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Development of Methods for Purification of Membrane Associated Gonadotropin‐Releasing Hormone Binding Proteins
Author(s) -
Flanagan Colleen A.,
Tetaz Tim,
Smith A. Ian,
Millar Robert P.
Publication year - 1996
Publication title -
biomedical chromatography
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.4
H-Index - 65
eISSN - 1099-0801
pISSN - 0269-3879
DOI - 10.1002/(sici)1099-0801(199603)10:2<83::aid-bmc563>3.0.co;2-z
Subject(s) - chemistry , agonist , gonadotropin releasing hormone , chromatography , gel electrophoresis , polyacrylamide gel electrophoresis , membrane , receptor , gonadotropic cell , anterior pituitary , biochemistry , luteinizing hormone , hormone , enzyme
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 ‐Lys 6 ,Pro 9 ‐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 an 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.