The vacuolar H+-ATPase of clathrin-coated vesicles is reversibly inhibited by S-nitrosoglutathione.
It has been previously demonstrated that the vacuolar H+-ATPase (V-ATPase) of clathrin-coated vesicles is reversibly inhibited by disulfide bond formation between conserved cysteine residues at the catalytic site on the A subunit (Feng, Y., and Forgac, M. (1994) J. Biol. Chem. 269, 13224-13230). Proton transport and ATPase activity of the purified, reconstituted V-ATPase are now shown to be inhibited by the nitric oxide-generating reagent S-nitrosoglutathione (SNG). The K0.5 for inhibition by SNG following incubation for 30 min at 37 degreesC is 200-400 microM. As with disulfide bond formation at the catalytic site, inhibition by SNG is reversed upon treatment with 100 mM dithiothreitol and is partially protected in the presence of ATP. Also as with disulfide bond formation, treatment of the V-ATPase with SNG protects activity from subsequent inactivation by N-ethylmaleimide, as demonstrated by restoration of activity by dithiothreitol following sequential treatment of the V-ATPase with SNG and N-ethylmaleimide. Moreover, inhibition by SNG is readily reversed by dithiothreitol but not by the reduced form of glutathione, suggesting that the disulfide bond formed at the catalytic site of the V-ATPase may not be immediately reduced under intracellular conditions. These results suggest that SNG inhibits the V-ATPase through disulfide bond formation between cysteine residues at the catalytic site and that nitric oxide (or nitrosothiols) might act as a negative regulator of V-ATPase activity in vivo.
Forgac
M
M
Department of Cellular and Molecular Physiology, Tufts University School of Medicine, Boston, Massachusetts 02111, USA.
D000818:Animals; D020134:Catalytic Domain; D002417:Cattle; D018699:Coated Vesicles / Q000201:enzymology*; D004220:Disulfides / Q000378:metabolism; D004229:Dithiothreitol / Q000494:pharmacology; D005033:Ethylmaleimide / Q000494:pharmacology; D005978:Glutathione / Q000031:analogs & derivatives* / Q000494:pharmacology; D006861:Hydrogen Peroxide / Q000378:metabolism; D009603:Nitroso Compounds / Q000494:pharmacology*; D006180:Proton-Translocating ATPases / Q000378:metabolism*; D026422:S-Nitrosoglutathione; D025262:Vacuolar Proton-Translocating ATPases
D004220:Disulfides; D009603:Nitroso Compounds; D026422:S-Nitrosoglutathione; D006861:Hydrogen Peroxide; D025262:Vacuolar Proton-Translocating ATPases; D006180:Proton-Translocating ATPases; D005978:Glutathione; D005033:Ethylmaleimide; D004229:Dithiothreitol