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Development ofp-Benzoylbenzoylated [N,C,rANP(1-28)]pBNP32 (pBNP1) Derivatives and Affinity Photolabeling of the Bovine NPR-A Receptor

1999; Elsevier BV; Volume: 258; Issue: 1 Linguagem: Inglês

10.1006/bbrc.1999.0597

1090-2104

Martin Coupal, André De Léan, Normand McNicoll, Alain Fournier,

Receptor Mechanisms and Signaling

Two Nα-benzophenone-substituted photoprobes, derived from the high affinity NPR-A chimeric agonist [N, C, rANP(1-28)]pBNP32 (pBNP1) were assembled by solid-phase peptide synthesis. [Nα-p-benzoylbenzoyl, Tyr2]pBNP1 (probe A), and [Nα-p-benzoylbenzoyl, Tyr18]pBNP1 (probe B) were synthesized and their affinity was tested on bovine zona glomerulosa membrane preparations. Both were found to exert ANP-type high affinities (Kd= 20 pM) with Kdof 10 pM and 30 pM for probe A, and probe B, respectively. Photolabeling of NPR-A with both analogs cross-linked specifically the 130 kDa monomeric NPR-A. The maximal irreversible ligand incorporations were estimated at 18% and 41% for probe A, and probe B, respectively. These results show that the N-terminus of the chimeric compound can be acylated with a large chemical function, such as the benzophenone moiety, without loosing its affinity for the NPR-A receptor. Furthermore, Leu2or Leu18can be substituted with tyrosine without disturbing the binding capacity of the ligand. Finally, it appears that the pBNP1 N-terminus is close to the receptor structure as irreversible incorporation is observed after photolabeling.