(Hydroxyproline9) luteinizing hormone-releasing hormone: A novel peptide in mammalian and frog hypothalamus

Abstract

Luteinizing hormone-releasing hormone (LHRH)-like immunoreactive material was detected in mammalian and frog hypothalamic extracts after high performance liquid chromatography. Radioimmunological detection was performed with three different antibodies directed respectively against the N-(N-antibody) or the C-terminus (C-antibody) as well as the conformational structure (W-antibody) of synthetic mammalian LHRH (mLHRH). Under the chromatographic conditions used, two immunoreactive peptides could be separated. One of those was detected in a homothetic manner by the three antibodies. It coeluted with synthetic mLHRH, had the same amino acid composition as mLHRH, and was shown to correspond to authentic endogenous LHRH. The other peptide was more efficiently detected by the N- than by the two other antibodies. It was eluted prior to mLHRH on reverse phase chromatography but did not correspond to LHRH fragments or to any other known LHRH sequences. Its molecular weight was 1198 Da. Amino acid analysis and enzymatic digestion indicated a very close LHRH sequence homology, with a substitution of proline (Pro) by a hydroxyproline (Hyp) residue in position 9. The new peptide was found in extracts of human, rodent, ovine and amphibian hypothalamus, with a markedly higher concentration in fetal than in adult rats. Various extraction and chromatographic control procedures led to the conclusion that the new peptide was genuinely endogenous and did not result from artifactual modification(s) of LHRH during the purification process. It is concluded that, in the mammalian as in the frog hypothalamus, LHRH-like material corresponds to at least two native molecular forms: (Pro(9)) and (Hyp(9))LHRH.