Immunocytochemical localization of cytochrome P-450scc in cultured rat oligodendrocytes

Primary cultures of glial cells from newborn rat forebrain were tested after 3 to 4 weeks. Oligodendrocytes and astrocytes were characterized by immunofluorescence with monoclonal antibodies to galactocerebroside and glial fibrillary acidic protein, respectively. The cytoplasm of oligodendrocytes was specifically and intensely immunostained with monospecific polyclonal antibodies to the cytochrome P-450scc involved in the synthesis of pregnenolone from cholesterol. This observation brings additional support to the concept of "neurosteroids".     

Further characterization of RLM2 and comparison with a related form of cytochrome P-450, RLM2b

We have extended the characterization of RLM2, a constitutive form of rat liver cytochrome P-450, using immunochemical means to quantitate its presence in microsomes, to follow its development in maturing male and female rats, and to determine its response to prototypical P-450 inducers. In addition, RLM2 is compared to RLM2b, a form of P-450 with similar migration on SDS-PAGE and NH2-terminal amino acid sequence. RLM2b is expressed in both sexes at a level of 0.08 nmol/mg microsomal protein at 2 weeks of age. In female rats, this level is unchanged with maturation. However, in the male, the level declined with maturation to reach 0.02 nmol/mg protein by 12 weeks of age. RLM2 is a male-specific form of cytochrome P-450. Originally absent in the 2-week-old rat, it reached a level of 0.03 nmol/mg protein in the adult male, its appearance and increase coinciding with the onset of puberty. Both phenobarbital and 3-methylcholanthrene induced microsomal levels of RLM2b in the adult male and female rat. RLM2, however, was suppressed in the male rat, 58 and 42%, respectively, by the same treatments. RLM2b and RLM2 each catalyze a unique spectrum of hydroxytestosterone metabolites. RLM2b is highly site specific. In contrast, RLM2 produces several isomeric products in the same region of the testosterone molecule. Substitution of the acetyl group of progesterone for the 17-hydroxy group of testosterone did not alter the site specificity of RLM2b, but did alter it for RLM2, indicating, further, a difference in the active site conformation of the two enzymes. Although RLM2b and RLM2 responded differently to inducers and to a changing physiology during maturation, and were functionally quite distinct, the proteins showed a high degree of immunologic relatedness which is suggestive of significant structural similarities. Structural differences do exist, however, as alpha-chymotryptic digestion formed a number of peptide fragments that differed between the two proteins.     

Purification and characterization of a new form (RLM2) of liver microsomal cytochrome P-450 from untreated rat

A new cytochrome P-450 isozyme (RLM2) has been purified to electrophoretic homogeneity from liver microsomes of the untreated rat. It has an apparent minimum molecular weight on sodium dodecyl sulfate-polyacrylamide gel electrophoresis of 49,000. Absolute spectrum of the oxidized form indicates that this isozyme is essentially all in the low spin state. The maximum of the reduced CO complex is at 449 nm. Amino-terminal partial amino acid sequence and amino acid composition are different from those of RLM3 and RLM5, two other native forms of cytochrome P-450 previously reported from this laboratory as well as other forms reported in the literature. RLM2 is capable of oxidizing a variety of drug substrates, like benzphetamine and aminopyrine, and to a lesser extent ethoxycoumarin. With the steroid substrate multiple isomeric products are formed differentially. Progesterone is preferentially hydroxylated at the 15-position (15 beta-hydroxylation (34%) and 15 alpha-hydroxylation (13%) of the total) and at the 6 beta-position (21%). The major metabolite when testosterone was the substrate, 15 alpha-hydroxytestosterone, comprised 43% of the total, while a modest amount of 6 beta-hydroxytestosterone (12%) is formed. Another major metabolite (31%) has yet to be unequivocally identified, but is suggested to be 7 beta-hydroxytestosterone. Examination of the substrate dependence of major and minor isomeric metabolites provides evidence for a single substrate-binding site on RLM2. Regardless of the position hydroxylated, a common Km value was obtained. It is suggested that differences in formation of the isomeric and epimeric products relate to differences in distance from the active oxygen center and the position of attack.     

