Plant Physiol. (1998) 116: 845-851

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

Cristina Bonza^*, Antonella Carnelli, Maria Ida De Michelis², and Franca Rasi-Caldogno³

Dipartimento di Biologia L. Gorini, Università di Milano, via G. Celoria 26, 20133 Milano, Italy (C.B., A.C., F.R.-C.); and Istituto Botanico Hanbury ed Orto Botanico dell'Università, corso Dogali 1, 16136 Genova, Italy (M.I.D.M.)

	ABSTRACT

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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 -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.

	INTRODUCTION

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Ca²⁺-pumping ATPases play a crucial role in maintaining Ca²⁺ homeostasis and in restoring it after the increase of cytosolic Ca²⁺ concentration brought about by different stimuli. Given the limited capacity of internal stores, the PM Ca²⁺-ATPase is particularly important in long-term control, especially to extrude Ca²⁺ entering the cell due to the opening of PM Ca²⁺ channels (Briskin, 1990; Evans et al., 1991; De Michelis et al., 1992; Poovaiah and Reddy, 1993; Ranjeva et al., 1993; Askerlund and Sommarin, 1996).

The PM Ca²⁺-ATPase is a P-type ATPase that catalyzes an H⁺/Ca²⁺ exchange (Rasi-Caldogno et al., 1987; Briskin, 1990; Evans et al., 1991; De Michelis et al., 1992; Askerlund and Sommarin, 1996). The PM Ca²⁺-ATPase activity is stimulated by CaM, which binds to an autoinhibitory domain of the enzyme, causing a strong increase in the V_max and a decrease in the apparent K_m for free Ca²⁺ (Malatialy et al., 1988; Robinson et al., 1988; Williams et al., 1990; Erdei and Matsumoto, 1991; Rasi-Caldogno et al., 1992, 1993, 1995; De Michelis et al., 1993; Kurosaki and Kaboraki, 1994). All of these characteristics resemble those of the PM Ca²⁺-ATPase of mammals (Carafoli, 1991). However, in contrast to mammals, plant PM-type Ca²⁺-ATPases are also present in endomembranes, and are often more abundant in endomembranes than in the PM (for review, see Evans, 1994; Askerlund and Sommarin, 1996). Moreover, the Ca²⁺-ATPase of the PM of plant cells has a very high affinity for CaM, so that stimulation by exogenous CaM is low or undetectable unless endogenous CaM is stripped by drastic treatments with Ca²⁺-chelating agents (Williams et al., 1990; Evans et al., 1992; Rasi-Caldogno et al., 1993).

CaM-stimulated Ca²⁺-ATPases of endomembranes and of PM have similar biochemical characteristics, and cannot be easily discerned (Hsieh et al., 1991; Askerlund and Evans, 1993; Thomson et al., 1993, 1994; Askerlund, 1996; Askerlund and Sommarin, 1996; Hwang et al., 1997). The main reported differences between the two types of plant CaM-stimulated Ca²⁺-ATPases are: (a) the sensitivity of the endomembrane enzymes to inhibition by fluorescein derivatives is slightly lower than that of the PM enzyme (Thomson et al., 1993; Bush and Wang, 1995; Askerlund and Sommarin, 1996), and (b) the molecular weight of the PM enzyme is higher than that of endomembrane CaM-stimulated Ca²⁺-ATPases (Thomson et al., 1993; Askerlund, 1996; Askerlund and Sommarin, 1996; Hwang et al., 1997).

Since the first report by Dieter and Marmè (1981), CaM-stimulated Ca²⁺-ATPases have been purified from different plant materials by CaM-affinity chromatography, but all of them were localized on endomembranes (Briars et al., 1988; Evans et al., 1989, 1992; Askerlund and Evans, 1992; Theodoulou et al., 1994; Askerlund, 1996; Hwang et al., 1997).

Germinating radish seeds are an experimental system particularly suitable for studying the PM-localized Ca²⁺-ATPase (Rasi-Caldogno et al., 1995). In fact, the endomembrane system is poorly developed, and all of the Ca²⁺-ATPase activity is localized on the PM (Rasi-Caldogno et al., 1987, 1989). Furthermore, highly purified PM vesicles are easily obtained by the aqueous two-phase partitioning technique, with quite a high yield (De Michelis et al., 1991; Rasi-Caldogno et al., 1995). Ca²⁺-ATPase activity in the PM is among the highest reported and can be easily monitored both as nucleoside-triphosphate-dependent Ca²⁺ transport and as Ca²⁺-dependent ITPase activity (Carnelli et al., 1992).

Here we show that affinity chromatography on CaM-agarose with a batch procedure purifies the PM Ca²⁺-ATPase of radish seeds by 70-fold, with a recovery of about 25%.

	MATERIALS AND METHODS

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Preparation of PM Vesicles

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Solubilization of PM Ca²⁺-ATPase

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CaM-Affinity Chromatography

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Assay of PM Ca²⁺-ATPase Activity

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Treatment of the PM with FITC

Protein Assay

SDS-PAGE and Western Analysis

Statistics

	RESULTS

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Purification of the PM Ca²⁺-ATPase by CaM-Agarose-Affinity Chromatography

1

View this table: [in this window] [in a new window]   Table I. Purification of the PM Ca2+-ATPase by CaM-agarose affinity chromatography PM proteins were solubilized with n-dodecyl -d-maltoside (4:4 mg detergent mL1: mg protein mL1) and purified by CaM-agarose-affinity chromatography as described in ``Materials and Methods''. The first wash was performed in the presence of 100 µm CaCl2 and 100 µm MgSO4; the second one was performed in the absence of added divalent cations. Data in the brackets represent the percent stimulation by CaM (20 µg mL1). Results are from one experiment, which is representative of more than 10 experiments.

