Purification and analysis of erythrocyte membrane Ca(2+)-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 Ca(2+)-ATPase from small samples (7 mL) of whole human blood. Ca(2+)-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 (VCA2+) = 15.5 +/- 1.2 mumol ATP hydrolysed/mg/min, and reciprocal of apparent affinity (KCa2+) = 0.73 +/- 0.15 microM free calcium (mean +/- SEM; n = 9). Using the isolation procedure described, purified Ca(2+)-ATPase could be prepared and assayed in a single working day. When the calcium-activation kinetics of cystic fibrosis erythrocyte membrane Ca(2+)-ATPase were reassessed using enzyme purified by this technique, VCa2+ and KCa2+ were not significantly different from normal values.