Transmembrane carboxyl residues are essential for cation-dependent function in the gastric H,K-ATPase.
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 [14C]DCCD-modified H,K-ATPase exhibited various K+ sensitivities with peptides at 23, 9.6, 8.2, 7.1, and 6.1 kDa containing 10-78%, 23-52%, 24-36%, 2%, and 3-4% K+-sensitivity, respectively. The N-terminal sequence of the K+-sensitive, approximately 23- and 9.6-kDa peptides was LVNE857, a C-terminal fragment of the ATPase alpha-subunit. The mass of the smaller peptide limited the residue assignment to the transmembrane M7/M8 domains and an intervening extracytoplasmic loop. An N-terminal sequence, SD840IM, was obtained from a 3.3-kDa, [14C]DCCD-labeled peptide resolved from a V8 digest of the partially purified alpha-subunit. This mass was sufficient to include LVNE but would exclude M8 and the intervening loop between M7 and M8. Glu857 is a unique residue present in each of the proteolytic preparations of the H,K-ATPase modified by [14C]DCCD. These data provide functional evidence of the selective inactivation of the K+-dependent partial reactions of the H,K-ATPase and show that Glu857 located at the M7 boundary in the C terminus of the pump molecule is a significant site of DCCD modification. These data are interpreted to indicate that this carboxyl residue is important for cation binding function.
Rabon
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Tulane University Medical Center and Veterans Administration Center, New Orleans, Louisiana 70112, USA.
Hoggatt
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M
Smillie
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D000634:Aminoquinolines / Q000378:metabolism; D000818:Animals; D004024:Dicyclohexylcarbodiimide / Q000494:pharmacology; D004591:Electrophoresis, Polyacrylamide Gel; D005456:Fluorescent Dyes / Q000378:metabolism; D017506:H(+)-K(+)-Exchanging ATPase / Q000737:chemistry*; D010446:Peptide Fragments / Q000737:chemistry; D011189:Potassium Chloride / Q000378:metabolism; D011487:Protein Conformation; D012413:Rubidium / Q000378:metabolism; D013270:Stomach / Q000201:enzymology*; D014357:Trypsin / Q000378:metabolism
D000634:Aminoquinolines; D005456:Fluorescent Dyes; D010446:Peptide Fragments; C069657:1-(2-methylphenyl)-4-methylamino-6-methyl-2,3-dihydropyrrolo(3,2-c)quinoline; D004024:Dicyclohexylcarbodiimide; D011189:Potassium Chloride; D014357:Trypsin; D017506:H(+)-K(+)-Exchanging ATPase; D012413:Rubidium