High affinity binding of ramiprilat on isolated human glomeruli.

Evidence for angiotensin-converting enzyme (ACE) on isolated human glomeruli was furnished by specific binding of tritium-labeled ramiprilat, a potent inhibitor of ACE. 3H-ramiprilat bound to isolated glomeruli, depending on time and temperature displaying a KD of 3.8 nmol/l and a Bmax of 853 fmol/mg protein. Specific binding represented more than 90% of total binding. Dissociation occurred rapidly after dilution of the sample with incubation buffer or after addition of an excess of unlabeled inhibitor. Binding of 3H-ramiprilat was also inhibited by increasing concentrations of enalaprilat, another ACE-inhibitor or by preincubation of the glomeruli with polyclonal antibodies against ACE. ACE is a zinc-containing enzyme. Addition of EGTA to the assay, which chelates zinc ions, completely inhibited binding. This inhibitory effect of EGTA was reversed by divalent Zn2+ and Ca2+ ions but not by magnesium. Binding of 3H-ramiprilat to isolated glomeruli was maximal when the pH of the assay medium was brought to pH 8. In conclusion, the binding of 3H-ramiprilat to isolated human glomeruli is specific and resembles the characteristics which have been found earlier for enzyme activity of ACE. Thus, binding of 3H-ramiprilat to isolated glomeruli can be assumed to be directed to ACE.