IRREVERSIBLE MODIFICATION OF RED-CELL CA2+ TRANSPORT BY PHENYLGLYOXAL

Phenylglyoxal, a dicarbonyl modifier of arginyl residues with a high s electivity for anion substrate binding sites in active centers of prot eins, was shown to irreversibly modify and inhibit erythrocyte plasma membrane Ca2+ transport and (Ca2+ + Mg2+)-ATPase activity. A method th at 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 remo val of the inhibitor after preincubation. Analysis of both (Ca2+ + Mg2 +)-ATPase and inside-out vesicular Ca2+ transport inactivation rate co nstants using this approach yielded an irreversible inhibition pattern . This pattern was consistent with the interpretation that both activi ties 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 perme ability of the vesicles to Ca2+ and, in concurrently performed vesicul ar Ca2+ uptake and (Ca2+ + Mg2+)-ATPase measurements, exhibited IC50 v alues of 2.9 and 3.4 mM, respectively. These data support the evidence for a functionally essential arginyl residue in the active site of th e Ca2+ pump/ATPase and provide pharmacological evidence for a tightly coupled ion-motive enzyme complex responsible for Ca2+ efflux.