Substitution of D-Trp32 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-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-Tyr1N epsilon 4-proxyl]-NPY, the KD was 8 x 10(-10) M and koff was 2.7 x 10(-4) sec-1 yielding a value for kon of 3.3 x 10(5) sec-1 M-1. The [Ac-Tyr1, N epsilon 4-proxyl,-D-Trp32]-NPY antagonist ligand had a value of KD equal to 1.35 x 10(-7) M and koff was 1.7 x 10(-4) sec-1 leading to a value for kon of 1.2 x 10(3) sec-1 M-1. The difference in the kon rates of two orders of magnitude is interpreted as demonstrating the N-acetyl-N epsilon 4 proxyl-D-Trp32-NPY ligand binding transition state to be of higher energy then for the unmodified NPY amino acid sequence.
Zand
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Department of Biological Chemistry, University of Michigan, Ann Arbor 48109, USA. rzand@umich.edu
Marcelo
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MacKenzie
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Georgic
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Maclean
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Dunham
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D000595:Amino Acid Sequence; D000818:Animals; D002462:Cell Membrane / Q000378:metabolism; D004578:Electron Spin Resonance Spectroscopy; D019204:GTP-Binding Proteins / Q000378:metabolism; D006801:Humans; D008969:Molecular Sequence Data; D009447:Neuroblastoma / Q000378:metabolism*; D009478:Neuropeptide Y / Q000737:chemistry* / Q000378:metabolism*; D013113:Spin Labels; D013329:Structure-Activity Relationship; D013552:Swine; D014364:Tryptophan; D014407:Tumor Cells, Cultured
D009478:Neuropeptide Y; D013113:Spin Labels; D014364:Tryptophan; D019204:GTP-Binding Proteins