PURIFICATION AND ANALYSIS OF ERYTHROCYTE-MEMBRANE CA2+-ATPASE FROM SMALL SAMPLES OF PATIENT BLOOD - APPLICATION TO CYSTIC-FIBROSIS

A method is presented for the micro-scale isolation and characterization of erythrocyte membrane Ca2+-ATPase from small samples (7 mL) of whole human blood. Ca2+-ATPase isolated by this technique was more than 92% pure and showed calcium-activation characteristics similar to enzyme purified by standard macroscale procedures-viz maximal velocity of activation (V(Ca)2+) = 15.5 +/- 1.2-mu-mol ATP hydrolysed/mg/min, and reciprocal of apparent affinity (K(Ca)2+) = 0.73 +/- 0.15-mu-M free calcium (mean +/- SEM; n = 9). Using the isolation procedure described, purified Ca2+-ATPase could be prepared and assayed in a single working day. When the calcium-activation kinetics of cystic fibrosis erythrocyte membrane Ca2+-ATPase were reassessed using enzyme purified by this technique, V(Ca)2+ and K(Ca)2+ were not significantly different from normal values.