Measurement of sarcoplasmic reticulum Ca2+-ATPase activity and E-type Mg2+-ATPase activity in rat heart homogenates.
The presence of a high and nonlinear Ca2+-independent (or basal) ATPase activity in rat heart preparations makes difficult the reliable measurement of sarcoplasmic reticulum (SR) Ca2+-ATPase activity by usual methods. A spectrophotometric assay for the accurate determination of SR Ca2+-ATPase activity in unfractionated homogenates from rat heart is described. The procedure is based on that reported by Simonides and van Hardeveld (1990, Anal. Biochem. 191, 321-331) for skeletal muscle homogenates. To avoid overestimation of the Ca2+-ATPase activity of cardiac homogenates that occurs when sequential measurements of total and basal ATPase activities are performed, two parallel and independent assays are required: one with low (micromolar) and other high (millimolar) calcium concentration. Addition of thapsigargin (0.2 microM) blocked totally the activity considered as Ca2+-ATPase activity. Using this method, the rat heart homogenate Ca2+-ATPase activity was 10.5 +/- 2.0 micromol. min-1 x g-1 tissue wet weight (n = 8). Likewise, a spectrophotometric assay for measuring E-type Mg2+-ATPase activity in cardiac total homogenates has been developed, comparing the following characteristics of the enzymatic activity in homogenate and a membrane-enriched fraction: first-order rate constant for ATP-dependent inactivation, Km for ATP, and effects of concanavalin A, Triton X-100, and specific inhibitors.
Department of Biochemistry and Molecular Biology I, Complutense University, Madrid, 28040, Spain.
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Saborido
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Delgado
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Megías
D000818:Animals; D017301:Ca(2+) Mg(2+)-ATPase / Q000378:metabolism*; D000252:Calcium-Transporting ATPases / Q000378:metabolism*; D002462:Cell Membrane / Q000201:enzymology; D009206:Myocardium / Q000201:enzymology*; D051381:Rats; D012519:Sarcoplasmic Reticulum / Q000201:enzymology*; D013053:Spectrophotometry / Q000379:methods*
D017301:Ca(2+) Mg(2+)-ATPase; D000252:Calcium-Transporting ATPases