Isolation and characterization of a gonadotropin-releasing hormone binding protein in goldfish serum.
A binding protein (BP) specific for gonadotropin-releasing hormone (GnRH) was previously demonstrated in goldfish serum. In the present study the binding protein was isolated and further characterized. The GnRH-BP, partially purified from goldfish serum using polyacrylamide gel electrophoresis (PAGE) under nondenaturing conditions, was concentrated in a single band, separate from all major components of serum proteins. The binding ability of the partially purified GnRH-BP was conserved; the isolated GnRH-BP specifically bound salmon GnRH and chicken GnRH-II, the native forms of GnRH present in goldfish, but not other forms of GnRH. The relative binding affinity of the partially purified GnRH-BP was [D-Arg6,Pro9-NEt]-salmon GnRH greater than chicken GnRH-II greater than or equal to salmon GnRH. The GnRH-BP, in raw serum or partially purified by PAGE, was specifically covalently labeled using 125I-[D-Lys6,Pro9-NEt]-salmon GnRH and the bifunctional cross-linking reagent, disuccinimidyl suberate, and then subjected to sodium dodecyl sulfate-PAGE under reducing conditions. The location of the radiolabeled GnRH-BP on PAGE gels was determined by cutting gels into sections and counting the radioactivity, or by autoradiography; the molecular weight of the GnRH-BP was estimated to be 40 KD. The covalently labeled GnRH-BP extracted from SDS-PAGE was subjected to high pressure liquid chromatography, and it coeluted with a single protein peak of the GnRH-BP partially purified by PAGE under nonreducing conditions. These studies demonstrate that the GnRH-BP is a minor component of serum proteins in goldfish; it is a single nonglycoprotein of about 40 kDa.
Huang
Y P
YP
Department of Zoology, University of Alberta, Edmonton, Canada.
Guo
D C
DC
Peter
R E
RE
D000345:Affinity Labels; D000818:Animals; D002851:Chromatography, High Pressure Liquid; D003432:Cross-Linking Reagents; D004305:Dose-Response Relationship, Drug; D004591:Electrophoresis, Polyacrylamide Gel; D005260:Female; D006054:Goldfish / Q000502:physiology*; D007987:Gonadotropin-Releasing Hormone / Q000494:pharmacology; D008297:Male; D011966:Receptors, LHRH / Q000302:isolation & purification*
D000345:Affinity Labels; D003432:Cross-Linking Reagents; D011966:Receptors, LHRH; D007987:Gonadotropin-Releasing Hormone