Ksiezak-Reding H, Blass J P, Gibson G E
J Neurochem. 1982 Jun;38(6):1627-36. doi: 10.1111/j.1471-4159.1982.tb06643.x.
A spectrophotometric assay for the brain pyruvate dehydrogenase complex (PDHC) with arylamine acetyltransferase (ArAT; EC 2.3.1.5) to follow the production of acetyl-CoA has been standardized. Activity was proportional to time and protein. It depended completely on added pyruvate, CoA, NAD, and MgCl2, and partially on thiamine pyrophosphate. Triton X-100, and a sulfhydryl compound. The activities are the highest in the literature for brain PDHC (50 nmol/min/mg protein) and equal to maximum recorded rates of pyruvate flux for brain in vivo. Activities as low as 0.6 nmol/min could be measured. Use of ArAT at different purities (I--2-fold and II--55-fold) allowed convenient measurement of total PDHC (ArAT-I) and of the active form of PDHC (ArAT-II). The proportion of PDHC in the active form was 50% in mouse brain, 30% in brain, and 10% in mouse liver. Total PDHC activity was unchanged postmortem during storage of mouse brain in situ at +4 degrees C or at -20 degrees C for 3 days or at +20 degrees C for 24 h. The relative specific activity of PDHC in cytoplasmic or synaptoplasmic fractions was less than that of two other mitochondrial enzymes, fumarase (EC 4.2.1.2) and monoamine oxidase (EC 1.4.3.4), which argues strongly against the hypothesis of a cytoplasmic PDHC in cholinergic nerve endings.
一种用于检测脑丙酮酸脱氢酶复合体（PDHC）的分光光度法已标准化，该方法利用芳胺乙酰转移酶（ArAT；EC 2.3.1.5）来追踪乙酰辅酶A的生成。活性与时间和蛋白质成正比。它完全依赖于添加的丙酮酸、辅酶A、烟酰胺腺嘌呤二核苷酸（NAD）和氯化镁，部分依赖于硫胺焦磷酸。还需要Triton X - 100和一种巯基化合物。该方法所测脑PDHC的活性是文献中最高的（50 nmol/分钟/毫克蛋白质），且与脑在体内丙酮酸通量的最大记录速率相当。低至0.6 nmol/分钟的活性也可被检测到。使用不同纯度的ArAT（I型——2倍纯化和II型——55倍纯化）能够方便地检测总PDHC（ArAT - I）和PDHC的活性形式（ArAT - II）。PDHC活性形式在小鼠脑内的比例为50%，在大鼠脑中为30%，在小鼠肝脏中为10%。小鼠脑在原位于4℃或 - 20℃储存3天或在20℃储存24小时后，总PDHC活性在死后保持不变。PDHC在细胞质或突触质组分中的相对比活性低于另外两种线粒体酶，即延胡索酸酶（EC 4.2.1.2）和单胺氧化酶（EC 1.4.3.4），这有力地反驳了胆碱能神经末梢中存在细胞质PDHC的假说。
