Expression of alcohol-inducible rabbit liver cytochrome P-450 3a (P-450IIE1) in Saccharomyces cerevisiae with the copper-inducible CUP1 promoter.

The expression of the cDNA for alcohol-inducible rabbit liver microsomal cytochrome P-450 form 3a (P450IIE1) in Saccharomyces cerevisiae, with the use of the copper-inducible yeast metallothionein (CUP1) promoter and the ADH1 promoter, is described. Strains 50.L4 and PP1002 were compared for optimal levels of expressed protein. Immunoblot analysis showed that a much higher level of expression of cytochrome P-450 3a is obtained with strain 50.L4, and that the uninduced levels of expressed protein are similar with the two promoters. With the CUP1 promoter, transcription of the cDNA is strongly induced in the presence of cupric ions, and the amount of immunoreactive protein expressed in increased 20-fold in strain 50.L4, such that it constitutes 0.8% of the total cellular protein. The cytochrome P-450 holoenzyme content of these cells, calculated from the reduced CO difference spectrum, is about 0.02 nmole/mg of protein, or 0.1% of the total cellular protein. The holoenzyme content of microsomes prepared from these cells is up to 0.06 nmole/mg of protein, or 0.4% of the microsomal protein. Microsomal assays for ethylene formation from N-nitrosodiethylamine and for aniline p-hydroxylation, two reactions typical of purified rabbit cytochrome P-450 form 3a, showed that the cytochrome synthesized in yeast catalyzes both reactions. Furthermore, polyclonal anti-3a IgG completely inhibits the reactions with both substrates in yeast microsomes. A comparison of the product ratios from these substrates showed that the cytochrome P-450 3a expressed in yeast has catalytic activities similar to those of the authentic rabbit protein.

Organization and differential expression of two highly similar genes in the rabbit alcohol-inducible cytochrome P-450 subfamily.

The exon-intron organization of two rabbit genes that hybridize with cytochrome P-450 3a (P-450ALC) cDNA has been determined by restriction mapping and sequence analysis. Gene 1 encodes cytochrome P-450 3a as judged by the complete identity of its coding nucleotide sequence with P-450 3a cDNA. Gene 2 encodes a previously uncharacterized cytochrome P-450 that is 97% identical in primary structure to P-450 3a, with 16 amino acid differences scattered throughout the protein. Genes 1 and 2, which are 10 and 9 kilobases in length, respectively, are comprised of 9 exons with exon-intron junctions occurring at identical positions along the mRNA sequences. Each gene contains two transcription start sites approximately 27 and 33 nucleotides upstream from the translation initiation codon, as determined by primer extension and S1 nuclease protection experiments. The predicted lengths of gene 1 and 2 transcripts from the first transcription start site to the poly(A) attachment site are 1999 and 1660 nucleotides, respectively. This difference in size is primarily the result of a 338-base pair deletion in the 3' nontranslated portion of the gene 2 transcript relative to that of gene 1. The two genes show considerable similarity in their 5' flanking regions, including a "TATAA" transcriptional promoter element at position -28. However, a 32-base pair element that is repeated in gene 1 is present only as a single inexact copy in gene 2. By use of synthetic oligonucleotides as hybridization probes, gene 2 transcripts were shown to be present in poly(A)+ RNA from untreated rabbit liver at approximately 50% of P-450 3a mRNA levels. In kidney, however, no gene 2 mRNA was detected although 3a mRNA was present at approximately 10% of the level in liver.

cDNA and derived amino acid sequence of ethanol-inducible rabbit liver cytochrome P-450 isozyme 3a (P-450ALC).

Administration of ethanol to rabbits is known to induce a unique liver microsomal cytochrome P-450, termed isozyme 3a or P-450ALC, which is responsible for the increased oxidation of ethanol and other alcohols and the activation of toxic or carcinogenic compounds such as acetaminophen and N-nitrosodimethylamine. To further characterize this cytochrome P-450 we have identified cDNA clones to isozyme 3a by immunoscreening, DNA hybridization, and hybridization-selection. The cDNA sequence determined from two overlapping clones contains an open reading frame of 1416 nucleotides, and the first 25 amino acids of this reading frame correspond to residues 21-45 of cytochrome P-450 3a. The complete polypeptide, including residues 1 to 20, contains 492 amino acids and has a molecular weight of 56,820. Cytochrome P-450 3a is approximately 55% identical in sequence to P-450 isozymes 1 and 3b and 48% identical to isozyme 2. Hybridization of clone p3a-2 to electrophoretically fractionated rabbit liver poly(A)+ RNA revealed multiple bands, but, with a probe derived from the 3' nontranslated portion of this cDNA, only a 1.9-kilobase band was observed. Treatment of rabbits with imidazole, which increases the content of isozyme 3a, resulted in a transient increase in form 3a mRNA, but this was judged to be insufficient to account for the known 4.5-fold increase in form 3a protein. Genomic DNA analysis indicated that the cytochrome P-450 3a gene does not belong to a large subfamily.

