Transmembrane carboxyl residues are essential for cation-dependent function in the gastric H,K-ATPase.
Abstract
The K+-dependent ATPase activity of the H,K-ATPase was irreversibly inhibited by the carboxyl activating reagent, dicyclohexylcarbodiimide (DCCD). The inhibition was first order and displayed a concentration dependence with the K0.5 (DCCD) = 0.65 +/- 0.04 mM. KCl protected 70% of the ATPase activity from DCCD-dependent inhibition in a concentration-dependent manner with a K0.5 (K+) = 0.58 +/- 0.1 mM KCl. DCCD modification selectively inhibited the K+-dependent rather than ATP-dependent partial reactions including eosin fluorescence responses and ligand-stabilized initial tryptic cleavage patterns of the membrane-associated enzyme. DCCD modification also inhibited the binding of 86Rb+ and the fluorescent responses of the K+-competitive, fluorescent inhibitor 1-(2-methylphenyl)-4-methylamino-6-methyl-2, 3-dihydropyrrolo[3,2-c]quinoline. [14C]DCCD was incorporated into the H,K-ATPase in a time course identical to that describing the inactivation of the K+-dependent ATPase activity of the H,K-ATPase. A component of the [14C]DCCD incorporated into the H,K-ATPase was K+-sensitive where K+ reduced the [14C]DCCD incorporated into the enzyme by 1.6 nmol of [14C]DCCD/mg of protein. Membrane-associated tryptic peptides resolved from the [...Continue Reading
References
Citations
Related Concepts
Related Feeds
ASBMB Publications
The American Society for Biochemistry and Molecular Biology (ASBMB) includes the Journal of Biological Chemistry, Molecular & Cellular Proteomics, and the Journal of Lipid Research. Discover the latest research from ASBMB here.