title: Purification and characterization of Mycoplasma penetrans Ca2+/Mg2+-dependent endonuclease.
abstract: The major nuclease from Mycoplasma penetrans has been purified to homogeneity. The enzyme seems to be present as a membrane-associated precursor of 50 kDa and as a peripheral membrane monomeric polypeptide of 40 kDa that is easily removed by washing of cells with isotonic buffers and in the aqueous phase upon Triton partitioning of Triton X-114-solubilized protein. The 40-kDa nuclease was extracted from M. penetrans cells by Triton X-114 and phase fractionation and was further purified by chromatography on Superdex 75 and chelating Sepharose (Zn2+ form) columns. By gel filtration, the apparent molecular mass was 40 kDa. The purified enzyme exhibits both a nicking activity on superhelical and 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 activity. It was inhibited by Zn2+, Mn2+, heparin, sodium dodecyl sulfate, and chelator agents such EDTA and EGTA, but no effect was observed with ATP, 2-mercaptoethanol, N-ethylmaleimide, dithiothreitol, nonionic detergents, phenylmethylsulfonyl fluoride, and iodoacetamide. Nuclease activity was inhibited by diethylpyrocarbonate at both pH 6 and 8 and by pepstatin, suggesting the involvement of a histidine and an aspartate in the active site. When added to human lymphoblast nuclei, the purified M. penetrans endonuclease induced internucleosomal fragmentation of the chomatin into oligonucleosomal fragments. On the basis of this result, and taking into account the fact that M. penetrans has the capacity to invade eucaryotic cells, one can suggest, but not assert, that produced Ca2+/Mg2+-dependent endonuclease may alter the nucleic acid metabolism of host cells by DNA and/or RNA degradation and may act as a potential pathogenic determinant.
authors.affiliation: Laboratory of Immunovirology UFR SVT, University of Paul Sabatier, Toulouse, France.
authors.forename: M
authors.initials: M
authors.lastname: Bendjennat
authors.forename: A
authors.initials: A
authors.lastname: Blanchard
authors.forename: M
authors.initials: M
authors.lastname: Loutfi
authors.forename: L
authors.initials: L
authors.lastname: Montagnier
authors.forename: E
authors.initials: E
authors.lastname: Bahraoui
mesh_terms: D000942:Antigens, Bacterial / Q000302:isolation & purification; D002118:Calcium; D002412:Cations; D002843:Chromatin / Q000378:metabolism; D004278:DNA, Superhelical / Q000378:metabolism; D004720:Endonucleases / Q000302:isolation & purification*; D006679:HIV Seropositivity / Q000276:immunology; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007526:Isoelectric Point; D008274:Magnesium; D008970:Molecular Weight; D018661:Mycoplasma penetrans / Q000201:enzymology* / Q000276:immunology; D009994:Osmolar Concentration; D013379:Substrate Specificity; D013696:Temperature
chemical_list: D000942:Antigens, Bacterial; D002412:Cations; D002843:Chromatin; D004278:DNA, Superhelical; D004720:Endonucleases; C121440:endonuclease P40; D008274:Magnesium; D002118:Calcium