Concanamycin A, the specific inhibitor of V-ATPases, binds to the VO subunit c

Abstract

Vacuolar-type ATPase (V-ATPase) purified from the midgut of the tobacco hornworm Manduca sexta is inhibited 50% by 10 nM of the plecomacrolide concanamycin A, the specific inhibitor of V-ATPases. To determine the binding site(s) of that antibiotic in the enzyme complex, labeling with the semisynthetic 9-O-[p-(trifluoro-ethyldiazirinyl)-benzoyl]- 21,23-dideoxy-23-[125I]iodo-concanolide A (J-concanolide A) was performed, which still inhibits the V-ATPase 50% at a concentration of 15-20 μM. Upon treatment with UV light, a highly reactive carbene is generated from this concanamycin derivative, resulting in the formation of a covalent bond to the enzyme. In addition, the radioactive tracer 125I makes the detection of the labeled subunit(s) feasible. Treatment of the V1/Vo holoenzyme, the Vo complex, and the V-ATPase containing goblet cell apical membranes with concanolide resulted in the labeling of only the proteolipid, subunit c, of the proton translocating Vo complex. Binding of J-concanolide A to subunit c was prevented in a concentration-dependent manner by concanamycin A, indicating that labeling was specific. Binding was also prevented by the plecomacrolides bafilomycin A1 and B1, respectively, but not by the benzolactone enamide salicylihalamide, a member of a novel class of V-ATPase inhibitors.