Development of methods for purification of membrane associated gonadotropin-releasing hormone binding proteins.
Gonadotropin-releasing hormone (GnRH) is the primary regulator of mammalian reproduction. It stimulates the release of luteinizing hormone and follicle stimulating hormone via receptors on the cell membranes of pituitary gonadotrope cells. This paper describes the development of a protocol for purification of GnRH binding proteins from sheep pituitary membranes. Membranes were best solubilized using a zwitterionic detergent. Solubilized membranes were applied to an affinity column prepared with a GnRH analogue. The most effective analogue was the agonist [D-Lys6,Pro9-NHEt]-GnRH. The column was washed with a gradient of sodium chloride up to 0.4 M and GnRH binding activity was eluted from the column using the acidic buffer. Eluted fractions bound labelled GnRH agonist after neutralization of the buffer. Sodium dodecyl sulphate polyacrylamide gel electrophoresis analysis revealed a major protein band with a relative molecular weight of 67 kD. Amino acid sequence analysis showed that the protein is different from the cloned GnRH receptor, but homologous with a similar protein recently purified from bovine pituitary. This protein may have a function which is modulated by binding of GnRH, GnRH fragments or GnRH-related peptides.
Flanagan
C A
CA
Department of Chemical Pathology, University of Cape Town Medical School, Observatory, South Africa.
Tetaz
T
T
Smith
A I
AI
Millar
R P
RP
D000595:Amino Acid Sequence; D000818:Animals; D015153:Blotting, Western; D002417:Cattle; D002462:Cell Membrane / Q000737:chemistry; D002846:Chromatography, Affinity; D003902:Detergents; D004591:Electrophoresis, Polyacrylamide Gel; D008969:Molecular Sequence Data; D010902:Pituitary Gland / Q000737:chemistry; D011966:Receptors, LHRH / Q000737:chemistry / Q000302:isolation & purification*; D012756:Sheep
D003902:Detergents; D011966:Receptors, LHRH