Biomedical subjects

T D Porter

Publications and source records attributed to T D Porter.

27 records · Page 2Linked to original sources

PubMed1989

Structural analysis of the FMN binding domain of NADPH-cytochrome P-450 oxidoreductase by site-directed mutagenesis.

Comparison of the amino acid sequence of rat liver NADPH-cytochrome P-450 oxidoreductase with that of flavoproteins of known three-dimensional structure suggested that residues Tyr-140 and Tyr-178 are involved in binding of FMN to the protein. To test this hypothesis, NADPH-cytochrome P-450 oxidoreductase was expressed in Escherichia coli using the expression-secretion vector pIN-III-ompA3, and site-directed mutagenesis was employed to selectively alter these residues and demonstrate that they are major determinants of the FMN-binding site. Bacterial expression produced a membrane-bound 80-kDa protein containing 1 mol each of FMN and FAD per mol of enzyme, which reduced cytochrome c at a rate of 51.5 mumol/min/mg of protein and had absorption spectra and kinetic properties very similar to those of the rat liver enzyme. Replacement of Tyr-178 with aspartate abolished FMN binding and cytochrome c reductase activity. Incubation with FMN increased catalytic activity to a maximum of 8.6 mumol/min/mg of protein. Replacement of Tyr-140 with aspartate did not eliminate FMN binding, but reduced cytochrome c reductase activity about 5-fold, suggesting that FMN may be bound in a conformation which does not permit efficient electron transfer. Substitution of phenylalanine at either position 140 or 178 had no effect on FMN content or catalytic activity. The FAD level in the Asp-178 mutant was also decreased, suggesting that FAD binding is dependent upon FMN; FAD incorporation may occur co-translationally and require prior formation of an intact FMN domain.

Amino Acid Sequence↗

PubMed1989

Induction and tissue-specific expression of rabbit cytochrome P450IIE1 and IIE2 genes.

Treatment of rabbits with a variety of dissimilar chemicals, including ethanol, acetone, and imidazole, results in elevated levels of hepatic and renal cytochrome P-450 form 3a, also designated P-450ALC or P-450IIE. The P450IIE1 subfamily in rabbits is composed of two genes that encode proteins with 97% sequence identity; the mRNAs from these genes can be distinguished by their differing electrophoretic mobilities. In the present studies, examination of the expression of these genes revealed that P450IIE1 (gene 1) mRNA is present in greatest abundance in the liver, is present in kidney and nasal mucosa at approximately 10% of the level in liver, and is present in lung at approximately 5% of the level in liver. P450IIE2 (gene 2) mRNA is present in liver and lung at approximately 50% of the level of gene 1 mRNA in these tissues but cannot be detected in kidney or nasal mucosa. Neither gene is expressed in testis, ovary, small intestine, or adrenal tissue. Treatment of rabbits with acetone or imidazole results in elevated levels of P-450 3a-immunoreactive protein in liver and kidney without concomitant increases in P450IIE gene mRNAs. Moreover, various lengths of ethanol treatment elevated the level of immunoreactive protein in liver and kidney, with a rapid reduction of gene 1 mRNA and, at 14 days, gene 2 mRNA to approximately 50% of control levels. In contrast to these chemical inducers of 3a, fasting for 48 hr significantly increases gene 1 and 2 mRNA in liver but does not increase the level of immunoreactive protein. These results indicate that the rabbit P450IIE genes are not coordinately expressed or regulated and, as found with the rat ortholog P-450j, chemical inducers of 3a evidently act through changes in the rate of synthesis or degradation of the enzyme, rather than through increased gene transcription.

Animals↗

PubMed1988

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.

Amino Acid Sequence↗

PubMed1988

Isolation and partial characterization of the gene for cytochrome P-450 3a (P-450ALC) and a second closely related gene.

Two genes that hybridize to the cDNA for alcohol-inducible cytochrome P-450 form 3a (P-450ALC) have been isolated from a rabbit genomic library and characterized by restriction mapping, hybridization, and partial sequence analysis. The genes show extensive sequence similarity as judged by hybridization at high stringency to the coding region of P-450 3a cDNA. However, only gene 1 hybridizes under these conditions to the 3' nontranslated segment of P-450 3a cDNA. The hybridizing fragments derived from both cloned genes were found to be present in the genome of all rabbits examined by Southern blot analysis, indicating that the genes represent separate loci and are not polymorphic alleles. Partial sequence analysis indicated that gene 1 encodes P-450 3a. Gene 2, if transcribed, would encode a protein with greater than 96% sequence identity with P-450 3a in the NH2-terminal region.

Alleles↗

PubMed1987

Expression of a functional 78,000 dalton mammalian flavoprotein, NADPH-cytochrome P-450 oxidoreductase, in Escherichia coli.

