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A Mg²⁺- and Ca²⁺-stimulated Adenosine Triphosphatase at the Outer Surface of Ehrlich Ascites Tumor Cells¹

Institute of Medical and Physiological Chemistry, The University of Uppsala, Biomedical Center, Box 575, 751 23 Uppsala, Sweden

A Mg²⁺- and Ca²⁺-stimulated adenosine triphosphatase (ATPase) at the outer surface of intact Ehrlich ascites tumor cells is described.

A surface-bound adenosine triphosphate (ATP)-splitting activity at a lower rate was also demonstrated in the absence of Ca²⁺ but with Mg²⁺, Na⁺, and K⁺ present in the isotonic medium. Hence, when part of the Mg²⁺ was exchanged for Ca²⁺, a marked increase of the ATP-splitting activity was observed. The stimulatory effect of Ca²⁺ was seen only if both Na⁺ and K⁺ were present in the isotonic incubation medium.

Thus, the enzyme activity was Mg²⁺- and Ca²⁺-dependent. Ca²⁺, together with the monovalent cations was inhibitory compared with Mg²⁺ under similar conditions.

The apparent K_m for ATP for the Mg²⁺-stimulated ATPase is 0.05 mM, while that of the Mg²⁺- and Ca²⁺-stimulated enzyme is 0.10 mM. The V_max of the former is 0.8 µmole per 100 mg Schneider protein per 30 sec compared with 1.92 µmoles per 100 mg Schneider protein per 30 sec for the latter. The calculated K_m for the Mg²⁺- and Ca²⁺-stimulated ATPase after subtraction of the Mg²⁺-stimulated part is 0.22 mM.

Ethacrynic acid and N-ethylmaleimide both inhibited the Mg²⁺- and Ca²⁺-stimulated ATPase by about 10%, while the ouabain inhibition was 15%. Cytochalasin B did not influence the enzyme activity, whereas La³⁺ had a slight stimulatory effect.

¹ This investigation was supported by a grant from the Swedish Medical Research Council (Project B74-13X-228-OC).

Received 9/20/74. Accepted 2/28/75.