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. 2014 Apr 1;9(4):e87843. doi: 10.1371/journal.pone.0087843

Figure 6. The endogenous ACE inhibitor has higher affinity at the C-terminal active site of ACE.

Figure 6

Inhibition of serum ACE was tested by active site specific flourescent substrates: (Abz-SDK(Dnp)P-OH (triangles) for the N-terminal active site, Abz-LFK(Dnp)-OH (squares) for the C-terminal active site). Abz-FRK(Dnp)P-OH (circles) was used as non-site specific substrate. Captopril (0.01 nM–100 nM, A) concentration-dependently inhibited serum ACE activity determined by all three substrates with a similar affinity. In contrast, serum fraction containing 50–100 kDa components (0.02–20 mg/mL protein concentration, 20 mg/mL represents 2.34-fold dilution, B) had higher affinity at the C-terminal active site (determined by Abz-LFK(Dnp)-OH). Symbols represent means ± SEM of 3 independent determinations, values are given in the percentage of control (without ACE inhibitor). Inhibitory activity of the serum fractions (proteins below 50 kDa, or in the range of 50–100 kDa, 2-fold dilution) and captopril (1 µM) were selectively tested by the Abz-FRK(Dnp)P-OH substrate (non-site specific flourescent substrate, C). Bars represent means ± SEM of the recombinant ACE activities in the percentage of vehicle (n = 3).