A cDNA containing the complete coding nucleotide sequence for rat liver NADPH-cytochrome P-450 oxidoreductase was constructed from two overlapping cDNA clones. This full-length cDNA was inserted into the plasmid expression vector pCQV2, transfected into Escherichia coli, and expressed reductase was identified in cell lysates by electrophoresis followed by electrophoretic transfer to nitrocellulose and immunodetection. Various strains were screened for maximal expression and minimal intracellular degradation of the expressed protein, and strain C-1A was selected for preparation of the expressed enzyme. Induced cells from 12-liter cultures were pelleted, lysed in a French press, and the 50,000g supernate was fractionated by DEAE-cellulose and 2'5'-ADP agarose chromatography. Thirty-five grams of packed cells yielded approximately 2 mg of affinity-purified protein that was essentially free of E. coli proteins. The final preparation exhibited considerable proteolytic degradation and only an estimated 5-10% of the immunoreactive protein was undegraded. Four principal forms could be distinguished upon sodium dodecyl sulfate-polyacrylamide gel electrophoresis, with molecular weights of 65,000, 66,000, 74,000, and 78,000, the latter being equivalent to that of intact reductase. High-performance liquid chromatography with a Spherogel-DEAE column resolved these forms but resulted in the loss of the 78-kDa form; three peaks eluted with molecular weights of 65,000. Several of the HPLC fractions exhibited cytochrome c reductase activity, indicating correct incorporation of both flavin prosthetic groups, with the 66-kDa form showing the highest specific activity (44 mumol of cytochrome c reduced/mg reductase/min at 22 degrees C). HPLC assay of flavin content demonstrated equimolar FMN and FAD concentrations, and spectrophotometric analysis of the 66-kDa form revealed a spectrum essentially identical to that of reductase purified from rat liver. When the affinity-purified preparation was reconstituted with cytochrome P-450c, rates of benzo[a]pyrene metabolism approaching rates observed with liver reductase were obtained, indicating that the undegraded component in the affinity-purified preparation was able to interact with cytochrome P-450 and catalyze electron transfer from NADPH.

Animals↗

PubMed1987

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.

Amino Acid Sequence↗

PubMed1986

NADPH-cytochrome P-450 oxidoreductase: flavin mononucleotide and flavin adenine dinucleotide domains evolved from different flavoproteins.

The FMN-binding domain of NADPH-cytochrome P-450 oxidoreductase, residues 77-228, is homologous with bacterial flavodoxins, while the FAD-binding domain, residues 267-678, shows a high degree of similarity to two FAD-containing proteins, ferredoxin-NADP+ reductase and NADH-cytochrome b5 reductase. Comparison of these proteins to glutathione reductase, a flavoprotein whose three-dimensional structure is known, has permitted tentative identification of FAD- and cofactor-binding residues in these proteins. The remarkable conservation of sequence between NADPH-cytochrome P-450 oxidoreductase and ferredoxin-NADP+ reductase, coupled with the homology of the FMN-binding domain of the oxidoreductase with the bacterial flavodoxins, implies that NADPH-cytochrome P-450 oxidoreductase arose as a result of fusion of the ancestral genes for these two functionally linked flavoproteins.

Amino Acid Sequence↗

PubMed1986

Complementary DNA and amino acid sequence of rat liver microsomal, xenobiotic epoxide hydrolase.

The coding nucleotide sequence for rat liver microsomal, xenobiotic epoxide hydrolase was determined from two overlapping cDNA clones, which together contain 1750 nucleotides complementary to epoxide hydrolase mRNA. The single open reading frame of 1365 nucleotides codes for a 455 amino acid polypeptide with a molecular weight of 52,581. The deduced amino acid composition agrees well with those determined by direct amino acid analysis of the rat protein, and the amino acid sequence is 81% identical to that of rabbit epoxide hydrolase. Analysis of codon usage for epoxide hydrolase, and that of rabbit epoxide hydrolase. Analysis of codon usage for epoxide hydrolase, and comparison to codon usage for NADPH-cytochrome P-450 oxidoreductase and cytochromes P-450b, P-450d, and P-450PCN, suggest that epoxide hydrolase is more conserved than cytochromes P-450b and P-450PCN; comparison of the extent of sequence conservation for 12 homologous proteins between the rat and rabbit, including cytochrome P-450b, supports this hypothesis, and indicates that much of epoxide hydrolase is constrained to maintain its hydrophobic character, consistent with its intramembranous location. The predicted membrane topology of epoxide hydrolase delineates 6 membrane-spanning segments, less than the 8 or 10 predicted for two cytochrome P-450 isozymes; the lower number of membrane-spanning segments predicted for epoxide hydrolase correlates with its lesser dependence on the membrane for maintenance of its tertiary structure and catalytic activity.

Amino Acid Sequence↗

PubMed1985

Coding nucleotide sequence of rat NADPH-cytochrome P-450 oxidoreductase cDNA and identification of flavin-binding domains.

The coding nucleotide sequence of the mRNA for NADPH-cytochrome P-450 oxidoreductase (NADPH:ferricytochrome oxidoreductase, EC 1.6.2.4) from rat liver was determined from two overlapping cDNA clones, pOR-7 and pOR-8, which together contain 2401 nucleotides complementary to rat liver oxidoreductase mRNA. The single open reading frame of 2034 nucleotides spanning these cDNAs codes for a 678 amino acid polypeptide with a molecular weight of 76,962. The deduced amino acid composition is in excellent agreement with that determined by direct amino acid analysis of purified rat liver P-450 oxidoreductase, and the amino-terminal region (residues 1-80) largely coincides with the amino-terminal sequence of the oxidoreductase isolated from rabbit liver. Comparison of the amino acid sequence to those of other flavoproteins revealed two separate domains that are likely to be involved in flavin binding: a long segment (residues 77-228) homologous with Desulfovibrio vulgaris flavodoxin, an FMN-containing protein, and a shorter segment (residues 452-477) homologous with the FAD-binding segment of fumarate reductase from Escherichia coli.

Amino Acid Sequence↗