cl?// calcium (1995) 17, 14-20 0 Pearson Professional Ltd 1995 Thapsigargin protects human erythrocyte Ca*+-ATPase from proteolysis C.O. BEWAJI and A.P. DAWSON School of Biological Sciences, University of East Anglia, Norwich, UK Abstract - The effect of thapsigargin on the activation by partial proteolysis of the plasma membrane Ca2+-ATPase was studied in intact human erythrocyte membranes and in the purified enzyme. The enzyme was maximally activated in the absence of thapsigargin within 1 min of exposure to trypsin. However, in the presence of thapsigargin maximal activation was achieved only after 5 min trypsin digestion. Thapsigargin did not alter the pattern of proteolysis as revealed by SDS-PAGE of the tryptic fragments, although it slowed down the rate of appearance of the fragments. Thapsigargin also enhanced the activation of the enzyme by calmodulin. These findings suggest that, although thapsigargin at low concentrations has no effect on the catalytic activity of the Ca2+-ATPase in vitro in the absence of calmodulin, it could interfere with its regulation in vivo. A Ca2+-activated, Mg2+-dependent ATPase (Ca2+, Mg2+-ATPase) operates as a calcium pump in the plasma membrane of red cells [l,Z]. This enzyme belongs to the P-class of ATPases and is the largest of all P-type ion pumps [3]. Recent cloning work has confirmed that it is a single polypeptide with a molecular weight of about 134 kD [4]; its kinetic and enzymatic properties are generally believed to be different from those of the sarco-endoplasmic re- ticulum (SERCA) types of Ca*+-ATPases [4,5]. A number of investigators have reported that thapsigargin (TG), a plant-derived sesquiterpene lac- tone, is a potent and specific inhibitor of calcium transport (and Ca2+-ATPase) by the SERCA family of Ca*+ pumps [6-IO] but does not inhibit the plasma membrane Ca*+ pumps (PMCA) or other ion-motive ATPases [IO]. One major difference be- tween the properties of the PMCA and SERCA types of Ca2+-ATPases is that the former is acti- vated by calmodulin, acidic phospholipids and by limited proteolysis [ 111. The calmodulin-binding domain of the erythrocyte Ca2+-ATPase is at the C- terminal region of the enzyme and is cleaved off during proteolysis [ 121. In order to examine further whether or not TG interacts with the plasma membrane Ca2+-ATPase (but without inhibiting its catalytic activity), we have studied the effects of TG on some of the acti- vating treatments of the human erythrocyte Ca*+- ATPase. We report here that the enzyme is resistant to proteolysis in the presence of TG and is hence more slowly activated by this treatment. However, TG enhances the activation of the enzyme by cal- modulin. It therefore seems that this PMCA type 14