Characterization of gonadotropin-releasing hormone (GnRH)-immunoreactive protein in the rat pineal gland.

The aim of the present study was to characterize GnRH-like substance(s) in the rat pineal gland using a monoclonal antibody, LRH13, as a probe. The epitope of LRH13 is between 2nd and 5th amino acid residues of the mammalian GnRH, and its immunological characters were previously defined by us. LRH13 could show strong immunological signal on the rat pineal gland. Immunoblot after SDS-PAGE of the pineal gland preparations showed a LRH13 immunoreactive band with apparent mol wt 52 kilo-Dalton (kD), which is much bigger than that of hypothalamic GnRH precursor (10 kD). The 52 kD protein, however, was detected from insoluble fraction of the pineal homogenate and liberated from the fraction by Triton X-100 (2%) treatment. On the other hand, NaCl (140 mM and 500 mM) or EDTA (10 mM) treatment failed to liberate. Two-dimensional gel electrophoresis showed that the 52 kD protein is a mixture of two proteins with different isoelectric points (pI approximately 6.8 and 7.0). Both proteins showed identical patterns of peptide mapping by V8 protease digestion, and they might be originated from the same peptide. These results suggest that the rat pineal GnRH-immunoreactive substance has a unique property as a membrane associate protein.