3-O-Methylfluorescein phosphate as a fluorescent substrate for plasma membrane Ca2+-ATPase
| Autor: | Hector Barrabin, Paulo C. Carvalho-Alves, Julio A. Mignaco, Helena M. Scofano, Monica M. Freire |
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| Rok vydání: | 2002 |
| Předmět: |
Calmodulin
Protein Conformation Proteolysis Biophysics Calcium-Transporting ATPases Phosphatidylserines Fluorimetric assay 3-O-Methylfluorescein phosphate Binding Competitive Biochemistry Nitrophenols Calcium Chloride Plasma membrane Ca2+-ATPase Hydrolysis chemistry.chemical_compound Organophosphorus Compounds medicine Animals Trypsin Fluorescein isothiocyanate Egtazic Acid Fluorescent Dyes medicine.diagnostic_test biology Erythrocyte Membrane Substrate (chemistry) Cell Biology Fluoresceins Phosphate Enzyme Activation Erythrocyte Kinetics chemistry biology.protein Plasma membrane Ca2+ ATPase Sodium-Potassium-Exchanging ATPase Ca2+-ATPase Fluorescein-5-isothiocyanate medicine.drug |
| Zdroj: | Biochimica et Biophysica Acta (BBA) - Bioenergetics. 1553:238-248 |
| ISSN: | 0005-2728 |
| Popis: | 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 μM 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. |
| Databáze: | OpenAIRE |
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