on and off rates
NPY agonist and antagonist binding
activation energy difference in receptor binding
Springer Online Journal Archives 1860-2000
Abstract The retention rate of the spin label 3-isothiocyanto methyl-2,2,5,5-tetramethyl-1-pyrrolidinyl oxyl spin label (proxyl) attached to the porcine N-acetyl-NPY peptide and the porcine N-acetyl-D-Trp32-NPY peptide at Lys4 was investigated using SK-N-MC neuroblastoma cell membranes containing the Y1 receptor. The release rate of the spin labeled peptides was monitored by electron spin resonance and the KD was determined by a direct radiolabeled NPY displacement binding assay. The analyses show that for the porcine [Ac-Tyr1Nε4-proxyl]-NPY, the KD was 8 × 10−10 M and koff was 2.7 × 10−4 sec−1 yielding a value for kon of 3.3 × 105 sec−1 M−1. The [Ac-Tyr1, Nε4-proxyl,-D-Trp32]-NPY antagonist ligand had a value of KD equal to 1.35 × 10−7 M and koff was 1.7 × 10−4 sec−1 leading to a value for kon of 1.2 × 103 sec−1 M−1. The difference in the kon rates of two orders of magnitude is interpreted as demonstrating the N-acetyl-Nε4 proxyl-D-Trp32-NPY ligand binding transition state to be of higher energy then for the unmodified NPY amino acid sequence.
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