BRAIN MITOCHONDRIA I. Isolation of Bovine Brain Mitochondria

Two methods of preparing a mitochondrial fraction from beef brain cortex are described. Data are presented on the rate of oxidation of substrates, P/O and respiratory control ratios, cholinesterase activity, and DNA content. Electron micrographs of isolated mitochondria and mitochondria in situ are shown. Comparisons are drawn between these preparations and mitochondria prepared in 0.25 M sucrose. The data on enzymic properties and contamination by non-mitochondrial material indicate that mitochondrial fractions which compare favorably with those from other tissues can be prepared from brain tissue. There is a considerable amount of information on mitochondria from brain which suggests that in many ways they exhibit the same enzymic properties as mitochondria of other tissues. For example, mitochondrial fractions isolated from brain have been shown to carry out citric acid cycle oxidations (1~-) with concomitant phosphorylation (1, 5-7). On the other hand, there are varying reports in the literature regarding the relationship of the enzymes of glycolysis and the mitochondria of brain (3, 8-13). 1 Brody and Bain (1) and Abood et al. (4) appear to have been the first to prepare mitochondrial fractions from brain, using procedures based on the method developed by Schneider and Hogeboom (14) for the isolation of liver mitochondria. Most of the subsequent work on the enzymology of brain mitochondria has been carried out on mitochondrial fractions prepared by the methods described by these workers (5, 6, I1, 13, 15)or by direct application of the Schneider-Hogeboom method (2, 3, 8, 9). 1 For a discussion of the glycolytic capacity of brain mitochondria, see the following paper (26). Our early attempts to prepare mitochondria from beef brain by the methods of Abood et al. (4) or Brody and Bain (1) indicated that the mitoehondrial preparations were contaminated by non-mitochondrial particles, especially myelin fragments. This observation has also been made by Balfizs and Richter (16) using the method of Brody and Bain (1). Dahl et al. (17) separated a rat brain mitochondrial fraction into two subfractions, "P" which contained all of the oxidative capacity and "W" which was not characterized, by differential centrifugation in 0.25 M sucrose. Until recently, estimates of the purity of mitochondrial preparations from brain seem to have been based mainly on visual examination under the light or phase contrast microscope. However, Petrushka and Giuditta (18) have shown by electron microscopy that rat brain mitochondrial fractions prepared in 0.88 M sucrose by the method of Hogeboom, Schneider, and Palade (19) are grossly contaminated by non-mitochondrial material. Gray and Whittaker (20, 21) and De Robertis 293 on A uust 4, 2017 jcb.rress.org D ow nladed fom et al. (22, 23) showed that crude mitochondria l fractions of guinea pig or ra t brain, prepared in 0.32 M sucrose, contained myelin fragments and pinched-off nerve endings containing synaptic vesicles as well as mitochondria . The mitochondrial fraction could be separated by density gradient centr ifugation into subfractions which were relatively pure with respect to subcellular particle type. The mi tochondr ia l fractions obtained by sucrose density gradient centr ifugation were character ized by electron microscopy and succinic dehydrogenase content. O the r enzymic properties were not reported. It seemed worthwhile, therefore, to develop a method of fract ionation of b ra in homogenates designed to permit the separation of a mitochondrial fraction which (a) was relatively pure with respect to subcellular particle type and (b) contained mi tochondr ia which satisfied the criteria for enzymic and morphological integrity which have been established for mi tochondr ia from