Perdew G H
Department of Foods and Nutrition, Purdue University, West Lafayette, Indiana 47907.
Arch Biochem Biophys. 1991 Dec;291(2):284-90. doi: 10.1016/0003-9861(91)90136-7.
2-[125I]iodo-7,8-dibromo-p-dioxin ([125I]Br2DpD) and 2-[125I]iodo-3-azido-7,8-dibromo-p-dioxin ([125I]N3Br2-DpD) are both capable of binding to the Ah receptor (AhR) with a high degree of specificity in cultured Hepa 1c1c7 cells. After incubation with either [125I]N3Br2DpD or [125I]Br2DpD Hepa 1c1c7 cytosolic and high salt nuclear extracts were analyzed by sucrose density gradient analysis with the following results: (i) With both radioligands an approximately 9 S form of the AhR was observed in cytosolic extracts. (ii) Nuclear extracts labeled with [125I]N3Br2DpD revealed both approximately 6 S and approximately 9 S forms of the AhR. (iii) In contrast, analysis of nuclear extracts labeled with [125I]Br2DpD revealed only an approximately 6 S form of the AhR. The approximately 9 S [125I]N3Br2DpD-labeled AhR was preferentially extracted with 100 mM KCl from a nuclear fraction and mixed with monoclonal antibody 8D3, an anti-90-kDa heat shock protein antibody. Monoclonal antibody 8D3 was able to bind to the approximately 9 S nuclear form of the AhR and caused the receptor to sediment as a heavier complex on sucrose density gradients. This would indicate that the AhR can reside in the nucleus bound to 90-kDa heat shock protein. The [125I]N3Br2DpD-labeled approximately 6 S peak fractions were collected and subjected to denaturing two-dimensional gel electrophoresis. A comparison of [125I]N3Br2DpD-labeled cytosolic (9 S) AhR preparations with the nuclear (6 S) AhR by 2-D gel electrophoresis was performed. The cytosolic form of the AhR was present in the apparent pI range of 5.2-5.7; the nuclear form focused between 5.5 and 6.2. The [125I]N3Br2DpD-labeled nuclear extracts were incubated with ATP-agarose and 43% of the photoaffinity-labeled AhR bound to the affinity gel. In contrast, approximately threefold lower binding of [125I]N3Br2DpD-labeled receptor was obtained when GTP-, AMP-, or ADP-agarose was used. Only 2% of the [125I]N3Br2DpD-labeled cytosolic AhR was able to bind to ATP-agarose. These results suggest that after the AhR translocates into the nucleus the following biochemical changes occur: (i) The sedimentation value for the AhR changes from an approximately 9 S to an approximately 6 S species. (ii) The AhR attains the ability to bind with specificity to ATP. (iii) The AhR undergoes a shift to a more basic pI.
2-[125I]碘-7,8-二溴-对二噁英（[125I]Br2DpD）和2-[125I]碘-3-叠氮基-7,8-二溴-对二噁英（[125I]N3Br2-DpD）在培养的Hepa 1c1c7细胞中均能够以高度特异性与芳烃受体（AhR）结合。用[125I]N3Br2DpD或[125I]Br-2DpD孵育Hepa 1c1c7细胞的胞质溶胶和高盐核提取物后，通过蔗糖密度梯度分析得到以下结果：（i）两种放射性配体在胞质溶胶提取物中均观察到约9S形式的AhR。（ii）用[125I]N3Br2DpD标记的核提取物显示出约6S和约9S两种形式的AhR。（iii）相比之下，用[125I]Br2DpD标记的核提取物分析仅显示出约6S形式的AhR。用100 mM KCl从核部分优先提取约9S的[125I]N3Br2DpD标记的AhR，并与单克隆抗体8D3（一种抗90-kDa热休克蛋白抗体）混合。单克隆抗体8D3能够与约9S的核形式的AhR结合，并使该受体在蔗糖密度梯度上以较重的复合物形式沉降。这表明AhR可以与90-kDa热休克蛋白结合存在于细胞核中。收集[125I]N3Br2DpD标记的约6S峰级分，并进行变性二维凝胶电泳。通过二维凝胶电泳对[125I]N3Br2DpD标记的胞质溶胶（9S）AhR制剂与核（6S）AhR进行比较。AhR的胞质溶胶形式存在于表观pI范围5.2 - 5.7；核形式聚焦在5.5和6.2之间。将[125I]N3Br2DpD标记的核提取物与ATP-琼脂糖孵育，43%的光亲和标记的AhR与亲和凝胶结合。相比之下，当使用GTP-、AMP-或ADP-琼脂糖时，[125I]N3Br2DpD标记的受体的结合量降低约三倍。只有2%的[125I]N3Br2DpD标记的胞质溶胶AhR能够与ATP-琼脂糖结合。这些结果表明，AhR转运到细胞核后会发生以下生化变化：（i）AhR的沉降值从约9S变为约6S。（ii）AhR获得与ATP特异性结合的能力。（iii）AhR的pI向更碱性方向转变。
