Ca(2+)-ATPase from chicken (Gallus domesticus) erythrocyte plasma membrane: effects of calmodulin and taurine on the Ca(2+)-dependent ATPase activity and Ca2+ uptake.
In the present report we describe a method for purifying plasma membranes from chicken erythrocytes using sonication under conditions that facilitate preferential lysis of plasma membrane, followed by centrifugation through a sucrose gradient. The Ca(2+)-dependent ATP hydrolysis by plasma membranes is activated by nanomolar levels of calmodulin, similarly to that from anucleated erythrocytes. Inside-out vesicles display a calmodulin-activated Ca2+ uptake. Purified Ca(2+)-ATPase is obtained from the plasma membrane by Sepharose-calmodulin affinity chromatography, and exhibits an apparent molecular mass of 150 kDa on SDS-polyacrylamide gel electrophoresis, clearly showing that the enzyme is distinct from that described in anucleated erythrocytes (140 kDa). The enzyme is insensitive to physiological concentrations of taurine, a beta-amino acid that has been proposed to be involved in Ca2+ homeostasis of nucleated erythrocytes, suggesting that the effect of taurine is not mediated by the Ca(2+)-ATPase. Taken together, these data suggest that the enzyme may be an isoform that resembles the previously described plasma membrane Ca(2+)-ATPase from anucleated erythrocytes in its regulation by calmodulin, but differs in its apparent molecular weight.
Alves-Ferreira
M
M
Departamento de Bioquimica Médica-ICB/CCS, Universidade Federal do Rio de Janeiro, Brazil.
Scofano
H M
HM
Ferreira-Pereira
A
A
D000255:Adenosine Triphosphate / Q000378:metabolism; D000408:Alamethicin / Q000494:pharmacology; D000818:Animals; D002118:Calcium / Q000378:metabolism*; D000252:Calcium-Transporting ATPases / Q000037:antagonists & inhibitors / Q000187:drug effects* / Q000378:metabolism; D002147:Calmodulin / Q000378:metabolism / Q000494:pharmacology*; D002645:Chickens; D002853:Chromatography, Liquid; D004591:Electrophoresis, Polyacrylamide Gel / Q000379:methods; D004791:Enzyme Inhibitors / Q000494:pharmacology; D004910:Erythrocyte Membrane / Q000187:drug effects / Q000378:metabolism*; D005260:Female; D008297:Male; D012995:Solubility; D013654:Taurine / Q000378:metabolism / Q000494:pharmacology*; D019284:Thapsigargin / Q000494:pharmacology
D002147:Calmodulin; D004791:Enzyme Inhibitors; D013654:Taurine; D000408:Alamethicin; D019284:Thapsigargin; D000255:Adenosine Triphosphate; D000252:Calcium-Transporting ATPases; D002118:Calcium