Figure 1.
Endogenous inhibitor masks ACE activity in human sera.
Effects of dilution on the ACE activity were tested on purified renal ACE (Purified ACE) and human serum (A). Plots were fitted by means of linear regressions in case of purified ACE while nonlinear regression was used in case of serum ACE (indicated by the solid lines), while symbols denote the means ± SEM of at least 3 independent determinations. The increase of specific serum ACE activity (2.78-fold) upon dilution is also indicated. The hypothesis proposed to explain these findings is that an endogenous inhibitor is present in the sera, which inhibits ACE at low dilution and then dissociates at higher dilution, unmasking ACE activity (B).
Figure 2.
FAPGG hydrolysis proportional to angiotensin I conversion.
A representative ACE activity measurement is shown in A. Decomposition of FAPGG results in a decrease in optical density at 340 nm. The decreases in optical density were plotted as a function of the reaction time and fitted by linear regression. The kinetics of decomposition was followed for at least 90 min. The slope of the linear regression was taken as a measure of the ACE activity. Determinations were repeated at least 3 times to obtain activity values. The ACE activity was also determined by using its endogenous substrate angiotensin I, which is converted to angiotensin II by ACE (B). This reaction was followed by HPLC. Area under the curve was calculated to quantify the amount of the peptides. Calibration plots were constructed based on standard concentrations of the peptides. Angiotensin I conversion was followed by a kinetic assay, where angiotensin I and angiotensin II were determined after 0, 2, 4 and 8 h. The slope of the linear fit gave the ACE activity. A single representative experiment is illustrated (B). Parallel experiments were performed to compare FAPGG and angiotensin I-converting activities of the same sample (C). FAPGG converting activities were plotted as a function of angiotensin I converting activities. Plots were fitted by means of linear regression (solid line), the symbols denote means ± SEM of 3 independent determinations. The goodness of fit (r2) is also indicated on figure.
Figure 3.
The size of the endogenous ACE inhibitor is in the range of 50–100 kDa.
To determine the size of the endogenous ACE inhibitor, serum samples were filtered through filter devices (50 kDa pore size, red; and 100 kDa pore size, blue; A). 1 volume of serum was diluted to 250 volume by the buffer and then ultrafiltration was done until 249 volume of the diluted sample has flown through the indicated pore size membranes, yielding 1 volume of retained fraction (being the same as the volume of the initial serum sample). The ACE concentration was the same in the initial serum sample and in the retained fraction, suggesting maintained protein concentrations in the case of the proteins with higher molecular sizes than the pore size of the membranes (50 and 100 kDa). Both the initial sera and the retained fractions were diluted to the same extent to be able to compare ACE activities. Since ACE concentration was the same in these samples at any dilutions, the effects of filtration is the consequence of the loss of the inhibiting factors upon filtration. At least 4-fold dilution of the sera was necessary to measure ACE activity by FAPGG in the initial sera (high level of absorbance at 340 nm by the human serum itself), therefore both the initial samples and the retained fractions were diluted to the same level (4-fold) to compare the inhibited activities. Similarly, both samples were diluted to 32-fold to estimate the level of maximal ACE activity, when the inhibitor was dissociated. Symbols denote means ± SD of 4 independent determinations. Significant differences from the unfiltered serum (green, A) are indicated by asterisks. Serum ACE activities are also shown on the bar graph (B) at 4-fold dilution before (Control, green) and after filtering through 50 kDa (red) and 100 kDa pore size (blue) devices. Unfiltered serum ACE activity is also shown at 32-fold dilution (Control, green). Bars denote means ± SEM of 4 independent determinations, significant differences are indicated by p values.
Figure 4.
Non-competitive ACE inhibition by the endogenous serum factor.
The reaction kinetics of FAPGG hydrolysis (in nmole/min units) was determined at different FAPGG concentrations (750, 500, 250, 167 and 125 µM) to create a Lineweaver-Burk (double reciprocal) plot. The same experiments were performed in the absence (vehicle) and presence of captopril (50 nM) and the 50–100 kDa fraction of the human serum (4.5-fold dilution). Symbols represent means ± SEM of 3 independent determinations. Values were fitted by a linear regression.
