixed-function oxidase activity in the intact cell is regulated by substrate-binding to cytochrome P-450. In fasted, sodium phenobarbital-treated rats, a substantial oxidation of pyridine nucleotide was observed in the presence of hexobarbital. The increase of oxygen uptake was a 2- to 2.5-fold smaller rate than in livers from fed, sodium phenobarbital-treated rats. Infusion of sorbitol (2 mM), a glycogenic substrate in fasted rats, stimulated oxygen uptake about 3-fold. Furthermore, reduced (an oxygenated) cytochrome P-450 increased in the presence of sorbitol. These results indicate that the rate of NADPH supply is rate-limiting for mixed-function oxidation in livers of fasted, sodium phenobarbital-treated rats. Finally, the difference spectrum of cytochrome P-450, the surface fluorescence of pyridine nucleotide, and the oxygen uptake were compared using livers from untreated, alloxan-treated and sodium phenobarbital-treated rats.

The metabolism of benzo(alpha)pyrene by cytochrome P-450 in transformable and nontransformable C3H mouse fibroblasts.

The microsomal metabolism of benzo(a)pyrene and its enhancement by inducers of aryl hywdrocarbon hydroxylase were studied in three C3H mouse fibroblast cell lines and in C3H mouse liver and lung. The cell lines examined included C3H/10T 1/2 CL8, a line that can be transformed by treatment with benzo(a)pyrene; CVP3SC6, a line which cannot be transformed by treatment with benzo(a)pyrene; and C3H/10T 1/2 CL16, a transformed cell line derived from 3-methylcholanthrene-treated C3H/10T 1/2 CL8 cells. Of four polycyclic hydrocarbons examined, benz(a)anthracene was found to be the most effective inducer of aryl hydrocarbon hydroxylase activity, while 3-methylcholanthrene did not induce. The extent of this induction in all three cell lines was dependent upon the time after seeding that the inducer was added. The C3H/10T 1/2 CL8 cells were induced by benz(a)anthracene to a greater extent than the other two cell lines. The high pressure liquid chromatographic profile of benzo(a)pyrene metabolites was qualitatively identical for microsomes from all three cell lines and differed from the metabolism by liver and lung microsomes in that the cells did not produce detectable levels of benzo(a)pyrene-4,5-hydrodiol. The reduced CO cytochrome P-450 difference spectra of control and induced microsomes from all three cell lines showed a characteristic shiftmore » to shorter wavelengths after induction with benz(a)anthracene. The specific activity of cytochrome P-450 in the cell microsomes closely paralled the benzo(a)pyrene metabolizing activity as measured both by the aryl hydrocarbon hydroxylase and high pressure liquid chromatography assays.« less

An unusual effect of N-octylamine on the cytochrome b5 spectrum of insect and mammalian microsomes

The addition of n-octylamine to microsomes prepared from the midgut of tobacco hornworm ( Manduca sexta) larvae causes an unusual spectral interaction. The initial optical difference spectrum appears to be the sum of reduced cytochrome b 5 and a type II difference spectrum of cytochrome P-450. This initial spectrum is unstable and diminishes in size, with a concurrent shift in peak (424 to 428 nm) and trough (409 and 392 to approx. 400 nm) positions, to yield a stable spectrum identical to the type II spectrum of cytochrome P-450. Thus, in addition to its interaction with cytochrome P-450, n-octylamine causes a reduction of cytochrome b 5 which subsequently becomes reoxidized. The casual factor for this unusual spectral interaction occurs in the cytoplasm and appears to be protein-bound. It was also present in similar preparations from the tobacco budworm ( Heliothis virescens) but not in those from rat or mouse liver or abdomens from insecticide-resistant or susceptible houseflies ( Musca domestica). Microsomes from rat and mouse liver, but not those from housefly abdomens, exhibit similar unusual spectral interactions with n-octylamine when supplemented with the soluble factor from the hornworm.

INHIBITION BY CYANIDE OF DRUG OXIDATIONS IN RAT LIVER MICROSOMES

Cyanide inhibited microsomal activities of aniline hydroxylation and aminopyrine, ethylmorphine and codeine demethylations and produced a modified type II difference spectrum of cytochrome P-450 to give two spectral dissociation constants, 0.21mM and 1.05 mM. The binding of cyanide to cytochrome P-450 resulted in innhibition of NADPH-cytochrome P-450 reductase activity. The cyanide inhibition of drug oxidations was partially avoided by increasing oxygen tension. A possible mechanism for the inhibition of drug oxidations by cyanide is discussed.

