Concanamycin A, the specific inhibitor of V-ATPases, binds to the V(o) subunit c.
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-(trifluoroethyldiazirinyl)-benzoyl]-21,23-dideoxy-23-[(125)I]iodo-concanolide A (J-concanolide A) was performed, which still inhibits the V-ATPase 50% at a concentration of 15-20 microm. 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 (125)I makes the detection of the labeled subunit(s) feasible. Treatment of the V(1)/V(o) holoenzyme, the V(o) 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 V(o) 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 A(1) and B(1), respectively, but not by the benzolactone enamide salicylihalamide, a member of a novel class of V-ATPase inhibitors.
Huss
Markus
M
Universität Osnabrück, Fachbereich Biologie/Chemie, Abteilung Tierphysiologie, 49069 Osnabrück, Germany.
Ingenhorst
Gudrun
G
König
Simone
S
Gassel
Michael
M
Dröse
Stefan
S
Zeeck
Axel
A
Altendorf
Karlheinz
K
Wieczorek
Helmut
H
D000595:Amino Acid Sequence; D000818:Animals; D000900:Anti-Bacterial Agents / Q000378:metabolism / Q000494:pharmacology*; D001665:Binding Sites; D004591:Electrophoresis, Polyacrylamide Gel; D004791:Enzyme Inhibitors / Q000378:metabolism / Q000494:pharmacology*; D018942:Macrolides; D018402:Manduca; D008969:Molecular Sequence Data; D019920:Photoaffinity Labels; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D025262:Vacuolar Proton-Translocating ATPases / Q000037:antagonists & inhibitors* / Q000737:chemistry / Q000378:metabolism
D000900:Anti-Bacterial Agents; D004791:Enzyme Inhibitors; D018942:Macrolides; D019920:Photoaffinity Labels; C036978:concanamycin A; D025262:Vacuolar Proton-Translocating ATPases