Identification of structurally distinct catalytic intermediates of the H+-ATPase from yeast plasma membranes.

摘要: Mild trypsin proteolysis of the H+-ATPase from yeast plasma membranes has been used to identify structurally distinct catalytic intermediates. In absence substrate, treatment resulted in rapid inactivation enzyme activity. By contrast, presence MgATP or plus vanadate enhanced rates ATP hydrolysis accompanied by protection extensive inactivation. High concentrations Pi also induced strong trypsin-induced inactivation, although enhancement activity was not observed. Western blot analysis peptide fragment profiles following tryptic digestion indicated that at least 15 prominent fragments identical size, ranging Mr = 12,800 48,000, were generated irrespective conditions. However, protected resistant further proteolysis, whereas unprotected extensively degraded. These data have interpreted terms a published reaction pathway (Amory, A., Goffeau, McIntosh, D.B., and Boyer, P.D. (1982) J. Biol. Chem. 257, 12509-12516) are consistent with conformations representing E1 E2 X intermediates, respectively. Trypsin proved an effective tool for evaluating preferred conformational states this approach, it found ATPase inhibitors N-ethylmaleimide fluorescein isothiocyanate locked conformation. The rate trypsin-treated fully coupled proton transport, all firmly bound membrane. results, fact initial fragmentation independent conformation, suggest different states, E1, Pi, related gross changes overall structure but likely reflect localized intramolecular bonding.