Affinity purification of the Ca-ATPase from cardiac sarcoplasmic reticulum membranes.
We report the isolation of the functional form of the Ca-ATPase from porcine cardiac sarcoplasmic reticulum (SR) membranes, taking advantage of the ability of this enzyme to bind to the nucleotide site affinity dye, Reactive Red 120. Conditions that optimize the solubility and functional stability of the cardiac Ca-ATPase in detergent during the purification procedure are essential to its recovery. The purified Ca-ATPase migrates as a single band on Coomassie blue-stained polyacrylamide gels and exhibits high specific activity (2.5 IU at 25 degreesC) and functional stability. Similar enrichment of the Ca-ATPase estimated from either relative amounts of the 100-kDa protein band on polyacrylamide gels or steady-state concentrations of phosphorylated enzyme intermediate (E-P) demonstrate that neither nonfunctional Ca-ATPases nor non-Ca-ATPase proteins migrating with an apparent molecular weight of 100 kDa constitute a significant fraction of these preparations. Steady-state levels of E-P are 1.3 and 8.6 nmol/mg protein, respectively, for native cardiac SR membranes and the final purified fraction. These values, in comparison to the maximum value (9.1 nmol/mg) for the 110-kDa protein, agree well with estimates of total Ca-ATPase abundance from gel densitometry for both preparations and indicate full site reactivity, i.e., one phosphorylation site for each 110-kDa cardiac Ca-ATPase polypeptide chain.
Department of Biochemistry, University of Kansas, Lawrence, Kansas, 66045, USA.
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Yao
L T
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Chen
D J
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Bigelow
D000818:Animals; D000252:Calcium-Transporting ATPases / Q000302:isolation & purification*; D002846:Chromatography, Affinity / Q000379:methods*; D002847:Chromatography, Agarose; D003902:Detergents; D004795:Enzyme Stability; D006352:Heart Ventricles / Q000201:enzymology; D018656:Muscle Fibers, Fast-Twitch / Q000201:enzymology; D018482:Muscle, Skeletal / Q000201:enzymology; D009206:Myocardium / Q000201:enzymology*; D011817:Rabbits; D012519:Sarcoplasmic Reticulum / Q000201:enzymology*; D013552:Swine; D014227:Triazines
D003902:Detergents; D014227:Triazines; C042516:reactive red 120 dye; D000252:Calcium-Transporting ATPases