A 55-KDA ENDONUCLEASE OF MAMMALIAN MITOCHONDRIA - COMPARISON OF ITS SUBCELLULAR-LOCALIZATION AND ENDONUCLEOLYTIC PROPERTIES WITH THOSE OF ENDONUCLEASE

A novel endonuclease of 55-kDa was found in rat liver mitochondria by a zymographic assay, in addition to the 29 kDa enzyme that is well-kno wn as endonuclease G (Endo G). Subcellular localization of these enzym es in rat liver cells was examined by biochemical fractionation, Endo G was located in both nuclei and mitochondria as has been previously r eported, while the 55-kDa enzyme was only detected in the mitochondria l fraction, The levels of the endonucleases in the mitochondria varied greatly among the rat organs, and the activity in the heart was about 30 times higher than that in the liver. The 55-kDa enzyme and Endo G were extracted from bovine heart mitochondria with 0.4 M NaCl. During purification the 55-kDa enzyme and Endo G were copurified because of t heir similar chromatographic behavior, so they were separated by gel f iltration or electrophoresis in the presence of SDS and the proteins w ere then renatured, The nucleolytic properties of the 55-kDa enzyme re sembled those of Endo G and other known mitochondrial nucleases. The e nzyme degraded single-stranded DNA more rapidly than duplex DNA at a w eak alkaline pH, requiring Mg2+ or Mn2+ but not Ca2+ or Zn2+. Nicks ge nerated by the enzyme had 5'-P and 3'-OH ends. The 55-kDa enzyme, like Endo G, displayed an unusually strong preference to nick within a (dG )(n) .(dC)(n) tract.