Multiple forms of cytochrome P-450 in rabbit colon microsomes

Three cytochrome P-450 preparations, designated as cytochrome P-450ca, cytochrome P-450cb, and cytochrome P-448c fraction, were separated and purified about 23-, 50-, and 29-fold, respectively, from the cholate extracts of rabbit colon mucosa microsomes. Their specific contents were 1.2, 2.6, and 1.5 nmol of cytochrome P-450 per mg of protein, respectively. Cytochrome P-450ca and cytochrome P-450cb migrated as heme-containing polypeptide bands with molecular weights of about 53,000 and 57,000, respectively, on SDS-polyacrylamide gel electrophoresis. The CO-reduced difference spectra of cytochrome P-450ca, cytochrome P-450cb, and cytochrome P-448c fraction showed maxima at 451, 450, and 449 nm, respectively. Cytochrome P-450ca efficiently catalyzed the omega-hydroxylation of prostaglandin A1 (PGA1) and the omega- and (omega-1)-hydroxylation of caprate, laurate, and myristate in the reconstituted system containing cytochrome P-450ca, NADPH-cytochrome P-450 reductase, cytochrome b5, and phosphatidylcholine. In contrast, cytochrome P-450cb and cytochrome P-448c fraction had no detectable activity toward PGA1 and fatty acids. Both catalyzed aminopyrine and benzphetamine N-demethylation. Cytochrome P-448c fraction also hydroxylated benzo(a)pyrene, and phosphatidylinositol or phosphatidylserine exhibited a stimulatory effect on this activity. The results show that rabbit colon microsomes contain catalytically different cytochrome P-450, one of which is specialized for the omega-oxidation prostaglandins, the others being involved in the metabolism of exogenous compounds such as drugs and polycyclic hydrocarbons.     

Magnetic circular dichroism studies on microsomal aryl hydrocarbon hydroxylase: comparison with cytochrome b-5 and cytochrome P-450-cam.

Magnetic circular dichroism spectra are reported for the visible and near ultraviolet spectral regions of liver microsomes from dimethylbenzanthracene-treated rats. The sequential addition of NADH, dithionite, and carbon monoxide enables us to determine contributions to the magnetic circular dichroism by cytochromes b-5 and P-450, which dominate the spectra. The magnetic circular dichroism of the microsomal preparation is compared with that of purified oxidized and reduced cytochrome -b-5 from pig liver and with the camphor-complexed and camphor-free oxidized, reduced, and reduced carbonmonoxy cytochrome P-450-cam from Pseudomonas putida. The magnetic circular dichroism spectra of the membrane bound cytochrome -b-5 are similar to those of the purified protein, indicating that little or no alteration in the environment of the heme occurs during the isolation procedure. The soluble bacterial cytochrome P-450 also appears to be a suitable model for microsomal P-450, although differences in the magnetic circular dichroism intensity are observed for the two enzymes. No effect of dimethylbenzanthracene on the magnetic circular dichroism spectra of induced compared to control rat microsomes could be observed.     

Multiple forms of cytochrome P-450 in liver microsomes from beta-naphthoflavone-pretreated rats. Separation, purification, and characterization of five forms

Five forms of cytochrome P-450 have been purified from liver microsomes of beta-naphthoflavone-pretreated rats by chromatography on DEAE-Sephadex, DEAE-cellulose, and hydroxylapatite or CM-Sepharose columns. Over 50% of the starting cytochrome P-450 content can be accounted for in these five forms after resolution on the DEAE-cellulose column, and after further purification, the combined total recovery is 30%. The five forms have the following Mr: 47,000, 50,500, 51,500, 53,500, and 56,500. The absorption maxima in reduced carbon monoxide difference spectra are 452.5, 449, 449, 447.5, and 447.5 nm, respectively. Antibody has been prepared in rabbits to each of the five forms; each antibody reacts with the antigen for which it was prepared, but not with the other four heterologous antigens. In addition, each form gives a unique peptide map pattern when partially digested with Staphylococcus aureus V-8 protease and electrophoresed in sodium dodecyl sulfate gels. Each form also shows an individual pattern of catalytic activities when tested with benzphetamine, ethylmorphine, p-nitroanisole, benzo[alpha]pyrene, and 7-ethoxycoumarin as substrates. By all criteria examined, these five forms appear to be distinct forms of cytochrome P-450.     