Testosterone metabolism by cytochrome P-450 isozymes RLM3 and RLM5 and by microsomes. Metabolite identification

Testosterone metabolism by cytochrome P-450 isozymes RLM3 and RLM5 in a reconstituted system and by rat liver microsomes was examined. Eleven metabolites were detected. Two of these, found in spots 2 and 4 of a thin layer plate, were only formed by the rat liver microsomes and may represent reductive metabolites of testosterone. A number of monohydroxy metabolites were conclusively identified by gas chromatography-mass spectrometry. These include the 2-, 6 beta-, 7 alpha-, and 16 alpha-hydroxy isomers. Liver microsomes formed the 2 alpha- and 2 beta-epimers in a 1:2 ratio and both co-chromatographed with a third reduced metabolite in thin layer plate spot 4. In contrast with RLM5 about 90% of the 2-hydroxy isomer was the 2 alpha-epimer. RLM3 did not perform the 2-hydroxylation in detectable amounts. The 6 beta-isomer was a major metabolite of RLM3 and microsomes, but a minor product of metabolism by RLM5. In contrast, the 7 alpha-isomer was a minor metabolite of RLM3, was not formed by RLM5, and was a major microsomal metabolite. Hydroxylation at position 16 alpha was a major activity of RLM5 and the heterogeneous microsomal cytochromes, but with RLM3 it was a minor reaction. One new metabolite was found which appeared to be hydroxylated in the D-ring, had a mass spectrum different from both 16 alpha- and 16 beta-hydroxytestosterone, and was tentatively identified as a 15-hydroxy isomer. In agreement with the literature, androstene-3,17-dione was found to be an oxidative metabolite of testosterone by both microsomes and purified cytochrome P-450. It was a major metabolite of RLM5 but was not produced by RLM3. Studies with 18O2 and H218O conclusively show that oxidation of testosterone at C-17 does not involve transient incorporation of an oxygen atom in this position. A mechanism is suggested whereby cytochrome P-450 acts as a peroxidase in the formation of androstenedione.     

Immunochemical similarity of P-450 HFLa, a form of cytochrome P-450 in human fetal livers, to a form of rat liver cytochrome P-450 inducible by macrolide antibiotics

A protein immunochemically related to P-450 HFLa, a form of cytochrome P-450 purified from human fetal livers, was detected in rat liver microsomes. The content of the immunoreactive protein in rat liver microsomes was increased by treatments with phenobarbital, pregnenolone 16 alpha-carbonitrile (PCN), erythromycin, erythromycin estolate, and oleandomycin but not with 3-methylcholanthrene, imidazole, ethanol, isosafrole, josamycin, midecamycin, or miocamycin. The activity of erythromycin N-demethylase correlated with the content of the immunoreactive protein in rat liver microsomes (r = 0.72). In addition, anti-P-450 HFLa IgG inhibited erythromycin N-demethylase in liver microsomes from erythromycin- or oleandomycin-pretreated rats. Furthermore, the content of the immunoreactive protein highly correlated with that of P-450 PB-1, which is distinct from Waxman's terminology, and is one of the forms of PCN-inducible cytochrome P-450s (r = 0.95). From these results and the results reported so far, it seems possible that P-450 HFLa is one of the forms of cytochrome P-450 inducible by glucocorticoids.     

Responses to insulin by two forms of rat hepatic microsomal cytochrome P-450 that undergo major (RLM6) and minor (RLM5b) elevations in diabetes

Total cytochrome P-450 levels rise in diabetic rats. Two specific forms of cytochrome P-450 that are elevated have been isolated from liver microsomes of streptozotocin-induced idabetic male rats. One enzyme, termed RLM6, metabolizes aniline and acetol, but not testosterone, in a reconstituted system with NADPH-cytochrome P-450 reductase. RLM6 is isolated as a high spin cytochrome with a minimum molecular weight of 53,500. It has a unique amino-terminal amino acid sequence lacking methionine at the amino-terminal position. Polyclonal antibodies to RLM6 recognized most other forms of cytochrome P-450 in Western blots, but could be made monospecific by adsorption to cross-reacting proteins coupled to Sepharose 4B. Using the monospecific antibodies, RLM6 was estimated to be present in microsomes of untreated male rats at 0.04 nmol/mg protein (5% of total P-450). In chronically diabetic rats this level rose to 0.35 nmol/mg protein and 24% of the P-450 content. Immunoreactive protein of molecular weight identical to RLM6 was elevated in microsomes of non-diabetic rats treated with ethanol, acetone, or isoniazid as well as in rats starved for 48 h. Insulin treatment of diabetic rats for 1 week lowered the immunologically detectable levels of RLM6 to levels found in the untreated rat. The other form of cytochrome P-450, RLM5b, does not metabolize aniline and only poorly metabolizes acetol and testosterone. This 52.5-kDa protein is isolated as a predominantly (60%) high spin enzyme. It has a unique NH2-terminal amino acid sequence with methionine as the terminal residue, and is present in untreated male rat liver microsomes at 0.16 nmol/mg protein. It is elevated in diabetes, like RLM6, but treatment with insulin for 1 week does not completely restore the microsomal content to that of the non-diabetic rat.     

