The role of enzyme and substrate concentration in the evaluation of serum angiotensin converting enzyme (ACE) inhibition by enalaprilat in vitro.
The relationship between serum angiotensin converting enzyme (ACE) activity and concentration of the ACE inhibitor enalaprilat was determined in vitro in the presence of different concentrations (S = 4-200 mM) of the substrate Hip-Gly-Gly. From Henderson plots, a competitive tight-binding relationship between enalaprilat and serum ACE was found yielding a value of approximately 5 nM for serum ACE concentration (Et) and an inhibition constant (Ki) for enalaprilat of approximately 0.1 nM. A plot of reaction velocity (Vi) versus total inhibitor concentration (It) exhibited a non-parallel shift of the inhibition curve to the right with increasing S. This was reflected by apparent Hill coefficients greater than 1 when the commonly used inhibitory sigmoid concentration-effect model (Emax model) was applied to the data. Slopes greater than 1 were obviously due to discrepancies between the free inhibitor concentration (If) present in the assay and It plotted on the abscissa and could, therefore, be indicators of tight-binding conditions. Thus, the sigmoid Emax model leads to an overestimation of Ki. Therefore, a modification of the inhibitory sigmoid Emax model (called "Emax tight model") was applied, which accounts for the depletion of If by binding, refers to It and allows estimation of the parameters Et and IC50f (free concentration of inhibitor when 50% inhibition occurs) using non-linear regression analysis. This model could describe the non-symmetrical shape of the inhibition curves and the results for Ki and Et correlated very well with those derived from the Henderson plots. The latter findings confirm that the degree of ACE inhibition measured in vitro is, in fact, dependent on the concentration of substrate and enzyme present in the assay. This is of importance not only for the correct evaluation of Ki but also for the interpretation of the time course of serum ACE inhibition measured ex vivo. The non-linear model has some advantages over the linear Henderson equation: it is directly applicable without conversion of the data and avoids the stochastic dependency of the variables, allowing non-linear regression of all data points contributing with the same weight.
Weisser
K
K
Zentrum der Pharmakologie, Klinikum der J.W. Goethe-Universität, Frankfurt/Main, Federal Republic of Germany.
Schloos
J
J
D000595:Amino Acid Sequence; D000806:Angiotensin-Converting Enzyme Inhibitors / Q000494:pharmacology*; D004305:Dose-Response Relationship, Drug; D015773:Enalaprilat / Q000494:pharmacology*; D007700:Kinetics; D008433:Mathematics; D008962:Models, Theoretical; D008969:Molecular Sequence Data; D007703:Peptidyl-Dipeptidase A / Q000097:blood / Q000378:metabolism*; D011485:Protein Binding; D013053:Spectrophotometry
D000806:Angiotensin-Converting Enzyme Inhibitors; D007703:Peptidyl-Dipeptidase A; D015773:Enalaprilat