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A Raha

Publications and source records attributed to A Raha.

10 recordsLinked to original sources
PubMed1999

The existence of the 4S polycyclic aromatic hydrocarbon-protein binding in 14-day-old chick embryo liver.

Cytochrome P-450IA1, the isozyme most closely associated with aryl hydrocarbon hydroxylase (AHH), is regulated by two high-affinity binding proteins, the 4S polycyclic aromatic hydrocarbon (PAH)-binding protein which primarily binds PAHs and the 8S Ah (dioxin) receptor which binds 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) and like congeners. The present study was conducted to determine whether the 4S protein existed in 14-day-old chick embryo liver when AHH activity is maximal to determine if they are linked as is the 8S Ah receptor and to confirm the existence of the dioxin receptor by investigating their ligand binding characteristics in the presence and absence of sodium molybdate, an agent that stabilizes steroid hormone receptors and partially stabilizes the dioxin receptor. Competitive ligand binding studies were performed with liver cytosol from livers of male 14-day-old chick embryos using [3H]-benzo[a]pyrene (B[a]P) or [3H]-TCDD in the presence and absence of a 200-fold excess of B[a]P, benzo[e]pyrene (B[e]P), 3-methylcholanthrene (3-MC), and tetrachlorodibenzofuran (TCDBF). Specific PAH-binding activity was assayed using sucrose gradient analysis. In the absence of molybdate, the 4S PAH-binding protein had high affinity for B[a]P, B[e]P, 3-MC, but very low affinity for TCDBF; the Ah receptor exhibited high affinity for TCDBF. In the presence of sodium molybdate, the Ah receptor was stabilized while the 4S PAH-binding protein was relatively unaffected. These results affirm the existence of two distinct PAH-binding proteins in 14-day-old chick embryo liver cytosol and suggest a linkage of the 4S protein to AHH.

Animals↗
PubMed1995

Glycine N-methyltransferase is a mediator of cytochrome P4501A1 gene expression.

Cytochrome P4501A1, the isozyme most closely approximating aryl hydrocarbon hydroxylase activity under conditions of induction, is thought to be regulated by several trans-acting factors, including the 4S polycyclic aromatic hydrocarbon-binding protein; this protein has recently been identified as glycine N-methyltransferase (Raha et al. (1994) J. Biol. Chem. 269, 5750-5756). Previous studies had shown that partially purified liver preparations containing the 4S binding protein interacted with 5'-flanking regions of the cytochrome P4501A1 gene. Consequently, the ability of the 4S binding protein to serve as a mediator in the regulation of the cytochrome P4501A1 gene was investigated further. Introduction of an antisense 24-mer oligonucleotide to glycine N-methyltransferase cDNA into rat hepatoma H4IIE cells by lipofectin resulted in a 60% reduction in the benzo(a)pyrene-mediated induction of ethoxyresorufin-O-deethylase activity and protein over the sense and scrambled antisense oligonucleotide controls. In addition, the antisense oligonucleotide caused a marked reduction in the steady-state level of cytochrome P4501A1 mRNA; no such effect was observed with the sense oligonucleotide. Introduction of GNMT polyclonal antibodies into H4IIE cells by a streptolysin-O permeabilization technique markedly reduced both benzo(a)pyrene-binding and benzo(a)-pyrene-induced ethoxyresorufin-O-deethylase activities, but had no effect on 2,3,7,8-tetrachlorodibenzo-p-dioxin induction. Collectively, these findings suggest that, in addition to the Ah (dioxin) receptor, glycine N-methyltransferase appears to be both a polycyclic aromatic hydrocarbon-binding protein and a mediator of the induction of the cytochrome P4501A1 gene by polycyclic hydrocarbons such as benzo(a)pyrene.

Animals↗
PubMed1994

Rat liver cytosolic 4 S polycyclic aromatic hydrocarbon-binding protein is glycine N-methyltransferase.

