CHARACTERIZATION OF CYTOSOLIC GLUCOCORTICOID RECEPTOR OF FETAL-RAT EPIPHYSEAL CHONDROCYTES
CHARACTERIZATION OF CYTOSOLIC GLUCOCORTICOID RECEPTOR OF FETAL-RAT EPIPHYSEAL CHONDROCYTES
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DOI：
10.1016/0022-4731(90)90259-u
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发表时间：
1990-03-01
影响因子：
4.1
通讯作者：
BRENT, RL
中科院分区：
文献类型：
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作者：
CARBONE, JP;BALDRIDGE, RC;BRENT, RL
细胞溶质糖皮质激素受体或第21天妊娠大鼠骺软骨细胞进行了评估。受体，一类糖皮质激素结合组分接近饱和，利用[3 H]曲安奈德（[3 H]TA）作为放射性标记的配体，在约1.8-2.0 × 10 - 6浓度下。10-8 M.解离常数（Kd）反映了高亲和力结合，等于4.0 ±。1.43 .+-. 10-9[3H]TA的M（n = 7）。从Scatchard分析估计的受体浓度约为250 fmol/mg胞质蛋白，当以位点/细胞为基础计算时，等于5800位点/细胞。发现类固醇与受体的相对结合亲和力为曲安奈德>皮质酮>氢化可的松>孕酮>醋酸甲羟孕酮> 17 α-羟孕酮。羟孕酮>>睾酮> 17 β-雌二醇通过加温（25 ℃）体外活化的细胞溶质制剂。20分钟）显示出对DNA-纤维素的亲和力增加。总的特异性结合的活化配体-受体复合物的46%与DNA-纤维素结合。细胞溶质保持在0 - 4 ℃。C在10 mM β-内酰胺酶存在下或在β-内酰胺酶存在下体外活化，分别以8%和10%与DNA-纤维素结合。非活化受体的DEAE-Sephadex洗脱曲线表明在0.4 M KCl下从柱中洗脱的单一结合部分。活化受体的洗脱曲线表明活化诱导的受体不稳定性。0.4 M KCl峰减弱，而0.2 M KCl峰的伴随增加仅可轻微辨别。使用[甲基-14 C]酪蛋白作为底物评价软骨细胞胞质溶胶中的内源性蛋白水解活性显示出温度依赖性蛋白水解活性，最适pH为5.9-6.65。蛋白水解活性对热失活敏感，并且通过20 mM EDTA是可恢复的。非活化受体的沉降系数为9.3s（n = 6）的蔗糖密度梯度，并表现出类固醇的特异性和激活诱导的分子变化时，孵育在10 mM的dopamine的存在下，电阻。在体外受体活化，在不存在的情况下，诱导受体对蛋白水解攻击的敏感性增加和/或配体受体解离增强，如通过9.3s结合形式的减少而不伴随5s或3s受体片段的增加所证明的。
The cytosolic glucocorticoid receptor or 21st gestational day rat epiphyseal chondrocytes has been evaluated. The receptor, a single class of glucocorticoid binding component approached saturation, utilizing [3H]triamcinolone acetonide ([3H]TA) as the radiolabeled ligand, at approximately 1.8-2.0 .times. 10-8 M. The dissociation constant (Kd) reflected high-affinity binding, equaling 4.0 .+-. 1.43 .+-. 10-9 M (n = 7) for [3H]TA. The concentration of receptor estimated from Scatchard analysis was approximately 250 fmol/mg cytosolic protein and when calculated on a sites/cell basis equalled 5800 sites/cell. The relative binding affinites of steroid for receptor were found to be triamcinolone acetonide > corticosterone > hydrocortisone > progesterone > medroxyprogesterone acetate >> 17.alpha.-hydroxyprogesterone >> testosterone > 17.beta.-estradiol. Cytosolic preparations activated in vitro by warming (25.degree. C for 20 min) were shown to exhibit an increased affinity for DNA-cellulose. 46% of the total specifically bound activated ligand-receptor complex was bound to DNA-cellulose. Cytosol maintained at 0-4.degree. C in the presence of 10 mM molybdate or activated in vitro in the presence of molybdate, bound to DNA-cellulose at 8 and 10% respectively. DEAE-Sephadex elution profiles of the nonactivated receptor were indicative of a single binding moiety which eluted from the columns at 0.4 M KCl. Elution profiles of activated receptor were suggestive of an activation induced receptor lability. The 0.4 M KCl peak was diminished, while a concomitant increase in the 0.2 M KCl peak was only modestly discernible. Evaluation of endogenous proteolytic activity in chrondrocyte cytosol using [methyl-14C]casein as substrate show a temperature-dependent proteolytic activity with a pH optimum of 5.9-6.65. The proteolytic activity was susceptible to heat inactivation and was inhibitable, by 20 mM EDTA. The sedimentation coefficient of the nonactivated receptor was 9.3s (n = 6) on sucrose density gradients and exhibited steroid specificity and a resistance to activation induced molecular alterations when incubated in the presence of 10 mM molybdate. Receptor activation in vitro, in the absence of molybdate induced an increased receptor susceptibility to proteolytic attack and/or enhanced ligand receptor dissociation as evidenced by a diminution of the 9.3s binding form without a concomitant increase in 5s or 3s receptor fragments.