|Budget Amount *help
¥2,200,000 (Direct Cost : ¥2,200,000)
Fiscal Year 1992 : ¥700,000 (Direct Cost : ¥700,000)
Fiscal Year 1991 : ¥800,000 (Direct Cost : ¥800,000)
Fiscal Year 1990 : ¥700,000 (Direct Cost : ¥700,000)
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.