Aromatization and 19-hydroxylation of androgens by rat brain cytochrome P-450

The oxidative metabolism of androgens in the rat brain includes aromatization preceded by the requisite 19-hydroxylation. We have examined the transformation of [19-C3H3]androstenedione and [4-14C]testosterone by the semipurified cytochrome P-450 fraction of the rat brain. [19-C3H3]Androstenedione generated tritiated water and formic acid in a ratio of 8 to 1 indicating that 19-hydroxylation in the brain far exceeds that necessary for aromatization. This was confirmed by the results of the 14C-testosterone incubation in which the 14C labeled 19-hydroxy and 19-oxo derivatives which were isolated exceeded the yield of 14C-estrogens by several fold. Thus the rat brain has the capacity to form in situ 19-hydroxylated androgens which are not available to it from the circulation.     

Heterogeneity of cytochrome P-450 in rat testis microsomes.

Cytochrome P-450 appears to be a component of the steroid-coverting enzymes, 17alpha-hydroxylase and 17,20-lyase, which catalyze sequential steps in sex hormone synthesis. Further evidence indicates that the steroid substrates of these enzymes bind to cytochrome P-450 during catalysis. The present report deals with the problem of whether a single form of cytochrome P-450 mediates both enzyme reactions or whether two enzymes are involved. Both activities are competitively inhibited by a number of the same inhibitors. Because K1 values of competitive inhibitors are dissociated constants, and thus a property of the cytochrome, different magnitudes of K1, determined for the same inhibitor with each enzyme, are consistent with the participation of more than one form of cytochrome P-450. Differences in the K1 values were found to be statistically significant and varied from 3- to 10-fold. Two competitive inhibitors retarded velocities with one reaction but not the other. In addition, the enzyme activities were markedly different in their sensitivity to carbon monoxide inhibition. The conclusion based on these two lines of evidence is that separate enzymes and different forms of cytochrome P-450 are involved in each reaction.     

Gene structure of a major form of phenobarbital-inducible cytochrome P-450 in rat liver

The gene structure of cytochrome P-450b, a major form of phenobarbital-inducible cytochrome P-450 in rat livers was elucidated by sequence analysis of the cloned genomic DNAs and was compared with the previously determined gene structures of cytochrome P-450e, a minor form of phenobarbital-inducible cytochrome P-450 and two forms of 3-methylcholanthrene-inducible cytochrome P-450 (P-450c and -d). The gene for cytochrome P-450b is 23 kilobase pairs (kb) long and is separated into 9 exons by 8 intervening sequences. This gene structure is very similar to that of cytochrome P-450e except for the first intron, the first intron being much longer in cytochrome P-450b gene (approximately 12 kb) than in cytochrome P-450e gene (3.2 kb), but differs greatly from the gene structures of two 3-methylcholanthrene-inducible cytochrome P-450s as pointed out previously (Sogawa, K., Gotoh, O., Kawajiri, K. & Fujii-Kuriyama, Y. (1984) Proc. Natl. Acad. Sci. U.S.A. 81, 5066-5070). The nucleotide sequences in all 9 exons and their flanking regions in introns show very close homology between the two phenobarbital-inducible cytochrome P-450 genes. Forty base substitutions are found in approximately 1900 nucleotides of all exonic sequences, and 15 of them result in 14 amino acid replacements. These base substitutions occur in relatively limited regions of the gene sequences. Most of them are found in exons 6, 7, 8, and 9, most frequently in exon 7 as described previously (Mizukami, Y., Sogawa, K., Suwa, Y., Muramatsu, M. & Fujii-Kuriyama, Y. (1983) Proc. Natl. Acad. Sci. U.S.A. 80, 3958-3962). The close sequence homology between the two phenobarbital-inducible cytochrome P-450 genes is also found to extend to the promoter region with one notable exception. The simple repeated sequences of (CA)n which is present at -254 position in cytochrome P-450e gene is also observed at the equivalent position in cytochrome P-450b gene, but the repetitiveness is greatly reduced in cytochrome P-450b gene ((CA)5 for P-450b versus (CA)19 for P-450e), and this may somehow be related to the difference in the level of cytochrome P-450b and P-450e in the inductive phase of phenobarbital administration.     

