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

B. S. Karon, E. R. Nissen, John C Voss, D. D. Thomas

Research output: Contribution to journalArticle

17 Citations (Scopus)

Abstract

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.

Original languageEnglish (US)
Pages (from-to)328-333
Number of pages6
JournalAnalytical Biochemistry
Volume227
Issue number2
DOIs
StatePublished - May 1995
Externally publishedYes

Fingerprint

Sarcoplasmic Reticulum
Adenosine Triphosphatases
Hydrolysis
Assays
Adenosine Triphosphate
Calcium
Phosphates
Oxalates
Egtazic Acid
NAD
Radioactivity
Enzymes

ASJC Scopus subject areas

  • Molecular Biology
  • Cell Biology
  • Biophysics
  • Biochemistry

Cite this

A Continuous Spectrophotometric Assay for Simultaneous Measurement of Calcium Uptake and ATP Hydrolysis in Sarcoplasmic Reticulum. / Karon, B. S.; Nissen, E. R.; Voss, John C; Thomas, D. D.

In: Analytical Biochemistry, Vol. 227, No. 2, 05.1995, p. 328-333.

Research output: Contribution to journalArticle

@article{b5e9c6bdc87f4867a833dfe2e5c5113a,
title = "A Continuous Spectrophotometric Assay for Simultaneous Measurement of Calcium Uptake and ATP Hydrolysis in Sarcoplasmic Reticulum",
abstract = "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.",
author = "Karon, {B. S.} and Nissen, {E. R.} and Voss, {John C} and Thomas, {D. D.}",
year = "1995",
month = "5",
doi = "10.1006/abio.1995.1288",
language = "English (US)",
volume = "227",
pages = "328--333",
journal = "Analytical Biochemistry",
issn = "0003-2697",
publisher = "Academic Press Inc.",
number = "2",

}

TY - JOUR

T1 - A Continuous Spectrophotometric Assay for Simultaneous Measurement of Calcium Uptake and ATP Hydrolysis in Sarcoplasmic Reticulum

AU - Karon, B. S.

AU - Nissen, E. R.

AU - Voss, John C

AU - Thomas, D. D.

PY - 1995/5

Y1 - 1995/5

N2 - 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.

AB - 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.

UR - http://www.scopus.com/inward/record.url?scp=0029008270&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=0029008270&partnerID=8YFLogxK

U2 - 10.1006/abio.1995.1288

DO - 10.1006/abio.1995.1288

M3 - Article

C2 - 7573954

AN - SCOPUS:0029008270

VL - 227

SP - 328

EP - 333

JO - Analytical Biochemistry

JF - Analytical Biochemistry

SN - 0003-2697

IS - 2

ER -