Phosphorylation of Androgen Sensitive Chromosomal Proteins in Mouse Submandibular Gland

1雄激素受体结合位点在核内的分布将丙酸睾丸酮处理后的小鼠下颌下腺细胞核用微球菌核酸酶消化，甘油梯度离心分析活性染色质组分。使用[ ^3H]米勃龙（合成雄激素）作为配体，通过体外交换试验检测与染色质结合位点相关的内源性雄激素受体复合物。睾酮处理后1h，双核体雄激素受体复合物水平升高，3 h单核体和三核体雄激素水平升高;这表明雄激素受体与染色质相关，并且这种相关性发生在对微球菌核酸酶优先敏感的转录活性染色质区域。和活性染色质分数 ...更多信息 丙酸睾酮从下颌下腺制备核染色质和活性染色质组分，并与[γ-^<32>P]ATP孵育。细胞核组分在体外迅速将<32>[γ-^<32>P]ATP中的^ P掺入内源性染色体蛋白。替吉奥导致磷酸化的速率在3小时增加，并在6小时降低到对照水平，表明核相关蛋白被细胞核固有的蛋白激酶磷酸化。睾酮处理后3 h，活性染色质磷酸化水平增加，这种刺激作用与~（13）<32>P掺入核磷蛋白的作用相似; 3核染色质和活性染色质中蛋白激酶的活性用组蛋白作为标记物测定蛋白激酶的活性。衬底在睾酮处理后3 h，在不透明部分中的蛋白激酶活性受到刺激，在活性染色质部分中观察到雄激素的类似作用;表明活性染色质相关蛋白磷酸化的雄激素反应。4磷酸化核蛋白的SDS-Page分析从磷酸化核蛋白中分离非组蛋白，并通过SDS-Page分离，然后进行放射自显影。放射自显影<32>显示磷酸化的非组蛋白条带，分子量在20 ~ 40 KD之间。少

1 Intranuclear distribution of androgen receptor binding sites Nuclei from mouse submandibular gland after administration of testosterone propionate were digested with micrococcal nuclease and resulting active chromatin fraction was analyzed by glycerol gradient centrifugation. Endogenous androgen receptor complexes associated with chromatin binding sites were detected by an in vitro exchange assay using a [ ^3H]mibolerone (synthetic androgen) as a ligand. The level of the androgen receptor complex in dinucleosomes increased 1 h after the testosterone treatment and the androgen level in the mono- and trinucleosomes increased 3 h after the testosterone treatment ; indicating that the androgen receptor associated with chromatin and this association occurred in transcriptionally active chromatin regions which were preferentially sensitive to micrococcal nuclease.2 Phosphorylation of chromosomal proteins in nuclear fraction and active chromatin fractionFemale mice were injected with testos … More terone propionate. Nuclear and active chromatin fractions were prepared from submandibular glands and incubated with [gamma-^<32>P]ATP. The nuclear fraction rapidly incorporated ^<32>P from [gamma-^<32>P]ATP Bin vitro into endogenous chromosomal proteins. Testosterone resulted in an increase in the rate of phosphorylation at 3 h and reduced to the control level at 6 h ; indicating that nuclear-associated proteins were phosphorylated by protein kinase intrinsic to nuclei. The level of phosphorylation in the active chromat in fraction increased 3 h after the testosterone treatment and this stimulation was similar to that of ^<32>P incorporation into nuclear phosphoproteins ; suggesting that the protein kinase which was sensitive to androgen was present in the active chromatin fraction.3 Activities of protein kinase in nuclear and active chromatin fractionsThe activities of protein kinase were measured using histone as a substrate. The activities of protein kinase in the unclear fraction stimulated at 3 h after testosterone treatment and a similar effect of androgen was observed in the active chromatin fraction ; indicating the androgenic response of phosphorylation of active chromatin-associated proteins.4 SDS-Page analysis of phosphorylated nuclear proteinsNonhistone proteins were isolated from phosphorylated nuclear proteins and separated by SDS-Page followed by autoradiography. Autoradiograms of ^<32>P labeled proteins showed phosphorylated nonhistone proteins bands with molecular mass ranging from 20 KD to 40 KD. Less