1 . Heart mitochondria from rats at 3 months and 23 months of age were investigated for their capacity to generate oxygen radicals and hydrogen peroxide.2. Highest values for 0;-formation were obtained with submitochondrial particles freed from superoxide dismutase after the addition of succinate and antimycin A to start the reaction. Under these conditions superoxide-radical formation with mitochondria from old rats (2.54 nmol x niin-'x m g -' ) exceeded formation rates in the young controls (1.9 nmol x min ~' x mg-') by 25 ",,.3. A constant fraction of 20 ' l o of the radicals produced escaped quenching by intraniitochondrial superoxide dismutase. This fraction was independent of the rate of radical formation ; its magnitude was deduced from generation rates of hydrogen peroxide in the presence and absence of exogenous superoxide dismutase.4. Free oxygen radicals could be obtained with intact rat-heart mitochondria as well following the addition of ATP. a system which is closer to physiological states. Formation rates of oxygen radicals observed under these conditions were similar to those seen in the particulate fractions which escaped quenching by mitoc1iondri:il superoxide dismutase.5. Peroxidative degradation o f membrane lipids was found t o parallel steady-state concentrations of free oxygen radicals.6. The results are discussed in terms of a radical-generating mechanism which functions in rive a t the level of the mitochondrial electron-transferring system.
1.The earlier observation that aging promotes peroxidation of lipids within inner mitochondrial membranes by a radical-generating mechanism has provided a model for studying the relationship of membrane alterations to energy-linked respiration.2. Electron transport was impaired as a function of age only in the presence of substrates oxidized within the inner mitochondrial membrane. Respiratory control ratios and P : 0 ratios decreased regardless of substrate identity.3. Arrhenius kinetic data revealed age-dependent shifts of temperature breaks only for membranebound enzymes including oligomycin-sensitive ATPase. Specific activities decreased for all enzymes studied except cytochrome oxidase and the glutamate oxidase system. 4. Molecular motion data of spin-labelled fatty acid analogues associated with phospholipid bilayer from inner mitochondrial membranes exhibited a decrease in lipid fluidity in membranes from aged rats.5. The exposure of isolated mitochondria to a 0; -radical generating system brought about identical changes in energy-linked respiration and oxidative phosphorylation as did aging in the animal. The effect was completely inhibited only in the presence of both superoxide dismutase and catalase, indicating the participation of 0 2 -radicals and H202 in causing the observed structural and functional changes.6. The results support the concept of an active mechanism in mitochondria of living tissues which can contribute to pathophysiological processes by a sequential reaction involving radicalinduced changes in the hydrophobic bonding of lipid-dependent enzyme systems.
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