Preparation and characterization of photoreactive derivatives of GnRH

Photoreactive derivatives of GnRH and its analogues were prepared by incorporation of the 2-nitro-4(5)-azidophenylsulfenyl [2,4(5)-NAPS] group into amino acid residues at positions 1, 3, 6, or 8 of the decapeptide sequence. The modification of Trp3 by the 2,4-NAPS group led to a complete loss of the luteinizing hormone (LH) releasing as well as LH-release-inhibiting activity of the peptide. The [D-Lys(2,4-NAPS)]6 analogue was a very potent agonist that, after covalent attachment by photoaffinity labeling, caused prolonged LH secretion at a submaximal rate. [Orn(2,4-NAPS)]8-GnRH, a full agonist with a relative potency of 7% of GnRH, after photoaffinity labeling caused prolonged maximal LH release from cultured pituitary cells. In contrast, [Orn(2,5-NAPS)]8-GnRH, although being equipotent with the 2,4-NAPS isomer in terms of LH releasing ability, was unable to cause prolonged LH release after photoaffinity labeling. Thus, [Orn(2,4-NAPS)]8-GnRH is a very effective photolabeling ligand of the functionally significant pituitary GnRH receptor. Based on this compound, a pituitary peptidase resistant derivative, D-Phe6,[Orn(2,4-NAPS)]8-GnRH-(1-9)-ethylamide, was synthesized. This derivative showed high-affinity binding to pituitary membranes with a Kd comparable to those of other GnRH analogues. A radioiodinated form of this peptide was used for pituitary GnRH-receptor labeling. This derivative labeled 59- and 57-kDa proteins in rat and 58- and 56-kDa proteins in bovine pituitary membrane preparations, respectively. This peptide also labeled pituitary GnRH receptors in the solubilized state and therefore appears to be a suitable ligand for the isolation and further characterization of the receptor.

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Preparation and characterization of photoreactive derivatives of GnRH

Cited by 4 publications

References 26 publications

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[11] General principles for photoaffinity labeling of peptide hormone receptors

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