title: Chemical modification reveals involvement of different sites for nucleotide analogues in the phosphatase activity of the red cell calcium pump.
abstract: The calcium pump of plasma membranes catalyzes the hydrolysis of ATP and phosphoric esters like p-nitrophenyl phosphate (pNPP). The latter activity requires the presence of ATP and/or calmodulin, and Ca2+ [22, 25]. We have studied the effects of nucleotide-analogues and chemical modifications of nucleotide binding sites on Ca2+-pNPPase activity. Treatment with fluorescein isothiocyanate (FITC), abolished Ca2+-ATPase and ATP-dependent pNPPase, but affected only 45% of the calmodulin-dependent pNPPase activity. The nucleotide analogue eosin-Y had an inhibitory effect on calmodulin-dependent pNPPase (Kieosin-Y = 2 microM). FITC treatment increased Kieosin-Y 15 times. Acetylation of lysine residues with N-hydroxysuccinimidyl acetate inactivates Ca2+-ATPase by modifying the catalytic site, and impairs stimulation by modulators by modifying residues outside this site [9]. Acetylation suppressed the ATP-dependent pNPPase with biphasic kinetics. ATP or pNPP during acetylation cancels the fast component of inactivation. Acetylation inhibited only partially the calmodulin-dependent pNPPase, but neither ATP nor pNPP prevented this inactivation. From these results we conclude: (i) ATP-dependent pNPPase depends on binding of ATP to the catalytic site; (ii) the catalytic site plays no role in calmodulin-dependent pNPPase. The decreased affinity for eosin-Y of the FITC-modified enzyme, suggests that the sites for these two molecules are closely related but not overlapped. Acetimidation of the pump inhibited totally the calmodulin-dependent pNPPase, but only partially the ATP-pNPPase. Since calmodulin binds to E1, the E1 conformation or the E2 if E1 transition would be involved during calmodulin-dependent pNPPase activity.
authors.affiliation: Renal Unit, Massachusetts General Hospital/Harvard Medical School, Charlestown, MA 02129, USA.
authors.forename: C
authors.initials: C
authors.lastname: Donnet
authors.forename: A J
authors.initials: AJ
authors.lastname: Caride
authors.forename: S
authors.initials: S
authors.lastname: Talgham
authors.forename: J P
authors.initials: JP
authors.lastname: Rossi
mesh_terms: D009597:4-Nitrophenylphosphatase / Q000187:drug effects / Q000378:metabolism*; D000255:Adenosine Triphosphate / Q000031:analogs & derivatives* / Q000378:metabolism*; D001665:Binding Sites; D000252:Calcium-Transporting ATPases / Q000097:blood* / Q000187:drug effects; D002384:Catalysis / Q000187:drug effects; D004789:Enzyme Activation / Q000187:drug effects; D004801:Eosine Yellowish-(YS) / Q000494:pharmacology; D004910:Erythrocyte Membrane / Q000201:enzymology*; D016650:Fluorescein-5-isothiocyanate / Q000494:pharmacology; D006801:Humans; D007096:Imidoesters / Q000494:pharmacology; D007700:Kinetics; D013388:Succinimides / Q000494:pharmacology
chemical_list: D007096:Imidoesters; D013388:Succinimides; D000255:Adenosine Triphosphate; C010222:methyl acetimidate; D009597:4-Nitrophenylphosphatase; D000252:Calcium-Transporting ATPases; D016650:Fluorescein-5-isothiocyanate; D004801:Eosine Yellowish-(YS)