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

Abstract

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 ␤-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 fluoresceinisothiocyanate labeling. The PM Ca 2؉ -ATPase cross-reacted with an antiserum against a putative Ca 2؉ -ATPase of the Arabidopsis thaliana chloroplast envelope.

Identification of a Calmodulin-Regulated Soybean Ca2+-ATPase (SCA1) That Is Located in the Plasma Membrane

“…Calmodulin stimulated ATPase activity by approximately sixfold in a dose-dependent manner, showing a half-maximum activation at ‫01ف‬ nM for SCaM-1 (Figure 9) or bovine calmodulin (not shown). This value represents a higher K 1/2 than that reported for ACA2p (K 1/2 ϭ 30 nM bovine calmodulin), in agreement with prior findings on the biochemical properties of enzymes purified from plant tissues, that is, that Ca 2ϩ pumps from the plasma membrane have a stronger binding affinity for calmodulin than do pumps from the tonoplast (Askerlund, 1996(Askerlund, , 1997 or other endomembranes (Dainese et al, 1997;Bonza et al, 1998). The greater calmodulin binding affinity of SCA1p also might be attributable to the The presence of an N-terminal regulatory domain in SCA1p was demonstrated by an N-terminal deletion that resulted in a calmodulin-independent (i.e., deregulated) pump with kinetic properties similar to those of a full-length pump stimulated by calmodulin.…”

“…Biochemical evidence previously suggested the presence of a calmodulin-regulated Ca 2ϩ pump in the plant cell plasma membrane (Evans, 1994;Bonza et al, 1998), but the corresponding gene for this was not identified until now. In this study, both membrane fractionation and cytology indicated that SCA1p was located in the plasma membrane, and biochemical and genetic evidence demonstrated that SCA1p was a calmodulin-stimulated Ca 2ϩ pump.…”

“…Second, unlike type IIB pumps from animals, which are thought to be located exclusively in the plasma membrane, the plant isoforms have been found only in non-plasma membrane locations, such as ACA2p in the ER membrane (Hong et al, 1999), BCA1p in the tonoplast (Malmstrom et al, 1997), and ACA1p, which is thought to reside in the chloroplast inner membrane (Huang et al, 1993). Despite the biochemical evidence for a calmodulin-regulated Ca 2 ϩ -ATPase in the plant cell plasma membrane (Evans, 1994;Askerlund, 1997;Dainese et al, 1997;Hwang et al, 1997;Bonza et al, 1998), the corresponding gene for this has not been cloned, and it is not known whether the anticipated pump is more similar to an animal type IIB pump or to the calmodulin-regulated pumps found in plant cell endomembranes.In this article, we report the isolation and characterization of a gene, SCA1 (for soybean Ca 2 ϩ -ATPase 1), encoding a calmodulin-regulated Ca 2 ϩ -ATPase that is located at the plasma membrane in soybean cells. Both biochemical and genetic evidence demonstrate that SCA1p has an N-terminal calmodulin-regulated autoinhibitor.…”

An electron microscopic-cytochemical localization of plasma membrane Ca2+-ATPase activity in poplar apical bud cells during the induction of dormancy by short-day photoperiods

“…The PM Ca 2+ -ATPase has been studied extensively in both animal and plant cells using either biochemical or cytochemical methods [8][9][10], [13], [14], [18], [25], [26]. In biochemi-cal assay with cell free preparation, Mg 2+ ions must be added to the reaction medium in addition to the Ca 2+ for the activation of the Ca 2+ -ATPase [8], [9], [13].…”

“…Plasma membrane (PM) Ca 2+ -ATPase plays a crucial role in controlling cytosolic Ca 2+ concentration [8][9][10][11][12][13][14]. The PM calcium-pumping, ATPase uses the energy supplied by the hydrolysis of ATP to transport calcium ions from the cytoplasm to the extracellular space.…”