[72c] Separation and some enzymatic properties of the inner and outer membranes of rat liver mitochondria

TLDR: A combined procedure results in morphologically well defined outer and inner membrane fractions with a quantitative recovery that makes it suitable for the study of intramitochondrial distribution of enzymes and other chemical constituents.

Abstract: Publisher Summary This chapter describes the separation and enzymatic properties of the inner and outer membranes of rat liver mitochondria. A brief exposure of isolated rat liver mitochondria to sonic oscillation, followed by centrifugation on a sucrose gradient, resulted in the separation of a particulate light subfraction from the bulk of the mitochondria, which exhibited a high rotenone-insensitive NADH-cytochrome c reductase activity, but was devoid of rotenone-sensitive NADH-cytochrome c reductase and other respiratory chain-linked enzyme activities. A similar subfraction was obtained when the mitochondria were subjected to swelling and contraction—rather than sonication—prior to density gradient centrifugation. As both the sonication and the swelling-contraction procedure yielded only partial separation of the rotenone-insensitive NADH-cytochrome c reductase, a combined procedure is described. This results in morphologically well defined outer and inner membrane fractions with a quantitative recovery that makes it suitable for the study of intramitochondrial distribution of enzymes and other chemical constituents. Electron microscopic examination of the heavy and light submitochondrial fractions reveals the following: Osmium-fixed specimens of the heavy subfraction consist of relatively large vesicles bordered by a single membrane. Negatively stained specimens of the same subfraction show mitochondrial images in the stage of bursting, with protrusions of unfolding cristae. The osmium-fixed light subfraction consists of relatively small vesicles (average diameter 0.2 μ ) bordered by a single membrane and devoid of inner structures.