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I Sagami

Publications and source records attributed to I Sagami.

At least 37 records · Page 2Linked to original sources
PubMed1997

Interaction of Angeli's salt with cytochrome P450 1A2 distal mutants: an optical absorption spectral study.

Angeli's salt, Na2N2O3 or O-N=N+-(OH)(O-) in aqueous solution, is known to release NO- or NO., which relaxes vascular tissue and lowers blood pressure. In the liver, the most abundant heme enzyme is cytochrome P450. In the present study, we studied the effect of rat liver cytochrome P450 1A2 (P450 1A2) in regard to its catalysis of the N=N bond scission of Angeli's salt with optical absorption spectra. Also, we examined the contribution of putative distal amino acids of P450 1A2 to the reaction with the salt. We found that wild-type Fe3+ P450 1A2 markedly enhances the N=N scission of the salt up to 100 fold in terms of absorption spectroscopy. A Fe3+ P450 1A2-NO complex with an absorption peak at 435 nm was formed when the salt was added and the complex was then changed to a 6-coordinated Fe2+-NO complex having a 440-nm peak. Glu318Asp, Glu318Ala and Thr319Ala mutants at the putative distal site of P450 1A2 formed a 5-coordinated Fe2+-NO complex having a 400-nm absorption, that was not formed with the wild type. The Glu318Ala mutant, in particular, did not form the Fe3+-NO complex with the addition of Angeli's salt. The presence of L-Cys, reduced glutathione, catalase or superoxide dismutase markedly stabilized the Fe3+ wild type-NO complex. Thus, our data suggests that the N=N bond of Angeli's salt is cleaved with the P450 1A2 active site and NO- or NO. is released. We discuss mechanisms of redox and ligand changes of the P450 heme.

Animals↗
PubMed1997

Distal site and surface mutations of cytochrome P450 1A2 markedly enhance dehalogenation of chlorinated hydrocarbons.

Chlorinated compounds such as chlorinated ethylenes and ethanes are serious environmental pollutants. In the present study, we examined whether or not a recombinant strain of Saccharomyces cerevisiae that expresses rat liver cytochrome P450 1A2 (P450 1A2) wild-type and mutant proteins can efficiently catalyze oxidative and reductive dehalogenations of trichloroethylene, pentachloroethane, and hexachloroethane. Mutations at putative heme distal and protein surface sites of P450 1A2 greatly enhanced turnover values toward those substrates under both aerobic and anaerobic conditions. For example, a Thr319Ala mutation at the putative heme distal site enhanced the degradation rate of trichloroethylene and pentachloroethane by 2- and 2.7-fold, respectively, under aerobic conditions. The Thr319Ala mutation also strongly facilitated the reaction with hexachloroethane up to 13- and 4.5-fold under aerobic and anaerobic conditions, respectively. The Thr319Ala mutation increased dechlorinated over protonated product ratios by 3-fold as well when either pentachloroethane or hexachloroethane was used as a substrate. A Lys250Leu mutation on the putative protein surface site enhanced the dehalogenation rate of hexachloroethane up to 4.8-fold under anaerobic conditions. In contrast, a Glu318Ala mutation at the putative distal site markedly decreased the activities with trichloroethylene and pentachloroethane substrates under aerobic conditions. Conserved amino acids Thr319 and Glu318 at the heme distal site have been suggested to be important in the O2 activation during monooxidation reactions of P450s. However, the present study indicates that Thr319 is likely to be an inhibitor of dechlorination of trichloroethylene and penta- and hexachloroethanes. The roles of Thr319, Glu318, and Lys250 in the catalysis with chlorinated hydrocarbons are discussed in association with reaction mechanisms.

Animals↗
PubMed1995

Identification of cellular protein that can interact specifically with the basic helix-loop-helix domain of the aromatic hydrocarbon receptor.

The Ah receptor is a basic helix-loop-helix (bHLH)-PAS protein and functions as a ligand-activated DNA binding protein directly interacting with target genes by binding to xenobiotic responsive elements. We have sought to identify possible cellular proteins that can interact with the Ah receptor. The bHLH domain of the Ah receptor was fused to glutathione-S-transferase (GST), and the resulting fusion protein was used as a probe to help us to identify receptor associated protein(s). At least one such protein, 45kDa (p45), was detected in mouse liver extracts, but it does not bind to the bHLH domain of the Ah receptor nuclear translocator, nor to the transactivation domain of Ah receptor or GST alone.

