The cDNA and protein sequence of a phenobarbital-induced chicken cytochrome P-450

Several cDNA clones complementary to a chicken phenobarbital-inducible cytochrome P-450 have been isolated and sequenced, representing the first non-mammalian eukaryotic cytochrome P-450 sequence to be analyzed. The cDNA clones hybridized to two mRNAs of 3.5 and 2.5 kilobases in length, but further analysis indicated that the clones were derived from the larger mRNA. The sequence contains a 5'-noncoding region of 39 nucleotides and an open reading frame of 1473 nucleotides. The remainder of the sequence is due to the 3'-noncoding region and poly(A) tail. The open reading frame encodes a protein of 491 amino acids with a molecular weight of 56,196. The chicken cytochrome P-450 shows an overall homology of 45-54% compared with the mammalian phenobarbital-induced cytochrome P-450s. The degree of homology is not uniform, with some short regions showing much greater levels of sequence conservation. In particular, the chicken cytochrome P-450 contains the conserved cysteinyl domain near the carboxyl terminus, found in all cytochrome P-450s and which is thought to be involved in heme binding. Using the chicken sequence, a more accurate estimate of the evolutionary rates of cytochrome P-450s has been made. It is suggested that the phenobarbital-, 3-methylcholanthrene, and pregnenolone 16 alpha-carbonitrile-induced cytochrome P-450 gene families diverged from a common ancestral gene 600 million years ago. Furthermore the phenobarbital-inducible gene apparently underwent gene duplication events at about the time of the divergence of the chicken and mammalian lineages. The results imply that most mammals should have at least four rather distantly related phenobarbital-inducible gene subfamilies.     

P-450(11beta)-dependent conversion of androgen to estrogen, the aromatase reaction

Purified bovine adrenal P-450(11)beta has been shown to act as an aromatase which catalyzes conversion of 19-oxoandrostenedione to estrone. No conversions took place when any one of the required components such as NADPH, NADPH:adrenodoxin reductase, adrenodoxin and P-450(11)beta was omitted from the complete reconstituted system. P-450scc, another mitochondrial P-450 obtained from adrenal cortex, did not substitute for the P-450(11)beta in the aromatase reaction. These results show that P-450(11)beta is able to catalyze a series of reaction which can generate adrenal estrogen through androstenedione and its 19-hydroxy- and 19-oxo-derivatives. The P-450(11)beta-dependent reaction appears to be quite different from the placental aromatase reaction in that the latter is catalyzed by a microsomal P-450.     

Stereochemistry of estrogen biosynthesis by a reconstituted aromatase cytochrome P-450 preparation from human placenta

According to the literature, the multistep reaction mechanism of estrogen biosynthesis proceeds with stereospecific loss of the equatorial 1 beta-, and axial 2 beta-protons. These results were deduced from experiments carried out, either with crude microsomes, or at best with impure enzyme extracts. However, when [1,2- 3H]4-androstene-3,17-dione of known absolute 3H-label distribution was incubated with a reconstituted enzyme system, consisting of homogeneous NADPH-cytochrome P-450 reductase and highly purified aromatase, we obtained results that can only be logically explained by a trans- and antiparallel elimination reaction of both the axially oriented C-2 beta-, and C-1-alpha protons. We further demonstrate that the reconstituted enzyme has an aromatase activity optimum at pH 7.2, and an apparent Km of 0.66 microM for NADPH and of 0.24 microM for 4-androstene-3,17-dione. Also, the enzyme requires 3 nmoles of NADPH for each nmole of estrogen that is formed.     

