Purification and characteristics of a mitochondrial endonuclease from the yeast Saccharomyces cerevisiae.
Saccharomyces cerevisiae contains a membrane-bound mitochondrial nuclease. The enzyme was purified nearly 500-fold from sphaeroplasts of the organism by differential centrifugation, differential solubilization, heparin-agarose chromatography, and gel filtration. A final specific activity of 98 mumol min-1 (mg of protein)-1 was obtained. The enzyme required further purification to achieve homogeneity. Two peaks of activity were obtained after gel filtration with apparent molecular weights of 140000 and 57000. Otherwise, these two components have nearly identical characteristics. Without detergent the enzyme is insoluble and has very low activity. Zwittergent 3-14 or Triton X-100 in the presence of KCl could be used to solubilize and activate the enzyme. A number of other detergents were much less effective in solubilizing or activating the nuclease. The enzyme requires Mg2+ for activity, and this can be replaced to some degree by Mn2+ but not by Ca2+ or Zn2+. It is most active at pH 6.5-7.0 and degrades the substrate to small oligonucleotides with 5'-phosphate ends. The relative rates of hydrolysis were 100 for poly(A), 31 for ssDNA, 19 for RNA, 2.1 for dsDNA, and less than or equal to 0.2 for poly(C). Under the assay conditions used the enzyme appears to constitute about 90% of the total nuclease activity of the cell. The enzyme is unstable, especially at neutral and alkaline pH.
D002118:Calcium / Q000378:metabolism; D002850:Chromatography, Gel; D004720:Endonucleases / Q000302:isolation & purification* / Q000378:metabolism; D008274:Magnesium / Q000378:metabolism; D008345:Manganese / Q000378:metabolism; D008928:Mitochondria / Q000201:enzymology; D008970:Molecular Weight; D012441:Saccharomyces cerevisiae / Q000201:enzymology*; D015032:Zinc / Q000378:metabolism
D008345:Manganese; D004720:Endonucleases; D008274:Magnesium; D015032:Zinc; D002118:Calcium