Purification and characterization of the potent endonuclease in extracts of bovine heart mitochondria.
A potent endonuclease identified in a crude fraction of soluble proteins from bovine heart mitochondria has been purified 2500-fold and partially characterized. Physical studies of the enzyme indicate a Stokes radius of 30.3 A and a sedimentation coefficient, S20 degrees, w, of 4.1 yielding a native molecule weight of 59,000 and a frictional coefficient of 1.2. Analysis of extensively purified fractions by sodium dodecyl sulfate-polyacrylamide gel electrophoresis reveals a major band at 29,000 Da accounting for 50% of the total protein and suggesting a dimeric subunit structure. The endonuclease maintains two distinct pH optima: pH 5.1-5.5 and 7-8. Both acid and neutral activities nick supercoiled M13 circular double-stranded replicative form I DNA and fragment single-stranded DNA templates to generate 5'-phosphoryl-3'-hydroxyl breaks. The endonuclease requires a divalent cation (preferring Mn2+ over Mg2+) and is sensitive to N-ethylmaleimide and moderate levels of salt. Analysis of the digestion products of double-stranded DNA after prolonged nuclease treatment yields a mixture of oligonucleotides, 13% of which are di- and trinucleotides. Despite the enzyme's ability to degrade DNA to oligonucleotides under some conditions, a strikingly nonrandom pattern of cleavage is observed when a restriction fragment composed of bovine D-loop DNA is used as a template. In this case, a strong preference for guanine tracts is seen.
D000818:Animals; D001483:Base Sequence; D002417:Cattle; D004277:DNA, Single-Stranded / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D004706:Endodeoxyribonucleases / Q000302:isolation & purification*; D005033:Ethylmaleimide / Q000494:pharmacology; D006863:Hydrogen-Ion Concentration; D046911:Macromolecular Substances; D008274:Magnesium / Q000378:metabolism; D008345:Manganese / Q000378:metabolism; D008929:Mitochondria, Heart / Q000201:enzymology*; D008970:Molecular Weight
D004277:DNA, Single-Stranded; D046911:Macromolecular Substances; D008345:Manganese; D004706:Endodeoxyribonucleases; C054195:bovine mitochondrial endonuclease; D008274:Magnesium; D005033:Ethylmaleimide