Chazot P L, Fotherby A, Stephenson F A
Department of Pharmaceutical Chemistry, School of Pharmacy, London, U.K.
Biochem Pharmacol. 1993 Feb 9;45(3):605-10. doi: 10.1016/0006-2952(93)90133-h.
A series of protein modifying reagents were tested for their effects on the specific binding of [3H]MK801 to adult rat brain membranes. N-Bromosuccinimide, acetyl imidazole, 2,3-butanedione, 5,5'-dithiobis-(2-nitrobenzoic acid) and dithiothreitol all had no significant effect on binding. The carboxylic acid residue modification reagent, 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDAC), inhibited [3H]MK801 specific binding in a dose-dependent manner with an IC50 = 1.9 mM. The inhibition by EDAC was due to a decrease in the Bmax with no change in KD. The inhibition of [3H]MK801 binding by EDAC was not prevented by prior incubation with competitive antagonists. Protection against EDAC inactivation was obtained, however, in a dose-dependent manner by preincubation with the divalent cations, Ca2+ and Mg2+, but not Zn2+. These results suggest that EDAC modifies an important carboxyl group located within the voltage-dependent Mg2+ binding site of the N-methyl-D-aspartate receptor. This modification yields a decrease in the specific [3H]MK801 binding activity thus demonstrating a close association between the two allosteric regulatory sites.
测试了一系列蛋白质修饰试剂对[3H]MK801与成年大鼠脑膜特异性结合的影响。N-溴代琥珀酰亚胺、乙酰咪唑、2,3-丁二酮、5,5'-二硫代双-(2-硝基苯甲酸)和二硫苏糖醇对结合均无显著影响。羧酸残基修饰试剂1-乙基-3-(3-二甲基氨基丙基)碳二亚胺(EDAC)以剂量依赖性方式抑制[3H]MK801特异性结合，IC50 = 1.9 mM。EDAC的抑制作用是由于Bmax降低而KD不变。EDAC对[3H]MK801结合的抑制作用不能被竞争性拮抗剂预先孵育所阻止。然而，通过与二价阳离子Ca2+和Mg2+预先孵育，而不是Zn2+，可以以剂量依赖性方式获得对EDAC失活的保护。这些结果表明，EDAC修饰了位于N-甲基-D-天冬氨酸受体电压依赖性Mg2+结合位点内的一个重要羧基。这种修饰导致[3H]MK801特异性结合活性降低，从而证明了两个变构调节位点之间的紧密联系。
