人大脑皮层膜谷氨酸受体的结合特性

Characterization of [3￣H] DL-glutamate binding to the human cerebral cortical membrane preparations

用放射性配体受体结合实验研究了人大脑皮层膜与［３￣Ｈ］ＤＬ－谷氨酸结合的特性。人大脑皮层与谷氨酸特异性结合的最适条件是在４℃，ＨＥＰＥＳ缓冲液（ｐＨ６．４－８．６）中孵温３０－４０ｍｉｎ。结合实验前洗涤膜样品至为重要。人大脑皮层膜与谷氨酸特异性结合是可饱和的，其ＫＤ和Ｂｍａｘ值分别为１０３和１．６７ｎｍｏｌ·Ｌ－１蛋白，Ｈｉｌｌ系数１．０６．谷氨酸受体激动剂对人大脑皮层膜与谷氨酸结合的抑制强度依次为Ｌ－谷氨酸＞ＤＬ－谷氨酸＞红藻氨酸＞Ｎ－甲基－Ｄ－天冬氨酸（ＮＭＤＡ）＞Ｄ－谷氨酸。谷氨酸受体亚型拮抗剂２－氨基－５－磷酸基戊酸和６，７－二硝基唑喔啉－２，３－二酮（１ｎｍｏｌ·Ｌ－１）分别取代了５４％和５６％的特异性谷氨酸结合。１．２５ｎｍｏｌ·Ｌ－１镁离子抑制了５５％的特异性谷氨酸结合。人大脑皮层膜与谷氨酸的特异性结合不依赖钠离子，部分依赖于钙离子。人大脑皮层膜与谷氨酸特异性结合至少涉及到ＮＭＤＡ和红藻氨酸两个谷氨酸受体亚型。

Charactenstics of [3H]DL-glutamate(Glu) binding to human cerebral cortical membranepreparations were studied by a radiolabelled ligandbinding test. The optimum condition for the specificbinding of [3H]DL-Glu was established at 4℃HEPES buffer (pH 6.4-8.4) and 30-40 min forincubation. Extensive membrane washing before usewas a critical factor for the specific Glu binding. Thespecific binding was saturable through a singlehigh-affinity process with a KD of 103 nmol·L-1.a Bmax 1.67 nmol·g-1 protein and a Hill coefficientof 1.06. Glu receptor agonists showed increasingpotency of binding inhibition in the order ofL-Glu > DL-Glu > kainate > N-methyl-D-aspartate (NMDA)> D-Glu. Antagonists of NMDAand kainate/α-amino 3-hydroxy 5 methyl-4-D-aspartate receptor subtypes, α-amino-5-phosphono-pentanoate and 6,7-dinitroquinoxaline-2,3-dione (1μmol·L-1) displaced 54% and 56% of specific bind-ing respectively. Magnesium 1.25 mmol·L-1 inhib-ited 55% of the Glu binding. The specific binding of[3H]DL-Glu to the human cerebral cortical membranewas Na+-independent and partially Ca2+-dependent.The binding of [3H]DL-Glu to human cerebral corticalmembrane preparation involves at least two subtypesof Glu receptor, NMDA and KA /AMPA.