Purification and analysis of erythrocyte membrane Ca2+-ATPase from small samples of patient blood: Application to cystic fibrosis

Abstract

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_ca2+) = 15.5 ± 1.2 μmol ATP hydrolysed/mg/min, and reciprocal of apparent affinity (K_ca2⁺) = 0.73 ± 0.15 μM 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_ca2+ and K_ca2+ were not significantly different from normal values.