Hendry W J, Danzo B J
J Steroid Biochem. 1986 Sep;25(3):433-43. doi: 10.1016/0022-4731(86)90258-x.
The nucleomyofibrillar fraction of mature rabbit epididymides contains a salt-extractable and leupeptin-sensitive protease that alters the sedimentation coefficient of cytosolic steroid receptors. We refer to this modification as receptor conversion. The substrate used in these studies was cytosolic estrogen receptor obtained from frozen rabbit uteri. The unactivated form of the receptor exists as an oligomer under hypotonic (0.01 M KCl) conditions (S20,w congruent to 9.6, Stokes radius (Rs) congruent to 7.4 nm, Mr congruent to 320,000) and dissociates under hypertonic (0.4 M KCl) conditions to yield the steroid-binding monomer (S20,w congruent to 4.7, Rs congruent to 5.1 nm, Mr congruent to 104,000). According to analysis under hypotonic conditions, the epididymal protease disrupts the oligomeric architecture of the receptor and reduces the size of the steroid-binding monomer (S20,w congruent to 3.2, Rs congruent to 3.0 nm, Mr congruent to 42,000). The epididymal protease had no detectable effect on the structure of the proteins used as standards for the ultracentrifugal or gel filtration analyses. Although inhibited by leupeptin, the epididymal enzyme is not a typical thiol protease since it was unaffected by thiol-blocking agents (iodoacetamide and N-ethylmaleimide), and was partially inhibited by thiol-reducing agents (monothioglycerol and dithiothreitol). Calcium and magnesium ions alone, or in combination with ATP, had no effect on the activity of the protease. However, both cations selectively suppressed recovery of the oligomeric receptor form. These results, in conjunction with those from previous studies, serve to distinguish the epididymal protease from receptor-active proteases described in extracts of other animal tissues. Molybdate, at a concentration of 50 mM, blocked receptor conversion. The ability of the receptor to be stabilized by molybdate was lost following conversion. Finally, the epididymal protease appears to remove a portion of the estrogen receptor that is necessary for nucleotide-binding.
成熟兔附睾的核肌原纤维部分含有一种可被盐提取且对亮抑酶肽敏感的蛋白酶，该蛋白酶会改变胞质类固醇受体的沉降系数。我们将这种修饰称为受体转化。这些研究中使用的底物是从冷冻兔子宫中获得的胞质雌激素受体。在低渗（0.01 M KCl）条件下，未活化形式的受体以寡聚体形式存在（S20,w约为9.6，斯托克斯半径（Rs）约为7.4 nm，相对分子质量（Mr）约为320,000），在高渗（0.4 M KCl）条件下会解离，产生类固醇结合单体（S20,w约为4.7，Rs约为5.1 nm，Mr约为104,000）。根据低渗条件下的分析，附睾蛋白酶会破坏受体的寡聚结构，并减小类固醇结合单体的大小（S20,w约为3.2，Rs约为3.0 nm，Mr约为42,000）。附睾蛋白酶对用作超速离心或凝胶过滤分析标准的蛋白质结构没有可检测到的影响。尽管附睾酶受到亮抑酶肽的抑制，但它不是典型的巯基蛋白酶，因为它不受巯基阻断剂（碘乙酰胺和N - 乙基马来酰亚胺）的影响，且受到巯基还原剂（单硫甘油和二硫苏糖醇）的部分抑制。单独的钙和镁离子，或与ATP结合，对蛋白酶的活性没有影响。然而，这两种阳离子都选择性地抑制了寡聚受体形式的恢复。这些结果与先前研究的结果相结合，有助于将附睾蛋白酶与其他动物组织提取物中描述的受体活性蛋白酶区分开来。50 mM浓度的钼酸盐可阻断受体转化。受体被钼酸盐稳定的能力在转化后丧失。最后，附睾蛋白酶似乎会去除核苷酸结合所需的一部分雌激素受体。
