3-O-methylfluorescein phosphate as a fluorescent substrate for plasma membrane Ca2+-ATPase.
3-O-methylfluorescein phosphate hydrolysis, catalyzed by purified erythrocyte Ca2+-ATPase in the absence of Ca2+, was slow in the basal state, activated by phosphatidylserine and controlled proteolysis, but not by calmodulin. p-Nitrophenyl phosphate competitively inhibits hydrolysis in the absence of Ca2+, while ATP inhibits it with a complex kinetics showing a high and a low affinity site for ATP. Labeling with fluorescein isothiocyanate impairs the high affinity binding of ATP, but does not appreciably modify the binding of any of the pseudosubstrates. In the presence of calmodulin, an increase in the Ca2+ concentration produces a bell-shaped curve with a maximum at 50 microM Ca2+. At optimal Ca2+ concentration, hydrolysis of 3-O-methylfluorescein phosphate proceeds in the presence of fluorescein isothiocyanate, is competitively inhibited by p-nitrophenyl phosphate and, in contrast to the result observed in the absence of Ca2+, it is activated by calmodulin. In marked contrast with other pseudosubstrates, hydrolysis of 3-O-methylfluorescein phosphate supports Ca2+ transport. This highly specific activity can be used as a continuous fluorescent marker or as a tool to evaluate partial steps from the reaction cycle of plasma membrane Ca2+-ATPases.
Freire
Monica M
MM
Departamento de Bioquímica Médica, Instituto de Ciências Biomédicas, Centro de Ciências da Saúde, Universidade Federal do Rio de Janeiro, Cidade Universitária, CEP 21941-590, Rio de Janeiro, Brazil.
Mignaco
Julio A
JA
de Carvalho-Alves
Paulo C
PC
Barrabin
Hector
H
Scofano
Helena M
HM
D000818:Animals; D001667:Binding, Competitive; D002122:Calcium Chloride / Q000494:pharmacology; D000252:Calcium-Transporting ATPases / Q000737:chemistry / Q000378:metabolism*; D002147:Calmodulin / Q000494:pharmacology; D004533:Egtazic Acid; D004789:Enzyme Activation; D004910:Erythrocyte Membrane / Q000201:enzymology / Q000378:metabolism*; D016650:Fluorescein-5-isothiocyanate; D005452:Fluoresceins / Q000378:metabolism*; D005456:Fluorescent Dyes; D006868:Hydrolysis; D007700:Kinetics; D009596:Nitrophenols / Q000494:pharmacology; D009943:Organophosphorus Compounds / Q000494:pharmacology; D010718:Phosphatidylserines; D011487:Protein Conformation; D000254:Sodium-Potassium-Exchanging ATPase / Q000378:metabolism; D014357:Trypsin
C032079:3-O-methylfluorescein phosphate; D002147:Calmodulin; D005452:Fluoresceins; D005456:Fluorescent Dyes; D009596:Nitrophenols; D009943:Organophosphorus Compounds; D010718:Phosphatidylserines; C008644:nitrophenylphosphate; D004533:Egtazic Acid; D014357:Trypsin; C045349:3-O-methyl-fluorescein phosphatase; D000252:Calcium-Transporting ATPases; D000254:Sodium-Potassium-Exchanging ATPase; D016650:Fluorescein-5-isothiocyanate; D002122:Calcium Chloride