Further observations on the effects of trypsin on the volume and functions of mitochondria isolated from normal liver and AH-130 Yoshida ascites hepatoma.

Evidence is given indicating that the outer membrane of mitochondria from AH-130 Yoshida ascites hepatoma has low resistance to trypsin. The outer membrane of freshly-isolated hepatoma mitochondria is impermeable to cytochrome c and dextran, but it seems to be easily digested by trypsin. The proteolytic enzyme partially inactivates the adenylate kinase and cytochrome c oxidase of these mitochondria. Inhibition of both enzymes by trypsin is also observed in rat liver mitochondria, but only after the outer membrane has been made permeable to large molecules. The succinate-cytochrome c reducÃ-ase of both hepatoma and liver mitochondria is not inactivated by trypsin even if the outer membrane is made permeable to large molecules by digitonin. Inactivation of succinate-cytochrome c reductase occurs when trypsin is added after a strong sonic treatment of mitochondria. The adenosine triphosphatase of both types of mitochondria is resistant to trypsin also after the sonic fragmentation of the particles. A marked inhibition of adenosine triphosphatase by proteolysis takes place in phospholipase C-defatted mito chondria from hepatoma and liver when incubation with trypsin precedes the reconstitution of the enzyme by phospholipids extracted from liver. Swelling-inducing amounts of trypsin affect some inner membrane-linked functional activities of hepatoma and liver mitochondria; however, in liver mitochondria, the time of onset of swelling is much later than in hepatoma mitochondria, since the swelling in the former is preceded by a lag phase during which no functional impairment occurs. These results are interpreted as indicative of structural alterations of the outer membrane of hepatoma mitochondria, whereas no differences with regard to sensitivity of trypsin seem to exist between the inner membrane of liver and hepatoma mitochondria.