天竺鼠大腦神經鍵胞內含物之研究

Abstract

為研究由天竺鼠大腦中分出的各個部分的內含物，本實驗使用了三種方法： 1. 乙醯膽鹼及膽鹼的放射性—酵素檢驗法 2. 新腎上腺素及腎上腺素的螢光檢驗法 3. 胺基酸的Dansylation法 這三種方法均有極高的敏感度，乙醯膽鹼用第一種方法可決定到10 ng的量，新腎上腺素及腎上腺素可定至100pg 的程度，Dansylation的方法雖然很敏感但只能適於定性不能定量。 在神經鍵胞中，每一毫克的蛋白質含有新腎上腺素6.6ng，其含量比在大腦的懸浮液中增加了7倍；腎上腺素則含有0.94ng。雖然比大腦懸浮液中增加但增加的比率不多。很明顯的新腎上腺素這種神經傳遞物質可以集中在我們所分離出來的神經鍵胞的部分中，但非神經傳遞物質則不能。在本實驗中只能決定乙醯膽鹼及膽鹼共同的含量，其含量為每一毫克蛋白質中含有311 ng。另外我們發現幾乎所有的胺基酸都出現在我們所分離的神經鍵胞的部分中，這其中並且包括可能作為示神經傳遞物質的胺基酸如GABA,Glutamate, Aspartate．除此以外還有很多未被確定的點，其中可能含有一些被懷疑是神經傳遞物質的蛋白?。

To study the content of neurotransmitters in subcellular fractions from guinea pig brain, three approachs were used. 1. Radioenzvmatic assay of ACh and choline 2. Fluorescent determination of NE and E 3. Amino acids determination by dansylation All these methods have the advantage of high senstivity. ACh could be determined to 10 ng level. NE and E both as low as 100 pg could be detected. Although higly sensitive , dansylation of amino acids is only suitable for qualitative analysis. In the vesicle fraction cbtained after a series of centrifugation, the NE content was 6.6 ng/mg of protein, a 7-fold enrichment over that of the whole homogenate of the brain; the content of E 0.94 ng/mg of protein was a1so higher than that in the homogenate, but only a very small percentage of E was in the vesicle fraction. An obvious accumulation of NE (a neurctransmitter) in the fraction was observed but not that of a non-transmitter. In this study, only the amount of ACh and choline combined could be correctly given by the radioenzymatic method, a value of 311 ng/mg of protein was obtained for the vesicle fraction. Almost all common amino acids were found by the dansylation method to be present in vesicle fraction, including putative neurotransmitters, glutamate, aspartate and GABA. In addition many unidentified dansylated products were also present, some of these may come from suspected peptide transmitters.