Modulation of an intracellular calmodulin-stimulated Ca2+-pumping ATPase in cauliflower by trypsin: The use of calcium green-5N to measure Ca2+ transport in membrane vesicles

Abstract

The effect of controlled trypsin digestion of a calmodulin-stimulated Ca2+-ATPase in low-density intracellular membranes from cauliflower (Brassica oleracea L.) inflorescences was investigated. Ca2+ uptake into vesicles was measured either continuously with the fluorescent Ca2+ indicator Calcium Green-5N or with a radioactive filter technique. Trypsin treatment of vesicles resulted in a 3-fold activation of Ca2+ uptake and loss of calmodulin sensitivity, Immunoblotting experiments with an antiserum raised against the Ca2+-ATPaSe showed that the trypsin activation was accompanied by a decrease in the amount of intact Ca2+-ATPase (111 kD) and by successive appearances of polypeptides of 102 and 99 to 84 kD. 125I-Calmodulin overlays showed that only the intact Ca2+-ATPase bound calmodulin. Removal of the calmodulin-binding domain (about 9 kD) was not enough to obtain full activation. Trypsin proteolysis resulted in a Ca2+ concentration necessary for half-maximal activity of 0.5 μM, whereas a value of about 2 μM was obtained with untreated membranes in the presence of calmodulin. Without trypsin treatment or calmodulin the activity was not saturated even at 57 μM free Ca2+. The data suggest that trypsin digestion and calmodulin activate the cauliflower Ca2+-ATPase by at least partly different mechanisms.