Animals↗
PubMed1995

Identification of a 120-kDa protein associated with aromatic hydrocarbon receptor nuclear translocator.

The aromatic hydrocarbon receptor nuclear translocator (ARNT) is a basic helix-loop-helix-PAS protein which forms a heterodimer with aromatic hydrocarbon receptor (AHR), this heterodimer mediating the signal transduction in response to the various xenobiotics such as 2,3,7,8-tetrachlorodibenzo-p-dioxin and directly interacting with target genes by binding to xenobiotic responsive elements. An anti-ARNT antibody was raised in rabbits against the bacterially expressed ARNT of amino acids 21-328 from the N-terminal. Using this antibody, besides ARNT itself, we detected at least one protein, 120 kDa, in the immunoprecipitate of anti-ARNT antibodies in HepG2 cells as well as in Hepa-1 cells. However, this protein is not present in the immunoprecipitate of the anti-AHR antisera nor in that of the preimmune sera of the rabbits used for the immunization.

Animals↗
PubMed1995

Assessment of cancer susceptibility in humans by use of genetic polymorphisms in carcinogen metabolism.

Prevention is an important and effective measure for reducing death caused by cancer. Thus information on individual susceptibility to cancer is valuable in suggesting high risk individuals to avoid intake of carcinogenic substances and receive frequent physical screening. To this end, polymorphisms found within cytochrome P450 (CYP) genes implicated in the metabolism of procarcinogens are expected to be good genetic targets in assessing human cancer susceptibility. We have found polymorphisms in the CYP2E1 and CYP1A1 genes associated with lung cancer susceptibility, though there were some discrepancies from observations made by other investigators. Discrepancies among investigators from different regions, however, are very common in these pharmacogenetic studies. We present an explanation for these discrepancies, difficulties associated with prediction of relative risk of individuals, and future directions.

Carcinogens↗
PubMed1994

Characterization of hamster CYP1A1 gene: inducible expression and negative regulation.

A genomic clone encoding the hamster CYP1A1 gene was isolated from a hamster EMBL-3 genomic library and characterized. The CYP1A1 gene contained seven exons including the noncoding first exon as determined for CYP1A1 of other species. DNA sequence analysis up to -2307 bp of the CYP1A1 gene revealed the occurrence of five consensus xenobiotic responsive elements (XREs) and one basal transcription element (BTE) in addition to the canonical TATA box. For functional analysis, transfection experiments were performed in human hepatoma HepG2 cells with reporter gene constructs consisting of fragments with various lengths of the 5'-flanking region of the CYP1A1 gene and bacterial chloramphenicol acetyltransferase (CAT) gene. External deletion of the upstream region from the reporter gene resulted in a stepwise decrease of the CAT activity, suggesting that XREs were responsible for inducible expression of CYP1A1 gene by 3-methylcholanthrene (MC). A negative regulatory element (NRE) was also identified in the 5'-flanking region at -833 to -642. Removal of the NRE from the CYP1A1-CAT fusion gene resulted in about 3-fold increase of MC-inducible CAT activity. Using gel retardation assays with HepG2 nuclear extract, we demonstrated the presence of a specific protein which bound to the NRE fragment. Further competition analysis and methylation interference assays revealed that the nuclear protein bound to a 22-base fragment (from -688 to -709) of the NRE region, whose sequences were conserved among hamster, human, and rat CYP1A1 genes.

Animals↗
PubMed1994

Restriction fragment length polymorphism of the human CYP2E1 (cytochrome P450IIE1) gene and susceptibility to lung cancer: possible relevance to low smoking exposure.

