Kinetics of the acid pump in the stomach. Proton transport and hydrolysis of ATP and p-nitrophenyl phosphate by the gastric H,K-ATPase.
Hydrolysis of adenosine 5'-triphosphate (ATP) and p-nitrophenyl phosphate by the hydrogen ion-transporting potassium-stimulated adenosine triphosphatase (H,K-ATPase) was investigated. Hydrolysis of ATP was studied at pH 7.4 in vesicles treated with the ionophore nigericin. The kinetic analysis showed negative cooperativity with one high affinity (Km1 = 3 microM) and one low affinity (Km2 = 208 microM) site for ATP. The rate of hydrolysis decreased at 2000 microM ATP indicating a third site for ATP. When the pH was decreased to 6.5 the experimental results followed Michaelis-Menten enzyme kinetics with one low affinity site (Km = 116 microM). Higher concentrations than 750 microM ATP were inhibitory. Proton transport was measured as accumulation of acridine orange in vesicles equilibrated with 150 mM KCl. The transport at various concentrations of ATP in the pH interval from 6.0 to 8.0 correlated well with the Hill equation with a Hill coefficient between 1.5-1.9. The concentration of ATP resulting in half-maximal transport rate (S0.5) increased from 5 microM at pH 6.0 to 420 microM at pH 8.0. At acidic pH the rate of proton transport decreased at 1000 microM ATP. The K+-stimulated p-nitrophenylphosphatase (pNPPase) activity resulted in a Hill coefficient close to 2 indicating cooperative binding of substrate. The pNPPase was noncompetitively inhibited by ATP and ADP; half-maximal inhibition was obtained at 2 and 100 microM, respectively. Phospholipase C-treated vesicles lost 80% of the pNPPase activity, but the Hill coefficient did not change. These kinetic results are used for a further development of the reaction scheme of the H,K-ATPase.
Ljungström
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M
Mårdh
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D000251:Adenosine Triphosphatases / Q000378:metabolism*; D000255:Adenosine Triphosphate / Q000378:metabolism*; D000818:Animals; D001693:Biological Transport, Active; D005753:Gastric Mucosa / Q000201:enzymology*; D017506:H(+)-K(+)-Exchanging ATPase; D006868:Hydrolysis; D007700:Kinetics; D008433:Mathematics; D009596:Nitrophenols / Q000378:metabolism*; D009943:Organophosphorus Compounds / Q000378:metabolism*; D011522:Protons; D013552:Swine
D009596:Nitrophenols; D009943:Organophosphorus Compounds; D011522:Protons; C008644:nitrophenylphosphate; D000255:Adenosine Triphosphate; D000251:Adenosine Triphosphatases; D017506:H(+)-K(+)-Exchanging ATPase