Binding of L-[3H]glutamate to fresh or frozen synaptic membrane and postsynaptic density fractions isolated from cerebral cortex and cerebellum of fresh or frozen canine brain.
Binding of L-[3H]glutamate to fresh or frozen synaptic membrane and postsynaptic density fractions isolated from cerebral cortex and cerebellum of fresh or frozen canine brain.
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L-[3H]谷氨酸与从新鲜或冷冻犬脑的大脑皮层和小脑分离的新鲜或冷冻突触膜和突触后密度部分的结合。
DOI：
10.1111/j.1471-4159.1986.tb13047.x
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发表时间：
1986
影响因子：
4.7
通讯作者：
Siekevitz,P
中科院分区：
文献类型：
--
作者：
Wu,K;Carlin,R;Siekevitz,P
发现从犬脑的大脑皮层（CTX）和小脑（CL）分离的突触膜（SPM）和突触后密度（PSD）组分（新鲜或冷冻）以及从新鲜或冷冻组织分离的组分均含有L-[3 H]谷氨酸结合位点。结果发现，在CTX-PSD和CL-PSD中，L-谷氨酸结合位点在各自的膜级分上存在浓度，CL-PSD的Bmax值（92.0 pmol/mg蛋白质）约为CTX-PSD（28.9 pmol/mg）的3倍。这些结果与其他结果一起表明，薄的CL‐PSD可能来自分子层的兴奋性突触。发现L-谷氨酸与犬CTX-SPM组分结合的离子依赖性与大鼠脑SPM组分的离子依赖性相似：（a）Cl−增加了L-谷氨酸结合位点的数量，Ca 2+增强了该作用;单独的Ca 2+没有显著影响;（B）Cl−/Ca 2+敏感性结合位点被2-氨基-4-膦酰基丁酸酯（AP B）或冻融消除：（c）Na+离子的作用是双相的：低浓度的Na+（< 5 mM）降低了依赖于Cl− 7 Ca 2+的L-谷氨酸结合位点，而在较高浓度的Na+下，发现谷氨酸的结合在存在或不存在Ca 2+和Cl−的情况下都增加。此外，发现K+离子（50 mM）使L-谷氨酸盐与新鲜CTX-SPM的Na+-非依赖性和Cl-/Ca 2--非依赖性结合降低18%，但使Na--依赖性和Cl-/Ca 2 +-非依赖性L-谷氨酸盐结合降低93%;在Cl-/Ca 2+存在下，K+离子使Na+-依赖性结合降低78%。在不存在Ca 2+和Cl−的情况下，CTX-SPM的冷冻和解冻导致Na+依赖性L-谷氨酸结合位点损失50%。CL-SPM组分显示出类似的L-谷氨酸结合的离子依赖性，除了不存在Na−依赖性谷氨酸结合位点。CTX-PSD组分不含Na+依赖性或APB（或Cl−/Ca 2+）敏感性L-谷氨酸结合位点，其L-谷氨酸结合不受冷冻和解冻的影响，与使用大鼠脑PSD制备物报告的结果一致。L-谷氨酸与CTX-SPM或CTX-PSD组分的结合不受10 mM L-谷氨酸预处理的影响，也不受与钙调蛋白同时孵育的影响。此外，CTX-SPM或CTX-PSD组分的磷酸化，无论是同时孵育还是在去除磷酸化试剂后孵育，对L-谷氨酸的结合均无影响。此外，发现L-谷氨酸与CTX-SPM或CTX-PSD的结合对组分的后续磷酸化没有显著影响。用0.5%脱氧胆酸盐、1.0% N-月桂酰肌氨酸盐、4 M盐酸胍（pH 7.0）、0.5 M KCl和1.0 M KCl处理CTX-PSD组分，可将PSD中的L-谷氨酸受体去除25%、44%、40%、8%和11%。分别这些试剂溶解的总蛋白的相应百分比相似，表明受体没有优先解离，并表明L-谷氨酸受体是内在PSD组分。目前的发现，以及早期显示分离的PSD组分中存在γ-氨基丁酸和氟硝西泮结合位点、Ca 2+依赖性K+通道和电压依赖性Ca 2+通道蛋白的发现，表明许多（如果不是全部）神经递质受体蛋白和离子通道蛋白在突触处锚定在PSD中，因此PSD可能在突触后部位的神经传递中起重要作用。
Synaptic membrane (SPM) and postsynaptic density (PSD) fractions isolated from cerebral cortex (CTX) and cerebellum (CL) of canine brain, either fresh or frozen and isolated from either fresh or frozen tissue, were found to contain L‐[3H]glutamate binding sites. It was found that there was a concentration of L‐glutamate binding sites in CTX‐PSD and CL‐PSD over the respective membrane fractions, and the Bmaxvalue of CL‐PSD (92.0 pmol/mg protein) was about three times that of CTX‐PSD (28.9 pmol/mg). The results, together with those of others, suggest that the thin CL‐PSD are probably derived from the excitatory synapses in the molecular layer. The ion dependency of L‐glutamate binding to canine CTX‐SPM fraction was found to be similar to that reported for a rat brain SPM fraction: (a) Cl−increased the number of L‐glutamate