Polymorphic metabolism of certain chemical carcinogens may result in differences in susceptibility to cancers. Human CYP2E1 (cytochrome P450IIE1) is an enzyme involved in the metabolic activation of precarcinogens such as nitrosamines. We detected a restriction fragment length polymorphism (RFLP) of the human CYP2E1 gene for the restriction endonuclease Dra I. The distribution of this polymorphism was examined among lung cancer patients (n = 91), patients with cancer of the digestive tract (n = 45) and controls (n = 76). A significant difference in the distribution was observed between lung cancer patients and controls (chi 2 = 11.4 with 2 df; p < 0.005). On the other hand, there was no significant difference between patients between cancer of the digestive tract and controls (chi 2 = 4.87 with 2 df; NS). This finding suggests that the Dra I polymorphism of the CYP2E1 gene is associated with susceptibility to lung cancer. In addition, an association was found between the amount of lifelong smoking exposure and the distribution of the genotypes of the RFLP among lung cancer patients. The distribution pattern seemed deviated from that of controls especially in the population of low smoking exposure. Our Northern blot analysis data using RNA from human liver autopsy samples suggest that the Dra I polymorphism might be associated with the gene expression of CYP2E1 at mRNA level.

Aged↗
PubMed1994

Aberrant CYP1A1 induction: discrepancy of CYP1A1 mRNA and aryl hydrocarbon hydroxylase activity in mutant cells of mouse hepatoma line, Hepa-1.

We have isolated new benzo[a]pyrene-resistant clones, cl-21 and cl-32, of the mouse hepatoma line, Hepa-1. CYP1A1-dependent aryl hydrocarbon hydroxylase activity is not inducible by 2,3,7,8-tetrachlorodibenzo-p-dioxin or 3-methylcholanthrene in these two cell lines. However, mRNA of CYP1A1 is inducible in cl-21 and cl-32 cells, as in the wild-type cells, in spite of an undetectable level of cytosolic Ah receptor. The cl-21 cDNA of Cyp1a-1 was found to have a single mutation leading to an amino acid substitution from Leu (118) to Arg (118). However, the CYP1A1 protein band was not detected on Western immunoblots. The cDNA of cl-32 was found to have a single mutation leading to an amino acid change from Arg (359) to Trp (359). The presence of the mature protein in cl-32 was confirmed by Western blot analysis. Somatic cell hybridization experiments demonstrated that the phenotype of cl-21 and cl-32 is recessive and that these clones belong to the same complementation group. These data suggest that there may be a non-Ah receptor-mediated mechanism of CYP1A1 induction.

Animals↗
PubMed1992

Human cytochrome P450IIE1 gene: DraI polymorphism and susceptibility to cancer.

Human cytochrome P450IIE1 (CYP2E) is involved in the metabolic activation of procarcinogens such as N-nitrosodimethylamine, benzene and ethyl carbamate. We screened DNA from 28 individuals for restriction fragment length polymorphisms (RFLPs) is the human P450IIE1 gene and detected an RFLP for the restriction endonuclease DraI. The distribution of the genotypes of this polymorphisms among lung cancer patients (n = 74) differed from that among controls (n = 73) with statistical significance of p < 0.05. In addition, the distribution among patients with cancers of the digestive system (n = 38) was also different from that among controls. Our findings indicate an association between the DraI polymorphism of the IIE1 gene and susceptibility to cancers of the lung and the digestive system.

Cytochrome P-450 CYP2E1↗
PubMed1991

Hamster cytochrome P-450 IA gene family, P-450IA1 and P-450IA2 in lung and liver: cDNA cloning and sequence analysis.

Two cDNA clones, 2C19 and 4C1, were isolated from a lung cDNA library of 3-methylcholanthrene (MC)-treated hamster by using rat P-450c cDNA as a probe. The cDNA determined from 2C19 and 4C1 was 2,916 bp long and contained an entire coding region for 524 amino acids with a molecular weight of 59,408. The deduced amino acid sequence showed a 85% identity with that of rat P-450c indicating 2C19 and 4C1 encode the hamster P-450IA1 protein. Another cDNA clone, designated H28, was isolated from a MC-induced hamster liver cDNA library by using the hamster lung 2C19 or 4C1 cDNA clone as a probe. H28 was 1,876 bp long and encoded a polypeptide of 513 amino acids with a molecular weight of 58,079. The N-terminal 20 residues deduced from nucleotide sequence of H28 were identical to those determined by sequence analysis of purified hamster hepatic P-450MCI. The high similarity of the nucleotide and deduced amino acid sequences between H28 and P-450IA2 of other species indicated that H28 encoded a P-450 protein which belongs to the P-450IA2 family. Northern blot analysis revealed that the mRNAs for hamster P-450IA1 and IA2 were about 2.9 and 1.9 kb long, respectively. Hamster P-450IA1 mRNA was induced to the same level in lungs as in livers by MC treatment, whereas hamster P-450IA2 mRNA was induced and expressed only in hamster liver.

