Legay F, Pelhate S, Tappaz M L
J Neurochem. 1986 May;46(5):1478-86. doi: 10.1111/j.1471-4159.1986.tb01765.x.
Brain high-speed supernatants from various lower and higher vertebrates were subjected to sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis, electroblot on nitrocellulose membranes, and immunolabelling using an anti-glutamic acid decarboxylase (anti-GAD) antiserum prepared from rat antigen. Rat brain extracts showed two distinct immunolabelled bands (MW 59,000 and 62,000 daltons). The molecular weight of the native enzyme was 120,000 daltons. The immunoblot pattern was not affected by a 3-h incubation of the homogenate. In the substantia nigra, the decrease in the immunolabelling of both bands corresponded very closely to the decrease of GAD activity following lesioning of the striato-nigral pathway. Moreover, experiments with preadsorbed antiserum showed that both subunits have common antigenic determinants. The immunolabelling was consistently more intense over the lightest band. The autoradiography of immunoprecipitated rat brain GAD, iodinated prior to electrophoresis, revealed two radiolabelled bands corresponding to the two immunolabelled ones. Their radioactivity was found in a one-to-five ratio which closely paralleled their respective immunolabelling intensity. Thus, the two subunits recognized by the antiserum are not present in stoichiometric proportions in the rat brain high-speed supernatant. These findings suggest the existence of two homodimeric GAD with common antigenic determinants which are present in different amounts. Immunoprecipitation curves of brain GAD from rat, mouse, rabbit, monkey, human, quail, frog, and trout were similar, with a less than 10-fold maximum shift in affinity for GAD. GAD immunoblots from the various higher vertebrates showed a pattern similar to that obtained in rat.(ABSTRACT TRUNCATED AT 250 WORDS)
对来自各种低等和高等脊椎动物的脑高速上清液进行十二烷基硫酸钠（SDS）-聚丙烯酰胺凝胶电泳、硝酸纤维素膜电印迹，并使用由大鼠抗原制备的抗谷氨酸脱羧酶（抗GAD）抗血清进行免疫标记。大鼠脑提取物显示出两条明显的免疫标记带（分子量分别为59,000和62,000道尔顿）。天然酶的分子量为120,000道尔顿。匀浆3小时的孵育对免疫印迹模式没有影响。在黑质中，两条带的免疫标记减少与纹状体-黑质通路损伤后GAD活性的降低非常密切相关。此外，用预吸附抗血清进行的实验表明，两个亚基具有共同的抗原决定簇。免疫标记在最浅的条带上始终更强烈。对电泳前碘化的大鼠脑GAD进行免疫沉淀的放射自显影显示，两条放射性标记带对应于两条免疫标记带。它们的放射性比例为1:5，与各自的免疫标记强度密切平行。因此，抗血清识别的两个亚基在大鼠脑高速上清液中不存在化学计量比例。这些发现表明存在两种具有共同抗原决定簇的同型二聚体GAD，其含量不同。来自大鼠、小鼠、兔子、猴子、人类、鹌鹑、青蛙和鳟鱼的脑GAD免疫沉淀曲线相似，对GAD的亲和力最大变化小于10倍。来自各种高等脊椎动物的GAD免疫印迹显示出与大鼠相似的模式。（摘要截断于250字）