Figure 5.
Efficacy of the ACE inhibition by human serum fractions and captopril.
Inhibition of recombinant ACE was measured alone (vehicle) or in the presence of four-fold diluted human serum fractions containing components below 50 kDa (<50 kDa), in the range of 50–100 kDa and 1 µM captopril. Experiments were done first using the physiological substrate angiotensin I (A). Bars represent means ± SD of 2 independent determinations, values are expressed in the percentage of vehicle. The effects were also determined using the artificial substrate FAPGG (B). In this latter case serum fractions were also combined with captopril (<50 kDa+captopril and 50–100 kDa+captopril), in addition to the above mentioned conditions (<50 kDa, 50–100 kDa and 1 µM captopril). Bars represent means ± SD of 3 independent determinations, values are given in the percentage of vehicle, significant differences are indicated by p values.
Figure 6.
The endogenous ACE inhibitor has higher affinity at the C-terminal active site of ACE.
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).
Figure 7.
Endogenous serum ACE inhibition is evolutionary conserved.
Effects of dilution on serum ACE activity were tested in different species. Specific ACE activities were plotted as a function of dilution levels. Symbols denote means ± SEM of at least 3 independent determinations. Significant differences from ACE activity values measured at 4-fold dilution are indicated by asterisks.
Figure 8.
Differences in pH, ionic strength and buffer concentration do not effect the increase in ACE activities upon dilution.
Effects of dilution (4 and 20-fold) were tested on ACE activity under different assay conditions. Increase in specific ACE activity was present at physiological pH (A), at physiological Cl− (B) and at higher buffer concentrations (C). Blue bars represent optimal assay conditions used in the previous experiments. Bars denote means ± SEM of at least 3 determinations. Significant differences are indicated by the p values. The increase in ACE activities for each pairs are also shown within the bars.
Figure 9.
Effects of various protease inhibitors on FAPGG hydrolysis by human serum.
Human serum (8-fold dilution) was incubated with Z-Prolyl-prolinal (prolyl-endopeptidase inhibitor, 1 µM), Apstatin (aminopeptidase P inhibitor, 10 µM), Amastatin (an inhibitor of various aminopeptidases, 10 µM), Bestatin (leucin aminopeptidase and aminopeptidase B inhibitor, 1 µM), E-64 (cysteine protease inhibitor, 1 µM), Leupeptin (serine and cysteine protease inhibitor, 10 µM), PMSF (serine and cysteine protease inhibitor, 100 µM), DX-600 (ACE2 inhibitor, 1 µM) and captopril (an ACE inhibitor, 1 µM) for 15 min. ACE activity measurement was initiated by the addition of FAPGG. Enzyme activity was measured for 120 min and apparent enzyme activities were expressed and plotted in absolute (U/L) units. Bars represent mean ± SEM of three independent determinations. Significant difference (p<0.001) from the control (vehicle) is indicated by the asterisk.
Figure 10.
Hypothetical function of the endogenous angiotensin converting enzyme (ACE) inhibitor.
The clinical success of ACE inhibitory drugs prove that ACE is a physiologically important angiotensin I converting enzyme. Here we propose the existence of an endogenous ACE inhibitor in human sera (Inh) which provide an evolutionary conserved mechanism for the suppression of circulating ACE activity. We hypothesize that ACE is catalytically active in the tissues, where it converts angiotensin I to angiotensin II. The formed angiotensin II then binds to its resident receptors (ATR) and activates intracellular signal transduction leading to physiological responses. Soluble ACE is the result of the shedding of tissue-bound ACE mediated by the ACE secretase (S). Our data suggest that this soluble ACE activity is inhibited by an endogenous inhibitor (Inh), restricting ACE mediated angiotensin I conversion to the tissues, irrespectively to the concentration of the circulating ACE. ACE function appears to be quenched by the endogenous inhibitor when it is secreted into the systemic circulation. This mechanism may contribute to the confinement of angiontensin II mediated physiological responses.