Relation between phosphorylation and adenosine triphosphate-dependent Ca 2+ binding of swine and bovine erythrocyte membranes

Abstract

The correlation between the ATP-dependent Ca 2+ binding and the phosphorylation of the membranes from swine and bovine erythrocytes was studied. The Ca 2+ binding was measured by using 45CaCl 2, and the phosphorylation by [γ- 32P]ATP was studied with the technique of SDS polyacrylamide gel electrophoresis. 200 mM NaCl and KCl markedly repressed the Ca 2+ binding of swine erythrocyte membranes. The radioactivity of 32P-labelled membranes was revealed mainly in 250 000 dalton protein and a lipid fraction. NaCl and KCl also repressed the phosphorylation of the lipid which was identified as triphosphoinositide by paper chromatography. The membranes prepared from trypsin-digested erythrocytes completely retained the Ca 2+-binding activity, and lost 30% of (Ca 2+ + Mg 2+)-ATPase activity. The Ca 2+-binding and ATPase activity of isolated membranes decreased to 55% and to 0%, respectively, by tryptic digestion. Neither the Ca 2+ binding nor the phosphorylation of polyphosphoinositides were detected in bovine erythrocyte membranes. These results suggest that the formation of triphosphoinositide rather than the (Ca 2+ + Mg 2+)-ATPase of membranes is linked to the ATP-dependent Ca 2+ binding of erythrocyte membranes.