A Continuous Spectrophotometric Assay for Simultaneous Measurement of Calcium Uptake and ATP Hydrolysis in Sarcoplasmic Reticulum

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 nm, in EGTA-buf...

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Published inAnalytical biochemistry Vol. 227; no. 2; pp. 328 - 333
Main Authors Karon, B.S., Nissen, E.R., Voss, J., Thomas, D.D.
Format Journal Article
LanguageEnglish
Published United States Elsevier Inc 20.05.1995
Subjects
Adenosine Triphosphate - metabolism
Animals
Benzofurans
Calcium - metabolism
Calcium-Transporting ATPases - analysis
Fluorescent Dyes
Hydrolysis
Imidazoles
Muscle, Skeletal - enzymology
Muscle, Skeletal - ultrastructure
Myocardium - enzymology
Myocardium - ultrastructure
Rabbits
Sarcoplasmic Reticulum - metabolism
Spectrophotometry - methods
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Summary: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 nm, in EGTA-buffered solutions containing initial free ionized calcium concentrations of 300 nM, 500 nM, 700 nM, and 2 μM, 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 nm, coupled to the hydrolysis of ATP by an enzyme-linked, continuous ATPase assay. This new method, unlike the standard 45Ca-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.
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ISSN:0003-2697
1096-0309
DOI:10.1006/abio.1995.1288