2 SIMULTANEOUS BINDING-SITES FOR NUCLEOTIDE ANALOGS ARE KINETICALLY DISTINGUISHABLE ON THE SARCOPLASMIC-RETICULUM CA2-ATPASE()

Erythrosin B and eosin Y stimulate p-nitrophenyl phosphate hydrolysis by purified sarcoplasmic reticulum Ca2+-ATPase by nearly 2-3-fold in t he presence of Ca2+. This stimulation is not due to a change on the ap parent affinity for substrate but is indeed due to acceleration of the turnover rate of the enzyme. Stimulation reaches a maximum at approxi mately 5 mu M erythrosin or 20 mu M eosin and is strictly dependent on the presence of Ca2+ in reaction media, while higher concentrations o f dye progressively inhibit phosphatase activity. Labeling with fluore scein isothiocyanate (FITC) largely shifts the K-m for p-nitrophenyl p hosphate (pNPP) and completely abolishes the stimulation of phosphatas e activity induced by erythrosin in the presence of Ca2+, apparently b y FITC impairing dye binding to an activator site and allowing only ma nifestation of an inhibitory binding site. In the absence of Ca2+, bot h erythrosin and eosin inhibit pNPP hydrolysis with Ic(50) values 3-4- fold higher than the maximally stimulatory concentrations for Ca2+-pho sphatase. This inhibitory effect is not modified by previous labeling of the enzyme with FITC, which by its turn does not affect pNPPase act ivity in absence of Ca2+. It is suggested that stimulation and inhibit ion of phosphatase activity are related to two simultaneous and physic ally different nucleotide analog binding sites.