Giambalvo C T, Rosenberg P
Biochim Biophys Acta. 1976 Jul 15;436(4):741-56. doi: 10.1016/0005-2736(76)90403-x.
A preparation enriched in junctional complexes, as judged by marker enzymes and electron microscopy, was prepared from rat cerebellum. The junctional complexes were incubated with gamma-amino [14C]butyric acid at 25degreesC for 10 min, using [3H]sucrose as a marker for entrapped space, Total binding was determined in the absence of, and non-specific binding in the presence of, and excess of unlabelled gamma-aminobutyric acid. The difference bewteen the two binding values, i.e. the specific binding, was saturable and reversible, and showed positive cooperativity with a Hill number of about 2. The specific binding was inhibited by N-methylbicuculline, picrotoxinine and imidazole-4-acetic acid, but not by curare, strychnine or L-2,4-diaminobutyric acid. The above compounds had little effect on the non-specipic binding, but addition of ethylenediaminetetraacetic acid decreased non-specific binding by 80%. Trypsin, pronase, phospholipase A2 (EC 3.1.1.4), lysolecithin and sodium dodecyl sulfate decreased binding. Phospholipase C (EC 3.1.4.3) increased the specific binding by 260%. Phospholipids competed with gamma-aminobutyric acid for binding, with phosphatidylethanolamine being more potent than phosphatidylcholine. These results lend support for Watkins' hypothesis that phosphatidylethanolamine competes with gamma-aminobutyric acid for binding to the receptor protein.
通过标记酶和电子显微镜判断，从大鼠小脑中制备出一种富含连接复合体的制剂。将连接复合体与γ-氨基[¹⁴C]丁酸在25℃下孵育10分钟，使用[³H]蔗糖作为截留空间的标记物。在不存在未标记γ-氨基丁酸的情况下测定总结合，在存在过量未标记γ-氨基丁酸的情况下测定非特异性结合。两种结合值之间的差异，即特异性结合，是可饱和且可逆的，并显示出正协同性，希尔系数约为2。特异性结合受到N-甲基荷包牡丹碱、苦味毒和咪唑-4-乙酸的抑制，但不受箭毒、士的宁或L-2,4-二氨基丁酸的抑制。上述化合物对非特异性结合影响很小，但添加乙二胺四乙酸可使非特异性结合降低80%。胰蛋白酶（EC 3.1.1.4）、链霉蛋白酶、磷脂酶A₂、溶血卵磷脂和十二烷基硫酸钠会降低结合。磷脂酶C（EC 3.1.4.3）可使特异性结合增加260%。磷脂与γ-氨基丁酸竞争结合，其中磷脂酰乙醇胺比磷脂酰胆碱更有效。这些结果支持了沃特金斯的假说，即磷脂酰乙醇胺与γ-氨基丁酸竞争结合受体蛋白。
