Bousfield G R, Liu W K, Ward D N
Department of Biochemistry and Molecular Biology, University of Texas M.D. Anderson Cancer Center, Houston 77030.
Endocrinology. 1989 Jan;124(1):379-87. doi: 10.1210/endo-124-1-379.
Residues 121-149 of equine LH beta (eLH beta) were removed by a simple mild acid treatment procedure. The modified eLH beta, des(121-149)eLH beta, was isolated by gel permeation chromatography on Sephacryl S-200. Recombination of des(121-149)eLH beta with eLH alpha and ovine LH alpha (oLH alpha) produced LH derivatives as efficiently as recombination with native eLH beta. In rat testicular LH radioligand assay systems employed in this study the potencies of the resulting LH preparations were, in order of decreasing potency: des(121-149)eLH beta:eLH alpha hybrid greater than eLH greater than eLH alpha + beta greater than oLH greater than des(121-149)eLH beta:oLH alpha greater than oLH alpha + eLH beta (1:0.82:0.67:0.15:0.02:0.006, eLH tracer; 1:0.88:0.67:0.21:0.02:0.006, hCG tracer). In a horse testicular LH radioligand assay with eLH tracer, only the equine LH derivatives were active, and the order of potencies was the same: des(121-149)eLH beta:eLH alpha hybrid greater than eLH greater than eLH alpha + beta (1:0.58:0.46). In a rat testicular Leydig cell steroidogenesis assay, eLH was the most active preparation, but the relative potencies of the other preparations remained unchanged: eLH greater than des(121-149)eLH beta:eLH alpha greater than eLH alpha + beta greater than oLH greater than des(121-149)eLH beta:oLH alpha greater than oLH alpha + eLH beta (1:0.61:0.55:0.27:0.004:0.003). We have previously reported that the hybrid consisting of native eLH beta and oLH alpha was inactive (less than 1%) in LH receptor and steroidogenesis assays. The data reported herein confirm this observation and demonstrate that the absence of LH activity in eLH beta:oLH alpha cannot be attributed to the C-terminal extension on eLH beta, since the des(121-149)eLH beta:oLH alpha hybrid LH is also inactive. Examination of the intrinsic FSH activity of eLH in both rat and chicken testicular FSH radioligand assays produced the following results; eLH, recombined eLH subunits, and des(121-149)eLH beta:eLH alpha were all of the same potency (13% and 0.9% as active as eFSH in rats and chickens, respectively). We conclude that the C-terminal extension on eLH and eCG beta-subunits is not involved in subunit association, LH receptor binding, or FSH receptor binding. The derivative des(121-149)eLH beta:eLH alpha provides a model compound that may be useful in determining the role, if any, of the glycoprotein hormone C-terminal extension that appears to have arisen independently at least twice in mammalian evolution.
通过简单的温和酸处理程序去除了马促黄体生成素β（eLHβ）的121 - 149位氨基酸残基。经修饰的eLHβ，即缺失（121 - 149）的eLHβ（des(121 - 149)eLHβ），通过在Sephacryl S - 200上进行凝胶渗透色谱法分离得到。缺失（121 - 149）的eLHβ与eLHα以及羊促黄体生成素α（oLHα）重组产生促黄体生成素衍生物的效率与与天然eLHβ重组的效率相同。在本研究中使用的大鼠睾丸促黄体生成素放射性配体测定系统中，所得促黄体生成素制剂的效力按效力递减顺序排列为：缺失（121 - 149）的eLHβ:eLHα杂交体大于eLH大于eLHα + β大于oLH大于缺失（121 - 149）的eLHβ:oLHα大于oLHα + eLHβ（以eLH为示踪剂时为1:0.82:0.67:0.15:0.02:0.006；以hCG为示踪剂时为1:0.88:0.67:0.21:0.02:0.006）。在以eLH为示踪剂的马睾丸促黄体生成素放射性配体测定中，只有马促黄体生成素衍生物具有活性，效力顺序相同：缺失（121 - 149）的eLHβ:eLHα杂交体大于eLH大于eLHα + β（1:0.58:0.46）。在大鼠睾丸间质细胞类固醇生成测定中，eLH是最具活性的制剂，但其他制剂的相对效力保持不变：eLH大于缺失（121 - 149）的eLHβ:eLHα大于eLHα + β大于oLH大于缺失（121 - 149）的eLHβ:oLHα大于oLHα + eLHβ（1:0.61:0.55:0.27:0.004:0.003）。我们之前报道过，由天然eLHβ和oLHα组成的杂交体在促黄体生成素受体和类固醇生成测定中无活性（小于1%）。本文报道的数据证实了这一观察结果，并表明eLHβ:oLHα中促黄体生成素活性的缺失不能归因于eLHβ的C末端延伸，因为缺失（121 - 149）的eLHβ:oLHα杂交促黄体生成素也无活性。在大鼠和鸡睾丸促卵泡激素放射性配体测定中对eLH的内在促卵泡激素活性进行检测，得到以下结果；eLH、重组的eLH亚基以及缺失（121 - 149）的eLHβ:eLHα效力均相同（在大鼠和鸡中分别为eFSH活性的13%和0.9%）。我们得出结论，eLH和eCGβ亚基的C末端延伸不参与亚基缔合、促黄体生成素受体结合或促卵泡激素受体结合。缺失（121 - 149）的eLHβ:eLHα衍生物提供了一种模型化合物，可能有助于确定糖蛋白激素C末端延伸的作用（如果有），这种延伸在哺乳动物进化过程中似乎至少独立出现了两次。
