title: Essential sulfhydryl groups in the catalytic center of the tonoplast H(+)-ATPase from coleoptiles ofZea mays L. as demonstrated by the biotin-streptavidin-peroxidase system.
abstract: Functional properties and the localization of essential SH-groups of the tonoplast H(+)-ATPase fromZea mays L. were studied. In contrast to the pyrophosphate-dependent H(+)-translocation activity of the tonoplast, the H(+)-ATPase activity was inhibited by SH-blocking agents, such as N-ethylmaleimide and iodoacetic acid. In the case ofp-hydroxymercuribenzoate, HgCl2 and oxidized glutathione, the inhibition could be reversed by adding reduced glutathione or dithiothreitol.Incubation of tonoplast vesicles with oxidized glutathione or N-ethylmaleimide in the presence of Mg·ADP-a competitive inhibitor of the ATP-dependent H(+) pump-avoided the inhibition of the H(+)-pumping activity. This effect is an indication for the occurrence of essential SH-groups at the catalytic site of the H(+)-ATPase.In order to characterize the active center these thiols were specifically labeled with maleimidobutyrylbiocytin. Subsequently, the membrane proteins were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred to an immobilizing membrane. The maleimidobutyrylbiocytin-labeled active-center protein was detected by a biotin-streptavidin-peroxidase staining system and was shown to be a 70-kDa subunit of the tonoplast H(+)-ATPase. It is suggested that the oxidation state of the critical sulfhydryl groups within the active center of the enzyme and their reversible blocking by endogenous compounds might be of great importance for the regulation of the enzyme activity in vivo.
authors.affiliation: Institut für Biologie I, Universität Tübingen, Auf der Morgenstelle 1, D-7400, Tübingen, Germany.
authors.forename: A
authors.initials: A
authors.lastname: Hager
authors.forename: C
authors.initials: C
authors.lastname: Lanz