Metyrapone - reduced cytochrome P-450 complex: A specific method for the determination of the phenobarbital inducible form of rat hepatic microsomal cytochrome P-450

Using homogeneous cytochrome P-450, we have shown that the well-known metyrapone-dithionite reduced cytochrome P-450 complex is specific for the cytochrome P-450b induced by phenobarbital. A linear relationship was observed between the absorbance of metyrapone-reduced cytochrome P-450 complex and the one of CO-reduced cytochrome P-450 complex, the usual method for the determination of cytochrome P-450. A method has been proposed for the specific determination of the cytochrome P-450b.     

Rotation of cytochrome P-450. II. Specific interactions of cytochrome P-450 with NADPH-cytochrome P-450 reductase in phospholipid vesicles

Purified rat liver microsomal cytochrome P-450 and NADPH-cytochrome P-450 reductase were co-reconstituted in phosphatidylcholine-phosphatidylethanolamine-phosphatidylserine vesicles using a cholate dialysis technique. The co-reconstitution of the enzymes was demonstrated in proteoliposomes fractionated by centrifugation in a glycerol gradient. The proteoliposomes catalyzed the N-demethylation of a variety of substrates. Rotational diffusion of cytochrome P-450 was measured by detecting the decay of absorption anisotropy r(t), after photolysis of the heme.CO complex by a vertically polarized laser flash. The rotational mobility of cytochrome P-450, when reconstituted alone, was found to be dependent on the lipid to protein ratio by weight (L/P450) (Kawato, S., Gut, J., Cherry, R. J., Winterhalter, K. H., and Richter, C. (1982) J. Biol. Chem. 257, 7023-7029). About 35% of cytochrome P-450 was immobilized and the rest was rotating with a mean rotational relaxation time phi 1 of about 95 mus in L/P450 = 1 vesicle. In L/P450 = 10 vesicles, about 10% of P-450 was immobile and the rest was rotating with phi 1 congruent to 55 mus. Co-reconstitution of equimolar amounts of NADPH-cytochrome P-450 reductase into the above vesicles results in completely mobile cytochrome P-450 with a phi 1 congruent to 40 mus. Only a small decrease in the immobile fraction of cytochrome P-450 is observed when the molar ratio of cytochrome P-450 to the reductase is 5. The results suggest the formation of a monomolecular 1:1 complex between cytochrome P-450 and NADPH-cytochrome P-450 reductase in the liposomes.     

Hepatic mitochondrial cytochrome P-450 system. Identification and characterization of a precursor form of mitochondrial cytochrome P-450 induced by 3-methylcholanthrene

Hepatic mitoplasts from 3-methylcholanthrene-treated rats contain cytochrome P-450 which can metabolize polycyclic aromatic hydrocarbons like benzo(a)pyrene. Mitochondrial cytochrome P-450 was partially purified and reconstituted in vitro using adrenodoxin and the adrenodoxin reductase electron transfer system and [3H]benzo(a)pyrene as the substrate. A polyclonal antibody to purified microsomal P-450c (a major 3-methylcholanthrene-inducible form) inhibited the activity of mitochondrial enzyme in a concentration-dependent manner and also reacted with a 54-kDa protein on the immunoblots. A monoclonal antibody having exclusive specificity for P-450c, on the other hand, did not inhibit the aryl hydrocarbon hydroxylase activity of the mitochondrial enzyme and showed no detectable cross-reaction with the 54-kDa mitochondrial protein. Similarly, two-dimensional analysis and immunodetection using the polyclonal antibody showed distinct molecular properties of the mitochondrial enzyme different from the similarly induced microsomal P-450c with respect to the isoelectric pH. In vitro translation of free polysomes from 3-methylcholanthrene-induced liver, transport of precursor proteins by isolated mitochondria in vitro, and immunoprecipitation with the polyclonal antibody showed the presence of a 57-kDa putative precursor which is transported and processed into mature 54-kDa species. These results present evidence for the true intramitochondrial location of the P-450c-antibody reactive isoform detected in 3-methylcholanthrene-induced rat liver mitochondria.     

Circadian rhythm of rat hepatic cytochrome P-450 reductase.

