Purification, characterization, and reconstitution of the Ca2+-transport system (high-affinity Ca2+, Mg2+-ATPase) of the human erythrocyte membrane.
The Ca2+-transport system of human erythrocyte membranes was solubilized by deoxycholate in the presence of the nonionic detergent Tween 20 and was purified by calmodulin affinity chromatography. The method yields a functional enzyme, which as compared with the erythrocyte membrane was purified 207-fold based on specific activity, and about 330-fold based on protein content. The activity of the isolated enzyme can be increased about 9-fold by the addition of calmodulin, resulting in a specific activity of 10.1 mumoles/mg . min at 37 degrees C. Triton X-100 and deoxycholate stimulate the calmodulin-deficient Ca2+-ATPase in a concentration dependent manner, which results in a loss of the calmodulin-sensitivity. The Ca2+-transport ATPase could be reconstituted after solubilization of the ATPase by deoxycholate and controlled dialysis near room temperature. The system was reconstituted to form membraneous vesicles capable of energized Ca2+ accumulation. The membrane vesicles showed a protein to lipid ratio (approx. 60% protein and 40% lipid) similar to that of the original erythrocyte membrane. The stimulation by calmodulin of the calmodulin-depleted membrane-bound and partially purified Ca2+-ATPase is strongly time dependent. At a Ca2+-concentration of 40 microM and low calmodulin concentrations, approx. 120 min are required to regain full activity. This time period is decreased to about 15 min in the presence of a high excess of calmodulin. Vice versa, at fixed concentrations of calmodulin, the time necessary for regain of full activity is decreased as the Ca2+ concentrations is increased. The dependence of the Ca2+-ATPase activity on the calmodulin concentration shows strong deviation from Michaelis-Menten kinetics at Ca2+ concentrations below (4--10 microM) and above (200 microM) the optimum concentration of 40 microM. Mathematical analysis of the results at 200 microM Ca2+ leads to the assumption that 4 calmodulin molecules interact with one oligomer of Ca2+-ATPase consisting of 4 identical subunits.
D001693:Biological Transport, Active / Q000187:drug effects; D017301:Ca(2+) Mg(2+)-ATPase; D002118:Calcium / Q000097:blood*; D000252:Calcium-Transporting ATPases / Q000097:blood* / Q000302:isolation & purification; D003840:Deoxycholic Acid / Q000494:pharmacology; D003902:Detergents / Q000494:pharmacology; D004910:Erythrocyte Membrane / Q000201:enzymology* / Q000648:ultrastructure; D004912:Erythrocytes / Q000201:enzymology*; D006801:Humans; D007700:Kinetics; D008854:Microscopy, Electron; D017830:Octoxynol; D011092:Polyethylene Glycols / Q000494:pharmacology
D003902:Detergents; D003840:Deoxycholic Acid; D011092:Polyethylene Glycols; D017830:Octoxynol; D017301:Ca(2+) Mg(2+)-ATPase; D000252:Calcium-Transporting ATPases; D002118:Calcium