Purification of gonadotropin releasing hormone receptors using the avidin-biotin technique.
The avidin-biotin technique has been applied to the purification of gonadotropin releasing hormone (GnRH) receptors from other solubilized membrane proteins. The following steps were involved in this approach: (a) solubilization of rat pituitary GnRH receptor with the zwitterionic detergent CHAPS, 3-[(3-cholamidopropyl)-di-methylammonio]-1-propane sulfonate, (b) equilibration of the solubilized GnRH receptor with [biotinyl-D-Lys6]GnRH immobilized on avidin-agarose; and (c) elution of the receptors with high salt and GnRH analogues. Following two cycles of affinity chromatography the GnRH receptor was purified to homogeneity. The overall recovery of the purified receptor was 4-10% of the initial activity in the CHAPS extract and the calculated purification was approximately 10,000 to 15,000 fold. The development of a two step affinity chromatography for the purification of GnRH receptors can be used for detailed studies on the structure and function of the receptor. These studies will advance our understanding of the molecular basis of GnRH action.
Hazum
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Department of Hormone Research, Weizmann Institute of Science, Rehovot, Israel.
D000818:Animals; D001360:Avidin; D001710:Biotin; D002478:Cells, Cultured; D002793:Cholic Acids; D002846:Chromatography, Affinity; D010902:Pituitary Gland / Q000166:cytology / Q000378:metabolism; D010906:Pituitary Hormone-Releasing Hormones / Q000138:chemical synthesis; D051381:Rats; D011967:Receptors, Gonadotropin / Q000302:isolation & purification*; D011966:Receptors, LHRH / Q000302:isolation & purification*
D002793:Cholic Acids; D010906:Pituitary Hormone-Releasing Hormones; D011967:Receptors, Gonadotropin; D011966:Receptors, LHRH; D001360:Avidin; D001710:Biotin; C028213:3-((3-cholamidopropyl)dimethylammonium)-1-propanesulfonate