Multiple forms of cytochrome P-450. Characteristics of isolated aminopyrine-N-demethylase (cytochrome P-450AP)

A previously unidentified cytochrome P-450AP possessing the highest aminopyrine-N-demethylase activity has been isolated from liver microsomes of 4-isopropylaminoantipyrine-induced rats, using affinity chromatography in combination with ion-exchange chromatography with subsequent separation on hydroxyl apatite. Using radioisotope techniques, it was found that 4-isopropylaminoantipyrine induces cytochrome P-450AP synthesis de novo. The isolated cytochrome P-450AP has the following characteristics: Mr = 49,000 Da. CO-peak maximum at 450.5 mm, rate of aminopyrine demethylation in a reconstituted system-20 nmol HCHO/min/nmol of cytochrome P-450, benzphetamine-15. The hemoprotein synthesis is paralleled with the synthesis of a protein with Mr of 51,000 Da. Immunochemical analysis permitted to identify the latter protein as cytochrome P-450b. It was demonstrated that cytochrome P-450AP does not interact with the antibodies to the major phenobarbital-induced form, i.e., with cytochrome P-450b.     

Monoclonal antibody-directed phenotyping of cytochrome P-450-dependent aryl hydrocarbon hydroxylase and 7-ethoxycoumarin deethylase in mammalian tissues

The distribution of cytochromes P-450 that catalyze aryl hydrocarbon hydroxylase and 7-ethoxycoumarin O-deethylase were studied with monoclonal antibody (MAb) 1-7-1 which completely inhibits these activities of a purified 3-methylcholanthrene-induced rat liver cytochrome P-450. The degree of inhibition by MAb 1-7-1 quantitatively assesses the contribution of different cytochromes P-450 in the liver, lung, and kidney microsomes from untreated, 3-methylcholanthrene- and phenobarbital (PB)-treated rats, mice, guinea pigs, and hamsters. Enzyme sensitivity to MAb 1-7-1 inhibition defines two types of cytochrome P-450 contributing to aryl hydrocarbon hydroxylase and 7-ethoxycoumarin O-deethylase. The MAb 1-7-1-sensitive cytochrome P-450 is a major contributor to aryl hydrocarbon hydroxylase in rat liver, lung, and kidney of 3-methylcholanthrene-treated rats, C57BL/6 mice, guinea pigs, and hamsters; this type is also present in lesser amounts in the extrahepatic tissues of the control and PB-treated animals, and in the lungs of the relatively "noninducible" DBA/2 mice treated with 3-methylcholanthrene. This form however makes little or no contribution to liver aryl hydrocarbon hydroxylase of control or PB-treated animals. 7-Ethoxycoumarin O-deethylase is also a function of both the MAb 1-7-1-sensitive and insensitive classes of cytochrome P-450. The ratio of the classes contributing to aryl hydrocarbon hydroxylase and 7-ethoxycoumarin O-deethylase differs in the various tissues and species and after inducer treatment. All of the 7-ethoxycoumarin O-deethylase activity in guinea pigs and hamsters is a function of cytochromes P-450 different than the MAb 1-7-1-sensitive cytochrome P-450 responsible for aryl hydrocarbon hydroxylase activity. Thus, the MAb 1-7-1 antigenically defines the type of cytochromes P-450 contributing to each reaction. Cytochromes P-450 can be viewed as paradigmatic for enzyme systems in which the nature and amount of product is regulated by multiple isoenzymic forms. Analyses using monoclonal antibodies to specific isoenzymes may thus have broad application to a variety of other complex systems which are composed of multiple isoenzymes.     

