Sims N R, Blass J P
J Neurochem. 1986 Aug;47(2):496-505. doi: 10.1111/j.1471-4159.1986.tb04529.x.
Respiratory studies of brain mitochondria have, in general, been limited to purified preparations. Conventional procedures for mitochondrial isolation yield relatively small and potentially selected subfractions of mitochondria. Examination of respiratory responses of homogenates of rat forebrain indicated that key respiratory properties of mitochondria are fully expressed in these preparations. In a high K+ buffer, comparable to those commonly used for purified mitochondria, forebrain homogenates exhibited many of the characteristics of oxygen uptake by "free" mitochondria: requirement for both pyruvate and malate for maximal respiration, stimulation (over threefold) by ADP, stimulation by uncoupling agent [carbonyl cyanide m-chlorophenylhydrazone (CCCP)], but little effect of digitonin. In a modified Krebs-Ringer phosphate buffer (a physiological buffer), respiratory responses were primarily due to mitochondria enclosed in synaptosomes: respiration with glucose was markedly stimulated by CCCP, further stimulated by pyruvate, and extensively inhibited by digitonin (which disrupts the cholesterol-rich synaptosomal membranes). Studies with purified mitochondria and synaptosomes supported the specificity of these responses. These data indicate that classical mitochondrial responses are expressed in whole brain homogenates and, under appropriate conditions, provide functional measures of the total pools of free and synaptosomal mitochondria.
一般来说，对脑线粒体的呼吸研究仅限于纯化的制剂。传统的线粒体分离程序会产生相对较小且可能经过筛选的线粒体亚组分。对大鼠前脑匀浆呼吸反应的检查表明，线粒体的关键呼吸特性在这些制剂中得到了充分表达。在与常用于纯化线粒体的缓冲液相当的高钾缓冲液中，前脑匀浆表现出许多“游离”线粒体摄取氧气的特征：最大呼吸需要丙酮酸和苹果酸，ADP刺激（超过三倍），解偶联剂[羰基氰化物间氯苯腙（CCCP）]刺激，但洋地黄皂苷影响很小。在改良的 Krebs-Ringer 磷酸盐缓冲液（一种生理缓冲液）中，呼吸反应主要归因于突触体包裹的线粒体：葡萄糖呼吸受到 CCCP 的显著刺激，丙酮酸进一步刺激，并受到洋地黄皂苷的广泛抑制（洋地黄皂苷会破坏富含胆固醇的突触体膜）。对纯化线粒体和突触体的研究支持了这些反应的特异性。这些数据表明，经典的线粒体反应在全脑匀浆中得到表达，并且在适当条件下，可以提供游离和突触体线粒体总库的功能指标。
