Active Calcium Ion Uptake by Inside-Out and Right Side-Out Vesicles of Red Blood Cell Membranes

Abstract

The relationship between active extrusion of Ca++ from red cell ghosts and active uptake of Ca++ by isolated red cell membrane fragments was investigated by studying the Ca++ uptake activities of inside-out and right side-out vesicles. Preparations A and B which had mainly inside-out and right side-out vesicles, respectively, were isolated from red cell membranes and were compared with respect to Ca++ adenosine triphosphatase (ATPase) and ATP-dependent Ca++ uptake activities. Preparation A had nearly eight times more inside-out vesicles and took up eight times more 45Ca in the presence of ATP compared to preparation B. Separation of the 45Ca-labeled membrane vesicles by density gradient centrifugation showed that the 45Ca label was localized to the inside-out vesicle fraction. In addition, the 45Ca taken up in the presence of ATP was lost during a subsequent incubation in the absence of ATP. The rate of 45Ca loss was not influenced by the presence of EGTA, but was slowed in the presence of La+8 (0.1 mM) in the efflux medium. The results presented here support the thesis that the active uptake of Ca++ by red cell membrane fragments is due to the active transport of Ca++ into inside-out vesicles.