Han Xia, Tomitori Hideyuki, Mizuno Satomi, Higashi Kyohei, Füll Christine, Fukiwake Tomohide, Terui Yusuke, Leewanich Pathama, Nishimura Kazuhiro, Toida Toshihiko, Williams Keith, Kashiwagi Keiko, Igarashi Kazuei
Graduate School of Pharmaceutical Sciences, Chiba University, Chiba, Japan.
J Neurochem. 2008 Dec;107(6):1566-77. doi: 10.1111/j.1471-4159.2008.05729.x. Epub 2008 Nov 5.
The binding of spermine and ifenprodil to the amino terminal regulatory (R) domain of the N-methyl-D-aspartate receptor was studied using purified regulatory domains of the NR1, NR2A and NR2B subunits, termed NR1-R, NR2A-R and NR2B-R. The R domains were over-expressed in Escherichia coli and purified to near homogeneity. The K(d) values for binding of [(14)C]spermine to NR1-R, NR2A-R and NR2B-R were 19, 140, and 33 microM, respectively. [(3)H]Ifenprodil bound to NR1-R (K(d), 0.18 microM) and NR2B-R (K(d), 0.21 microM), but not to NR2A-R at the concentrations tested (0.1-0.8 microM). These K(d) values were confirmed by circular dichroism measurements. The K(d) values reflected their effective concentrations at intact NR1/NR2A and NR1/NR2B receptors. The results suggest that effects of spermine and ifenprodil on NMDA receptors occur through binding to the regulatory domains of the NR1, NR2A and NR2B subunits. The binding capacity of spermine or ifenprodil to a mixture of NR1-R and NR2A-R or NR1-R and NR2B-R was additive with that of each individual R domain. Binding of spermine to NR1-R and NR2B-R was not inhibited by ifenprodil and vice versa, indicating that the binding sites for spermine and ifenprodil on NR1-R and NR2B-R are distinct.
使用纯化的NR1、NR2A和NR2B亚基的氨基末端调节（R）结构域（分别称为NR1-R、NR2A-R和NR2B-R），研究了精胺和艾芬地尔与N-甲基-D-天冬氨酸受体的结合情况。这些R结构域在大肠杆菌中过表达，并纯化至接近均一状态。[¹⁴C]精胺与NR1-R、NR2A-R和NR2B-R结合的解离常数（K(d)）值分别为19、140和33微摩尔。[³H]艾芬地尔与NR1-R（K(d)，0.18微摩尔）和NR2B-R（K(d)，0.21微摩尔）结合，但在测试浓度（0.1 - 0.8微摩尔）下不与NR2A-R结合。这些K(d)值通过圆二色性测量得到了证实。这些K(d)值反映了它们在完整的NR1/NR2A和NR1/NR2B受体上的有效浓度。结果表明，精胺和艾芬地尔对N-甲基-D-天冬氨酸受体的作用是通过与NR1、NR2A和NR2B亚基的调节结构域结合而发生的。精胺或艾芬地尔与NR1-R和NR2A-R或NR1-R和NR2B-R混合物的结合能力与每个单独R结构域的结合能力具有加和性。艾芬地尔不抑制精胺与NR1-R和NR2B-R的结合，反之亦然，这表明NR1-R和NR2B-R上精胺和艾芬地尔的结合位点是不同的。
