Abstract
中樞神經系統的神經突觸接合區(synaptic junction, SJ )包含了前突觸 膜(presynaptic membrane)，後突觸膜( postsynaptic membrane)，後突 觸緻密體(postsynaptic density)，與兩末梢間的區域。生化分析結果指 出，SJ中含有細胞骨架蛋白，神經傳導物質受器，蛋白激 ，磷酸化蛋白 質和醣蛋白等。有些間接的實驗證據指出，SJ可能直接或間接的調節神經 衝動的傳導，但至目前為止，SJ的蛋白質組成和功能仍不十分清楚，因此 為進一步了解神經突觸接合區未知的組成蛋白種類及已知組成蛋白之功能 ，將實驗分為兩部份進行。第一部份利用蛋白質之等電點與分子量大小的 不同，以二維電泳分離SJ之蛋白質並純化其中之84kDa蛋白質。利用 trypsin將84kDa蛋白質截切成片段，進而做胺基酸序列分析。所得的胺基 酸序列和 SWISSPROT資料庫中的蛋白質序列相比對後，部份 peptides序 列和已知蛋白質序列並無相似性，另有一部份 peptides序列則與多種已 知的蛋白質序列有些微之相似性。由目前所獲得的片段胺基酸序列並不能 對84kDa蛋白質的種類或功能做任何的定論。以peptides序列合成對應之 核甘酸為探針(probe)可於豬腦反訊息DNA集合庫(cDNA library)中尋找帶 有84kDa蛋白質遺傳訊息之cDNA，以期對84kDa蛋白質做進一步的了解。此 外，麩胺酸受器是位於神經突觸接合區上已知參與神經訊號傳遞之蛋白質 。為糷F解魚腦內是否存在AMPA-subtype glutamate receptor，第二部份 實驗則以一段攜帶AMPA-subtype glutamate receptor之transmembrane（ TM）region遺傳訊息的DNA片段，於魚腦cDNA library中篩選cDNA獲得一 株純化之cDNA，經DNA定序分析比對後可歸為rat GluR4一類，命名為 pC8 。分析此cDNA之序列發現其中並無起始譯碼，停止譯碼或poly(A)尾部存 在，所以並不是一個包含完整開放譯讀區之cDNA，此段cDNA和老鼠之 GluR1∼GluR4之序列比對後，可知此cDNA包含了TM region前N-terminal 的400個胺基酸，三段TM domains（TMI, TMII和TMIII），及TMIII和TMIV 間的85個胺基酸的譯碼(codon)。雖然目前此株 cDNA並不完整，但可為尋 找完整cDNA之有利工具。 The synaptic junction(SJ)of the central nervous system is composed of presynaptic, postsynaptic membranes, and the area in between. Biochemical analyses indicate that the basic compositions of the SJ include cytoskeletal proteins, neurotrans- mitter receptors, ion channels, phosphoproteins, and protein kinases. Glutamate receptor is one of the receptors located on SJ and is known to be the major constitute of the excitatory transmitter system. It has been suggested that SJ proteins may be directly or indirectly involved in the regulation of neurotransmission. The functions and compositions of SJ proteins have not been fully explored. The first part of my work is to characterize a 84kDa protein of the SJ from porcine brains. This protein was purified, digested with trypsin, and partially sequenced. Partial of amino acid sequence of the 84kDa protein was obtained and compared to protein sequences deposited to the SWISSPROT data bank. Degenerate oligonucleotides were synthesized according to the amino acid sequences and used to screen a cDNA library. The second part of my work is to isolate cDNA clone encoding the subunits of fish glutamate receptor. This work was performed by using a DNA fragment encoding transmembrane region of an AMPA-subtype glutamate receptor. One of the isolated clones, names pC8, was sequenced and was classified as GluR4-homolog. This pC8, however, does not include the initiation and stop codons, and thus, is incomplete. Comparisons between amino acid sequence of the pC8 and that of rat AMPA-subtype receptor suggest that the pC8 contains a portion of the N-terminal, three TM domains (TMD), and a portion of the loop between TMDIII and TMDIV.