Cytochrome P-450 isozymes 2 and 5 in rabbit lung and liver. Comparisons of structure and inducibility.

Rabbit cytochrome P-450 isozymes 2 and 5 were purified from pulmonary and hepatic microsomal preparations. Purification of isozyme 5 was monitored by immunochemical methods so that contamination by isozymes 2, 4, and 6 could be avoided. Partial proteolysis of hepatic and pulmonary isozyme 5 showed minor differences in peptide formation when analyzed by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate and visualized by the silver staining method. In contrast, identical patterns were observed when the peptides were transferred to nitrocellulose paper and visualized immunochemically. The differences observed between the results obtained with the two methods was apparently caused by differences in small amounts of contaminants present in both preparations. HPLC profiles of peptides formed by treatment of pulmonary and hepatic isozyme 5 with trypsin appeared to be the same. In addition, it was found that the pulmonary and hepatic isozymes had identical sequences for the first 20 NH2-terminal amino acids. Three distinct fractions of hepatic cytochrome P-450 isozyme 2 were obtained when chromatography on DEAE-cellulose was used as the final step in the purification procedure. In contrast, only a single fraction of purified pulmonary isozyme 2 was isolated by the same method. Analysis of the pulmonary and three hepatic preparations of isozyme 2 by partial proteolysis and visualization of peptides by silver staining or immunoblotting showed no differences. Analysis of tryptic digests by HPLC also produced the same results for each of the four preparations. The first 24 NH2-terminal amino acids were identical for all four preparations of isozyme 2.(ABSTRACT TRUNCATED AT 250 WORDS)

On the amino acid sequence of cytochrome P-450 isozyme 4 from rabbit liver microsomes.

Isozyme 4 of rabbit liver microsomal cytochrome P-450 was shown earlier in this laboratory to contain multiple NH2-terminal residues, whereas isozymes 2, 3a, 3b, and 3c have single, unique NH2-terminal sequences. Similar results were obtained with isozyme 4 obtained from animals that were untreated, treated with phenobarbital (which does not induce this isozyme), or induced with beta-naphthoflavone or isosafrole. With the use of selective chemical blocking at seryl or at nonprolyl residues, the complexity of the NH2-terminal sequence has now been shown to be due to the presence of three forms of the cytochrome differing only in the absence of the first or the first two residues: NH2-Ala-Met-Ser-Pro-Ala-Ala-Pro-, NH2-Met-Ser-Pro-Ala-Ala-Pro-, and NH2-Ser-Pro-Ala-Ala-Pro-. These forms may result from variable biological processing. Peptides containing the seven cysteine residues were sequenced and compared with similar peptides reported for other P-450 cytochromes; homology was extensive with respect to two of the cysteine regions in isozyme 4, and a third cysteine region showed partial identity. The sequence of peptides representing about two-thirds of the amino acids in isosafrole-induced cytochrome P-450 isozyme 4 was determined. Comparison with phenobarbital-induced rabbit cytochrome P-450 isozyme 2 indicated about 25% homology. In contrast, comparison of isozyme 4 with rat cytochrome P-450d, which is also induced by isosafrole and for which the sequence has recently been deduced from cDNA [Kawajiri, K., Gotoh, O., Sogawa, K., Tagashira, Y., Muramatsu, M. & Fujii-Kuriyama, Y. (1984) Proc. Natl. Acad. Sci. USA 81, 1649-1653], showed about 70% homology.

Complete amino acid sequence and predicted membrane topology of phenobarbital-induced cytochrome P-450 (isozyme 2) from rabbit liver microsomes.

The complete amino acid sequence of phenobarbital-induced isozyme 2 of rabbit liver microsomal cytochrome P-450 (P-450LM2) is presented. The polypeptide consists of 491 residues with a calculated Mr of 55,755. The rabbit isozyme is 77% identical to the corresponding rat cytochrome, P-450b, as deduced from cDNA, with 96% of the hydrophobic, 88% of the anionic, and 83% of the cationic positions conserved. The secondary structure of isozyme 2 was predicted and a model was developed for the membrane topology of this cytochrome. Of the two highly conserved cysteinyl peptides in P-450LM2, P-450b, and bacterial P-450cam, we favor, on the basis of our model, the one nearer the NH2 terminus (Cys-152 in P-450LM2) as the source of the thiolate ligand to the heme iron atom. The recently reported sequence of the apparently identical protein [Heinemann, F. S. & Ozols, J. (1983) J. Biol. Chem. 258, 4195-4201] has two fewer residues and differs in 14 other amino acid assignments.