Amino Acid Sequence↗
PubMed1991

Association between restriction fragment length polymorphism of the human cytochrome P450IIE1 gene and susceptibility to lung cancer.

Cytochrome P450IIE1 (P450IIE1) is involved in metabolic activation of carcinogenic nitrosamines, aniline and benzene. We detected a restriction fragment length polymorphism of the human P450IIE1 gene with the restriction endonuclease DraI. The population was thus divided into three genotypes, namely, heterozygotes (CD) and two forms of homozygotes (CC and DD). The distribution of these genotypes among lung cancer patients differed from that among controls with statistical significance of P less than 0.05 (chi 2 = 7.01 with 2 degrees of freedom). This result strongly suggests that host susceptibility to lung cancer is associated with the DraI polymorphism of the P450IIE1 gene.

Adenocarcinoma↗
PubMed1990

Complementary DNA sequence of 3-methylcholanthrene-inducible P-450 from the rat lung.

Cytochrome P-450MC was induced in pulmonary microsomes of 3-methylcholanthrene-treated rats and also at low level in that of isosafrole-treated rats. Cytochrome P-450d was not detected in the lungs of 3-methylcholanthrene- or isosafrole-treated rats by the method of Western blot analysis with a polyclonal antibody raised against cytochrome P-450c which is equally effective against P-450d, nor by the method of Northern hybridization probed with pcP450mc3 (P-450d probe). Complementary DNA of P-450MC was isolated from rat pulmonary cDNA library and the nucleotide sequence of pulmonary cDNA was compared with that of hepatic P-450c cDNA reported by Yabusaki et al. There was no gross change in nucleotide sequences of cDNA between pulmonary P-450MC and hepatic P-450c.

Amino Acid Sequence↗
PubMed1989

Purification and characterization of cytochromes P-450 from liver microsomes of 3-methylcholanthrene-treated crab-eating monkeys.

Two forms of cytochrome P-450 (P-450MC1 and P-450MC2) were purified from liver microsomes of crab-eating monkeys (Macaca irus) treated with 3-methylcholanthrene (MC). Monkey P-450MC1 preparation had a specific content of 14.0 nmol/mg protein and showed a main protein band with a minimum molecular weight of 52,000 on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Monkey P-450MC2 preparation had a specific content of 12.1 nmol/mg protein and a minimum molecular weight of 54,000. The carbon monoxide-reduced difference spectral peaks of monkey P-450MC1 and P-450MC2 were at 448 and 447 nm, respectively. In the reconstituted system, monkey P-450MC2 had high activities for benzo[a]pyrene 3-hydroxylation and 7-ethoxycoumarin O-deethylation. Monkey P-450MC1 had low activities toward these two substrates and a high activity for benzphetamine N-demethylation. Monkey P-450MC1 and P-450MC2 were detected by immunoblotting using an antibody prepared against rat cytochrome P-450c, which is a major form of cytochrome P-450 in liver microsomes of MC-treated rats. These results suggested that the molecular properties of cytochrome P-450 in liver microsomes of crab-eating monkeys treated with MC are similar to those in rats.

Animals↗
PubMed1988

The studies of COUP transcription factor from HeLa cells: purification and partial characterization.

A transcription factor COUP, which is responsible for the recognition of the Chicken Ovalbumin Upstream Promoter sequence between -70 and -90, is required for efficient transcription of the ovalbumin gene. This COUP combination of conventional column and sequence specific DNA affinity column chromatography. A few polypeptides were identified in the purified preparation on an SDS-gel. Upon renaturation, all the major polypeptides in the molecular size range between 43 to 53 kD bound specifically to the COUP sequence. Furthermore, at least one of the renatured polypeptides in the region of 43-45 kD retained transcriptional activity. Using band-shifting assays and 3' and 5' deletion mutants and oligonucleotide mutants, the sequence important for binding was mapped. Methylation and ethylation interference assays revealed the contact sites for the purine base and the phosphate backbone. These studies indicate that the COUP transcription factor interacts with the base residue in the major groove and wraps around the DNA helix in one full turn.

Animals↗