The enzyme (native or recombinant) was incubated (FALC Thermoblock) in a buffer solution at 37°C and the reaction was started by addition of 1 mM ATP. For SERCA, the buffer solution contained 80 mM KCl, 25 mM MOPS (pH 7.0 by TRIS), 3 mM MgCl2, 5 mM sodium azide, 0.2 mM EGTA, 0.2 mM CaCl2 (about 10 µM free calcium) and 2 µM A23187. In the case of Na,K-ATPase, the buffer solution was composed by 20 mM KCl, 100 mM NaCl, 25 mM MOPS (pH 7.0 by TRIS) and 3 mM MgCl2. Blank samples were prepared in the absence of calcium ions and in the presence of 2 mM EGTA (SERCA) or in the absence of sodium ions (Na,K-ATPase). Control experiments were performed in the presence of 1 µM thapsigargin (SERCA) or 50 µM ouabain (Na,K-ATPase). The total protein concentration was 10 µg/mL for native and recombinant (WT) SERCA, 1 µg/mL for Na,K-ATPase and 4.5 µg/mL for mutant (D351N) SERCA.
After incubation, aliquots were taken at subsequent times and immediately added to the coloring solution contained in glass disposable test tubes (Corning, mod. 99445-12). Usually, aliquots of 100 µL were added to 900 µL of coloring solution, giving a total final volume of 1 mL. This addition suddenly interrupts the enzymatic ATP hydrolysis, and, therefore, Pi release, due to enzyme denaturation produced by the strong acid conditions.
For each protein, five aliquots, each as triplicate, were taken at different times to plot an activity curve (Pi vs time). Blank was also evaluated as a triplicate. Where indicated, sodium citrate was added from a 10% (w/w) stock 10 minutes after aliquot addition.
The calibration curve was determined by preparing standard solutions of Pi from 1 or 0.1 mM aqueous stock solutions.
All measurements were carried out by an UV/Vis spectrophotometer (Jasco, mod. V-560) provided with a stirring and thermostatable holder (Jasco, mod. EHC-477S). The bandwidth was settled to 2 nm. Acquisition data pitch was 1 s for kinetic measurements and 0.5 nm for spectra registration. Quartz semimicro cuvettes (Hellma, 109.004F-QS) were used to allow continuous stirring of the solution. The temperature was always maintained at 20°C by a Peltier temperature controller (Jasco, mod. EHC-477T).
After incubation, aliquots were taken at subsequent times and immediately added to the coloring solution contained in glass disposable test tubes (Corning, mod. 99445-12). Usually, aliquots of 100 µL were added to 900 µL of coloring solution, giving a total final volume of 1 mL. This addition suddenly interrupts the enzymatic ATP hydrolysis, and, therefore, Pi release, due to enzyme denaturation produced by the strong acid conditions.
For each protein, five aliquots, each as triplicate, were taken at different times to plot an activity curve (Pi vs time). Blank was also evaluated as a triplicate. Where indicated, sodium citrate was added from a 10% (w/w) stock 10 minutes after aliquot addition.
The calibration curve was determined by preparing standard solutions of Pi from 1 or 0.1 mM aqueous stock solutions.
All measurements were carried out by an UV/Vis spectrophotometer (Jasco, mod. V-560) provided with a stirring and thermostatable holder (Jasco, mod. EHC-477S). The bandwidth was settled to 2 nm. Acquisition data pitch was 1 s for kinetic measurements and 0.5 nm for spectra registration. Quartz semimicro cuvettes (Hellma, 109.004F-QS) were used to allow continuous stirring of the solution. The temperature was always maintained at 20°C by a Peltier temperature controller (Jasco, mod. EHC-477T).
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