Separation of two forms of cytochrome P-450 with aryl hydrocarbon hydroxylase activity from intestinal mucosa microsomes of rabbits treated with 3-methylcholanthrene

Two forms of cytochrome P-450, designated P-448a and P-448b, were purified from intestinal mucosa microsomes of rabbits treated with 3-methylcholanthrene. Both the cytochromes had absorption maxima at 448 nm in the carbon monoxide-reduced difference spectra. They exhibited comparable catalytic activities with benzo(a)pyrene, 7-ethoxycoumarin, and 7-ethoxyresorufin, when reconstituted with hepatic NADPH-cytochrome c reductase and phosphatidylserine. P-448a was apparently homogeneous on sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and its monomeric molecular weight was estimated to be 58,000. The oxidized form had absorption maxima at 416, 512 and 571 nm, indicative of the low spin state. Thus P-448a appeared to be similar to one form of P-450, which was induced in rabbit liver by 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). On the other hand, SDS-PAGE of P-448b gave a single major protein band with a monomeric molecular weight of 55,500, indicating that P-448b can be distinguished from P-448a.     

Differential expression of cytochrome P-450 1 and related forms in rabbit liver and kidney

Monoclonal antibodies developed to cytochrome P-450 1, some of which react with proteins in addition to P-450 1, were used to investigate the differential expression of P-450 1 dependent 21-hydroxylase activity in renal tissue of rabbits exhibiting differences in hepatic 21-hydroxylase activity. Using immunohistochemical techniques, the monoclonal antibodies, 2F5 and 3C3, localized protein in the S2 and S3 segments of the proximal tubule in the renal cortex. These two monoclonal antibodies, 2F5 and 3C3, reacted with a kidney protein that migrated on sodium dodecyl sulfate-polyacrylamide gel electrophoresis with a relative electrophoretic mobility that did not correspond to known rabbit hepatic isozymes and was termed P-450 K. Antibodies specific for P-450 1 and 3b, 1F11 and 8-27, respectively, produced no staining in kidney. The protein recognized by the 2F5 and 3C3 antibodies is immunologically distinct from cytochrome P-450s 1, 2, and 3b. The rate of 21-hydroxylation of progesterone was shown to be approximately 100-fold less in kidney than liver microsomes where this pathway is largely catalyzed by P-450 1. The activity of the kidney microsomes was not inhibited by antibodies directed to P-450 1. In addition, the variation observed for the 21-hydroxylase activity in the hepatic microsomal fraction of outbred New Zealand white rabbits was not evident in kidney microsomes from these same animals. The 2F5 antibody was found, however, to be inhibitory (about 50%) of the 11-hydroxylation of lauric acid in kidney microsomes. This suggests that P-450 K participates in lauric acid 11-hydroxylase activity. The treatment of rabbits with phenobarbital, but not 2,3,7,8-tetrachlorodibenzo-p-dioxin, was found to induce the levels of P-450 K.     

Beta-naphthoflavone induction of a cytochrome P-450 arachidonic acid epoxygenase in chick embryo liver distinct from the aryl hydrocarbon hydroxylase and from phenobarbital-induced arachidonate epoxygenase.

