High-Affinity Binding of the Converting Enzyme Inhibitor, Ramiprilat, to Isolated Human Glomeruli

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Abstract
Evidence for effects of angiotensin converting enzyme (ACE) on isolated human glomeruli was provided using 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. ACE is a zinc-containing enzyme. Addition of EGTA to the assay, which chelates zinc ions, completely prevented binding. This was reversed by divalent Zn2+ and Ca2+ ions, but not by magnesium. Binding of [3H]ramiprilat to isolated glomeruli was maximal at pH 8, which also is optimal for ACE activity. 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.
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