Electrogenic partial reactions of the gastric H,K-ATPase.
The fluorescent styryl dye RH421 was used to identify and investigate electrogenic reaction steps of the H,K-ATPase pump cycle. Equilibrium titration experiments were performed with membrane vesicles isolated from hog gastric mucosa, and cytoplasmic and luminal binding of K(+) and H(+) ions was studied. It was found that the binding and release steps of both ion species in both principal conformations of the ion pump, E(1) and P-E(2), are electrogenic, whereas the conformation transitions do not contribute significantly to a charge movement within the membrane dielectric. This behavior is in agreement with the transport mechanism found for the Na,K-ATPase and the sarcoplasmic reticulum Ca-ATPase. The data were analyzed on the basis of the Post-Albers reaction cycle. For proton binding, two pK values were found in both conformations: 6.7 and </=4.5 in the E(1) conformation; 6.7 and </=2 in the P-E(2) conformation. The equilibrium dissociation constants for K(+) binding on the cytoplasmic side were 11 and 16 mM. The respective equilibrium dissociation constants on the luminal side were obtained via K(+) concentration dependence of the enzyme activity and determined to be 0.11 mM for both luminal binding sites.
Diller
Anna
A
Department of Biology, University of Konstanz, 78457 Konstanz, Germany.
Vagin
Olga
O
Sachs
George
G
Apell
Hans-Jürgen
HJ
D000255:Adenosine Triphosphate / Q000737:chemistry; D000818:Animals; D001665:Binding Sites; D001703:Biophysics / Q000379:methods*; D000252:Calcium-Transporting ATPases / Q000737:chemistry; D003593:Cytoplasm / Q000378:metabolism; D005456:Fluorescent Dyes / Q000494:pharmacology; D017506:H(+)-K(+)-Exchanging ATPase / Q000737:chemistry*; D006863:Hydrogen-Ion Concentration; D006868:Hydrolysis; D007477:Ions / Q000737:chemistry; D007700:Kinetics; D008024:Ligands; D008954:Models, Biological; D008956:Models, Chemical; D010766:Phosphorylation; D011188:Potassium / Q000737:chemistry; D011189:Potassium Chloride / Q000737:chemistry; D011522:Protons; D011726:Pyridinium Compounds / Q000494:pharmacology; D012519:Sarcoplasmic Reticulum / Q000201:enzymology; D013050:Spectrometry, Fluorescence; D013270:Stomach / Q000201:enzymology*; D013343:Styrenes / Q000494:pharmacology; D013552:Swine; D013997:Time Factors
D005456:Fluorescent Dyes; D007477:Ions; D008024:Ligands; D011522:Protons; D011726:Pyridinium Compounds; D013343:Styrenes; C070534:RH 421; D011189:Potassium Chloride; D000255:Adenosine Triphosphate; D017506:H(+)-K(+)-Exchanging ATPase; D000252:Calcium-Transporting ATPases; D011188:Potassium