A Continuous Spectrophotometric Assay for Simultaneous Measurement of Calcium Uptake and ATP Hydrolysis in Sarcoplasmic Reticulum
✍ Scribed by B.S. Karon; E.R. Nissen; J. Voss; D.D. Thomas
- Publisher
- Elsevier Science
- Year
- 1995
- Tongue
- English
- Weight
- 504 KB
- Volume
- 227
- Category
- Article
- ISSN
- 0003-2697
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✦ Synopsis
A continuous, spectrophotometric assay to simultaneously measure Ca uptake and ATP hydrolysis has been developed, in order to assess the function of the Ca-ATPase in skeletal and cardiac sarcoplasmic reticulum (SR) vesicles. The absorbance of Fura Red was measured continuously at (490 \mathrm{~nm}), in EGTA-buffered solutions containing initial free ionized calcium concentrations of (300 \mathrm{~nm}, 500 \mathrm{nM}, 790 \mathrm{~nm}), and (2 \mu \mathrm{M}), during assays of oxalate-facilitated or phosphate-facilitated active calcium uptake in skeletal SR. Simultaneous measurement of ATP hydrolysis during the measurement of phosphate-facilitated Ca uptake was accomplished by measuring the disappearance of NADH at (340 \mathrm{~nm}), coupled to the hydrolysis of ATP by an enzymelinked, continuous ATPase assay. This new method, unlike the standard ({ }^{45} \mathrm{Ca})-filtration assay, measures calcium uptake in real time and eliminates the need for radioactivity. Moreover, the rates of calcium uptake and ATP hydrolysis are measured simultaneously, allowing the direct quantitative comparison of the two parameters. This assay will facilitate the characterization of Ca-ATPase function and malfunction in skeletal and cardiac SR and advances the methodology for comparison of normal and physically, chemically, or biologically altered Ca-ATPase. 1995 Academic Press, Inc.