Abstract
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.
Highlights
ATP diphosphohydrolase (EC 3.6.1.5)catalyzesthe a cation-substrate complexwhich is likely to be abetter hydrolysisofdiphospho- and triphosphonucleosides substrate
Considering its sen- membrane proteins were solubilized in 0.2% (w/v) Triton X-100, 20 sitivity to divalent cations and its cellular localization, we have suggested that the ATP diphosphohydrolase might be involvedinsome manner along the route leading tothe secretion of digestive enzymes.We have investigated the role ofCa2’ and Mg2+in the activation of this enzyme
Purification of the ATP Diphosphohydrolase-Purification of the enzyme wasfollowedby measuring the rate of hydrolysis of both ATP and ADP
Summary
ATP diphosphohydrolase (EC 3.6.1.5)catalyzesthe a cation-substrate complexwhich is likely to be abetter hydrolysisofdiphospho- and triphosphonucleosides substrate. These possibilities are not mutually exclusive, and and is sensitive to divalent cations. We in many studies, both binding to theenzyme and to one of the investigated the dependence ofATP hydrolysis on the substrate have been reported [8,9,10,11].In thepresent study, we concentration of freMe&+ and Ca2+ anthde cation ATP showed that free ATP4-, Ca-ATP2-, and Mg-ATP2- are subcomplexes. The enzyme was isolated from porcine zy- strates of the enzyme and that there is no activation of the mogen granule membranes, solubilized in Triton X-1e0n0z,yme by free Ca2’ and Mg2+ and purified on a 5’-AMP-Sephar4oBseaffinity column resulting in a 1500-fold purification.
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