Purification and characterization of an Ah receptor binding factor in chromatin.
Purification and characterization of an Ah receptor binding factor in chromatin.
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染色质中 Ah 受体结合因子的纯化和表征。
DOI：
10.1016/0006-2952(95)02199-x
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发表时间：
1996
影响因子：
5.8
通讯作者：
Ruh,MF
中科院分区：
文献类型：
--
作者：
Dunn2nd,RT;Ruh,TS;Burroughs,LK;Ruh,MF
二恶英通过与特定的细胞内受体（Ah 受体）相互作用以及随后 Ah 受体与染色质的相互作用来诱导生物反应。我们之前报道了从兔肝脏中部分纯化的 Ah 受体与染色质中的受体结合因子（称为 AhRBF）的结合。通过将染色质吸附到羟基磷灰石上，然后用 3 M NaCl 和 1–8 M 盐酸胍 (GdnHCl) 连续提取来分离兔肝染色质蛋白 (CP)。在本研究中，我们继续纯化 CP5 级分，该级分表现出 AhRBF 活性。 CP5 中的蛋白质通过 CL-Sepharose 6B 柱色谱分离，分离出较低分子量的组分。为了测定受体结合，使用 7.5 至 0.0 M GdnHCl 的反向梯度透析将每个 CL-Sepharose 6B 级分的一部分重构为兔双链 DNA (dsDNA)。然后通过链霉素过滤结合测定检查这些重构的染色质与[3H]-2,3,7,8-四氯二苯并-p-二恶英([3H]TCDD)-受体复合物的结合。两种分子量在 10,000-14,000 范围内的蛋白质片段表现出与 Ah 受体的高亲和力结合。通过与未标记的 TCDD (TCDD-R) 结合的 Ah 受体的竞争实验表明，重组的 AhRBF 与 dsDNA 的结合对 [3H]TCDD-R 具有 > 90% 的特异性。通过制备型 SDS-PAGE 进一步纯化，AhRBF 活性归因于分子量在 12,000 至 10,000 之间的两个级分。发现具有 AhRBF 活性的 12 kDa 蛋白的等电点 (pI) ⩾ 10。12 kDa AhRBF 在溴化氰裂解后通过 Edman 降解进行测序，并鉴定为组蛋白 H4。尽管组蛋白 H4 已被假定与多种系统中的转录因子相互作用，但这是 AhR 与组蛋白 H4 特异性相互作用的首次报道。
Dioxin induces biological responses through interaction with a specific intracellular receptor, the Ah receptor, and the subsequent interaction of the Ah receptor with chromatin. We previously reported the binding of the Ah receptor, partially purified from rabbit liver, to receptor binding factors (termed AhRBFs) in chromatin. Rabbit liver chromatin proteins (CP) were isolated by adsorption of chromatin to hydroxylapatite followed by sequential extraction with 3 M NaCl and 1–8 M guanidine hydrochloride (GdnHCl). In the present study, we continued the purification of the CP5 fraction, which exhibited AhRBF activity. The proteins in CP5 were separated by CL-Sepharose 6B column chromatography resolving lower molecular weight fractions. To assay for receptor binding, a portion of each CL-Sepharose 6B fraction was reconstituted to rabbit doublestranded DNA (dsDNA) using a reverse gradient dialysis of 7.5 to 0.0 M GdnHCl. These reconstituted chromatins were then examined for binding to [3H]-2,3,7,8- tetrachlorodibenzo -p- dioxin ([3H]TCDD)-receptor complexes by the streptomycin filter binding assay. Two protein fractions with a molecular weight in the range of 10,000–14,000 demonstrated high affinity binding to the Ah receptor. The binding of AhRBFs reconstituted to dsDNA was shown, by competition experiments with Ah receptor bound by unlabeled TCDD (TCDD-R), to be > 90% specific for [3H]TCDD-R. Further purification was achieved by preparative SDS-PAGE, and AhRBF activity was attributed to two fractions with molecular weights between 12,000 and 10,000. A 12 kDa protein with AhRBF activity was found to have an isoelectric point (pI) of ⩾ 10. The 12 kDa AhRBF was sequenced by Edman degradation after cyanogen bromide cleavage and identified as histone H4. Although histone H4 has been postulated to interact with transcription factors in a variety of systems, this is the first report of a specific interaction of AhR with histone H4.