Novel ligands for the affinity labelling of luteinizing hormone releasing hormone receptors.
A number of novel luteinizing hormone releasing hormone (LHRH) analogues incorporating biotin together with potential covalent attachment sites have been synthesized. Those based on the des-Gly10-[D-Lys6]-LHRH ethylamide peptide backbone resulted in the most useful characteristics of binding to the LHRH receptor in rat anterior pituitary gland membranes. Of these, des-Gly10-[biotinyl-aminoethylglycyl-D-Lys6]-LHRH ethylamide (XBAL) gave the best specific: non-specific binding ratio, with 44 +/- 6% (+/- S.E.M.) of total binding being specific with a Kd of 131 +/- 16 pM (+/- S.E.M., n = 4) as determined by Scatchard analysis. Two methods have been used to covalently crosslink these analogues with the LHRH receptor; photoaffinity labelling and the use of homobifunctional N-hydroxysuccinimide ester crosslinkers. The photoaffinity analogues gave poor specific: non-specific binding ratios. Of the chemical crosslinkers tested, ethylene glycolbis(succinimidylsuccinate) (EGS) was found to be the most efficient at covalently linking the 125I-XBAL bound to the LHRH receptor site. At an EGS concentration of 5 mM, 23 +/- 3% (+/- S.E.M.) of the specific binding of 125I-XBAL was covalently crosslinked.
Ogier
S A
SA
MRC Brain Metabolism Unit, Edinburgh, Scotland.
Mitchell
R
R
Bladon
C M
CM
D000345:Affinity Labels; D000818:Animals; D003432:Cross-Linking Reagents; D007987:Gonadotropin-Releasing Hormone / Q000031:analogs & derivatives; D008024:Ligands; D008297:Male; D008566:Membranes / Q000378:metabolism; D010903:Pituitary Gland, Anterior / Q000378:metabolism; D051381:Rats; D011919:Rats, Inbred Strains; D011966:Receptors, LHRH
D000345:Affinity Labels; D003432:Cross-Linking Reagents; D008024:Ligands; D011966:Receptors, LHRH; D007987:Gonadotropin-Releasing Hormone