Ca2+/Mg(2+)-dependent endonuclease from human spleen: purification, properties, and role in apoptosis.
A major event in apoptosis is the digestion of chromatin into oligonucleosomal fragments. However, the enzymes responsible for the DNA degradation have not been well characterized. Here we report the purification of an endonuclease from human spleen cell nuclei that is likely to be responsible for DNA digestion in apoptosis. Enzyme activity was measured by a sensitive fluorometric assay, which assesses the conversion of plasmid DNA from a supercoiled to an open form. The endonuclease was extracted from isolated nuclei with NaCl between 100 and 350 mM and was further purified by chromatography on columns of phosphocellulose, Superdex 75, and chelating Sepharose (Zn2+ form). By gel filtration, the apparent molecular mass was 22-26 kDa; on SDS-polyacrylamide gel electrophoresis, the purified enzyme showed a single 27-kDa band. The enzyme required both Mg2+ (optimum, 5 mM) and Ca2+ (optimum, 2 mM) for activity. It was inhibited by Zn2+ (100% inhibition at 50 microM) and by high (> 10 mM) concentrations of Ca2+. Aurintricarboxylic acid, spermine, p-(hydroxymercuri)benzoate, and N-ethylmaleimide were also endonuclease inhibitors. No inhibition was observed with iodoacetamide, G-actin, or nucleoside 3',5'-bisphosphates. An optimum pH of 8.0 was found. When added to human CCRF-CEM lymphoblast nuclei, that do not contain the endonuclease, the purified splenic enzyme digested the chromatin into the mono- and oligonucleosomal fragments that are characteristic of apoptosis. On the basis of this result, and the observation that the activators and inhibitors of the purified endonuclease closely parallel those that affect apoptosis, it seems likely that this enzyme is involved in the apoptotic degradation of DNA in human lymphocytes.
Department of Medicine, University of California, San Diego, La Jolla 92093-0663.
J M
JM
Ribeiro
D A
DA
Carson
D017209:Apoptosis / Q000502:physiology*; D001312:Aurintricarboxylic Acid / Q000494:pharmacology; D002118:Calcium / Q000494:pharmacology; D002467:Cell Nucleus / Q000201:enzymology*; D002729:Chloromercuribenzoates / Q000494:pharmacology; D002843:Chromatin / Q000378:metabolism; D004720:Endonucleases / Q000037:antagonists & inhibitors / Q000187:drug effects / Q000302:isolation & purification / Q000378:metabolism*; D006801:Humans; D006863:Hydrogen-Ion Concentration; D008274:Magnesium / Q000494:pharmacology; D008970:Molecular Weight; D013154:Spleen / Q000166:cytology / Q000201:enzymology*; D020245:p-Chloromercuribenzoic Acid
D002729:Chloromercuribenzoates; D002843:Chromatin; D001312:Aurintricarboxylic Acid; D020245:p-Chloromercuribenzoic Acid; D004720:Endonucleases; D008274:Magnesium; D002118:Calcium