Abstract
The H+,K+-ATPase of intact gastric vesicles has two Km values for ATP hydrolysis, 7 and 80 microM. Irradiation of vesicles with ultraviolet light in the presence of 1 mM ATP resulted in K+-ATPase activity that shows only the low affinity ATP binding. The irradiation stimulated or inhibited proton uptake rate compared with control vesicles at high or low ATP concentrations, respectively. The relation between proton uptake rate and K+-ATPase activity at different ATP concentrations was linear with irradiated vesicles and nonlinear with control vesicles. These results indicate that hydrolysis at the high affinity ATP binding site regulates the energy-transport coupling in negative and positive manners at high and low ATP concentrations, respectively. The complete inhibition of K+-ATPase by a specific proton pump inhibitor E3810 (rabeprazole) (2-([4-(3-methoxypropoxy)-3-methylpyridin-2-yl]methylsulf i nyl)-1H-benzimidazole sodium salt) occurred when E3810 bound to half of the alpha-subunit of H+,K+-ATPase in unirradiated vesicles at both 200 and 10 microM ATP, whereas the complete inhibition of proton uptake occurred when E3810 bound to half or a quarter of the alpha-subunit at 200 or 10 microM ATP, respectively. These results suggest that dimeric interaction between the alpha-subunits is necessary for the enzyme activity at all ATP concentrations and that dimeric or tetrameric interaction is necessary for proton transport at high or low ATP concentrations, respectively.
Highlights
The Hϩ,Kϩ-ATPase is involved in the final step of gastric acid secretion and secretes Hϩ in exchange for Kϩ [1,2,3,4,5,6]
We studied the biochemical basis of two Km values for ATP hydrolysis by gastric Hϩ,Kϩ-ATPase and found that ATP hydrolysis at the high affinity site regulated proton uptake with negative and positive cooperativity at high and low ATP concentrations, respectively
From measurements of the relations between ATP hydrolysis, proton transport rate and the amount of specific binding of a proton pump inhibitor E3810,1 we found that at high ATP concentrations the functions of ATP hydrolysis and proton transport require the dimeric interaction between ␣-subunits, but at low ATP concentrations, the function of ATP hydrolysis requires the dimeric interaction, whereas that of proton uptake requires the tetrameric interaction
Summary
The Hϩ,Kϩ-ATPase is involved in the final step of gastric acid secretion and secretes Hϩ in exchange for Kϩ [1,2,3,4,5,6]. Several different explanations are possible for the presence of high and low Km values [9]. We studied the biochemical basis of two Km values for ATP hydrolysis by gastric Hϩ,Kϩ-ATPase and found that ATP hydrolysis at the high affinity site regulated proton uptake with negative and positive cooperativity at high and low ATP concentrations, respectively. From measurements of the relations between ATP hydrolysis, proton transport rate and the amount of specific binding of a proton pump inhibitor E3810,1 we found that at high ATP concentrations the functions of ATP hydrolysis and proton transport require the dimeric interaction between ␣-subunits, but at low ATP concentrations, the function of ATP hydrolysis requires the dimeric interaction, whereas that of proton uptake requires the tetrameric interaction
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