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+.
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