Cytochrome P-450-mediated arachidonic acid metabolism in chick embryo liver microsomes was increased by both Ah receptor-dependent (2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) and beta-naphthoflavone) and independent (phenobarbital) P-450 inducers. Arachidonic acid epoxides and monohydroxyeicosatetraenoic acids were increased 9-12-fold. omega-1-OH arachidonic acid was also significantly increased by TCDD and beta-naphthoflavone while omega-OH arachidonic acid, the main metabolite in uninduced livers, was decreased by all three agents. The P-450s catalyzing the enhanced arachidonate metabolism in beta-naphthoflavone- and phenobarbital-treated liver were investigated in reconstituted systems containing wholly or partially purified P-450s. beta-Naphthoflavone induced formation of a 55-kDa P-450 selective for arachidonate metabolism and for epoxygenation in particular. This P-450 was purified (beta NFAA). It was found to be distinct from a 54.5-kDa beta-naphthoflavone-induced P-450 catalyzing aryl hydrocarbon hydroxylase and 7-ethoxyresorufin deethylase (designated NF1). Mean turnover numbers for arachidonate epoxygenase, aryl hydrocarbon hydroxylase, and 7-ethoxyresorufin deethylase were 11.2, 0.56, and 0.04, respectively, for reconstituted beta NFAA and 0.33, 11.8, and 2.4 for NF1. beta NFAA and NF1 also differed in chromatography elution characteristics and N-terminal amino acid sequences. Both were low spin, with carbon monoxide binding peaks at 448 nm. The phenobarbital-induced arachidonate epoxygenation was catalyzed by P-450 fractions containing the main 48- and 49-kDa phenobarbital-induced P-450s; fractions in which the 49-kDa P-450 predominated were the most active. Turnover numbers for arachidonic acid epoxygenation were not correlated with those for aminopyrine demethylation or 7-ethoxycoumarin deethylation for P-450s from phenobarbital-treated livers or with aryl hydrocarbon hydroxylase, 7-ethoxyresorufin deethylase, or 7-ethoxycoumarin deethylase for P-450s from beta-naphthoflavone-treated livers. Also, different P-450s catalyzed the epoxygenation and the omega-hydroxylation of arachidonic acid in both beta-naphthoflavone- and phenobarbital-treated livers. The findings support a physiologic role for P-450-induced arachidonate metabolism and provide a basis for a possible link between TCDD's induction of P-450 and alterations of cellular homeostasis.     

Simultaneous purification of multiple forms of rat liver microsomal cytochrome P-450 by high-performance liquid chromatography

14 microsomal cytochromes P-450 were purified from the liver of untreated and phenobarbital- or 3-methylcholanthrene-treated male rats. Following solubilization of microsomes with sodium cholate, poly(ethylene glycol) fractionation and aminohexyl-Sepharose 4B chromatography, cytochromes P-450 were purified by high-performance liquid chromatography (HPLC), using a preparative DEAE-anion-exchange column. The pass-through fraction was further purified by HPLC using a cation-exchange column. Other fractions eluted on preparative DEAE-HPLC were further applied onto an HPLC using a DEAE-column. Five kinds (P-450UT-2-6), four kinds (P-450PB-1,2,4 and 5) and five kinds (P-450MC-1-5) of cytochromes P-450 were purified from untreated rats or rats treated with phenobarbital or 3-methylcholanthrene, respectively. HPLC profiles of tryptic peptides of cytochromes P-450UT-2 and P-450MC-2 were identical and the other profiles obtained from seven purified cytochromes P-450 were distinct from each other. Amino-terminal sequences of eight forms of cytochrome P-450 (UT-2, UT-5, PB-1, PB-2, PB-4, PB-5, MC-1 and MC-5) were distinct except for cytochromes P-450PB-4 and P-450PB-5.     

Resolution of two forms of cytochrome P-450 from liver microsomes of rabbit treated with 2,3,7,8-tetrachlorodibenzo-p-dioxin.

Two forms of cytochrome P-450 with different substrate specificities were isolated from liver microsomes of rabbits treated with 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). A specific antibody was produced toward the major form of the cytochrome. The antibody inhibits microsomal acetanilide hydroxylation (80%). It does not cross-react with the minor fraction of the cytochrome or inhibit the hydroxylation of 3,4-benzpyrene or coumarin, the N-demethylation of aminopyrine or the O-deethylation of 7-ethoxycoumarin catalyzed by rabbit liver microsomes. The major form has an estimated Mr = 54,000 and displays an n-octylamine difference spectrum with an absorption maximum at 426 nm and a minimum at 391 nm. When reconstituted, this cytochrome catalyzes acetanilide hydroxylation at a higher rate than microsomes or the minor fraction. The n-octylamine difference spectrum of the minor fraction displays an absorption maximum at 431 nm and a minimum at 410 nm. When reconstituted, this fraction catalyzes the hydroxylation of 3,4-benzpyrene and the O-deethylation of 7-ethoxycoumarin. The two cytochromes appear to be distinct entities and function in different catalytic pathways.     

