An alkaline thiol proteinase in the liver mitochondria of bullfrog, Rana catesbeina

Abstract (1) The mitoplasts were prepared from bullfrog ( Rana catesbeiana ) liver mitochondria by treatment with digitonin and were then separated into the matrix and inner membrane fractions. The matrix fraction thus obtained was free of lysosomal contaminations and exhibited a distinct proteinase activity. (2) pH dependency of the matrix proteinase activity measured in the presence and absence of iodoacetamide revealed that the matrix contained at least two kinds of proteinase, a major alkaline thiol proteinase having an optimal pH at 8.5 and a minor neutral proteinase having an optimal pH at 7.5. (3) The major matrix proteinase activity was strongly inhibited by leupeptin, chymostatin, antipain and E64-C, an inhibitor of Ca 2+ -dependent thiol proteinase, while it was scarcely affected by diethylpyrocarbonate. The activity was also inhibited by DTNB and p -chloromercuribenzoate. (4) Addition of hydrocarbon compounds such as ethylene glycol, glycerol, Triton X-100 and poly(ethylene glycol) to the reaction mixture was found to decrease the matrix proteinase activity. (5) Neither cytochrome c nor glutamate dehydrogenase was hydrolyzed when subjected to the matrix proteinase activity in vitro. On the other hand, cytochrome c oxidase was effectively hydrolyzed, and the enzyme associated with the mitochondrial innermembrane fragments was partially hydrolyzed by the major matrix proteinase activity.