The formation of cytochrome P-450 from cytochrome P-420 is promoted by spermine

This paper is concerned with camphor-bound bacterial cytochrome P-450 and processes that alter its spin-state equilibrium and influence its transition to the nonactive form, cytochrome P-420, as well as its renaturation to the native camphor-bound cytochrome P-450. Spermine, a polycation carrying a charge of 4 +, and potassium, a monovalent cation, were shown to differently cause an increase of high-spin content of camphor-bound cytochrome P-450. The spermine-induced spin transition saturates around 75% of the high spin; a further addition of KCl to the spermine-containing sample shifted the spin state to 95% of the high spin. The volume change of these spin transitions as measured by the use of high pressure indicated an excess of -40 mL/mol for the sample containing potassium as compared to that containing spermine. These results suggest that the proposed privileged site for potassium has not been occupied by spermine and that pressure forces both the camphor and the potassium ion from its sites, allowing solvent movement into the protein as well as ordering of solvent by the excluded camphor and potassium. Cytochrome P-420 was produced from cytochrome P-450 by hydrostatic pressure in the presence of potassium, spermine, and cysteine. Potassium cation shows a bigger effect on the stability of cytochrome P-450 than spermine or cysteine, as revealed by a higher value of the pressure of half-inactivation, P1/2, and a bigger inactivation volume change. However, potassium cation did not promote renaturation of cytochrome P-420 to cytochrome P-450 while the presence of spermine did.(ABSTRACT TRUNCATED AT 250 WORDS)     

Nucleotide sequence of a cDNA encoding porcine testis 17 alpha-hydroxylase cytochrome P-450.

We describe the isolation and characterization of a cDNA encoding the complete porcine neonatal testis 17 alpha-hydroxylase/C-17,20-lyase cytochrome P-450. The deduced amino acid sequence is 509 amino acids in length.     

Isolation of a cytochrome P-450 that catalyzes the 25-hydroxylation of vitamin D3 from rat liver microsomes

A molecular species of cytochrome P-450 that catalyzes the 25-hydroxylation of cholecalciferol (P-450cc25) was purified from rat liver microsomes on the basis of its catalytic activity. The purification procedure consisted of polyethylene glycol fractionation, and column chromatographies on octylamino Sepharose 4B, hydroxylapatite, DEAE-Sepharose CL-6B, and CM-Sepharose CL-6B. The specific cytochrome P-450 content of the final preparation was 17.0 nmol/mg of protein. The enzymatic activity was reconstituted with the purified cytochrome P-450, NADPH-cytochrome P-450 reductase, an NADPH-generating system, and dilauroylglyceryl-3-phosphorylcholine, the specific activity obtained being 3.7 nmol/min/mg of protein, which was 4,000 times as high as that in microsomes. The apparent molecular weight of the P-450cc25 was 50,000, based on the results of sodium dodecyl sulfate polyacrylamide gel electrophoresis. The absorption spectra of the oxidized form of the enzyme showed a Soret band at 416 nm, which is typical of the low spin state of cytochrome P-450, and alpha and beta bands at 570 and 536 nm, respectively. The Soret peak of the reduced cytochrome P-450-CO complex was at 450 nm. The purified enzyme not only catalyzed the 25-hydroxylation of cholecalciferol but also showed hydroxylation activity toward a variety of substrates, i.e. 1 alpha-hydroxycholecalciferol (at 25), testosterone (at 2 alpha and 16 alpha) and dehydroepiandrosterone (at 16 alpha). Amino terminal sequence of the purified cytochrome P-450 was determined by the manual sequence method to be H2N-Met-Asp-Pro-Val-leu-Val-Leu-Val-. The antibody elicited against the purified enzyme in a rabbit inhibited the cholecalciferol 25-hydroxylation activity by more than 90% with a concentration of 2 mg of immunoglobulin per nmol of cytochrome P-450.     

A form of rabbit liver cytochrome P-450 that catalyzes the 7 alpha-hydroxylation of cholesterol

A form of cytochrome P-450 which comigrates with cytochrome P-450LM4 (molecular weight, 55 000) on SDS-polyacrylamide gel was purified from liver microsomes of cholestyramine-treated rabbits. This form of cytochrome P-450 catalyzed the 7 alpha-hydroxylation of cholesterol with an activity of 37.5 pmol/min per nmol cytochrome P-450 in the reconstituted enzyme system containing cytochrome P-450 and NADPH-cytochrome P-450 reductase. The substrate specificity of this form of cytochrome P-450 was compared with cytochrome P-450LM4 isolated from phenobarbital- and beta-naphthoflavone-treated rabbit liver microsomes. The latter two isoenzymes do not catalyze 7 alpha-hydroxylation of cholesterol, but are more active in O-deethylation of 7-ethoxycoumarin and p-nitrophenetole. Ouchterlony double diffusion revealed cross-reactivity between anti-P-450LM4 (phenobarbital) IgG and cytochrome P-450 isolated from cholestyramine- or beta-naphthoflavone-treated rabbit liver microsomes. A two-dimensional iodinated tryptic peptide fingerprint indicated only minor structural differences among these three cytochrome P-450LM4 preparations.     

