Bousfield G R, Ward D N
Department of Biological Sciences, Wichita State University, Kansas 67260-0026.
Endocrinology. 1992 Dec;131(6):2986-98. doi: 10.1210/endo.131.6.1280209.
Ovine (o) and equine (e) LH alpha-subunits were reduced and reoxidized using conditions known to be effective for bovine and human alpha-subunits. The major product of oLH alpha refolding was alpha-subunit monomer. In contrast, eLH alpha formed a 121,000 mol wt aggregate. Monomeric eLH alpha was recovered, but in greatly reduced yield. To test the effects of carbohydrate variation on the aggregation of equine alpha-subunits, all of the equine gonadotropin alpha-subunits (eFSH alpha, eCG alpha, eLH alpha, and free alpha-subunit) were reduced and reoxidized. In each case, the major product was the 121,000 mol wt aggregate accompanied by monomeric equine alpha. Removal of carbohydrate by trifluoromethane sulfonic acid hydrolysis accentuated the tendency to aggregation during reoxidation. Most reduced-reoxidized deglycosylated eLH alpha did not enter a 12% sodium dodecyl sulfate-polyacrylamide gel. The highest LH receptor-binding activities were found in the alpha-subunit preparations, eLH alpha itself and pituitary free alpha-subunit. Operationally, the latter was separated from eLH in the last step of the eLH purification procedure; thus, LH contamination in this preparation is likely. Reduction and reoxidation reduced the LH receptor-binding activity of these two preparations to the level of LH activity observed in the eFSH alpha and eCG alpha preparations. We concluded that the majority of the LH receptor-binding activity observed in equine alpha-subunit preparations was due to contamination with eLH. We also obtained preliminary evidence that the amino-terminal and carboxy-terminal fragments of proteolytically "nicked" equine alpha-subunits refolded properly to form alpha monomer.
使用已知对牛和人α亚基有效的条件，对绵羊（o）和马（e）的促黄体生成素α亚基进行还原和再氧化。oLHα重折叠的主要产物是α亚基单体。相比之下，eLHα形成了分子量为121,000的聚集体。单体eLHα得以回收，但产率大幅降低。为了测试碳水化合物变化对马α亚基聚集的影响，对所有马促性腺激素α亚基（eFSHα、eCGα、eLHα和游离α亚基）进行还原和再氧化。在每种情况下，主要产物都是分子量为121,000的聚集体，并伴有单体马α亚基。用三氟甲磺酸水解去除碳水化合物会加剧再氧化过程中的聚集倾向。大多数还原再氧化的去糖基化eLHα无法进入12%的十二烷基硫酸钠-聚丙烯酰胺凝胶。在α亚基制剂、eLHα本身和垂体游离α亚基中发现了最高的促黄体生成素受体结合活性。实际上，后者是在eLH纯化过程的最后一步从eLH中分离出来的；因此，该制剂中可能存在LH污染。还原和再氧化使这两种制剂的促黄体生成素受体结合活性降低到在eFSHα和eCGα制剂中观察到的LH活性水平。我们得出结论，在马α亚基制剂中观察到的大多数促黄体生成素受体结合活性是由于eLH污染所致。我们还获得了初步证据，即经蛋白水解“切口”的马α亚基的氨基末端和羧基末端片段能正确重折叠形成α单体。
