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.
Bridges
M A
MA
Division of Clinical Pharmacy, Faculty of Pharmaceutical Sciences, University of British Columbia, Vancouver, Canada.
Katz
S
S
D000328:Adult; D002118:Calcium / Q000378:metabolism; D000252:Calcium-Transporting ATPases / Q000032:analysis / Q000302:isolation & purification*; D002147:Calmodulin / Q000494:pharmacology; D002846:Chromatography, Affinity; D003550:Cystic Fibrosis / Q000097:blood*; D003902:Detergents; D004591:Electrophoresis, Polyacrylamide Gel; D004789:Enzyme Activation / Q000187:drug effects; D004910:Erythrocyte Membrane / Q000201:enzymology*; D006801:Humans; D007700:Kinetics; D008846:Micromanipulation / Q000379:methods*; D010743:Phospholipids / Q000494:pharmacology
D002147:Calmodulin; D003902:Detergents; D010743:Phospholipids; D000252:Calcium-Transporting ATPases; D002118:Calcium