Open AccessInactivation of chloroplast H + ‐ATPase by modification of Lysβ359, Lysα176 and Lysα266
Author(s) -
HORBACH Martina,
MEYER Helmut E.,
BICKELSANDKÖTTER Susannę
Publication year - 1991
Publication title -
european journal of biochemistry
Language(s) - English
Resource type - Journals
eISSN - 1432-1033
pISSN - 0014-2956
DOI - 10.1111/j.1432-1033.1991.tb16203.x
Subject(s) - pyridoxal , pyridoxal phosphate , dithiothreitol , cyanogen bromide , chemistry , enzyme , biochemistry , protein subunit , atpase , chloroplast , gamma subunit , stereochemistry , binding site , g alpha subunit , cofactor , peptide sequence , gene
Treatment of isolated, latent chloroplast ATPase with pyridoxal‐5‐phosphate (pyridoxal‐ P ) in presence of Mg 2+ causes inhibition of dithiothreitol‐activated plus heat‐activated ATP hydrolysis. The amount of [ 3 H]pyridoxal‐ P bound to chloroplast coupling factor 1 (CF 1 ) was estimated to run up to 6 ± 1 pyridoxal‐ P / enzyme, almost equally distributed between the α‐ and β‐subunits. Inactivation, however, is complete after binding of 1.5–2 pyridoxal‐ P /CF 1 , suggesting that two covalently modified lysines prevent the activation of the enzyme. ADP as well as ATP in presence of Mg 2+ protects the enzyme against inactivation and concomittantly prevents incorporation of a part of the 3 H‐labeled pyridoxal‐ P into β‐ and α‐subunits. Phosphate prevents labeling of the α‐subunit, but has only a minor effect on protection against inactivation. The data indicate a binding site at the interface between the α‐ and β‐subunits. Cleavage of the pyridoxal‐ P ‐labeled subunits with cyanogen bromide followed by sequence analysis of the labeled peptides led to the detection of Lysβ359, Lysα176 and Lysα266, which are closely related to proposed nucleotide‐binding regions of the α‐ and β‐subunits.