Membrane-bound Adenosine Triphosphatase of Escherichia coliI. Partial Purification and Properties

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Abstract
A simple procedure was developed for releasing membrane-bound adenosine triphosphatase [EC 3. 6. 1. 3] from the membrane fraction of Escherichia coli. This involved washing the fraction with dilute buffer solution. The partial purification of the enzyme is described. The purified enzyme was nearly homogeneous as judged by gel electrophoresis on polyacrylamide and histochemical examination of the activity on the gel. The molecular weight of the enzyme was estimated to be more than 400,000. The enzyme is cold-labile but can be stabilized by keeping it in buffer solution containing 20% methanol or 50% glycerol. It has a pH optimum at 9.5 and requires magnesium ions, whose optimal molar ratio to ATP is 2: 5. Sodium ions slightly activate the enzyme and a combination of sodium and potassium ions is inhibitory. The enzyme hydrolyzes ATP stoichiometrically to ADP and inorganic phosphate. The only other substrate of the enzyme was GTP. The Km for ATP hydrolysis was 6.0×10−4 M. ADP competitively inhibited the enzyme with a Ki value of 3.0×10−4 M. The enzyme activity was strongly inhibited by sodium azide. Oligomycin, dinitrophenol or ouabain showed no significant effect on the activity.

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