Reduction and reoxidation of equine gonadotropin alpha-subunits.
Reduction and reoxidation of equine gonadotropin alpha-subunits.
复制标题
马促性腺激素 α 亚基的还原和再氧化。
作者：
G. Bousfield;D. N. Ward
在已知对牛和人的α-亚基有效的条件下，对绵羊(O)和马(E)的lhα亚基进行还原和再氧化。OLHα复性的主要产物是α-亚基单体。相反，ELHα形成了121,000摩尔重量的聚集体。单体Elhα被回收，但产率大大降低。为了测试碳水化合物变化对马促性腺激素α亚基聚集的影响，将马促性腺激素α亚基(eFSHα、CGα、ELHα和游离α亚基)还原并重新氧化。在每种情况下，主要产物都是121,000摩尔重量的聚集体，并伴随着单体马阿尔法。用三氟甲烷磺酸水解法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-再氧化还原糖基化的Elhα没有进入12%十二烷基硫酸钠-聚丙烯酰胺凝胶。α-亚基制剂、ELH-α本身和垂体游离α-亚基的黄体生成素受体结合活性最高。在操作上，后者是在ELH纯化程序的最后一步从ELH中分离出来的；因此，该制剂中可能存在黄体生成素污染。还原和再氧化将这两种制剂的促黄体生成素受体结合活性降低到促卵泡刺激素α和心电α制剂中观察到的促黄体生成素活性水平。我们的结论是，在马的α-亚基制剂中观察到的大部分促黄体生成素受体结合活性是由于受到ELH的污染。我们还获得了初步的证据，表明蛋白质水解性“刻痕”的马α亚基的氨基末端和羧基末端片段能够正确地折叠，形成α单体。
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.
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DOI：
--
发表时间：
1987
期刊：
The Journal of biological chemistry
影响因子：
--
作者：
Sugino,H;Bousfield,GR;MooreJr,WT;Ward,DN
通讯作者：
Ward,DN
位点特异性诱变定义了绒毛膜促性腺激素β亚基的天冬酰胺连接寡糖的细胞内作用。
DOI：
--
发表时间：
1988
期刊：
The Journal of biological chemistry
影响因子：
--
作者：
Matzuk,MM;Boime,I
通讯作者：
Boime,I
用无细胞系统硫酸化促黄体素寡糖。
DOI：
10.1073/pnas.81.17.5320
发表时间：
1984
影响因子：
11.1
作者：
Green,ED;Gruenebaum,J;Bielinska,M;Baenziger,JU;Boime,I
通讯作者：
Boime,I
单个基因编码马黄体生成素和绒毛膜促性腺激素的β亚基。
DOI：
10.1210/mend.6.6.1379674
发表时间：
1992
期刊：
Molecular endocrinology (Baltimore, Md.)
影响因子：
--
作者：
Sherman,GB;Wolfe,MW;Farmerie,TA;Clay,CM;Threadgill,DS;Sharp,DC;Nilson,JH
通讯作者：
Nilson,JH
使用离子色谱法分析糖蛋白和其他生物化合物中的硫酸盐和磷酸盐。
DOI：
10.1016/s0021-9673(01)96511-2
发表时间：
1987
期刊：
Journal of chromatography
影响因子：
--
作者：
Ward,DN;Wen,T;Bousfield,GR
通讯作者：
Bousfield,GR