In the rat, cytochrome P-450IA1 gene expression, which is most closely associated with aryl hydrocarbon hydroxylase activity, is thought to be regulated by several trans-acting factors, including the 4 S polycyclic aromatic hydrocarbon (PAH)-binding protein. This protein has been purified to homogeneity from rat liver using ion exchange, gel permeation, hydrophobic interaction, and affinity chromatographies. Partial sequencing of the 33-kDa band indicated its identity as glycine N-methyltransferase (GNMT). Polyclonal antibodies to GNMT immunoprecipitated PAH-binding activity from rat liver cytosol. Methyltransferase and PAH-binding activities copurified during the course of protein purification. GNMT protein and PAH binding activity were colocalized in various cytosolic fractions. These data all indicate that the 4 S PAH-binding protein and GNMT are one and the same protein or very similar proteins. Western blot analyses yielded a positive signal under denaturing (33 kDa) and nondenaturing (150 kDa, tetramer) conditions; the PAH-binding protein also was an oligomer. GNMT was detected by immunohistochemistry in nuclei from H4IIE rat hepatoma cells and rat liver. The localization of GNMT in liver nuclei is in accordance with a role in modulating cytochrome P-450IA1 gene expression.

Amino Acid Sequence↗
PubMed1994

Induction of CYP1A1 gene expression in mouse hepatoma cells by benzo[e]pyrene, a ligand of the 4S polycyclic hydrocarbon-binding protein.

Hepa 1c1c7 (WT), TAOc1BPrc1 (CI), and BPrc1 (CII) mouse hepatoma cells were exposed to benzo[e]pyrene (B[e]P) or benzo[a]pyrene (B[a]P). B[e]P induced activity of a rat CYP1A1 reporter gene construct (-3015 to +2545 bp) by 1.8- to 2-fold and 5-fold in WT and CI cells, respectively. B[e]P caused a 2-fold induction of a truncated CYP1A1 reporter gene construct (-658 to +2545 bp) in WT cells and induced ethoxyresorufin O-deethylase (EROD) activity by 24- and 13-fold in WT and CI cells. B[a]P also induced CYP1A1 reporter gene and EROD activity in these cells. WT and CII cells had both 8S (Ah) receptor and 4S polycyclic hydrocarbon (PAH)-binding activity, while CI cells exhibited a lower 4S binding activity; 8S binding activity was not detected in CI cells under two separate binding conditions. 8S binding activity in the presence of sodium molybdate was 60-fold greater in WT cells than in CII cells. The absence of sodium molybdate resulted in a dramatic decrease of 8S binding activity in WT cells. The ability of B[e]P to induce CYP1A1 promoter-reporter gene activity and EROD activity in WT and CI cells suggested a role for the 4S PAH-binding protein in the induction of CYP1A1. The lack of detectable 8S binding activity in CI cells was in concert with this role.

Animals↗
PubMed1992

Induction of CYP1A1 gene expression in H4-II-E rat hepatoma cells by benzo[e]pyrene.

In the rat, expression of the CYP1A1 gene is closely associated with arylhydrocarbon hydroxylase (AHH) enzyme activity. AHH is an inducile enzyme activity known to play an important role in the bioactivation of polycyclic aromatic hydrocarbons (PAHs) to mutagenic and carcinogenic metabolites. PAH-induced expression of the CYP1A1 gene appears to be regulated by several trans-acting factors, including the Ah receptor and the 4S PAH-binding protein. In this study, we used the PAH isomers benzo[a]pyrene (BaP) and benzo[e]pyrene (BeP) to further evaluate the role of the 4S PAH-binding protein in induction of the CYP1A1 gene in H4-II-E rat hepatoma cells. Although BaP is believed to bind to both the Ah receptor and the 4S protein, BeP has been reported to bind exclusively to the 4S protein. The results of the study presented here indicate that BaP and BeP induce the expression of the CYP1A1 gene, as measured by ethoxyresorufin O-deethylase (EROD) activity, in a concentration-dependent manner. However, BaP is about 25 times as potent as BeP in inducing EROD activity in these cells. Slot-blot analysis of total RNA isolated from these cells indicated that BeP, BaP, and 3-methylcholanthrene increased the level of CYP1A1 mRNA expression. Sucrose-gradient analysis of BeP binding activity indicated that BeP bound with high affinity to the 4S PAH-binding protein, but not to the Ah receptor. These results suggest that the 4S protein may play a role in the PAH-induced expression of the CYP1A1 gene in rat H4-II-E cells.

