Purification of the Plasma Membrane Ca(2+)-ATPase from Radish Seedlings by Calmodulin-Agarose Affinity Chromatography

The Ca(2+)-ATPase of the plasma membrane (PM) of germinating radish (Raphanus sativus L.) seeds was purified by calmodulin (CaM)-affinity chromatography using a batch procedure. PM purified by aqueous two-phase partitioning was solubilized with n-dodecyl β-d-maltoside and applied to a CaM-agarose matrix. After various washings with decreasing Ca(2+) concentrations, the Ca(2+)-ATPase was eluted with 5 mm ethylenediaminetetraacetate (EDTA). The EDTA-eluted fraction contained about 25% of the loaded Ca(2+)-ATPase activity, with a specific activity 70-fold higher than that of the starting PM fraction. The EDTA-eluted fraction was highly enriched in a 133-kD polypeptide, which was identified as the PM Ca(2+)-ATPase by (125)I-CaM overlay and fluorescein-isothiocyanate labeling. The PM Ca(2+)-ATPase cross-reacted with an antiserum against a putative Ca(2+)-ATPase of the Arabidopsis thaliana chloroplast envelope.