The activity of cytochrome P-450 reductase was measured in liver microsomes prepared from adult male rats which had been surgically adrenalectomized, pinealectomized, pinealectomized-adrenalectomized, or sham adrenalectomized-pinealectomized and from intact controls. Rats of each class were killed at 1, 4, 6, 10 hours after the beginning of the light period and 1, 4, 6, 10 hours after the lights were turned off (dark period). The activity of cytochrome P-450 reductase shows a significant diurnal variation in the control group with minimum and maximum at 1 and 10 hours after dark, respectively. The rhythm was altered in the animals surgically treated and the average reductase activity was decreased.     

The active form of cytochrome P-450 from bovine adrenocortical mitochondria.

Cytochrome P-450 from bovine adrenocortical mitochondria exists in three forms of molecular weight: 850,000 (protein 16), of one-half (protein 8), and of one-quarter of this value (protein 4). The forms of the enzyme are named according to the number of subunits and all appear to be active in converting cholesterol to 3beta-hydroxy-5-pregnen-20-one (side chain cleavage) (Shikita, M., and Hall, P.F. (1973) J. Biol. Chem. 248, 5606). To determine whether all three forms are active at their characteristic molecular weights, the three cytochromes were each layered onto separate sucrose density gradients and centrifuged at 49,000 rpm for 60 min; the gradients contained all the factors necessary for side chain cleavage including one of the following substrates: cholesterol, 20S-hydroxycholesterol, and 20S,22R-dihydroxycholesterol. Regardless of the form of P-450 layered onto the gradient and regardless of the substrate, enzyme activity (side chain cleavage) was observed only in fractions corresponding to a sedimentation coefficient of 20 to 22 S which is that for protein 16. No activity was observed at S values corresponding to either protein 8 or protein 4. These findings indicate that the active form of cytochrome P-450 from adrenocortical mitochondria is that containing 16 subunits, i.e. the form in which the cytochrome is normally isolated from adrenal mitochondria. Forms consisting of eight and four subunits which can be prepared from protein 16 become active only by forming protein 16, at least in an aqueous medium in vitro.     

Segmental homologies in the coding and 3' non-coding sequences of rat liver cytochrome P-450e and P-450b cDNAs and cytochrome P-450e-like genes

The nucleotide sequence of a cloned cDNA insert carried by pHDQ14 was determined and found to code for the 107 C-terminal amino acids of rat liver cytochrome P-450e. Comparison of the pHQ14 cDNA sequence with those of cloned cDNAs for cytochrome P-450b and of 2 P-450e-like genes revealed segmental homologies that may have resulted from gene conversion. These results suggest that gene conversion may generate sequence variants of genes for rat liver cytochrome P-450s.     

Neonatal imprinting and hepatic cytochrome P-450 II. Partial purification of a sex-dependent and neonatally imprinted form(s) of cytochrome P-450

A new form of cytochrome P-450 was partially purified from hepatic microsomes of neonatally imprinted rats (adult male and adult male castrated at four weeks of age). This new form of cytochrome P-450 appears to have an apparent molecular weight of approximately 50,000 daltons as judged by sodium dodecyl sulfate polyacrylamide gel electrophoresis. It appears that this form of cytochrome P-450 is either absent or present in low concentrations in cytochrome P-450 preparations isolated from neonatally nonimprinted rats (adult female and adult male castrated at birth). Reconstitution of testosterone hydroxylase and benzphetamine N-demethylase activities of this partially purified cytochrome P-450 revealed that the presence of testosterone 16α-hydroxylase activity, an imprintable microsomal enzyme, was in parallel with the imprinting status of the animals; a significantly higher activity was detected in the neonatally imprinted than that of the nonimprinted animals. This was in contrast to the nonimprintable benzphetamine N-demethylase, testosterone 7α-and 6β-hydroxylase activities which exhibited no correlation with the imprinting status of the animals. We have prepared antisera from rabbits using the partially purified cytochrome P-450 preparations from adult male rats as antigens. These antisera inhibited microsomal testosterone 16α- and 7α-hydroxylase activities in a concentration-dependent manner, without impairing 6β-hydroxylase activity. These data suggest that the partially purified cytochrome P-450 from adult male rats consists of both imprintable (16α-) and nonimprintable (7α-) testosterone hydroxylase activities. The antisera formed immunoprecipitant lines in the Ouchterlony double diffusion plates with partially purified cytochrome P-450 from both neonatally imprinted and nonimprinted adult rats. The immunoprecipitant lines, as stained by coomassie blue, suggest the homology of the cytochrome P-450 preparations from neonatally imprinted and nonimprinted rats. Immunoabsorption of the antisera against neonatally nonimprinted, partially purified cytochrome P-450 completely removed the immunoprecipitant lines without appreciably impairing the inhibitory effects of antisera on the microsomal testosterone 16α-and 7α-hydroxylase activities. In contrast, immunoabsorption of the antisera against partially purified cytochrome P-450 from adult male rats (imprinted) abolished completely both the immunoprecipitant lines and the inhibition on microsomal testosterone hydroxylation reaction (16α and 7α). The inhibitory actin of antisera on testosterone hydroxyulation was also abolished upon boiling the antisera at 100°C for 5 minutes. The biochemical and immunochemical data in this study suggest that the neonatally imprintable form or forms of hepatic microsomal cytochrome P-450 accounts for a small fraction of the bulk of total cytochrome P-450. However, the existence of this form of cytochrome P-450 is regulated by gonadal hormones during the neonatal period and accounts for the major imprintable sex difference in drug and steroid metabolism in adulthood.     

