Studies on the Active Transport of Calcium in Human Red Cells

Abstract

The Ca ++ transport mechanism in the red cell membrane was studied in resealed ghost cells. It was found that the red cell membrane can transport Ca ++ from inside the cell into the medium against great concentration gradient ratios. Tracing the movement of 45Ca infused inside red cells indicated that over 95 % of all Ca ++ in the cells was transported into media in 20 min incubation under the opdmum experimental conditions. The influence of temperature on the rate constant of transport indicated an activation energy of 13,500 cal per mole. The optimum pH range of media for the transport was between 7.5 and 8.5. As energy sources, ATP, 1 CTP, and U T P were about equally effective, G T P somewhat less effective, and ITP least effective among the nucleotides tested. The Ca ++ transport does not appear to involve exchange of Ca ++ with any monovalent or divalent cations. Also, it is not influenced by oligomycin, sodium azide, or ouabain in high concentrations, which inhibit the Ca ++ transport in mitochondria or in sarcoplasmic reticulum. In these respects, the Ca ++ transport mechanism in the red cell membrane is different from those of mitochondria and the sarcoplasmic reticulum.T h e energy-linked transport of Ca ++ in phosphorylating m i t o c h o n d r i a and in the sarcoplasmic r e t i c u l u m has been well-established d u r i n g the past d e c a d e (Brierley, 1963;Brierley et al., 1964; E n g s t r o m and D e Luca, 1964; D e L u c a and Engstrom, 1961;Chance, 1965;Hasselbach, 1964; Hasselbach a n d Makinose, 1961; Ebashi and L i p m a n n , 1962; Lee, 1965; Lee et al., 1966). Since the free Ca ++ concentration gradient between extra-and intracellular fluid is e x t r e m e l y large in tissues such as muscle and nerve, the existence of a similar Ca ++ p u m p m e c h a n i s m in the cell m e m b r a n e which is responsible for Ca ++ extrusion f r o m these cells against an electrochemical gradient has been suggested ( R e u t e r and Seitz, 1968;Baker et al., 1969). H o w e v e r , no direct evidence was available for an active, o u t w a r d Ca ++ transx ATP, adenosinetriphosphate; CTP, cytidine triphosphate; ITP, inosine triphosphate; GTP, guanosine triphosphate; UTP, uridine triphosphatc.

Phosphorylation of the red blood cell membrane during the active transport of C++.

“…Previously, it was found that when red blood cells are fragmented, red blood cell membrane fragments (RBCMF) were found to take up Ca ++ and this Ca ++ uptake is accompanied by ATP hydrolysis by Mg++-activated ATPase (Lee and Shin, 1969;Cha et al 1971). It has been suggested that this Ca ++ uptake is due to the presence of inside out vesicles in the present system although not all of the vesicles are inside out (Steck et al, 1970;Weiner and Lee, 1972).…”

“…It has been established that Na+-K+-activated ATPase and Ca++-activated ATPase are intimately associated with outward extrusion of Na + and Ca 2+, respectively (Skou, 1957 and1965;Hoffman, 1961;Schatzmann, 1966;Albers, 1967;Post et al, 1969;Schatzmann and Vincenzi, 1969;Lee and Shin, 1969). Since the present RBCMF has both of the above two ATPase activities, an attempt has been made to investigate the similarity of these two active transport systems in molecular mechanisms.…”

“…It has been suggested that this Ca ++ uptake is due to the presence of inside out vesicles in the present system although not all of the vesicles are inside out (Steck et al, 1970;Weiner and Lee, 1972). RBCMF were found to have both Na+-K+-ATPase and Ca++-ATPase which are intimately associated with the sodium and calcium transport mechanisms, respectively (Schatzmann, 1966;Skou, 1965;Lee and Shin, 1969).…”

“…It has been shown previously that the red blood cell membrane has an active transport mechanism for outward extrusion of Ca ++ (Schatzmann and Vincenzi, 1969;Lee and Shin, 1969), and ATP is the specific energy source of the Ca ++ transport system (Vincenzi and Schatzmann, 1967;Cha et al, 1971).…”

Active Uptake of Ca++ and Ca++-Activated Mg++ ATPase in Red Cell Membrane Fragments

“…It has been previously shown that the red cell membrane has an active Ca++ extrusion mechanism (Schatzmann, 1966;Schatzmann and Vincenzi, 1969;Lee and Shin, 1969). Also, it was found that the fragmented red cell membrane splits ATP in the presence of Mg + + and this Mg++ ATPase activity is greatly stimulated by Ca + + .…”