Development of p-benzoylbenzoylated [N,C,rANP(1-28)]pBNP32 (pBNP1) derivatives and affinity photolabeling of the bovine NPR-A receptor.
Two Nalpha-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. [Nalpha-p-benzoylbenzoyl, Tyr2]pBNP1 (probe A), and [Nalpha-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 Kd of 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, Leu2 or Leu18 can 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.
Coupal
M
M
INRS/Institut Armand-Frappier, Université du Québec, Pointe-Claire, Québec, Canada.
De Léan
A
A
McNicoll
N
N
Fournier
A
A
D000595:Amino Acid Sequence; D000818:Animals; D002417:Cattle; D006162:Guanylate Cyclase / Q000737:chemistry* / Q000378:metabolism; D008969:Molecular Sequence Data; D009419:Nerve Tissue Proteins / Q000235:genetics*; D019920:Photoaffinity Labels / Q000737:chemistry* / Q000378:metabolism; D011869:Radioligand Assay; D017461:Receptors, Atrial Natriuretic Factor / Q000737:chemistry* / Q000378:metabolism; D015384:Zona Glomerulosa / Q000737:chemistry / Q000378:metabolism
D009419:Nerve Tissue Proteins; D019920:Photoaffinity Labels; D006162:Guanylate Cyclase; D017461:Receptors, Atrial Natriuretic Factor; C091715:atrial natriuretic factor receptor A