STUDIES ON THE LHRH-LIKE IMMUNOREACTIVE SUBSTANCE IN THE PINEAL GLAND AND ITS BIOLOGICAL ROLE.

在牛、大鼠和羊的松果体中有免疫反应性和生物活性GnRH的报道。我们试图用抗gnrh抗体来表征大鼠松果体中的这种gnrh样物质。本实验室制备的单克隆抗体LRH13是一种具有良好特征的GnRH特异性抗体，该抗体识别在各种动物中高度保守的n端3-5个氨基酸序列。结合不受N端和c端结构的影响，表明LRH13不仅可以识别加工过的GnRH，也可以识别未加工过的GnRH前体分子。使用该抗体，我们可以在大鼠松果体中识别出强烈的免疫组织化学信号，而与固定液的pH无关，尽管Piekut（1982,1983）报道免疫组织化学信号依赖于固定液的酸度。经SDS-聚丙烯酰胺凝胶电泳（SDS- page）和免疫印迹检测，gnrh免疫反应物质的表观分子量为52kD。52kD蛋白的免疫化学信号是LRH13特异性的，因为0.5mg LHRH在10ml LRH13溶液（1:100稀释）中完全阻断。信号仅在膜组分中检测到，而在松果体匀浆的细胞质上清中检测不到。在pH为7.4的50mM Tris-HCl缓冲液中，NaCl（140和500mM）、Mg^<++>(5mM)和EDTA（10mM）等溶解剂不能释放这种52kD膜结合的gnrh样物质。而Triton-X-100（2%）从膜组分中释放52kD蛋白。提示大鼠松果体GnRH免疫反应物质是一种膜蛋白。二维电泳和免疫印迹显示两种gnrh免疫反应物质具有不同的等电点；6.8和7.0。对52kD蛋白的v8蛋白酶进行多肽定位实验，结果表明，这两个蛋白的氨基酸序列完全相同。

Immunoreactive and bioactive GnRH in the pineal gland has been reported in cattle, rat, and sheep. We tried to characterize this GnRH-like substance in the rat pineal gland using an anti-GnRH antibody. A monoclonal antibody, LRH13, prepared in our laboratory, is a well-characterized GnRH specific antibody which recognizes the n-terminal 3-5 amino acid sequence which is highly conserved in various animals. the binding is not affected by the N- and C-terminal structures, indicating that LRH13 can recognize not only processed GnRH, but also unprocessed GnRH precursor molecule.Using this antibody, we could identify strong immunohistochemical signal in the rat pineal gland irrespective of pH of the fixatives though Piekut(1982,1983) reported that the immunohistochemical signal was dependent on the acidity of the fixative. By SDS- polyacrylamide gel electrophoresis (SDS-PAGE) and immunoblot, the GnRH-immunoreactive substance showed an apparent molecular weight of 52kD. The immunochemical signal of 52kD protein was LRH13-specific because of the complete blocking by 0.5mg of LHRH in 10ml of LRH13 solution (diluted 1:1000). The signal was detected only in the membrane fraction, and not in the cytosolic supernatant of the pineal homogenate. This 52kD membrane bound GnRH-like substance could not be released by the treatments with some agents dissolved in the 50mM Tris-HCl buffers, pH 7.4, like NaCl (140 and 500mM), Mg^<++>(5mM) and EDTA(10mM). But Triton-X-100 (2%) released the 52kD protein from the membrane fraction. This result suggests that the rat pineal GnRH- immunoreactive substance is a membrane protein. Second-dimensional electrophoresis and immunoblot revealed two GnRH-immunoreactive substances with different isoelectric points; 6.8 and 7.0. Peptide mapping experiment with V8-protease of the 52kD proteins showed that these two proteins are identical in their amino acid sequences.