INHIBITION OF THE RED-BLOOD-CELL CALCIUM-PUMP BY EOSIN AND OTHER FLUORESCEIN ANALOGS

This paper addresses the mechanism of inhibition of the plasma membran e Ca pump by fluorescein analogues and their isothiocyanate derivative s. Eosin (i.e., tetrabromofluorescein) was found to be one of the most potent reversible inhibitors of the erythrocyte Ca pump [half-maximal inhibitory concentration (IC50) <0.2 muM]; fluorescein itself was abo ut four orders of magnitude less potent (IC50 approximately 1,000 muM) . Eosin decreased the maximum influx and thus did not compete with ATP for the Ca pump. Irreversible inhibition produced by the isothiocyana te analogues of eosin and fluorescein [eosin 5-isothiocyanate (EITC) a nd fluorescein 5-isothiocyanate (FITC), respectively] was also studied . While EITC bound reversibly at the eosin site, two results suggest t hat EITC does not react covalently at this site: 1) eosin did not alte r the time course of the EITC irreversible reaction, and 2) the concen tration dependence for reversible EITC inhibition was different from t he concentration dependence for irreversible EITC inhibition. ATP did slow the rate of inactivation of both EITC and FITC consistent with th e idea that EITC and FITC bind to the ATP site. Our results are consis tent with eosin and ATP binding to separate sites and EITC reacting co valently at the ATP site, but not the eosin site.