Phosphorylation of Androgen Sensitive Chromosomal Proteins in Mouse Submandibular Gland

用微球菌核酸酶消化小鼠颌下腺核素后的雄激素受体结合位点的核内分布，用甘油梯度离心分析所得活性染色质部分。内源性雄激素受体复合物与染色质结合位点通过体外交换测定使用[^3H]米波内酮（合成雄激素）作为配体。睾酮处理后1 h二核小体雄激素受体复合物水平升高，3 h单核小体和三核小体雄激素水平升高；这表明雄激素受体与染色质相关，这种关联发生在转录活跃的染色质区域，这些区域对微球菌核酸酶优先敏感核区和活性染色质区染色体蛋白的磷酸化研究雌性小鼠注射睾酮和丙酸酮。从下颌腺中提取核染色质和活性染色质，用[γ -^<32>P]ATP孵育。核部分迅速将体外[γ -^<32>P]ATP Bin中的^<32>P并入内源性染色体蛋白。睾酮导致磷酸化率在3小时增加，在6小时降低到对照水平；表明核相关蛋白被核固有的蛋白激酶磷酸化。在睾酮处理后3 h，活性染色质部分磷酸化水平升高，这种刺激类似于^<32>P掺入核磷酸化蛋白；提示活性染色质部分存在对雄激素敏感的蛋白激酶细胞核和活性染色质部分蛋白激酶的活性用组蛋白作为底物测定蛋白激酶的活性。在睾酮治疗后3小时，蛋白激酶活性在不清楚部分受到刺激，雄激素在活性染色质部分也有类似的作用；表明活性染色质相关蛋白磷酸化的雄激素反应磷酸化核蛋白的SDS-Page分析从磷酸化核蛋白中分离出nonhistone蛋白，用SDS-Page进行分离，然后进行放射自显影。^<32>P标记蛋白的x线自显像显示磷酸化的非组蛋白带，分子质量在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