The localization of proteolytic activity in rat liver mitochondria and its relation to mitochondrial swelling and aging.

1. On storage of rat liver mitochondria at 0 degrees , water content, total amino acid content and leakage of protein all rose steadily over a 72hr. period. The initial ratio of intramitochondrial to extramitochondrial amino acid concentration lay between 18 and 24. Initially this rose, but it then fell to 1.9 at the end of storage. The concentration gradient between internal and external amino acids was relatively constant throughout the period. These processes were accentuated at 22 degrees and 40 degrees , the concentration gradient reaching 70mumoles/ml., water content rising to 8.3mg./mg. dry wt. and protein leakage reaching 42% of total mitochondrial protein. ;Swelling agents' produced no correlated changes in amino acid production and swelling. 2. Added glutamate was not concentrated within the pellet of whole or disrupted mitochondria. Endogenous amino acids were distributed evenly between the pellet and the supernatant of disrupted mitochondria. It is concluded that amino acids are produced within mitochondria and that adsorption and uptake from the medium do not contribute significantly to amino acids in the pellet. 3. beta-Glycerophosphate, a lysosome protectant, increased amino acid production by rat liver mitochondria. Treatment with Triton X-100 and disruption by freezing and thawing showed that 56% of proteolytic activity was ;free' in whole mitochondria, whereas only 11% of acid phosphatase activity, a lysosomal enzyme, was ;free'. 4. ;Light' mitochondria contained 30% more neutral proteolytic activity but 300% more acid phosphatase activity than ;heavy' mitochondria. 5. Electron micrographs of mitochondrial preparations showed less than one particle in 500 that could be identified as a lysosome. Treatment with Triton X-100 disrupted the structure of roughly 50% of the mitochondria; the rest appeared to retain their membrane, cristae and ground substance. Freezing and thawing caused gross swelling and loss of ground substance and rupture of external membranes. 6. Of the recovered proteolytic activity, 81% at pH7.4 and 70% at pH5.8 were found in the high-speed supernatant of broken mitochondria. A further fivefold increase in specific activity was found in the first protein fraction obtained by Sephadex G-50 gel filtration. 7. Between 60 and 80% of proteolytic activity was found in the 40-60%-saturated ammonium sulphate precipitate. Almost all of the soluble-fraction proteolytic activity could be recovered in a pH5.0 supernatant. 8. The results give no support to the view that mitochondrial neutral proteolytic activity reflects lysosomal content. 9. The possible role of intramitochondrial amino acid production and the proteolysis of internal barriers in passive swelling of mitochondria is discussed.