权益分类	功能权益	普通用户	{{item.name}}会员
{{category.name}}	{{benefitItem.name}}

Phosphorylation of Androgen Sensitive Chromosomal Proteins in Mouse Submandibular Gland

小鼠颌下腺雄激素敏感染色体蛋白的磷酸化

基本信息

批准号：
01571025
负责人：
SATO Nobuko
金额：
$ 1.28万
依托单位：
Iwate Medical University
依托单位国家：
日本
项目类别：
Grant-in-Aid for General Scientific Research (C)
财政年份：
1989
资助国家：
日本
起止时间：
1989 至 1990
项目状态：
已结题

项目摘要

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

1注射丙酸睾丸酮后小鼠颌下腺雄激素受体结合部位的核内分布，用微球核酸酶消化后，用甘油梯度离心法分析活性染色质组分。用体外交换实验检测与染色质结合部位相关的内源性雄激素受体复合体。二核小体中雄激素受体复合体的水平在睾酮处理后1小时增加，单核和三核小体中的雄激素水平在睾酮处理后3小时增加；这表明雄激素受体与染色质相关，这种联系发生在转录活跃的染色质区域，该区域对微球菌核酶优先敏感。2核部分和活性染色质部分染色体蛋白的磷酸化。雌性小鼠注射睾丸…更多的丙酸特龙。从颌下腺制备核染色质组分和活性染色质组分，并与[Gamma-^&lt；32&gt；P]ATP孵育。核组分迅速地将来自体外的[Gamma-^&lt；32&gt；P]ATP B的^&lt；32&gt；P结合到内源染色体蛋白中。睾酮使核相关蛋白的磷酸化速率在3h时升高，6h时降至对照水平，说明核相关蛋白被细胞核内固有的蛋白激酶所磷酸化。睾酮处理3h后，活性染色质组分的磷酸化水平增加，这种刺激作用类似于~(32)&gt；P掺入核磷蛋白的过程，提示活性染色质组分中存在对雄激素敏感的蛋白激酶。3核染色质组分和活性染色质组分的蛋白激酶活性。在睾酮处理3h后刺激的不明部分的蛋白激酶活性，在活性染色质部分也观察到雄激素的类似作用，表明活性染色质相关蛋白的磷酸化的雄激素反应。4磷酸化核蛋白的SDS-PAGE分析从磷酸化的核蛋白中提取非组蛋白，经SDS-PAGE分离，然后进行放射自显影。~&lt；32&gt；P标记蛋白质的放射自显影显示非组蛋白的磷酸化条带，其分子质量在20~40 kD之间。较少