Animals↗
PubMed1991

Presence of the 4 S polycyclic hydrocarbon-binding protein in H4-II-E cells.

The 4 S polycyclic aromatic hydrocarbon (PAH)-binding protein had been implicated in regulating the expression of rat cytochrome P450IA1 which is most closely associated with aryl hydrocarbon hydroxylase (AHH). We have now investigated the presence of both the 4 S PAH-binding protein and the 8 S Ah receptor in rat hepatoma H4-II-E cells as well as the induction of P450IA1 upon their exposure to PAH's such as benzo[a]pyrene (BP) and 3-methylcholanthrene (3MC), and halogenated dioxins such as 2,3,7,8-tetrachlordibenzo-p-dioxin (TCDD) and 2,3,7,8-tetrachlorodibenzofuran (TCDBF). Sucrose density gradient analyses and hydroxylapatite assays indicate that, in addition to the 8 S protein, the 4 S PAH binding protein is present in these cells. This protein interacts in a saturable and high affinity manner with BP and 3MC, but not with TCDD or TCDBF. Using a P450IA1 probe, the induction of gene expression was observed by Northern blot analysis of total cellular RNA after exposure of the H4-II-E cells to BP, 3MC, or TCDBF. Since the 4 S protein was observed to interact only with BP and 3MC, these results suggest that this protein may also play a role in the PAH-induced expression of cytochrome P450IA1 gene expression in H4-II-E.

Animals↗
PubMed1990

Binding characteristics of 4S PAH-binding protein and Ah receptor from rats and mice.

Cytochrome P-450IA1 (Cyto-P450IA1) is the isozyme most closely associated with aryl hydrocarbon hydroxylase (AHH). At least two distinct high-affinity binding proteins may regulate its expression, the 4S protein that primarily binds polycyclic aromatic hydrocarbons (PAHs), and the 8S Ah receptor that binds 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) and like congeners. The present study was conducted to investigate ligand binding characteristics of the 4S and 8S binding proteins before and after separation from liver cytosol in the presence and absence of sodium molybdate. Liver cytosol and 4S and 8S receptor-enriched fractions from livers of male Sprague-Dawley rats (AHH-responsive), and from C57BL/6N (AHH-responsive) and DBA/2N and AKR/N mice (AHH-nonresponsive) served as sources of these proteins. Competitive binding studies were performed using 10 nM [3H]benzo[a]pyrene (BaP) or [3H]-TCDD in the presence and absence of a 200-fold excess of BaP, 3-methylcholanthrene (3-MC), and tetrachlorodibenzofuran (TCDBF). Specific PAH-binding activity was assayed by using either sucrose density gradient analysis or a hydroxylapatite assay. Our results indicate that before and after the separation of liver cytosol into 4S and 8S fractions, ligand binding characteristics were relatively unaltered for the 4S protein in comparison to that for the Ah receptor, particularly in the presence of molybdate. The 4S protein had high affinity for BaP and 3-MC but very low affinity for TCDBF; the 8S protein had high affinity for TCDBF, lesser affinity for 3-MC, and low affinity for BaP. In the presence of sodium molybdate, the Ah receptor fractions were significantly stabilized, whereas the 4S protein was relatively unaffected. After the separation of Ah receptor fraction from liver cytosol in the presence of molybdate, 3-MC consistently bound to a greater extent. These results affirm the existence of two distinct PAH-binding proteins.

Animals↗
PubMed1985

Locked twins.

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Female↗