ABSTRACT
Rat and hamster brain tissues were used to investigate the possible existence of a follicle stimulating hormone (FSH)-releasing factor with similar characteristics to the lamprey gonadotropin-releasing hormone III (lGnRH-III) form proposed in previous reports. The present studies involved isolation and purification of the molecule by high-performance liquid chromatography (HPLC), identification by radioimmunoassay, sequence analysis by automated Edman degradation, mass spectrometry and examination of biological activity. Hypothalamic extracts from both species contained an HPLC fraction that was immunoreactive to GnRH and coeluted with lGnRH-III and 9-hydroxyproline mGnRH ([Hyp(9)]GnRH). Determination of primary structure from purified total brain material demonstrated that the isolated molecule was [Hyp(9)]GnRH. This is the first report showing the presence of the posttranslationally modified form already known as [Hyp(9)]GnRH by primary sequence analysis. The biological activity of distinct GnRH peptides was also tested in vitro for gonadotropin release using rat pituitary primary cell cultures. The results showed that [Hyp(9)]GnRH stimulated both luteinizing hormone and FSH release, as already reported, whereas lGnRH-III had no action on the secretion of either gonadotropin.
Subject(s)
Brain/metabolism , Gonadotropin-Releasing Hormone/chemistry , Gonadotropin-Releasing Hormone/pharmacology , Amino Acid Sequence/genetics , Animals , Cricetinae , Female , Follicle Stimulating Hormone/metabolism , Gonadotropin-Releasing Hormone/analogs & derivatives , Gonadotropin-Releasing Hormone/genetics , Gonadotropin-Releasing Hormone/isolation & purification , Hydroxyproline/analogs & derivatives , Hydroxyproline/pharmacology , Hypothalamus/metabolism , Luteinizing Hormone/metabolism , Male , Mass Spectrometry , Mesocricetus , Pituitary Gland/cytology , Pituitary Gland/metabolism , Protein Isoforms/chemistry , Protein Isoforms/genetics , Protein Isoforms/isolation & purification , Protein Isoforms/pharmacology , Rats , Structure-Activity RelationshipABSTRACT
The melanin-concentrating hormone (MCH) is a cyclic neuropeptide which induces skin paling and may be involved in the control of the pituitary adrenal axis in teleost fishes. We have recently cloned and characterized the salmon and rat MCH mRNAs and we report in the present paper the cloning and sequencing of the human counterpart. The deduced human MCH (hMCH) precursor is 165 amino acids long and as for rat and salmon, encodes the MCH peptide at the C-terminus. The human and rat MCH precursors are very similar to one another but differ extensively from the salmon counterpart. Strong sequence conservation was found in the regions of mammalian prohormones encoding the novel putative neuropeptides named NGE and NEI which we had originally identified in the rat MCH precursor. Furthermore, sequence identities, with perhaps functional implications, were found among the MCH, human ANF, and aplysia peptide A hormone precursors.
Subject(s)
Hypothalamic Hormones , Melanins/genetics , Pituitary Hormones/genetics , Amino Acid Sequence , Animals , Aplysia/genetics , Atrial Natriuretic Factor/genetics , Base Sequence , DNA/genetics , Gene Library , Humans , Hypothalamus , Molecular Sequence Data , Neuropeptides/genetics , Protein Precursors/genetics , Rats/genetics , Salmon/genetics , Sequence Alignment , Sequence Homology, Nucleic Acid , Species SpecificityABSTRACT
A melanin-concentrating hormone (MCH)-like peptide was isolated from rat hypothalamus by acid extraction, gel filtration chromatography, immunoaffinity chromatography using antiserum directed against salmon MCH, and two steps of HPLC using octadecyl columns. Several zones of immunoreactivity were isolated, and Edman degradation in a gas phase sequencer indicated that the amino acid sequence of all zones was identical. Rat hypothalamic MCH is a nonadecapeptide which differs from salmon MCH by an N-terminal extension of two amino acids and four additional substitutions. Rat MCH has the following primary structure: Asp-Phe-Asp-Met-Leu-Arg-Cys-Met-Leu-Gly-Arg-Val-Tyr-Arg-Pro-Cys-Trp-Gln- Val.