Effect of calcium(ion) uptake by rat adrenal mitochondrial on pregnenolone formation and spectral properties of cytochrome P-450

The effect of calcium on pregnenolone formation from endogenous precursors has been studied in mitochondria from rat decapsulated and capsular adrenalgglands. In the presence of succinate, addition of calcium chloride in the concentration range 20–150 μM caused an inhibition of pregnenolone formation of added deoxycosticosterone in decapsulated adrenal mitochondria was also inhibited. Under these conditions, calcium inhibited the reduction of adrenodoxin, a component of the cytochrome P-450 reductase system, presumably because uptake of calcium by the mitochondria competes with energy-linked transhydrogenase for high-energy intermediates. For this reason, incubations were carried out in the presence of succinate plus isocitrate plus NADP +. Under these conditions, calcium chloride in the concentration range 120–875 μM caused a 2–4-fold stimulation of pregnenolone formation, but had no effect on corticosterone formation from added deoxycorticosterone. The effect of calcium on the optical spectra of cytochrome P-450 has also been examined in mitochondria from decapsulated and capsular rat adrenals. In the presence of succinate, calcium induced a spectral change resembling a type I difference spectrum of cytochrome P-450. Thus it appears that uptake of calcium the interaction of mitochondrial cytochrome P-450 with endogenous substrate.

Genetics of cytochrome P450 in two insecticide-resistant strains of the housefly, Musca domestica L.

A susceptible strain of Musca domestica containing visible mutant markers on chromosomes II, III, and V was crossed with multiresistant R-Fc and R-diazinon strains. F1 flies were backcrossed to the mutant parent, resultant progenies were isolated according to phenotype, and substrains were established. The level of resistance to diazinon, aldrin epoxidase activity, and cytochrome P450 difference spectra of microsomes from each substrain were measured. Titers of cytochrome P450, measured as CO spectra, as well as type I, type II, and type III cytochrome P450 substrate difference spectra were compared in microsomal preparations obtained from phenotypes containing vatious resistant chromosome combinations. In both resistant strains, high levels of cytochrome P450 were controlled by a gene(s) on chromosome II. In R-diazinon, qualitative spectral changes were also controlled by chromosome II, whereas in R-Fc both chromosomes II and V contributed to qualitative changes in cytochrome P450. Both quantitative and qualitative characteristics were intermediate in heterozygous flies, suggesting incomplete dominance for their inheritance. Findings are discussed in relation to known genetics of microsomal resistance to insecticides.

Cytochrome P-450 difference spectra of microsomes from several insecticide-resistant and -susceptible strains of the housefly, musca domestica L.

Cytochrome P-450 difference spectra were obtained with microsomes prepared from several strains of the housefly, Musca domestica, that were susceptible or resistant to insecticides. Based on Type I, Type II and Type III spectral interactions, both quantitative and qualitative variations are apparent among strains. Cytochrome P-450 from Fc and dimethoate strains had spectral characteristics which were most different from those of susceptible strains. No correlation could be made between strains having known high mixed-function oxidase activity and any single cytochrome P-450 spectral characteristic.

The production and modification of cytochrome P-450 difference spectra by in vivo administration of methylenedioxyphenyl compounds

The in vivo administration of piperonyl butoxide (PB) or propyl isome (PI), both methylenedioxyphenyl compounds, to mice affects the levels of several hepatic cytochrome P-450 spectral interactions. The difference spectrum produced by carbon monoxide (commonly employed as a measurement of cytochrome P-450 levels) is initially lowered in magnitude by both compounds. The reduction is followed by an increase with time indicative of cytochrome induction. This biphasic effect, when produced by piperonyl butoxide, is accompanied by specific changes in the levels of the cytochrome P-450 difference spectra produced by hexobarbital, a type-I substrate, and pyridine, a type-II substrate. In addition, the ethyl isocyanide (EtNC) equilibrium point, calculated from the pH effect on the 455-nm and 430-nm peaks of the ethyl isocyanide-cytochrome P-450 difference spectrum, undergoes a biphasic shift. In contrast, propyl isome has the same effect on the substrate difference spectra as it does on the cytochrome level and produces no change in the ethyl isocyanide equilibrium point with time. Piperonyl butoxide, administered in vivo, produces a cytochrome P-450 difference spectrum when dithionite-reduced microsomes from treated animals are compared at different pH values. This spectrum, when observed by comparing microsomes from treated and untreated animals, has two pH-dependent Soret peaks and is essentially identical to the NADPH-dependent piperonyl butoxide-cytochrome P-450 spectrum produced by in vitro treatment. This spectrum is also produced in vitro by propyl isome and sulfoxide.