Hepatic mitochondrial cytochrome P-450 system. Purification and characterization of two distinct forms of mitochondrial cytochrome P-450 from beta-naphthoflavone-induced rat liver

We have purified two distinct isoforms of mitochondrial cytochrome P-450 from beta-naphthoflavone (beta-NF)-induced rat liver to greater than 85% homogeneity and characterized their molecular and catalytic properties. One of these isoforms showing an apparent molecular mass of 52 kDa is termed P-450mt1 and the second isoform with 54-kDa molecular mass is termed P-450mt2. Cytochrome P-450mt2 comigrates with similarly induced microsomal P-450c (the major beta-NF-inducible form) on sodium dodecyl sulfate-polyacrylamide gels and cross-reacts with polyclonal antibody monospecific for cytochrome P-450c. Cytochrome P-450mt2, however, represents a distinct molecular species since it failed to react with a monoclonal antibody to P-450c and produced V8 protease fingerprints different from P-450c. Cytochrome P-450mt1, on the other hand, did not show any immunochemical homology with P-450c or P-450mt2 as well as partially purified P-450 from control mitochondria. Electrophoretic comparisons and Western blot analysis show that both P-450mt1 and P-450mt2 are induced forms not present in detectable levels in control liver mitochondria. A distinctive property of mitochondrial P-450mt1 and P-450mt2 was that their catalytic activities could be reconstituted with both NADPH-cytochrome P-450 reductase as well as mitochondrial specific ferredoxin and ferredoxin reductase electron transfer systems, while P-450c showed exclusive requirement for NADPH-cytochrome P-450 reductase. Cytochromes P-450mt1 and P-450mt2 were able to metabolize xenobiotics like benzo(a)pyrene and dimethyl benzanthracene at rates only one-tenth with cytochrome P-450c. Furthermore, P-450mt1, P-450mt2, as well as partially purified P-450 from control liver, but not P-450c, showed varying activities for 25- and 26-hydroxylation of cholesterol and 25-hydroxylation of vitamin D3. These results provide evidence for the presence of at least two distinct forms of beta-NF-inducible cytochrome P-450 in rat hepatic mitochondria.     

Structural analysis of cloned cDNAs for polycyclic hydrocarbon-inducible forms of rabbit liver microsomal cytochrome P-450

Two cDNA clones, pHPah1 and pHPah2, encoding polycyclic hydrocarbon-inducible forms of rabbit liver microsomal cytochrome P-450 were isolated and their nucleotide sequences were determined. The inserts of pHPah1 and pHPah2 contained open reading frames specifying the entire primary structures of cytochrome P-450s, consisting of 518 and 516 amino acid residues, respectively. The deduced amino acid sequences for pHPah1 and pHPah2 are 76 and 73% homologous with rat P-450c and P-450d, respectively, and 96% homologous with rabbit P-450 forms 6 and 4, respectively. We conclude that pHPah1 and pHPah2 encode the rabbit counterparts of rat P-450c and P-450d, respectively. A region highly conserved in all species of cytochrome P-450 so far examined, called the HR2 region, can be detected in the pHPah1 and pHPah2 primary structures, but another conserved region, HR1, cannot be observed. Northern hybridization analysis of total RNAs from livers of untreated and drug-treated rabbits demonstrated that the pHPah1 and pHPah2 genes are expressed in untreated animals, induced considerably by administration of 3-methylcholanthrene or beta-naphthoflavone, and suppressed by phenobarbital and isosafrole.