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

据报道，牛、大鼠和绵羊松果体中的GnRH具有免疫活性和生物活性。我们试图用一种抗GnRH抗体来鉴定这种类似于GnRH的物质在大鼠松果体中的分布。本实验室研制的单抗LRH13是一种特异性较强的GnRH特异性抗体，能识别在多种动物中高度保守的N端3-5氨基酸序列。这种结合不受N端和C端结构的影响，表明LRH13不仅可以识别处理过的GnRH，还可以识别未处理的GnRH前体分子。使用这种抗体，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，与固定剂的pH值无关，尽管Piekut(1982,1983)报道了免疫组织化学信号依赖于固定剂的酸度。经SDS-PAGE和免疫印迹分析，GnRH免疫反应物的表观分子量为52kD。52kD蛋白的免疫化学信号是LRH13特异的，因为在10ml的LRH13溶液中加入0.5 mg的LHRH(1：1000稀释)可以完全阻断LHRH。该信号仅在松果体匀浆的细胞膜部分中检测到，而在松果体匀浆的胞浆上清液中未检测到。溶于50 mM Tris-HCl缓冲液、pH 7.4的氯化钠(140 mM和500 mM)、镁离子(5 MM)、EDTA(10 MM)等试剂不能释放这种52kD膜结合的GnRH样物质。但Triton-X-100(2%)从膜组分中释放出52kD的蛋白。这一结果提示，大鼠松果体GnRH免疫反应物质为膜蛋白。双向电泳和免疫印迹显示两种不同等电点的GnRH免疫反应物质：6.8和7.0。用V8-蛋白酶对52kD蛋白进行的肽图谱实验表明，这两种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.