Page 25 - Demo

SECTION I: MOLECULAR GENETIC ENGINEERING AND BIOCHEMICAL TECHNOLOGY 29different concentrations and subjected to pronase digestion. Wild-type M. tuberculosis ClpC1 was protected against pronase digestion by ecumicin, and the protective effect increased with ecumicin concentration (Fig. 5). The protected protein band was excised and identified as ClpC1 by mass spectrometry. Such a protective effect was not observed for any of the three mutant ClpC1s. This result strongly suggests that ClpC1 is at least one of the molecular targets of ecumicin. Figure 5. Ecumicin protects wild-type but not mutated (L92F, L92S, L96P) M. tuberculosis ClpC1 from pronase digestion. Analysis was performed by SDS-PAGE (lanes 1 to 7) and Western blotting (lanes 8 to 14). Lanes: M, size marker; 1 and 8, whole-cell lysate without any treatment; 2 and 9, whole-cell lysate digested by pronase; 3 to 7 and 10 to 14, whole-cell lysates treated with ecumicin at increasing concentrations (0.04, 0.4, 4, 40, and 80 %u03bcg/ml) and subjected to pronase digestion3.6. Effect of ecumicin on the ATPase and proteolitic activity of ClpC1 ClpC1 is a hexameric ATPase belonging to the large AAA family of ATPases[8] and in M. tuberculosis, unlike in most