Identification of an extracytoplasmic region of H+,K(+)-ATPase labeled by a K(+)-competitive photoaffinity inhibitor.
The photoaffinity reagent 8-[(4-azidophenyl)-methoxy]-1-tritiomethyl-2, 3-dimethylimidazo-[1,2-alpha]pyridinium iodide ([3H]mDAZIP) has been synthesized and used to photoinactivate and label purified hog gastric H+,K(+)-ATPase. The specific (K(+)-sensitive) components of both photoinactivation and labeling showed dependences on inhibitor concentration consistent with covalent modification at an extracytoplasmic site of reversible K(+)-competitive binding in the dark. The maximum amount of specific labeling (1.2 nmol/mg) was similar to the number of phosphorylation sites measured (1.0 +/- 0.14 nmol/mg). Specific labeling was distributed 76% on the alpha chain, 18% on the beta chain, and 6% on undefined peptides. Various digestions with trypsin, protease V8, and thermolysin were employed to fragment the labeled enzyme. Gasphase sequencing of the radioactive peptides identified the major site of specific labeling to be within a region where only two stretches of amino acids (Leu105 to Ile126 and Leu139 to Phe155, designated H1 and H2, respectively) are predicted to span the membrane. This in turn suggested that the labeling site was located within or close to the proposed loop between them (Gln127 to Asn138). A computer-driven energy minimization protocol yielded a loop structure to which SCH 28080 (the parent structure of [3H]mDAZIP) could be docked. Conversely, modeling of the corresponding region of Na+,K(+)-ATPase (a homologous enzyme with much lower affinity for SCH 28080) yielded no apparent binding site. Similarities in the inhibition of H+,K(+)-ATPase by SCH 28080 and of Na+,K(+)-ATPase by ouabain lead to the hypothesis that, in each case, inhibitor binding to E2-P is associated with an increase in the hydrophobicity of the environment of the loop between H1 and H2.
Munson
K B
KB
Veterans Administration Medical Center, West Los Angeles, California 90072.
Gutierrez
C
C
Balaji
V N
VN
Ramnarayan
K
K
Sachs
G
G
D000251:Adenosine Triphosphatases / Q000037:antagonists & inhibitors / Q000737:chemistry*; D000345:Affinity Labels / Q000737:chemistry; D000595:Amino Acid Sequence; D001386:Azides / Q000378:metabolism; D001667:Binding, Competitive; D002850:Chromatography, Gel; D003198:Computer Simulation; D004591:Electrophoresis, Polyacrylamide Gel; D017506:H(+)-K(+)-Exchanging ATPase; D007093:Imidazoles / Q000378:metabolism; D008958:Models, Molecular; D008968:Molecular Conformation; D008969:Molecular Sequence Data; D010777:Photochemistry
D000345:Affinity Labels; D001386:Azides; D007093:Imidazoles; C071187:8-((4-azidophenyl)methoxy)-1,2,3-trimethylimidazo(1,2-a)pyridinium; D000251:Adenosine Triphosphatases; D017506:H(+)-K(+)-Exchanging ATPase