Irreversible modification of red cell Ca2+ transport by phenylglyoxal.
Phenylglyoxal, a dicarbonyl modifier of arginyl residues with a high selectivity for anion substrate binding sites in active centers of proteins, was shown to irreversibly modify and inhibit erythrocyte plasma membrane Ca2+ transport and (Ca(2+)+Mg2+)-ATPase activity. A method that allows the reaction to proceed in the presence of the modifier was used to analyze the kinetics of inactivation. This proved particularly important for the determination of the inactivation rate constant for Ca2+ transport, because inside-out vesicles cannot withstand the removal of the inhibitor after preincubation. Analysis of both (Ca(2+)+Mg2+)-ATPase and inside-out vesicular Ca2+ transport inactivation rate constants using this approach yielded an irreversible inhibition pattern. This pattern was consistent with the interpretation that both activities underwent noncomplexing nonsaturable reactions with the inhibitor. ATP was shown to compete with phenylglyoxal by reducing its effects on Ca2+ transport. Phenylglyoxal did not appear to alter passive permeability of the vesicles to Ca2+ and, in concurrently performed vesicular Ca2+ uptake and (Ca(2+)+Mg2+)-ATPase measurements, exhibited IC50 values of 2.9 and 3.4 mM, respectively. These data support the evidence for a functionally essential arginyl residue in the active site of the Ca2+ pump/ATPase and provide pharmacological evidence for a tightly coupled ion-motive enzyme complex responsible for Ca2+ efflux.
Department of Pharmacology, Indiana University School of Medicine, Evansville 47732.
B U
BU
Raess
D000255:Adenosine Triphosphate / Q000494:pharmacology; D001665:Binding Sites; D001693:Biological Transport, Active / Q000187:drug effects; D017301:Ca(2+) Mg(2+)-ATPase / Q000097:blood*; D002118:Calcium / Q000097:blood*; D000252:Calcium-Transporting ATPases / Q000187:drug effects*; D004910:Erythrocyte Membrane / Q000187:drug effects / Q000378:metabolism; D004912:Erythrocytes / Q000187:drug effects* / Q000378:metabolism; D006801:Humans; D010658:Phenylglyoxal / Q000494:pharmacology*; D013379:Substrate Specificity
D000255:Adenosine Triphosphate; D017301:Ca(2+) Mg(2+)-ATPase; D000252:Calcium-Transporting ATPases; D010658:Phenylglyoxal; D002118:Calcium