Isolation and characterization of erythrocyte membrane Ca2+-ATPase in cystic fibrosis.
Ca2+-ATPase was purified from erythrocyte membranes prepared from cystic fibrosis (CF) blood samples (n = 10) and from age/sex-matched control blood samples (n = 10). The kinetics of calcium activation of the purified enzyme was investigated in the presence of asolectin phospholipids and found to be virtually identical for both CF and control preparations: VCa2+ = 3.01 +/- 0.24 mumol ATP hydrolyzed/mg pure enzyme/min (mean +/- SE) and 3.09 +/- 0.20 for CF and control Ca2+-ATPase, respectively; KCa2+ = 0.328 +/- 0.046 mu molar free calcium and 0.333 +/- 0.040 for CF and control enzyme, respectively. The preparative procedure used (one-step purification by calmodulin-affinity chromatography) allowed quantitative recovery of essentially 100% of the Ca2+-ATPase present in detergent-solubilized erythrocyte membranes, enabling expression of the yield of purified enzyme in terms of the quantity of starting membrane protein: 0.127% +/- 0.006% (w/w) and 0.140% +/- 0.007% for CF and control enzyme preparations, respectively. None of the parameters evaluated showed a statistically significant difference (p less than 0.05) between the CF and control groups. Furthermore, when CF and control purified Ca2+-ATPase samples were analyzed by high-resolution gradient SDS-polyacrylamide gel electrophoresis, no differences in mobility were observed (mol wt = 128 kdaltons). Thus, Ca2+-ATPase purified from CF erythrocyte membranes and assayed in the presence of asolectin appears to be quantitatively similar to control purified enzyme in amount, molecular weight, and kinetics of activation by calcium. These data suggest that Ca2+-ATPase may not be the defective gene product in CF.(ABSTRACT TRUNCATED AT 250 WORDS)
D000328:Adult; D002118:Calcium / Q000494:pharmacology; D000252:Calcium-Transporting ATPases / Q000097:blood* / Q000302:isolation & purification; D003550:Cystic Fibrosis / Q000201:enzymology*; D004789:Enzyme Activation / Q000187:drug effects; D004910:Erythrocyte Membrane / Q000201:enzymology*; D005260:Female; D006801:Humans; D066298:In Vitro Techniques; D007700:Kinetics; D008297:Male; D008970:Molecular Weight
D000252:Calcium-Transporting ATPases; D002118:Calcium