PURIFICATION AND CHARACTERIZATION OF MYCOPLASMA PENETRANS CA2+ MG-2+-DEPENDENT ENDONUCLEASE/

The major nuclease from Mycoplasma penetrans has been purified to homo geneity, The enzyme seems to be present as a membrane-associated precu rsor of 50 kDa and as a peripheral membrane monomeric polypeptide of 4 0 kDa that is easily removed by washing of cells with isotonic buffers and in the aqueous phase upon Triton partitioning of Triton X-114-sol ubilized protein, The 40-kDa nuclease was extracted from hi, penetrans cells by Triton X-114 and phase fractionation and was further purifie d by chromatography on Superdex 75 and chelating Sepharose (Zn2+ form) columns, By gel filtration, the apparent molecular mass was 40 kDa, T he purified enzyme exhibits both a nicking activity on superhelical an d linear double-stranded DNA and a nuclease activity on RNA and single -stranded DNA. No exonuclease activity was found for this enzyme, This nuclease required both Mg2+ (optimum, 5 mM) and Ca2+ (optimum, 2 mM) for activity and exhibited a pH optimum between pH 7 and 8 for DNase a ctivity, It was inhibited by Zn2+, Mn2+, heparin, sodium dodecyl sulfa te, and chelator agents such EDTA and EGTA, but no effect was observed with ATP, 2-mercaptoethanol, N-ethylmaleimide, dithiothreitol, nonion ic detergents, phenylmethylsulfonyl fluoride, and iodoacetamide. Nucle ase activity was inhibited by diethylpyrocarbonate at both pH 6 and 8 and by pepstatin, suggesting the involvement of a histidine and an asp artate in the active site, When added to human lymphoblast nuclei, the purified M. penetrans endonuclease induced internucleosomal fragmenta tion of the chomatin into oligonucleosomal fragments. On the basis of this result, and taking into account the fact that ill, penetrans has the capacity to invade eucaryotic cells, one can suggest, but not asse rt, that produced Ca2+/Mg2+ dependent endonuclease may alter the nucle ic acid metabolism of host cells by DNA and/or RNA degradation and may act as a potential pathogenic determinant.