binding sites and the effect was enhanced by Ca2+; Ca2+alone had no significant effect; (b) the Cl−/Ca2+‐sensitive binding sites were abolished by 2‐amino‐4‐phosphonobutyrate (APB) or freezing and thawing: (c) the effect of Na+ion was biphasic: low concentration of Na+(< 5 mM) decreased Cl−7Ca2+‐de‐pendent L‐glutamate binding sites, whereas at higher concentrations of Na+the binding of glutamate was found to increase either in the presence or absence of Ca2+and Cl−. In addition, the K+ion (50 mM) was found to decrease the Na+‐independent and Cl−/Ca2‐‐independent binding of L‐glutamate to fresh CTX‐SPM by 18%, but it decreased the Na−‐dependent and Cl−/Ca2+‐independent L‐glutamate binding by 93%; in the presence of Cl,−/Ca2+, the K+ion decreased the Na+‐dependent binding by 78%. Freezing and thawing of CTX‐SPM resulted in a 50% loss of the Na+‐dependent L‐glutamate binding sites assayed in the absence of Ca2+and Cl−. The CL‐SPM fraction showed similar ion dependency of L‐glutamate binding except for the absence of Na−‐dependent glutamate binding sites. The CTX‐PSD fraction contained neither Na+‐dependent nor APB (or Cl−/Ca2+)‐sensitive L‐glutamate binding sites and its L‐glutamate binding was unaffected by freezing and thawing, in agreement with the reported findings using rat brain PSD preparation. L‐Glutamate binding to CTX‐SPM or CTX‐PSD fraction was not affected by pretreatment with 10 mM L‐glutamate, nor by simultaneous incubations with calmodulin. Also, phosphorylation of CTX‐SPM or CTX‐PSD fraction, whether incubated simultaneously or after removal of the phosphorylating reagents, had no effect on binding of L‐glutamate. Furthermore, binding of L‐glutamate to CTX‐SPM or CTX‐PSD was found to have no significant effect on subsequent phosphorylation of the fractions. Treatment of the CTX‐PSD fraction with 0.5% deoxycholate, 1.0% N‐lauroyl sarcosinate, 4 M guanidine‐HCl, pH 7.0, 0.5 M KCl, and 1.0 M KCl removed the L‐glutamate receptors from the PSD by 25%, 44%, 40%, 8%, and 11%. respectively. The respective percentages of total protein solubilized by these reagents were similar, indicating no preferential dissociation of the receptors, and suggesting that the L‐glutamate receptor is an intrinsic PSD component. The present findings, together with the earlier ones showing the presence of γ‐aminobutyric acid and flunitrazepam binding sites, of the Ca2+‐dependent K+channel, and of the voltage‐dependent Ca2+channel proteins in the isolated PSD fraction, suggest that many, if not all, neurotransmitter receptor proteins and ion channel proteins are anchored in the PSD at the synapse, and thus the PSD may play an important role in neurotransmission at the postsynaptic site.