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

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 (45)Ca in the presence of ATP compared to preparation B. Separation of the (45)Ca-labeled membrane vesicles by density gradient centrifugation showed that the (45)Ca label was localized to the inside-out vesicle fraction. In addition, the (45)Ca taken up in the presence of ATP was lost during a subsequent incubation in the absence of ATP. The rate of (45)Ca 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.