Journal: ACS chemical biology
Article Title: Active-site directed chemical tools for profiling mitochondrial Lon protease
doi: 10.1021/cb100408w
Figure Lengend Snippet: (a) Representative time course of peptide cleavage by human Lon in the presence of ATP and DBN93. Two rates of cleavage, vi and vss, and a rate constant of interconversion between the two, kinter, can be quantified from the experimental time course. (b) Reactions containing 150 nM human Lon were preincubated with 1 mM FRETN 89–98 prior to the addition of 1 mM ATP. After 90 s, varying amounts of DBN93 (0–2 µM) were added and peptide hydrolysis was monitored over 45 min. All experiments were done in at least duplicate and the vi and vss values determined by fitting the time courses as done previously for bacterial Lon (23). The averaged vi (■), vss (▲) in the presence of inhibitor normalized to 1 with the average velocity in the absence of inhibitor were plotted against corresponding inhibitor concentrations. The solid lines represent the best fit of the data, as described in Materials and Methods. (c) Experimental time courses were fit to both one-step and two-step inhibition mechanisms using the global non-linear fitting program, DynaFit (Biokin, Ltd.). Black lines represent the averaged experimental time courses at 150 nM hLon, 1 mM ATP, 1 mM peptide and varying concentrations of DBN93. Gray lines represent the best fit of the data to the two-step time-dependent mechanism which was most consistent with the experimental data. The kinetic parameters yielded from this fit are summarized and compared to those previously determined for bacterial Lon (23) in Table 1.
Article Snippet: The solid lines represent the best fit of the data, as described in Materials and Methods. ( c ) Experimental time courses were fit to both one-step and two-step inhibition mechanisms using the global non-linear fitting program, DynaFit (Biokin, Ltd.).
Techniques: Inhibition
