在神经肽Y（NPY）中用D-色氨酸32进行替换会使SK-N-MC细胞膜中Y1受体位点的结合过渡态不稳定。

Substitution of D-Trp32 in NPY destabilizes the binding transition state to the Y1 receptor site in SK-N-MC cell membranes.

作者信息

Zand R, Marcelo C L, MacKenzie R, Georgic L, Maclean D, Dunham W R

机构信息

Department of Biological Chemistry, University of Michigan, Ann Arbor 48109, USA.

出版信息

Neurochem Res. 1997 Apr;22(4):437-43. doi: 10.1023/a:1027307710425.

DOI:10.1023/a:1027307710425

PMID:9130254

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-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.

摘要

利用含有Y1受体的SK-N-MC神经母细胞瘤细胞膜，研究了与猪N-乙酰-NPY肽和猪N-乙酰-D-Trp32-NPY肽的Lys4连接的自旋标记物3-异硫氰酸根合甲基-2,2,5,5-四甲基-1-吡咯烷基氧基自旋标记物（proxyl）的保留率。通过电子自旋共振监测自旋标记肽的释放率，并通过直接放射性标记的NPY置换结合试验确定KD。分析表明，对于猪[Ac-Tyr1Nε4-proxyl]-NPY，KD为8×10⁻¹⁰ M，koff为2.7×10⁻⁴ s⁻¹，kon值为3.3×10⁵ s⁻¹ M⁻¹。[Ac-Tyr1,Nε4-proxyl,-D-Trp32]-NPY拮抗剂配体的KD值等于1.35×10⁻⁷ M，koff为1.7×10⁻⁴ s⁻¹，kon值为1.2×10³ s⁻¹ M⁻¹。kon速率两个数量级的差异被解释为表明N-乙酰-Nε4 proxyl-D-Trp32-NPY配体结合过渡态的能量高于未修饰的NPY氨基酸序列。