Physical Reactivity of Liver and Brain Cortex Mitochondria

Abstract

In recent investigations¹ we found that rat brain cortex mitochondria, isolated in 0.25 M sucrose by method II of Brody and Bain², had, in the same experimental conditions, certain physical properties different from those of rat liver mitochondria isolated by the method of Myers and Slater³. The incorporation of 0.01 M ethylenediaminetetraacetate in the 0.25 M sucrose, used by Slater⁴ in the preparation and stabilization of heart sarcosomes, has yielded some results concerning the apparent stability of brain granules at 25° C. The ethylenediaminetetraacetate used was the disodium salt, brought to pH 7.5 with potassium hydroxide, and the solution was made up with de-ionized water. Experiments were performed with mitochondria isolated from the liver and the brain cortex of normal white rats (Wistar strain), three months old. The tissues were rapidly homogenized in a Potter–Elvehjem homogenizer fitted with a ‘Teflon’ pestle, with ten volumes of the sucrose–ethylenediaminetetraacetate medium. Nuclei, erythrocytes, intact cells and debris were sedimented by centrifugation at 700g for 10 min., and the mitochondria then separated by centrifugation of the supernatant fluid at 8,500g for 10 min. (rate controlled by an electronic stroboscope, Philips PR 9103). The mitochondria were washed by resuspension and resedimentation in the same conditions. The final pellets were suspended in the sucrose–ethylenediaminetetraacetate medium, so that the mitochondria from 0.80 gm. of fresh liver or from 0.90 gm. of fresh brain cortex were suspended in 1.0 ml. All operations were carried out at 0° C. The purity of isolated fractions was controlled morphologically with phase microscopy and chemically by the colorimetric test of Potter, Recknagel and Hurlbert⁵ (reduction of janus green B to yield diethylsafranine at 37° C.). The mitochondria were used immediately following their isolation. Mitochondrial volume changes were studied at 25° C. by following the optical density of suspensions at 550 mµ, in 1-cm. cells, with a Meunier photometer (Jobin and Yvon, Arcueil), as described by Cleland⁶. The basic test system (volume 7 ml.) contained mitochondria (about 0.03 ml. of the stock suspension, giving an initial optical density of approximately 0.300) suspended in 0.05 M sucrose and 0.01 M tris (hydroxymethyl) aminomethane, pH 7.4 ; the solution was made up in de-ionized water. The first reading was taken 30 sec. after the addition of the mitochondria and subsequent readings at 2-min. intervals for 10 min.; the zero-time value was obtained by extrapolation.