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. 2014 Feb 4;9(2):e88001. doi: 10.1371/journal.pone.0088001

Figure 9. Characterization of recombinant mutant (S333W) ACE.

Figure 9

A. ACE activity of the membrane-bound form of ACE of CHO cells (lysates) transiently expressing WT and mutant ACE was determined as in Fig. 1. Ratio of hydrolysis of the two substrates (ZPHL/HHL ratio) in the tested samples are presented as mean (±SD) from 4 independent experiments. *p<0.05 vs. WT. B. Effect of ACE inhibitors and anti-catalytic mAbs on mutant ACE activity. Lysates form CHO cells expressing mutant and WT membrane-bound ACE were incubated with ACE inhibitor enalaprilat (100 nM). Residual ACE activity was determined with both substrates and ZPHL/HHL ratio was determined as in A. C–F. Conformational fingerprinting of mutant ACE. C–D. Membrane-bound WT and mutant ACE (lysates from corresponding CHO cells) were normalized to achieve 5 mU/ml ACE activity with Hip-His-Leu as substrate and incubated in microtiter plate wells covered with 16 mAbs to human ACE as in Fig. 4 Data (mean ± SD of 2–3 independent experiments in duplicate) are expressed as ratio of ACE activity with HHL (C) and ZPHL (D) precipitated by mAbs from mutant ACE to that of WT ACE. Bars highlighted with yellow – the values of precipitated ACE activity from mutant ACE was 20% lower than this value for WT. Bars highlighted with red- the values of precipitated ACE activity from mutant ACE was 20% higher than this value for WT. E–F. The data presented in C and D were expressed as the ZPHL/HHL ratio of ACE activity precipitated by each mAb. Data are mean ± SD of 6–8 independent experiments in duplicate. Bars highlighted with yellow – the values of ZPHL/HHL ratio of the precipitated ACE activity was 20% lower than this value for this type of ACE in solution. Bars highlighted with red - the values of ZPHL/HHL ratio of precipitated ACE activity was 20% higher than this value for this type of ACE in solution. * - p<0.05 vs. ZPHL/HHL ratio for corresponding values of WT and mutant ACE in solution (horizontal lines in E and F). The ratio for any duplicate samples of WT ACE or mutant ACE for each mAb was approximately 1.0 and the SD was less than 10%.