Occurrence of fragmentation of free and combined forms of the beta-subunit of human chorionic gonadotropin.
Occurrence of fragmentation of free and combined forms of the beta-subunit of human chorionic gonadotropin.
复制标题
人绒毛膜促性腺激素 β 亚基的游离形式和组合形式发生断裂。
作者：
A. Puisieux;D. Bellet;F. Troalen;A. Razafindratsita;C. Lhommé;C. Bohuon;J. Bidart
为了进一步研究生物体液中hCGβ的各种游离形式和结合形式的片段，我们进行了一维和二维十二烷基硫酸钠-聚丙烯酰胺凝胶电泳，然后使用针对hCGβ-(111-116)部分(单抗fb12)抗血清的抗肽抗体对hCGβ(8-16)部分或针对第47位结束的片段的抗血清进行免疫印迹。在一种粗制的尿hCG中观察到的结果表明，除了还原的hCGβNICK亚基(βNS)的羧基末端部分[hCGβ-(48-145)]外，在二硫键断裂后，还检测到另外三个摩尔质量分数为18,000(F1)、16,500(F2)和12,000(F3)的片段。免疫反应图谱和多肽序列分析表明，F1片段由hCGβ-(1-47)序列组成，而F2片段由6-47部分组成。然后，我们研究了孕妇或四名绒毛膜癌患者尿液中的β-NS。结果表明，hCG和游离的β-亚基均含有β-NS。此外，这些尿样中存在的游离hCGβ似乎被广泛地(如果不是全部)划破。通过两步层析法将hCG从其β亚基中分离出来，用特异的单抗免疫放射分析法鉴定hCG和hCGβ免疫反应峰，并在还原条件下通过一维电泳分析所得的制剂，然后用fb12进行Western免疫印迹，证实了尿液中观察到的结果。后一种方案也被用来研究4例绒毛膜癌肿瘤患者血清中β-NS的存在。在这些血清中，hCG似乎被切断了。这项研究证明hCG的β亚基被多个片段修饰。
In an attempt to further study various fragments of free and combined forms of hCG beta present in biological fluids, we performed one- and two-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis, followed by Western immunoblotting using antipeptide antibodies directed to the hCG beta-(111-116) portion (monoclonal antibody FB12) antiserum to the hCG beta(8-16) portion or antiserum which was specific for fragments ending at residue 47. Results observed in a crude preparation of urinary hCG demonstrated that in addition to the carboxyl-terminal part of the reduced hCG beta nicked subunit (beta NS) [hCG beta-(48-145)], three other fragments of mol wt 18,000 (F1), 16,500 (F2), and 12,000 (F3) were detectable after cleavage of disulfide bonds. Both the immunoreactivity pattern and peptide sequencing revealed that the F1 fragment was constituted of the hCG beta-(1-47) sequence, whereas the F2 fragment comprised the 6-47 portion. We then studied the beta NS in urine from either pregnant women or four patients with choriocarcinomas. Results showed that both hCG and the free beta-subunit contained beta NS. Furthermore, free hCG beta present in those urine samples appeared to be extensively, if not totally, nicked. Results observed in urine were confirmed using separation of hCG from its beta-subunit by a two-step chromatography procedure, identification of hCG and hCG beta immunoreactive peaks by specific monoclonal immunoradiometric assay, and analysis of resulting preparations by one-dimensional electrophoresis under reducing conditions, followed by Western immunoblotting with FB12. This latter protocol was also used to investigate the presence of beta NS in sera of four patients with choriocarcinoma tumors. In those sera, hCG appeared to be nicked. This study demonstrates that the beta-subunit of hCG is modified by multiple fragmentations.