AFFINITY PURIFICATION OF THE CA-ATPASE FROM CARDIAC SARCOPLASMIC-RETICULUM MEMBRANES

We report the isolation of the functional form of the Ca-ATPase from p orcine 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 fu nctional stability of the cardiac Ca-ATPase in detergent during the pu rification procedure are essential to its recovery. The purified Ca-AT Pase migrates as a single band on Coomassie blue-stained polyacrylamid e gels and exhibits high specific activity (2.5 IU at 25 degrees C) an d functional stability. Similar enrichment of the Ca-ATPase estimated from either relative amounts of the 100-kDa protein band on polyacryla mide gels or steady-state concentrations of phosphorylated enzyme inte rmediate (E-P) demonstrate that neither nonfunctional Ca-ATPases nor n on-Ca-ATPase proteins migrating with an apparent molecular weight of 1 00 kDa constitute a significant fraction of these preparations. Steady -state levels of E-P are 1.3 and 8.6 nmol/mg protein, respectively, fo r native cardiac SR membranes and the final purified fraction. These v alues, in comparison to the maximum value (9.1 nmol/mg) for the 110-kD a protein, agree well with estimates of total Ca-ATPase abundance from gel densitometry for both preparations and indicate full site reactiv ity, i.e., one phosphorylation site for each 110-kDa cardiac Ca-ATPase polypeptide chain. (C) 1998 Academic Press.