A calcium regulated adenosine triphosphatase in Entamoeba histolytica

Axenically grown trophozoites of Entamoeba histolytica (NIH-200 strain) contain an active ATPase (170 nmol PO4/min per mg protein) with maximal activity at pH 8.8, a high affinity for ATP (Km approximately 40 micro M) and an absolute and specific requirement for Ca2+. The activation by Ca2+ shows positive cooperativity (nH = 2.48) at calcium concentrations below 8 micro M and no cooperativity between 8 and 25 micro M. The latter concentration fully saturates the enzyme. The observed activity is insensitive to oligomycin, ouabain and ruthenium red and is unaffected by a range of inhibitors of electron transport and uncouplers of oxidative phosphorylation. The enzyme exhibits structure bound latency and is tightly bound to cellular membranes. It is sedimentable ( greater than 80%) by high speed centrifugation of cell homogenates which are either protected osmotically or in which subcellular structures are damaged by sonication or treatment with Triton X-100. Arrhenius plots of V in the temperature range of 0-38 degrees C are linear without breaks, similar to other pyrophosphatases of E. histolytica. The calculated activation energy is 14.8 kcal/mol. This finding as well as the failure of phospholipase treatment to affect activity indicate that interactions with membrane lipids play no role in the catalytic function of this tightly membrane-bound ATPase.