title: Identification of acidic, alkaline, and neutral sphingomyelinase activities in Mycobacterium tuberculosis.
abstract: BACKGROUND
  Sphingomyelinase enzymes are pathogenic factors of several intracellular bacteria species, which have been little studied in Mycobacterium tuberculosis.
  
  
  METHODS
  Cell free extracts from H37Rv and CDC-1551 M. tuberculosis strains were assayed for sphingomyelinase activity by using [N-methyl-14C]-sphingomyelin as substrate. Double-directional thin-layer chromatography was used to separate the substrate and hydrolysis product. Sphingomyelinase activity was analyzed as a function of incubation time, dose, pH and the presence of MgCl2, CaCl2, ZnSO4, HgCl2, MnCl2, CoCl2 and EDTA (1 or 10 mM).
  
  
  RESULTS
  Mycobacterial preparations hydrolyzed [14C]-sphingomyelin, in time- and dose-dependent manners, producing [14C]-phosphorylcholine as a unique product. The activity of H37Rv neutral sphingomyelinase at pH 7.5 was 2.15 times higher than that of CDC-1551. This activity was inhibited 21-82% by Ca2+, Hg2+ or Zn2+ and EDTA, and stimulated 40-117% by Mn2+ and Mg2+. In addition, preparations from both strains showed two peaks of sphingomyelinase, one at pH 5.5 and the other at pH 3.0. However, these activities were 4-22 times lower than that observed at pH 7.5 for strain H37Rv. Preparations from H37Rv, but not those of CDC-1551, hydrolyzed sphingomyelin at pH 8-9, with a specific activity similar to that of the neutral CDC-1551 enzyme.
  
  
  CONCLUSIONS
  Both strains H37Rv and CDC-1551 of M. tuberculosis have cation-dependent acidic and neutral sphingomyelinase-C enzymes, showing the neutral as the major activity. In addition, H37Rv has an alkaline sphingomyelinase-C. The importance of SMases in M. tuberculosis pathogenesis remains to be elucidated.
authors.affiliation: Centro de Investigación Biomédica del Noreste, Instituto Mexicano del Seguro Social, División de Biología Celular y Molecular, Monterey, N.L, Mexico.
authors.forename: Javier
authors.initials: J
authors.lastname: Vargas-Villarreal
authors.forename: Benito D
authors.initials: BD
authors.lastname: Mata-Cárdenas
authors.forename: Manon
authors.initials: M
authors.lastname: Deslauriers
authors.forename: Frederick D
authors.initials: FD
authors.lastname: Quinn
authors.forename: Jorge
authors.initials: J
authors.lastname: Castro-Garza
authors.forename: Herminia G
authors.initials: HG
authors.lastname: Martínez-Rodrĺguez
authors.forename: Salvador
authors.initials: S
authors.lastname: Said-Fernández
mesh_terms: D001426:Bacterial Proteins / Q000378:metabolism; D002474:Cell-Free System; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D009169:Mycobacterium tuberculosis / Q000201:enzymology* / Q000472:pathogenicity; D009994:Osmolar Concentration; D013108:Sphingomyelin Phosphodiesterase / Q000378:metabolism*; D014774:Virulence
chemical_list: D001426:Bacterial Proteins; C112980:acid sphingomyelinase-1; D013108:Sphingomyelin Phosphodiesterase