title: Two simultaneous binding sites for nucleotide analogs are kinetically distinguishable on the sarcoplasmic reticulum Ca(2+)-ATPase.
abstract: Erythrosin B and eosin Y stimulate p-nitrophenyl phosphate hydrolysis by purified sarcoplasmic reticulum Ca(2+)-ATPase by nearly 2-3 fold in the presence of Ca(2+). This stimulation is not due to the change on the apparent affinity for substrate but is indeed due to acceleration of the turnover rate of the enzyme. Stimulation reaches a maximum at approximately 5 microM erythrosin or 20 microM eosin and is strictly dependent on the presence of Ca(2+) in reaction media, while higher concentrations of dye progressively inhibit phosphatase activity. Labeling with fluorescein isothiocyanate (FITC) largely shifts the Km for p-nitrophenyl phosphate (pNPP) and completely abolishes the stimulation of phosphatase activity induced by erythrosin in the presence of Ca(2+), apparently by FITC impairing dye binding to an activator site and allowing only manifestation of an inhibitory binding site. In the absence of Ca(2+), both erythrosin and eosin inhibit pNPP hyrolysis with Ic50 values 3-4 fold higher than the maximally stimulatory enzyme with FITC, which by its turn does not affect pNPPase activity in absence of Ca(2+). It is suggested that stimulation and inhibition of phosphatase activity are related to two simultaneous and physically different nucleotide analog binding sites.
authors.affiliation: Departamento de Bioquímica Médica, ICB/CCS, Universidade Federal do Rio de Janeiro-Cidade Universitária, RJ, Brasil.
authors.forename: J A
authors.initials: JA
authors.lastname: Mignaco
authors.forename: O H
authors.initials: OH
authors.lastname: Lupi
authors.forename: F T
authors.initials: FT
authors.lastname: Santos
authors.forename: H
authors.initials: H
authors.lastname: Barrabin
authors.forename: H M
authors.initials: HM
authors.lastname: Scofano
mesh_terms: D009597:4-Nitrophenylphosphatase / Q000737:chemistry / Q000378:metabolism*; D000818:Animals; D001665:Binding Sites; D002118:Calcium / Q000378:metabolism; D000252:Calcium-Transporting ATPases / Q000737:chemistry* / Q000378:metabolism*; D004801:Eosine Yellowish-(YS) / Q000494:pharmacology; D004923:Erythrosine / Q000494:pharmacology; D006868:Hydrolysis; D007700:Kinetics; D018482:Muscle, Skeletal / Q000201:enzymology*; D009596:Nitrophenols / Q000378:metabolism; D009943:Organophosphorus Compounds / Q000378:metabolism; D011817:Rabbits; D012265:Ribonucleotides / Q000378:metabolism*; D012519:Sarcoplasmic Reticulum / Q000201:enzymology*; D013379:Substrate Specificity
chemical_list: D009596:Nitrophenols; D009943:Organophosphorus Compounds; D012265:Ribonucleotides; C008644:nitrophenylphosphate; D009597:4-Nitrophenylphosphatase; D000252:Calcium-Transporting ATPases; D004923:Erythrosine; D002118:Calcium; D004801:Eosine Yellowish-(YS)