MYCOPLASMA NUCLEASES ABLE TO INDUCE INTERNUCLEOSOMAL DNA-DEGRADATION IN CULTURED-CELLS POSSESS MANY CHARACTERISTICS OF EUKARYOTIC APOPTOTICNUCLEASES

It was previously shown (Paddenberg ef al (1996) fur J Cell Biol 69, 1 05 - 119) that cells of established lines like NIH3T3 fibroblasts and the human pancreatic adenocarcinoma PaTu 8902 line only degrade their chromatin at internucleosomal sites after an apoptotic stimulus when i nfected with Mycoplasma hyorhinis. In order to distinguish mycoplasma nucleases (M-r 47 - 54 kDa) from already described eukaryotic apoptoti c enzymes, the mycoplasma nucleases were partially purified from serum -free culture supernatants and further characterized. Here we demonstr ate directly that the enriched mycoplasma nucleases were able to fragm ent the DNA of nuclease-negative substrate nuclei at internucleosomal sites. The DNA degradation was accompanied by morphological changes ty pical of apoptosis like chromatin condensation and margination followe d by shrinkage of the nuclei, The biochemical characterization reveale d that the mycoplasma nucleases had a neutral to weakly basic pH-optim um. They required both calcium and magnesium in the mM range for maxim al activation and were inhibited by zinc chloride, EGTA and EDTA. In t wo dimensional zymograms they migrated as three spots with isoelectic points between 8.1 and 9.5. They were not inhibited by monomeric actin . Our data also demonstrate that nuclear extracts prepared from nuclei isolated from Mycoplasma hyorhinis infected cells contained the mycop lasma nuclease activities leading to their internucleosomal DNA-degrad ation after incubation in the presence of calcium and magnesium.