Chazot P L, Stephenson F A
Department of Pharmaceutical and Biological Chemistry, School of Pharmacy, London, England.
J Neurochem. 1997 Feb;68(2):507-16. doi: 10.1046/j.1471-4159.1997.68020507.x.
Optimum conditions were determined for the solubilisation of native NMDA receptors of adult mammalian brain with the retention of [3H]MK-801 radioligand binding activity. The most efficient conditions were 1% Triton X-100/1 M NaCl. The efficiency of solubilisation was as follows: cloned NMDA receptors expressed in mammalian cells > forebrain receptors > cerebellar receptors. Triton X-100/1 M NaCl-solubilised forebrain NMDA receptors had a molecular size of 710,000 daltons, but significant NR1 immunoreactivity (41%) migrated as a monomer of 125,000 daltons. Immunoaffinity purification of NMDA receptors from forebrain by anti-NR1 911-920 antibody affinity chromatography from 1% Triton X-100/1 M NaCl solubilised extracts yielded purification of the NR1 Mr 120,000 immunoreactive species, but no detectable NR2A or NR2B immunoreactivity. Immunoprecipitation of NMDA receptors from Triton X-100/1 M NaCl extracts with anti-NR1 911-920 antibodies also resulted in precipitation of NR1 subunits, but with no detectable NR2A or NR2B subunits. In contrast, by immunoprecipitation with anti-NR1 17-35 antibodies, which recognise all forms of NR1, NR1, NR2A, and NR2B immunoreactivities were detected in the immune pellets. Similarly, a co-association of NR1, NR2A, and NR2B subunits was demonstrated following extraction of forebrain membranes with 1% sodium deoxycholate (pH 9) and purification by anti-NR1 911-920 antibody affinity chromatography. These results are consistent with the identification of a pool of unassembled C2 exon-containing NR1 subunits, i.e., NR1-1a, NR1-1b, NR1-2a, and NR1-2b, selectively solubilised by 1% Triton X-100/1 M NaCl.
确定了成年哺乳动物脑内天然NMDA受体增溶并保留[3H]MK-801放射性配体结合活性的最佳条件。最有效的条件是1% Triton X-100/1 M NaCl。增溶效率如下：在哺乳动物细胞中表达的克隆NMDA受体>前脑受体>小脑受体。用Triton X-100/1 M NaCl增溶的前脑NMDA受体分子大小为710,000道尔顿，但显著的NR1免疫反应性（41%）以125,000道尔顿的单体形式迁移。通过抗NR1 911-920抗体亲和层析从1% Triton X-100/1 M NaCl增溶提取物中对前脑NMDA受体进行免疫亲和纯化，得到了Mr 120,000的NR1免疫反应性物种的纯化，但未检测到可检测的NR2A或NR2B免疫反应性。用抗NR1 911-920抗体对Triton X-100/1 M NaCl提取物中的NMDA受体进行免疫沉淀，也导致NR1亚基沉淀，但未检测到可检测的NR2A或NR2B亚基。相反，通过用识别所有形式NR1的抗NR1 17-35抗体进行免疫沉淀，在免疫沉淀中检测到了NR1、NR2A和NR2B免疫反应性。同样，在用1%脱氧胆酸钠（pH 9）提取前脑膜并用抗NR1 911-920抗体亲和层析纯化后，证明了NR1、NR2A和NR2B亚基的共缔合。这些结果与鉴定出一组未组装的含C2外显子的NR1亚基一致，即NR1-1a、NR1-1b、NR1-2a和NR1-2b，它们被1% Triton X-100/1 M NaCl选择性增溶。
