Pharmacological characterization of metabotropic glutamate receptor-mediated high-affinity GTPase activity in rat cerebral cortical membranes
Pharmacological characterization of metabotropic glutamate receptor-mediated high-affinity GTPase activity in rat cerebral cortical membranes
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DOI：
10.1038/sj.bjp.0703464
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发表时间：
2000-08-01
影响因子：
7.3
通讯作者：
Koyama, T
中科院分区：
文献类型：
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作者：
Nishi, N;Odagaki, Y;Koyama, T
1通过激动剂诱导的高亲和力GT3（EC3.6.1.-）活性在大鼠大脑皮层膜。2 L-谷氨酸（1 mM）刺激高亲和力的GT3活性在整个孵育期相同的程度上，高达20分钟，在Mg 2+依赖性的方式。添加1 mM L-谷氨酸可增加酶活性的V_（max（1670至3850 pmol mg（-1）蛋白15 min（-1）），K-M值略有增加（0.26至0.63 μ M）。3高亲和力GT β活性由以下化合物刺激，效力等级为（2S，2'R，3'R）-2-（2 '，3'-二羧基环丙基）甘氨酸（DCG-IV）>（2S，1'S，2'S）-2-（羧基环吡咯基）甘氨酸（L-CCG-I）> L-谷氨酸大于或等于2 R，4 R-4-氨基吡咯烷-2，4-二甲酸酯[（2 R，4 R）-APDC] > 1 S，3R-1-氨基环戊烷-1，3-二羧酸[（1 S，3R）-ACPD] >（S）-4-羧基-3-羟基苯甘氨酸[（S）-4C3HPG] >（S）-3-羧基-4-羟基苯甘氨酸[（S）-3C4HPG] >鹅膏蕈氨酸，但不受L-（+）-2-氨基-4-膦酰基丁酸（L-AP 4）、（RS）-3，5-二羟基苯甘氨酸[（RS）-3，5-DHPG]、使君子苷酸、或L-丝氨酸-O-磷酸（L-SOP），表明涉及II组mGluR，特别是mGluR2.4（2S）-α-乙基谷氨酸（EGLU），一种推测的II组mGluR的选择性拮抗剂，以竞争性方式抑制DCG-IV刺激的高亲和力GT3活性，表观K-B为220 μ M。通过用30-50 μ M的巯基烷基化剂N-乙基马来酰亚胺（NEM）预处理膜来消除刺激的活性，这些结果表明，mGluR激动剂诱导的高表达，大鼠大脑皮层膜中的亲和性G β活性可用于检测II组mGluR，特别是mGluR 2和NEM敏感性G（i）蛋白之间的功能性相互作用。
1 Activation of heterotrimeric guanine nucleotide-binding regulatory proteins (G-proteins) functionally coupled to metabotropic glutamate receptors (mGluRs) was assessed by agonist-induced high-affinity GTPase (EC3.6.1.-) activity in rat cerebral cortical membranes.2 L-Glutamate (1 mM) stimulated high-affinity GTPase activity to the same extent throughout the incubation period up to 20 min, in a Mg2+-dependent manner. The addition of 1 mM L-glutamate augmented V-max of the enzyme activity (1670 to 3850 pmol mg(-1) protein 15 min(-1)) with slight increase in K-M value (0.26 to 0.63 mu M).3 The high-affinity GTPase activity was stimulated by the following compounds with a rank order of potency of (2S,2'R,3'R)-2-(2',3'-dicarboxycyclopropyl)glycine (DCG-IV) > (2S,1'S,2'S)-2-(carboxycyclopyropyl)glycine (L-CCG-I) > L-glutamate greater than or equal to 2R,4R-4-aminopyrrolidine-2,4-dicarboxylate [(2R, 4R)-APDC] > 1S,3R-1-aminocyclopentane-1,3-dicarboxylate [(1S,3R)-ACPD] > (S)-4-carboxy-3-hydroxyphenylglycine [(S)-4C3HPG] > (S)-3-carboxy-4-hydroxyphenylglycine [(S)-3C4HPG] > ibotenate, but not by L-(+)-2-amino-4-phosphonobutyrate (L-AP4), (RS)-3,5-dihydroxyphenylglycine [(RS)-3,5-DHPG], quisqualate, or L-serine-O-phosphate (L-SOP), indicative of involvement of group II mGluRs, in particular mGluR2.4 (2S)-alpha-Ethylglutamate (EGLU), a presumably selective antagonist against group II mGluRs, inhibited DCG-IV-stimulated high-affinity GTPase activity in a competitive manner with an apparent K-B of 220 mu M.5 L-Glutamate-stimulated activity was eliminated by pretreatment of the membranes with sulfhydryl alkylating agent N-ethylmaleimide (NEM) at 30-50 mu M, indicating that G-proteins of the G(i) family are involved.6 These results indicate that mGluR agonist-induced high-affinity GTPase activity in rat cerebral cortical membranes may be used to detect the functional interaction between group II mGluRs, in particular mGluR2, and NEM-sensitive G(i) proteins.