Purification of the Plasma Membrane Ca2+-ATPase from Radish Seedlings by Calmodulin-Agarose Affinity Chromatography1 | Zendy

Maria Cristina Bonza | Zendy; Antonella Carnelli | Zendy; Maria Ida De Michelis | Zendy; Franca RasiCaldogno | Zendy

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Purification of the Plasma Membrane Ca2+-ATPase from Radish Seedlings by Calmodulin-Agarose Affinity Chromatography1

Author(s) -

Maria Cristina Bonza,

Antonella Carnelli,

Maria Ida De Michelis,

Franca RasiCaldogno

Publication year - 1998

Publication title -

plant physiology

Language(s) - English

Resource type - Journals

SCImago Journal Rank - 3.554

H-Index - 312

eISSN - 1532-2548

pISSN - 0032-0889

DOI - 10.1104/pp.116.2.845

Subject(s) - calmodulin , atpase , agarose , chromatography , chemistry , affinity chromatography , raphanus , elution , biochemistry , membrane , antiserum , biology , enzyme , botany , antibody , immunology

The Ca2+-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 withn-dodecyl β-d-maltoside and applied to a CaM-agarose matrix. After various washings with decreasing Ca2+ concentrations, the Ca2+-ATPase was eluted with 5 mm ethylenediaminetetraacetate (EDTA). The EDTA-eluted fraction contained about 25% of the loaded Ca2+-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 Ca2+-ATPase by 125I-CaM overlay and fluorescein-isothiocyanate labeling. The PM Ca2+-ATPase cross-reacted with an antiserum against a putative Ca2+-ATPase of the Arabidopsis thalianachloroplast envelope.

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