大鼠颗粒细胞中第二种雌激素结合物质的进一步特性研究。

Further characterization of the second oestrogen-binding species of the rat granulosa cell.

作者信息

Kudolo G B, Elder M G, Myatt L

出版信息

J Endocrinol. 1984 Jul;102(1):93-102. doi: 10.1677/joe.0.1020093.

DOI:10.1677/joe.0.1020093

Abstract

Rat granulosa cell cytosol contains a second oestrogen-binding species (SOB) distinguished from the classical oestrogen receptor by its lower dissociation constant (approx. 45 nmol/l) and the ability to bind oestrogens, antioestrogens, androgens and progesterone but not diethylstilboestrol. The SOB and the oestrogen receptor can be further distinguished by their differential adsorption to spheroidal hydroxylapatite and Concanavalin A-Sepharose. Addition of chaotropic salts or molybdate to granulosa cell cytosol did not alter the concentration of SOB or oestrogen receptor measured, indicating that there are no 'masked' binding sites in the two species caused by aggregation phenomena. The association rate of oestradiol with SOB at 4 degrees C (1.72 +/- 0.27(S.E.M.) X 10(8) mol/h) and 25 degrees C (4.50 +/- 0.36 X 10(8) mol/h) was faster than with the oestrogen receptor (7.20 +/- 0.15 X 10(7) mol/h and 1.23 +/- 0.15 X 10(8) mol/h respectively). The biphasic dissociation kinetics of [3H]oestradiol from the oestrogen receptor at 25 degrees C (rate constants k-1 = 0.30 +/- 0.07/min and k-2 = 3.73 +/- 0.57 X 10(-3)/min) were similar to those reported in other target tissues but the dissociation of [3H]oestradiol from SOB appeared to be much more rapid and could not be measured by the Sephadex LH-20 separation method employed for determining receptor kinetics. Using sucrose density-gradient (SDG) analysis and Sephacryl S-200 gel chromatography the oestrogen receptor fractionated in an aggregated form (10.3S, Stokes radius greater than 5.2 nm) in low ionic strength buffers and as a small species (4.4S, Stokes radius 3.5 nm) in buffers containing 0.4 M-KCl. However, the SOB fractionated as 2-3S, Stokes radius 3.7-4.0 nm at low ionic strength and as 5.8S, Stokes radius 3.5 nm in 0.4 M-KCl. In contrast to the receptor from other target tissues the granulosa cell oestrogen receptor did not bind to the artificial acceptor matrix oligo(dT)-cellulose and heat activation did not promote a 4S to 5S conversion when analysed on SDG. The salt-extracted form of nuclear receptor sedimented at 4.6S, mol. wt 69-72000 on SDG.

摘要

大鼠颗粒细胞胞质溶胶含有第二种雌激素结合物质（SOB），它与经典雌激素受体的区别在于其较低的解离常数（约45 nmol/l），以及能够结合雌激素、抗雌激素、雄激素和孕酮，但不能结合己烯雌酚。SOB和雌激素受体可通过它们对球状羟基磷灰石和伴刀豆球蛋白A-琼脂糖的不同吸附作用进一步区分。向颗粒细胞胞质溶胶中添加离液盐或钼酸盐不会改变所测得的SOB或雌激素受体的浓度，这表明这两种物质中不存在由聚集现象导致的“隐蔽”结合位点。在4℃（1.72±0.27（标准误）×10⁸ mol/h）和25℃（4.50±0.36×10⁸ mol/h）时，雌二醇与SOB的结合速率比与雌激素受体的结合速率快（分别为7.20±0.15×10⁷ mol/h和1.23±0.15×10⁸ mol/h）。在25℃时，[³H]雌二醇从雌激素受体上解离的双相动力学（速率常数k⁻¹ = 0.30±0.07/min和k⁻² = 3.73±0.57×10⁻³/min）与其他靶组织中报道的相似，但[³H]雌二醇从SOB上的解离似乎要快得多，并且无法通过用于测定受体动力学的Sephadex LH - 20分离方法来测量。使用蔗糖密度梯度（SDG）分析和Sephacryl S - 200凝胶色谱法，雌激素受体在低离子强度缓冲液中以聚集形式（10.3S，斯托克斯半径大于5.2 nm）分级分离，而在含有0.4 M - KCl的缓冲液中则以小物种（4.4S，斯托克斯半径3.5 nm）分级分离。然而，SOB在低离子强度下分级分离为2 - 3S，斯托克斯半径3.7 - 4.0 nm，在0.4 M - KCl中为5.8S，斯托克斯半径3.5 nm。与其他靶组织的受体不同，颗粒细胞雌激素受体不与人工受体基质寡聚（dT）-纤维素结合，并且在SDG分析时热激活不会促进4S到5S的转化。核受体的盐提取形式在SDG上沉降为4.6S，分子量69 - 72000。