Inhibition of membrane erythrocyte (Ca2+ + Mg2+)-ATPase by hemin.
Red blood cell lysis is a common symptom following severe or prolonged oxidative stress. Oxidative processes occur commonly in sickle cells, probably mediated through denatured hemoglobin and the accumulation of ferric hemes in the membranes. Calmodulin-stimulated (Ca2+ + Mg2+)-ATPase from sickle red cell membranes is partially inactivated (Leclerc et al. (1987) Biochim. Biophys. Acta 897, 33-40). In this study (Ca2+ + Mg2+)-ATPase activity from normal adult erythrocyte membranes was measured in the presence of hemin. We report a time- and concentration-dependent inhibition of the activity of the enzyme by hemin due to a decrease in the maximum velocity. Only a mild inhibitory effect was observed in the presence of iron-free protoporphyrin IX, indicating the catalytic influence of the iron. Experiments carried out with hemin (ferric iron) liganded with imidazole or with reduced protoheme (ferrous iron) liganded with carbon monoxide, demonstrated that the inhibition requires that hemin be capable of binding additional ligands. The inhibition was not influenced by the absence of oxygen but was prevented by addition of bovine serum albumin. Addition of butylated hydroxytoluene, a protective agent of lipid peroxidation, failed to prevent the inhibition of calmodulin-stimulated (Ca2+ + Mg2+)-ATPase. As dithiothreitol partially restores the enzyme activity, we postulated that hemin interacts with the thiol groups of the enzyme.
Leclerc
L
L
INSERM U 299, Hôpital de Bicêtre, Le Kremlin-Bicêtre, France.
Vasseur
C
C
Bursaux
E
E
Marden
M
M
Poyart
C
C
D017301:Ca(2+) Mg(2+)-ATPase / Q000037:antagonists & inhibitors / Q000097:blood*; D000252:Calcium-Transporting ATPases / Q000037:antagonists & inhibitors / Q000097:blood*; D002147:Calmodulin / Q000494:pharmacology; D004229:Dithiothreitol / Q000494:pharmacology; D004910:Erythrocyte Membrane / Q000187:drug effects / Q000201:enzymology*; D006418:Heme / Q000031:analogs & derivatives*; D006427:Hemin / Q000494:pharmacology*; D006801:Humans
D002147:Calmodulin; D006418:Heme; D006427:Hemin; D017301:Ca(2+) Mg(2+)-ATPase; D000252:Calcium-Transporting ATPases; D004229:Dithiothreitol