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.
Albus
U
U
Hoechst AG, Department of Pharmacology, Frankfurt, Federal Republic of Germany.
Kress
I
I
Linz
W
W
Vasmant
D
D
Delarue
F
F
Sraer
J D
JD
D000806:Angiotensin-Converting Enzyme Inhibitors / Q000378:metabolism*; D001667:Binding, Competitive; D002413:Cations, Divalent / Q000494:pharmacology; D004533:Egtazic Acid / Q000494:pharmacology; D006801:Humans; D066298:In Vitro Techniques; D007678:Kidney Glomerulus / Q000378:metabolism*; D007703:Peptidyl-Dipeptidase A / Q000378:metabolism*; D011485:Protein Binding; D011758:Pyrroles / Q000378:metabolism*; D017257:Ramipril / Q000031:analogs & derivatives*; D012263:Ribonucleosides / Q000378:metabolism / Q000494:pharmacology*; D015032:Zinc / Q000494:pharmacology
D000806:Angiotensin-Converting Enzyme Inhibitors; D002413:Cations, Divalent; D011758:Pyrroles; D012263:Ribonucleosides; D004533:Egtazic Acid; C052549:ramiprilat; C054665:N 10169; D007703:Peptidyl-Dipeptidase A; D015032:Zinc; D017257:Ramipril