Proton countertransport by the reconstituted erythrocyte Ca2+-translocating ATPase: evidence using ionophoretic compounds.
Human erythrocyte Ca2+-translocating ATPase was solubilized from calmodulin-depleted membranes using the detergent Triton X-100, and subsequently purified by calmodulin-affinity chromatography. The purified enzyme was reconstituted in artificial phospholipid vesicles using a cholate-dialysis method and various phospholipids. The reconstituted enzyme was able to translocate Ca2+ inside the vesicles, both in the absence and in the presence of the Ca2+-chelating agent, oxalate, inside the vesicles. The tightness of coupling between ATP hydrolysis and cation translocation was investigated by the use of different ionophoretic compounds. The efficiency of Ca2+ translocation was measured by the ability of the ionophores to stimulate ATP hydrolytic activity of the reconstituted enzyme. It was found that the maximum stimulation of the ATP hydrolytic activity was induced by the electroneutral Ca2+/2H+ ionophore A23187 (9 to 10-fold). A Ca2+ ionophore unable to translocate H+, CYCLEX-2E, was less efficient in stimulating the activity of the reconstituted enzyme (two- to threefold). However, the combined addition of CYCLEX-2E plus protonophores further increased the ATP hydrolytic activity (around fourfold), whereas, the protonophores did not further stimulate ATP hydrolysis in the presence of A23187. Furthermore, in the absence of Ca2+ ionophore, the electroneutral K+(Na+)/H+ ionophoretic exchanger, monensin, stimulated the rate of ATP hydrolysis in the reconstituted enzyme two- or threefold, respectively. These results suggest that the Ca2+-ATPase not only translocates Ca2+ but also H+ in the opposite direction.
D000001:Calcimycin / Q000494:pharmacology; D000252:Calcium-Transporting ATPases / Q000097:blood*; D004910:Erythrocyte Membrane / Q000201:enzymology*; D006801:Humans; D007476:Ionophores / Q000494:pharmacology; D007700:Kinetics; D008081:Liposomes; D010713:Phosphatidylcholines / Q000494:pharmacology; D010743:Phospholipids / Q000494:pharmacology; D011522:Protons
D007476:Ionophores; D008081:Liposomes; D010713:Phosphatidylcholines; D010743:Phospholipids; D011522:Protons; D000001:Calcimycin; C024990:asolectin; D000252:Calcium-Transporting ATPases