SUBSTITUTION OF D-TRP(32) IN NPY DESTABILIZES THE BINDING TRANSITION-STATE TO THE Y1 RECEPTOR-SITE IN SK-N-MC CELL-MEMBRANES

The retention rate of the spin label 3-isothiocyanto methyl-2,2,5,5-te tramethyl-1-pyrrolidinyl oxyl spin label (proxyl) attached to the porc ine N-acetyl-NPY peptide and the porcine N-acetyl-D-Trp(32)-NPY peptid e at Lys(4) was investigated using SK-N-MC neuroblastoma cell membrane s containing the Y1 receptor. The release rate of the spin labeled pep tides was monitored by electron spin resonance and the K-D was determi ned by a direct radiolabeled NPY displacement binding assay. The analy ses show that for the porcine [Ac-Tyr(1)N(epsilon 4)-proxyl]-NPY, the K-D was 8 x 10(-10) M and k(off) was 2.7 x 10(-4) sec(-1) yielding a v alue for k(on) of 3.3 x 10(5) sec(-1) M-1. The [Ac-Tyr(1), N-epsilon 4 -proxyl,-D-Trp(32)]-NPY antagonist ligand had a value of K-D equal to 1.35 x 10(-7) M and k(off) was 1.7 x 10(-4) sec(-1) leading to a value for k(on) of 1.2 x 10(3) sec(-1) M-1. The difference in the k(on) rat es of two orders of magnitude is interpreted as demonstrating the N-ac etyl-N-epsilon 4 proxyl-D-Trp(32)-NPY ligand binding transition state to be of higher energy then for the unmodified NPY amino acid sequence .