Affinity chromatography and the purification of estrogen receptors.
Affinity chromatography and the purification of estrogen receptors.
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亲和层析和雌激素受体的纯化。
DOI：
10.1016/s0021-9258(19)43479-0
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发表时间：
1973
期刊：
影响因子：
--
通讯作者：
P. Cuatrecasas
中科院分区：
文献类型：
--
作者：
V. Sica;I. Parikh;E. Nola;G. Puca;P. Cuatrecasas
介绍了从小牛子宫粗提物中提取的雌激素受体亲和层析用的各种含雌二醇琼脂糖载体的制备、测试和应用。在使用之前，凝胶必须在长时间内用有机溶剂彻底清洗。洗涤程序的充分性必须通过检查层析的子宫提取物的游离态雌二醇和可溶性雌二醇受体复合体的流出物来测试。在纯化过程中，即使完全去除非共价吸附的激素也不能防止激素的释放，因为某些含雌二醇的吸附剂具有化学键，容易通过减少子宫粗提物中存在的物质和亲核试剂而加速切割。激素从凝胶中释放到介质中并不一定排除受体在吸附剂上的有效吸附；这种情况是否发生将取决于凝胶结合和游离激素分子的相对亲和力和浓度。通过亲和吸附剂提取的受体分子可以通过在30°的含雌二醇缓冲液中孵育凝胶来高产率地洗脱出来；所利用的条件提高了凝胶结合受体的解离速度，并有利于这些受体与可溶性雌二醇分子的有效交换。各种含有雌二醇的衍生物通过类固醇分子的A环共价结合，作为受体的吸附剂是无效的。在玻璃和聚丙烯酰胺载体上制备的类似类型的吸附剂被证明比利用琼脂糖制得的吸附剂要差。虽然含有19-去甲睾酮17-琥珀酸酯的琼脂糖衍生物能选择性地与雌二醇受体结合，但柱容量较低，最有效的亲和吸附剂是将17-β-17-琥珀酸雌二醇酯连接到含有白蛋白的琼脂糖衍生物或支化的聚(L-赖氨酸)/聚(dl-丙氨酸)(侧臂)共聚物上。这些大分子“链”可能通过多点连接与琼脂糖基质结合，它们具有大量的功能基团，允许高度的配体取代，并且连接的配体离琼脂糖骨架非常远。其中一些吸附剂非常有效，即使用未取代的琼脂糖将凝胶稀释100倍，也能显著提取受体。用这种方法稀释的吸附剂可以在纯化过程中常规使用，因为可以实现对超过90%的受体的选择性吸附。此外，洗涤凝胶的并发症减少了，非受体蛋白的非特异性吸附最小，并促进了吸附受体的洗脱。从粗制的子宫上清液中提纯子宫雌二醇受体(S 4.5.5)可纯化10,000-100,000倍，一步总收率为30-50%。纯化的受体在分子大小(凝胶过滤)、沉淀系数、等电聚焦和热稳定性方面与前面描述的相同。
The preparation, testing, and use of a variety of estradiol-containing agarose supports for affinity chromatography of estrogen receptors from crude extracts of calf uterus are described. Before use the gels must be washed exhaustively with organic solvents over prolonged periods of time. The adequacy of the washing procedures must be tested by examining the effluents of chromatographed uterine extracts for free estradiol as well as for soluble estradiol-receptor complexes. Even complete removal of the non-covalently adsorbed hormone may not safeguard against the release of hormone during the purification procedures, since certain estradiol-containing adsorbents possess chemical linkages which are susceptible to accelerated cleavage by reducing substances and nucleophiles present in the crude uterine extracts. The release of hormone from the gel into the medium does not necessarily exclude effective adsorption of receptors onto the adsorbent; whether this occurs will depend on the relative affinities and concentrations of the gel-bound and free hormone molecules.Receptor molecules specifically extracted by affinity adsorbents can be eluted from the gels in high yield by incubating the gel, in suspension, with estradiol-containing buffers at 30°; the conditions utilized enhance the rate of dissociation of gel-bound receptors and favor an effective exchange of these receptors with the soluble estradiol molecules.Various agarose derivatives containing estradiol covalently bound through linkages with the A ring of the steroid molecules are ineffective as adsorbents for receptors. Similar kinds of adsorbents prepared on glass and polyacrylamide supports proved to be inferior to those which utilize agarose. Although agarose derivatives containing 19-nortestosterone 17-hemisuccinate can selectively bind estradiol receptors, the capacity of these columns is low.The most effective affinity adsorbents were prepared by attaching 17β-estradiol 17-hemisuccinate to agarose derivatives containing albumin or the branched copolymer of poly(l-lysine) (“backbone”) and poly(dl-alanine) (“side arms”). These macromolecular “leashes” are presumably bound to the agarose matrix by multipoint attachments, they possess numerous functional groups which permit a high degree of ligand substitution, and the attached ligand is very distant from the agarose backbone. Some of these adsorbents are so effective that significant extraction of receptors occurs even when the gels are diluted 100-fold with unsubstituted agarose. Adsorbents diluted in this way can be used routinely in purification procedures, since selective adsorption of receptors in excess of 90% can be achieved. Furthermore, the complications of washing the gels are diminished, nonspecific adsorption of non-receptor proteins is minimal, and elution of the adsorbed receptors is facilitated. Uterine estradiol receptors (4.5 S) from crude uterine supernatants can be purified between 10,000- and 100,000-fold in over-all yields of 30 to 50% in a single step. The purified receptors are identical with those described earlier by molecular size (gel filtration), sedimentation coefficient, isoelectric focusing, and heat stability.