Heterotropic “Macroeffector” for Cooperative Behavior of Ca2+-Adenosine Triphosphatase of Escherichia coli

The allosteric behavior of Ca2+-ATPase from Escherichia coli was studied in both soluble and membrane-bound states. At 36° the membrane-bound enzyme presents cooperativity toward two ligands: inhibitor and substrate. A break in the Hill plot for inhibition by Na+ appears at relatively high concentrations of Na+ (60 mm), with values of of 2.1 at lower Na+ and of 0.7 at higher concentrations. In the substrate saturation curves a break is also present with slopes of 0.8 and 2.0 under and over the transition. When the enzyme is released from the membrane, linear plots are obtained over the whole range with n of 1.0 for both inhibitor and substrate. If the temperature of assay is shifted to 19°, opposite effects are encountered: linear plots indicating a lack of cooperativity observed for both effectors in the membranebound enzyme. On the other hand, breaks are present in the case of the soluble enzyme with values of n of 1.8 and 0.8 for inhibitor and of 0.9 and 1.8 for substrate under and over the break point, respectively. The allosteric behavior inherent to the membrane-bound enzyme at both temperatures is regained when the membrane is reconstituted. The membrane reconstitution was carried out by passage through a column of Sephadex G-25 equilibrated with 1 mm Ca2+. It is clear from these experiments that the membrane acts as an macroeffector in both Na+ inhibition and substrate activation at 36 and 19°. At 36°, the membrane enhances cooperativity shown by the soluble enzyme; at 19° the opposite holds true.