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. 1998 Feb 1;116(2):845-51.
doi: 10.1104/pp.116.2.845.

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

C Bonza  1 , A Carnelli, Ida De Michelis M, F Rasi-Caldogno
Affiliations

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

C Bonza et al. Plant Physiol. .

Abstract

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 with n-dodecyl beta-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 thaliana chloroplast envelope.

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Figures

Figure 1
Figure 1
Silver stain of EDTA-eluted fractions from different purifications with different stimulation by CaM. Each lane was loaded with similar Ca2+-ATPase activity (approximately 0.2 μg of proteins per lane). Values below the picture represent the Ca2+-ATPase activities, expressed in micromoles per minute per milligram of protein.
Figure 2
Figure 2
A, Labeling of the PM Ca2+-ATPase of different fractions of the CaM-agarose purification procedure by 125I-CaM. Proteins were from solubilized PM (lane 1), unbound fraction (lane 2), EGTA-eluted fraction (lane 3), and EDTA-eluted fraction (lane 4). Each lane contained protein solubilized from 10 μL of the relevant fraction. B, Labeling of the PM Ca2+-ATPase of native PM (lane 1, 70 μg of proteins) and of the EDTA-eluted fraction (lane 2, 0.2 μg proteins) by FITC and immunodetection on western analysis with an anti-FITC antiserum.
Figure 3
Figure 3
Immunodecoration of the main fractions of the CaM-agarose purification procedure, with an antiserum against a putative Ca2+-ATPase of the A. thaliana plastid envelope. Lane 1 was loaded with proteins from native PM (70 μg), lane 2 with proteins from solubilized PM (32 μg), lane 3 with proteins from the unbound fraction (28 μg), and lane 4 with proteins from the EDTA-eluted fraction (0.23 μg).
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