Kinetic effects of Ca2+ and Mg2+ on ATP hydrolysis by the purified ATP diphosphohydrolase.
ATP diphosphohydrolase (EC 3.6.1.5) catalyzes the hydrolysis of diphospho- and triphosphonucleosides and is sensitive to divalent cations. In this paper, we investigated the dependence of ATP hydrolysis on the concentration of free Mg2+ and Ca2+ and the cation ATP complexes. The enzyme was isolated from porcine zymogen granule membranes, solubilized in Triton X-100, and purified on a 5'-AMP-Sepharose 4B affinity column resulting in a 1500-fold purification. Free unprotonated ATP4- was hydrolyzed in the presence of ethylene glycol bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid. When hydrolysis rate was measured at different concentrations of the cation-ATP complex at constant free cation concentrations, normal hyperbolic curves were obtained. In CaCl2, both Kapp and Vapp increased as free Ca2+ increased from 25 to 1000 microM. In MgCl2, Kapp increased and Vapp decreased as free Mg2+ increased from 25 to 500 microM. From the rapid equilibrium rate equation, Ks and Vmax values of the substrates were calculated. We found that free ATP4-, Ca-ATP2-, and Mg-ATP2- are substrates and free cations do not bind the enzyme.
D000255:Adenosine Triphosphate; D000818:Animals; D001081:Apyrase / Q000302:isolation & purification / Q000378:metabolism*; D002118:Calcium / Q000494:pharmacology*; D007700:Kinetics; D008274:Magnesium / Q000494:pharmacology*; D008433:Mathematics; D010179:Pancreas / Q000201:enzymology; D010744:Phosphoric Monoester Hydrolases / Q000378:metabolism*; D013552:Swine
D000255:Adenosine Triphosphate; D010744:Phosphoric Monoester Hydrolases; D001081:Apyrase; D008274:Magnesium; D002118:Calcium