cDNA cloning of cytochrome P-450 related to P-450p-2 from the cDNA library of human placenta. Gene structure and expression

We have isolated and analyzed cDNA (designated P-450HP cDNA) clones from a human placenta cDNA library, using the cDNA for rabbit pulmonary cytochrome P-450p-2, a prostaglandin omega-hydroxylase, as a hybridization probe. The cDNA obtained encoded a polypeptide comprising 511 amino acids with a calculated molecular mass of 58987 Da, and the amino acid sequence similarity with P-450p-2 and rat liver laurate omega-hydroxylase (P-450LA omega) was only about 50%. RNA blot analysis showed that the mRNA hybridizable with the human P-450HP cDNA was inducibly expressed 3-5-fold in rabbit small intestine and lung by gestation, but the expression remained constant in rabbit liver and kidney. This mode of expression was quite different from that of P-450p-2 and P-450LA omega. Interestingly, the mRNA hybridized with the cDNA of P-450HP was found to be expressed in all the human tumor tissues so far examined, in sharp contrast with the facts that almost all the other species of P-450s are known to disappear in the tumor tissues. Taken together, the deduced hemoprotein termed P-450HP dose not seem to be the human counterpart of rabbit P-450p-2 or rat P-450LA omega, and is presumably a new member of the P-450 family including P-450p-2 and P-450LA omega. Furthermore, the corresponding genomic DNA was also cloned and analyzed. The gene of P-450HP spanned 18.8 kb and was separated into 11 exons by 10 introns whose locations were completely different from those of P-450 genes so far determined.     

Isolation of a full-length cDNA encoding mouse aromatase P450

A full-length cDNA clone for aromatase P450 has been isolated from a pregnant mouse ovarian cDNA library. The insert of this clone (2394 bp) contains a 1509-bp open reading frame encoding 503 amino acid residues together with a 46-bp 5'-untranslated stretch and an 839-bp 3'-untranslated region to which a poly(A) tract is attached. Northern blot analysis of ovarian RNA from pregnant mice reveals a major mRNA band of 2.5 kb with a minor band of 2.1 kb. Comparison of mouse aromatase P450 with that of rat, human, and chicken shows 91, 81, and 69% identity in the nucleotide sequence and 92, 79, and 69% identity in the deduced amino acid sequence, respectively. The membrane-spanning domain of mouse aromatase P450 is estimated to be an extremely hydrophobic segment located within the N-terminal region of the molecule. Furthermore, a highly conserved heme-binding domain is noticed.     

The gene for aromatase (P450arom) in the chicken is located on the long arm of chromosome 1

An 125I-labeled partial cDNA for the chicken aromatase P450 was used for in situ hybridization to chromosomes from primary chicken embryo fibroblast cultures. The results indicate that the gene that encodes aromatase is located on the long arm of chromosome 1 at approximately position 0.16.     

Isolation and partial characterization of a cytochrome P-450 isoenzyme (cytochrome P-450tu) from mouse liver tumors

Experimental hepatomas induced with 5,9-dimethyldibenzo[c,g]carbazole in female XVIInc/Z mice display a strong microsomal steroid 15 alpha-hydroxylation activity. A cytochrome P-450 isoenzyme (cytochrome P-450tu), specific for this activity, has been isolated by an HPLC derived method using various Fractogel TSK and hydroxyapatite supports. On SDS polyacrylamide gel electrophoresis the purified protein appeared as one major band with an apparent Mr of 50,000. Its specific cytochrome P-450 content was 7.55 nmol/mg protein. As deduced from the visible spectrum, the heme iron of the isolated P-450tu was to 72% in the high-spin state. The CO-bound reduced form showed an absorption maximum at 450 nm. In addition to the stereospecific 15 alpha-hydroxylation of progesterone (2.3 min-1) and testosterone (2.5 min-1), the enzyme catalyzed also 7-ethoxycoumarin O-deethylation, benzphetamine N-demethylation and aniline 4-hydroxylation. Its N-terminal amino-acid sequence (21 residues) was identical to that of cytochrome P-450(15) alpha, isolated by Harada and Negishi from liver microsomes of 129/J mice. P-450tu differed from P-450(15) alpha by its higher molecular weight, its 40-times lower steroid 15 alpha-hydroxylation and its 4-times higher benzphetamine N-demethylation.     