Characterization and identification of a pyrazole-inducible form of cytochrome P-450

In vivo administration of the alcohol dehydrogenase inhibitor pyrazole induces a cytochrome P-450 isozyme. The pyrazole-inducible cytochrome P-450 has been purified from rat livers to electrophoretic homogeneity and its biochemical, spectral, and immunological properties characterized. The final preparation had a specific content of 11 nmol of cytochrome P-450/mg of protein. A single band with an apparent molecular weight of 52,000 was observed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The absolute spectrum of the isolated pyrazole cytochrome P-450 displayed peaks at 648 and 396 nm, suggestive of a high spin cytochrome. The ethylisocyanide difference spectrum exhibited two maxima, one at 457 nm, the other at 428 nm. Pyrazole and dimethyl sulfoxide produced binding spectra with the purified P-450, with peaks at 425 or 419 nm and troughs at 390 or 386 nm, respectively. K8 values for dimethyl sulfoxide and pyrazole were 21 and 0.04 mM, respectively. The catalytic activity of the pyrazole cytochrome P-450 was elevated with aniline and dimethylnitrosamine (low Km) but not with aminopyrine, benzphetamine, ethoxycoumarin, or ethoxyresorufin as substrates. An antibody against pyrazole cytochrome P-450 recognized a 52,000 molecular weight protein upon reaction with saline microsomes. The intensity of the immunoblot was increased when microsomes isolated from pyrazole, 4-methylpyrazole-, acetone-, or chronic ethanol-treated rats were utilized, but not after phenobarbital or 3-methylcholanthrene treatment. Homology at the amino terminus of 19 amino acids was observed between pyrazole P-450 and the isoniazid-inducible P-450j. Based upon the above catalytic, spectral, and immunological properties, it appears that pyrazole induces a form of cytochrome P-450 which is identical to that induced by ethanol and isoniazid.     

Identification of the cytochrome P-450 induced by macrolide antibiotics in rat liver as the glucocorticoid responsive cytochrome P-450p

We administered triacetyloleandomycin (TAO) to rats and found that this macrolide antibiotic is the most efficacious inducer of liver microsomal cytochrome P-450 (P-450) examined to date. Liver microsomes prepared from TAO-treated rats contained greater than 5.0 nmol of P-450/mg of protein and a single induced protein as judged by analysis on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. This protein comigrated with P-450p, the major form of P-450 induced in liver microsomes of rats treated with pregnenolone-16 alpha-carbonitrile (PCN) or dexamethasone (DEX). On immunoblots of such gels developed with antibodies to P-450p, the TAO-induced protein reacted strongly as a single band. There was strict parallelism between the amount of immunoreactive P-450p in liver microsomes prepared from untreated rats or from rats treated with phenobarbital, TAO, DEX, or PCN, the ability of these microsomes to catalyze conversion of TAO to a metabolite which forms a spectral complex, and the ethylmorphine and erythromycin demethylase activities. Antibodies to P-450p specifically blocked microsomal TAO metabolite complex formation and ethylmorphine and erythromycin demethylase activities. Moreover, anti-P-450p antibodies completely immunoprecipitated solubilized TAO metabolite complexes prepared by detergent treatment of liver microsomes obtained from TAO-treated rats. Finally, we found that the major form of P-450 isolated from liver microsomes of TAO-treated rats and purified to homogeneity was indistinguishable from purified P-450p as judged by molecular weights, spectral characteristics, enzymatic activities, ability to bind TAO, peptide maps, and amino-terminal amino acid sequences. We concluded that, in addition to glucocorticoids, macrolide antibiotics are specific inducers of P-450p.