Interaction of calcium and magnesium in activating and inhibiting the nucleoside triphosphatase of sarcoplasmic reticulum vesicles

Abstract

The dependence of ATP and ITP hydrolysis on the concentration of the free Mg + and Ca 2+ and of the metal · nucleotide complexes was investigated, using sarcoplasmic reticulum vesicles rendered leaky by treatment with ethyleneglycol-bis(aminoethyl)-tetraacetic acid at pH 9.5 or with diethylether. The Mg 2+ · ATP complex is the true substrate of the reaction ( K m = 18.5 μM for Mg 2+ · ATP concentrations higher than 6 μM), while the Ca 2+ · ATP complex is a potent competitive inhibitor ( K i = 2 μM ). At a fixed Mg 2+ μ ATP concentration, the optimal concentration of Ca 2+ varies with the Mg 2+ concentration. The inhibition in the presence of excess Ca 2+ is caused by the Ca 2+ · ATP complex. ITP hydrolysis, however, is not inhibited in the presence of excess Ca 2+ (up to 1 mM), the Ca 2+ · ITP complex not being inhibitory. At low Ca 2+ concentrations, in conditions which do not permit the formation of inhibitory levels of Ca 2+ · ATP, hydrolysis of either nucleotide is activated by Ca 2+, showing a sigmoidal dependence on Ca 2+ concentration. The apparent K m for Ca 2+ is not influenced by the concentration of Mg 2+ · ATP or Mg 2+ · ITP, and is practically the same in presence of either nucleotide (0.23–0.28 μM). Mg 2+ is a competitive inhibitor with respect to Ca 2+.