THE S U B M I T O C H O N D R I A L LOCALIZATION OF MONOAMINE OXIDASE An Enzymatic Marker for the Outer Membrane of Rat Liver Mitochondria

Controlled osmotic lysis (water-washing) of rat liver mitochondria results in a mixed population of small vesicles derived mainly from the outer mitochondrial membrane and of larger bodies containing a few cristae derived from the inner membrane. These elements have been separated on Ficoll and sucrose gradients. The small vesicles were rich in monoamine oxidase, and the large bodies were rich in cytochrome oxidase. Separation of the inner and outer membranes has also been accomplished by treating mitochondria with digitonin in an isotonic medium and fractionating the treated mitochondria by differential centrifugation. Treatment with low digitonin concentrations released monoamine oxidase activity from low speed mitochondrial pellets, and this release of enzymatic activity was correlated with the loss of the outer membrane as seen in the electron microscope. The low speed mitochondrial pellet contained most of the cytochrome oxidase and malate dehydrogenase activities of the intact mitochondria, while the monoamine oxidase activity could be recovered in the form of small vesicles by high speed centrifugation of the low speed supernatant. The results indicate that monoamine oxidase is found only in the outer mitochondrial membrane and that cytochrome oxidase is found only in the inner membrane. Digitonin treatment released more monoamine oxidase than cytochrome oxidase from sonic particles, thus indicating that digitonin preferentially degrades the outer mitochondrial membrane. I N T R O D U C T I O N Extensive studies of subcellular fractions from tissue homogenatcs have shown that many enzymes concerned with respiration and intermediary metabolism are associated with the mitochondria. However, because of the complexity of the mitochondrion itself, which consists of two membrane systems and at least two compartments, the precise intramitochondrial localization of enzymes and chemical components has remained somewhat speculative. Only recently have attempts been made to separate and characterize the inner and outer mitochondrial membranes. Green et al. (1-3) have examined membrane fractions obtained from beef heart mitochondria by sonication and enzymatic digestion and have concluded that the citric acid cycle enzymes are associated with the outer mitochondrial membrane. Parsons et al. (4, 5) have reported the separation of the inner and outer membranes of rat liver mitochondria follow719 on O cber 9, 2017 jcb.rress.org D ow nladed fom ing induced swelling, and Levy et al. (6) have used digitonin to remove the outer membranes of rat liver mitochondria. Each of these studies has employed the absence of respiratory chain enzymes or morphologically distinct structures such as inner membrane subunits in order to "identify" the outer membrane. In none of these studies has an enzymatic or chemical marker specific for the outer membrane been utilized to monitor the enrichment of this membrane in a given submitochondrial fraction. The problem of isolating the outer mitochondrial membrane is compounded by the fact that the inner membrane is extensively folded to form cristae and may comprise 75 % or more of the total membrane of the mitochondrion. Therefore, care must be taken to obtain preparations of the outer membrane which are not heavily contaminated with inner membrane. The present paper presents data which indicate that mitochondrial monoamine oxidase is localized in the outer membrane of rat liver mitochondria and that this enzyme provides a suitable biochemical marker for the outer membrane in submitochondrial fractionation procedures. Monoamine oxidase was selected as a possible outer membrane marker because it is presumably not linked to the respiratory chain or to the major synthetic and degradative pathways of the mitochondrion, e.g. citric acid cycle, fatty acid metabolism, and yet is a membrane-bound enzyme localized primarily in the mitochondria. Cotzias and Dole (7) first reported that rat liver monoamine oxidase activity is predominantly associated with the mitochondrial fraction. Rodriquez de Lores Arnaiz and De Robertis (8) have thoroughly investigated the distribution of monoamine oxidase in rat brain homogenates and concluded that this enzyme is localized exclusively in the mitochondria and is not found in synaptic vesicles or intact nerve endings. Baudhuin et al. (9) studied the distribution of this enzyme in rat liver homogenates by density gradient centrifugation and concluded that it is not associated with lysosomes or microbodies. Oswald and Strittmatter (10) found that the distributions of monoamine oxidase and succinate oxidase are similar in fractions of rat liver homogenates. Gorkin (11) showed that monoamine oxidase is firmly bound to sonic particles derived from rat liver mitochondria. Vasington and Greenawah (12) reported that rat liver mitochondria retained the capacity to oxidize succinate and ~-hydroxybutyrate and to accumulate Ca ++ and Pi after controlled osmotic lysis. The morphology and biochemical properties of this "water-washed" preparation have been studied in more detail by Caplan and Greenawalt (13), who showed that the water-washed preparation consists of two populations of particles: large LINEAR SUCROSE GRADIENT