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.