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The existence of the 4S polycyclic aromatic hydrocarbon-protein binding in 14-day-old chick embryo liver
A Raha1, J W Hamilton, E Bresnick
1Department of Pharmacology and Toxicology, Dartmouth Medical School, Hanover, New Hampshire 03755, USA.
Insights
This study confirms two distinct polycyclic aromatic hydrocarbon (PAH)-binding proteins in chick embryo liver, suggesting a link between the 4S PAH-binding protein and aryl hydrocarbon hydroxylase (AHH) activity.
Area of Science:
- Biochemistry
- Molecular Biology
- Toxicology
Background:
- Cytochrome P-450IA1, crucial for aryl hydrocarbon hydroxylase (AHH) activity, is regulated by two proteins: the 4S polycyclic aromatic hydrocarbon (PAH)-binding protein and the 8S Ah (dioxin) receptor.
- The 4S protein binds PAHs, while the 8S Ah receptor binds 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) and related compounds.
Purpose of the Study:
- To determine if the 4S PAH-binding protein exists in 14-day-old chick embryo liver during maximal AHH activity.
- To investigate potential linkage between the 4S protein and AHH, similar to the 8S Ah receptor.
- To confirm the dioxin receptor's existence by examining ligand binding in the presence and absence of sodium molybdate.
Main Methods:
- Competitive ligand binding assays using liver cytosol from 14-day-old male chick embryos.
- Radioactive ligands [3H]-benzo[a]pyrene (B[a]P) or [3H]-TCDD were used with excess competitor ligands.
- Sucrose gradient analysis was employed to determine specific PAH-binding activity.
Main Results:
- The 4S PAH-binding protein demonstrated high affinity for B[a]P, benzo[e]pyrene (B[e]P), and 3-methylcholanthrene (3-MC) but low affinity for tetrachlorodibenzofuran (TCDBF) in the absence of molybdate.
- The Ah receptor showed high affinity for TCDBF.
- Sodium molybdate stabilized the Ah receptor, with minimal effect on the 4S PAH-binding protein.
Conclusions:
- The study affirms the presence of two distinct PAH-binding proteins in chick embryo liver cytosol.
- Results suggest a functional linkage between the 4S PAH-binding protein and AHH activity.
- The distinct binding characteristics support the existence of separate PAH and dioxin receptors.
Abstract:
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.