Substitution of D-Trp 32 in NPY Destabilizes the Binding Transition State to the Y1 Receptor Site in SK-N-MC Cell Membranes

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-Trp 32 -NPY peptide at Lys 4 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 K D was determined by a direct radiolabeled NPY displacement binding assay. The analyses show that for the porcine [Ac-Tyr 1 N ε4 -proxyl]-NPY, the K D was 8 × 10 −10 M and k off was 2.7 × 10 −4 sec −1 yielding a value for k on of 3.3 × 10 5 sec −1 M −1 . The [Ac-Tyr 1 , N ε4 -proxyl,-D-Trp 32 ]-NPY antagonist ligand had a value of K D equal to 1.35 × 10 −7 M and k off was 1.7 × 10 −4 sec −1 leading to a value for k on of 1.2 × 10 3 sec −1 M −1 . The difference in the k on rates of two orders of magnitude is interpreted as demonstrating the N-acetyl-N ε4 proxyl-D-Trp 32 -NPY ligand binding transition state to be of higher energy then for the unmodified NPY amino acid sequence.