title: Purification of a candidate gonadotrophin surge-inhibiting/attenuating factor (GnSIF/AF) showing MAPK as a possible target.
abstract: Gonadotrophin surge-inhibiting/attenuating factor (GnSIF/AF) has been known for over two decades, but its molecular structure has not been completely characterized yet. In the last 20 years, five different putative GnSIF/AF sequences have been published. In this article, we describe a procedure to isolate and characterize GnSIF/AF from bovine follicular fluid, a GnSIF/AF-derived synthetic peptide (SP-GnSIF/AF) was produced, and the intracellular bioactivity of GnSIF/AF was tested for intracellular action with a MAPK-assay. Two different bioactive molecular weight forms of GnSIF/AF were isolated, a 160 kDa heteromeric and a monomeric 40 kDa protein. The 40 kDa form appeared to be a subunit of the 160 kDa protein. The synthetic peptide mimicked the actions of GnSIF/AF, such as inhibition of GnRH-induced LH secretion and attenuation of the MAPK phosphorylation. The two GnSIF/AF candidates do not show similarities with previously published GnSIF/AF sequences. These are the first data showing the influence of GnSIF/AF on intracellular processes involved in GnRH self-priming and that the biological action of GnSIF/AF was preserved in the produced synthetic peptide. The results provide strong evidence that the identified candidate proteins are the true GnSIF/AF.
authors.affiliation: Division of Reproductive Medicine, Department of Obstetrics and Gynaecology, VU University Medical Center, Amsterdam, The Netherlands. m.hendriks@vumc.nl
authors.forename: M L
authors.initials: ML
authors.lastname: Hendriks
authors.forename: C B
authors.initials: CB
authors.lastname: Lambalk
authors.forename: M N
authors.initials: MN
authors.lastname: Helder
authors.forename: J
authors.initials: J
authors.lastname: de Koning
mesh_terms: D000818:Animals; D002417:Cattle; D005260:Female; D015571:Follicular Fluid / Q000737:chemistry; D042341:Gonadal Hormones / Q000138:chemical synthesis / Q000302:isolation & purification / Q000502:physiology; D007987:Gonadotropin-Releasing Hormone / Q000032:analysis* / Q000037:antagonists & inhibitors*; D007986:Luteinizing Hormone / Q000032:analysis / Q000037:antagonists & inhibitors / Q000378:metabolism; D051379:Mice; D020928:Mitogen-Activated Protein Kinases / Q000378:metabolism*; D008970:Molecular Weight; D011506:Proteins / Q000138:chemical synthesis / Q000302:isolation & purification / Q000502:physiology; D051381:Rats; D017208:Rats, Wistar
chemical_list: D042341:Gonadal Hormones; D011506:Proteins; C080538:attenuin; D007987:Gonadotropin-Releasing Hormone; D007986:Luteinizing Hormone; D020928:Mitogen-Activated Protein Kinases