van der Walt L A, Wittliff J L
Laboratory, James Graham Brown Cancer Center, Louisville, KY.
J Chromatogr. 1988 Mar 18;425(2):277-91. doi: 10.1016/0378-4347(88)80032-x.
Molecular heterogeneity of the human uterine progestin receptor was investigated employing sucrose density gradient centrifugation and high-performance liquid chromatography (HPLC) in size-exclusion (HPSEC), ion-exchange (HPIEC) and chromatofocusing (HPCF) modes. Synthetic progestomimetic ligands, [3H]R5020 and [3H]ORG-2058, were used to identify these receptors. Rapid centrifugation with a vertical tube rotor showed both 8-9 S and 4-5 S receptor species in the presence of 10 mM sodium molybdate with a 90-96% recovery. [3H]R5020 displayed greater nonspecific binding than [3H]ORG-2058. When separated receptor preparations were labeled, each with a different ligand, mixed and separated on optimized gradients, at least two receptor isoforms were identified in the components sedimenting at 8-9 S. HPSEC confirmed the presence of receptor isoforms displaying different molecular size and shape dependent upon the progestin ligand used. When the surface charge properties were examined by HPIEC using AX-1000, two distinct species were observed irrespective of the radioactive ligand. The first peak appeared in the void volume similar to the position of free steroid, indicating the possibility of ligand stripping by the column. The second peak bound steroid specifically and eluted with 100 mM phosphate. If either 8-S or 4-S progestin receptors were first separated by gradient centrifugation then by HPIEC, both receptor isoforms eluted with 60 mM phosphate. Re-chromatography of these on HPIEC also gave the isoform eluting at 60 mM phosphate. HPCF of ligand-bound receptors on AX-500 columns also identified one isoform eluting at pH 5.6-6.1. Using a combination of HPLC techniques and sucrose gradient centrifugation, heterogeneity of the progestin receptor has been demonstrated.
采用蔗糖密度梯度离心法和高效液相色谱法（HPLC），在尺寸排阻（HPSEC）、离子交换（HPIEC）和色谱聚焦（HPCF）模式下，研究了人子宫孕激素受体的分子异质性。使用合成孕激素类似物配体[3H]R5020和[3H]ORG-2058来鉴定这些受体。在10 mM钼酸钠存在下，使用垂直管转子进行快速离心，结果显示存在8 - 9 S和4 - 5 S的受体种类，回收率为90 - 96%。[3H]R5020的非特异性结合比[3H]ORG-2058更强。当分离的受体制剂分别用不同的配体标记、混合并在优化的梯度上分离时，在8 - 9 S沉降的组分中鉴定出至少两种受体亚型。HPSEC证实，根据所使用的孕激素配体不同，存在显示不同分子大小和形状的受体亚型。当使用AX-1000通过HPIEC检查表面电荷性质时，无论放射性配体如何，均观察到两种不同的种类。第一个峰出现在空体积中，类似于游离类固醇的位置，表明存在被柱体剥离配体的可能性。第二个峰特异性结合类固醇，并用100 mM磷酸盐洗脱。如果首先通过梯度离心然后通过HPIEC分离8 - S或4 - S孕激素受体，则两种受体亚型均用60 mM磷酸盐洗脱。将这些在HPIEC上重新色谱分离，也得到在60 mM磷酸盐处洗脱的亚型。在AX-500柱上对配体结合受体进行HPCF分析，也鉴定出一种在pH 5.6 - 6.1处洗脱的亚型。通过结合HPLC技术和蔗糖梯度离心法，已证明孕激素受体具有异质性。