The alternative exons 1 of the mouse aromatase cytochrome P-450 gene

Aromatase cDNA clones were isolated from cDNA libraries of mouse hypothalamus, amygdala and ovary. Analysis of the nucleotide sequences of the 5′ regions of the obtained cDNAs suggested that the mouse aromatase gene is tissue-specifically regulated by alternative exons 1. There were obvious differences between the 5′ regions of the brain and ovary aromatase cDNAs, but no difference was found between the sequences of the hypothalamus and amygdala ones. We further isolated a mouse genomic DNA clone containing brain- and ovary-specific exons 1. The brain specific exons 1 and their promoters were highly homologous in the human and mouse aromatase genes. In contrast there were several differences in the sequences among the promoter regions of the ovary-specific exons 1 of the mouse, human and rat aromatase genes, significant homology between their sequences was also observed. The present results demonstrate that expression of the mouse aromatase gene is also tissue-specifically regulated through the use of alternative exons 1 and promoters, as reported for man.     

The induction of a specific form of cytochrome P-450 (P-450j) by fasting

In previous work we have demonstrated that liver microsomal N-nitrosodimethylamine demethylase (NDMAd) activity is increased in rats by fasting, and we have postulated that this is due to the induction of a specific form of cytochrome P-450. This communication provides evidence for such a hypothesis. Fasting for 24 and 48 h caused 59 and 116% increases, respectively, in NDMAd activity in male rats, and fasting for 48 h caused a 63% increase in female rats. These increases were accompanied by corresponding increases of cytochrome P-450j (P-450ac) determined by immunoblotting. Fasting for 24 and 48 h also increased the mRNA for P-450j by 153 to 250%, as determined by hybridization with a cDNA probe of this cytochrome. The results suggest that fasting affects the gene expression of P-450j.     

Isolation and characterization of a cytochrome P-450 from rat kidney mitochondria that catalyzes the 24-hydroxylation of 25-hydroxyvitamin D3

A cytochrome P-450 that catalyzes the 24-hydroxylation of 25-hydroxyvitamin D3 (P-450cc24: P-450cholecalciferol24) was purified to electrophoretic homogeneity from the kidney mitochondria of female rats treated with vitamin D3 (Ohyama, Y., Hayashi, S., and Okuda, K. (1989) FEBS Lett. 255, 405-408). The molecular weight was 53,000, and its absorption spectrum showed peaks characteristic of cytochrome P-450. The turnover number was 22 min-1 and the specific content was 2.8 nmol/mg protein. The N-terminal amino acid sequence, Arg-Ala-Pro-Lys-Glu-Val-Pro-Leu-, is different from the N-terminal sequence of any other cytochrome P-450s so far reported. Upon reconstitution with the electron-transferring system of the adrenal mitochondria, the enzyme showed a high activity in hydroxylating 25-hydroxyvitamin D3 as well as 1 alpha,25-dihydroxyvitamin D3 at position 24. However, the purified enzyme hydroxylated neither vitamin D3 nor 1 alpha-hydroxyvitamin D3. The enzyme was also inactive toward xenobiotics. The enzyme hydroxylated 25-hydroxyvitamin D3 at position 24 but not at 1 alpha, indicating that the enzyme is distinct from that catalyzing 1 alpha-hydroxylation. The reaction followed Michaelis-Menten kinetics, and the Km value for 25-hydroxyvitamin D3 was 2.8 microM. Both vitamin D3 and 1 alpha-hydroxyvitamin D3 inhibited the 24-hydroxylation of 25-hydroxyvitamin D3 in a competitive, concentration-dependent manner. 25-Hydroxyvitamin D3 24-hydroxylase activity was significantly inhibited by 7,8-benzoflavone, ketoconazole, and CO, whereas it was only slightly inhibited by aminoglutethimide, metyrapone, and SKF-525A. Mouse antibodies raised against the cytochrome P-450 inhibited the reaction about 70% and reacted with the P-450cc24 in immunoblotting but did not react with other kinds of cytochrome P-450 in rat liver microsomes and mitochondria.