Given the low concentration of Ca²⁺-ATPase in the PM, for the purification of the enzyme by affinity chromatography with CaM-agarose, we chose to adopt a batch procedure that allows recovery of the eluted fractions in relatively small volumes (details are given in ``Materials and Methods''). Preliminary experiments showed that no Ca²⁺-ATPase activity could be recovered unless phosphatidylcholine was included in the elution medium (data not shown; see also Hwang et al., 1997).

Table I shows the results of a typical purification procedure. Upon overnight incubation of PM protein solubilized with n-dodecyl -d-maltoside (4:4 mg of detergent:mg of protein) with CaM-agarose, the bulk of CaM-stimulated Ca²⁺-ATPase activity bound to the matrix: the unbound fraction contained the bulk of protein and most of the CaM-independent Ca²⁺-ATPase activity measured in the solubilized fraction, but stimulation by CaM of the Ca²⁺-ATPase activity was virtually undetectable.

Subsequent washes of the matrix with decreasing concentrations of Ca²⁺ released only traces of Ca²⁺-ATPase activity, which was virtually insensitive to CaM. Also, elution of CaM-bound proteins with 1 mm EGTA released very low amounts of Ca²⁺-ATPase activity, also only slightly stimulated by CaM.

EDTA (5 mm) eluted a substantial amount of CaM-stimulated Ca²⁺-ATPase activity: in 10 purifications performed independently, the activity in the EDTA-eluted fraction, measured in the presence of CaM, was 25 ± 2% of the loaded activity. The CaM-agarose purification procedure determined a marked increase of CaM stimulation of the PM Ca²⁺-ATPase activity. In the experiment shown in Table I, CaM stimulation in the EDTA-eluted fraction was about 260% (compared with 120% in the solubilized PM), but in some preparations CaM stimulation of the Ca²⁺-ATPase in the EDTA-eluted fraction was up to 600% (see Fig. 1 and Table II). The source of this variability will be discussed below. The EDTA-eluted fraction contained only about 0.5% of the loaded proteins, so that it was 65-fold enriched in Ca²⁺-ATPase activity (and 75-fold in CaM-dependent Ca²⁺-ATPase activity) compared with the native PM.

View larger version (69K): [in this window] [in a new window]   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.

In the experiment described in Table I, the PM Ca²⁺-ATPase activity was assayed as Ca²⁺-dependent ITPase activity, the procedure routinely used with native PM vesicles, to avoid interference due to the simultaneous activity of the much more abundant H⁺-ATPase (Rasi-Caldogno et al., 1989; Carnelli et al., 1992). Table II shows that in the EDTA-eluted fraction the nucleoside triphosphatase activity measured in the absence of Ca²⁺ was also extremely low when ATP was supplied as a substrate, indicating that the fraction was devoid of H⁺-ATPase activity.

Figure 1 shows the results of the electrophoretic separation of the EDTA-eluted fraction from four independent purification procedures. The EDTA-eluted fractions were highly enriched in a band of about 133 kD; two more bands at 180 and 120 kD were also visible. The intensity of the 120-kD band compared with the 133-kD band, was quite variable from experiment to experiment and was inversely related to the extent of CaM stimulation of Ca²⁺-ATPase activity.

Identification of the PM Ca²⁺-ATPase in the EDTA-Eluted Fraction

The Ca²⁺-ATPase is the major protein labeled by CaM overlay of western blots of PM proteins of radish seedlings (Rasi-Caldogno et al., 1995). Figure 2A shows the CaM overlay of a western blot of the main fractions of the CaM-agarose purification procedure. ¹²⁵I-CaM heavily labeled a 133-kD band in the solubilized PM fraction (lane 1) and in the EDTA-eluted lane (lane 4); labeling was much weaker both in the fraction that did not bind to CaM-agarose (lane 2), and in the EGTA-eluted fraction (lane 3).

View larger version (81K): [in this window] [in a new window]   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.

The PM Ca²⁺-ATPase is extremely sensitive to inhibition by fluorescein derivatives (Giannini et al., 1987; Rasi-Caldogno et al., 1987, 1989, 1995; Olbe and Sommarin, 1991), which act as competitive inhibitors with the nucleoside triphosphates (De Michelis et al., 1993). Treatment of the PM with low concentrations of FITC selectively labels the Ca²⁺-ATPase, which can be easily detected on western analysis with an anti-FITC antiserum (Rasi-Caldogno et al., 1995). Figure 2B shows the western blot of native PM and of the EDTA-eluted fraction, labeled with FITC under selective conditions. In agreement with previous observations, FITC labeled two major bands of 133 and 120 kD in native PM (lane 1), which have been identified, respectively, as the intact Ca²⁺-ATPase and a product of its proteolysis lacking the CaM-binding domain (Rasi-Caldogno et al., 1995). In the EDTA-eluted fraction (lane 2), FITC labeled the same two bands, but the signal was much stronger for the 133-kD protein. Conversely, in the fraction that did not bind to CaM-agarose, FITC labeling was much stronger for the lower-molecular-mass band (data not shown).

The first plant cDNA encoding a polypeptide with high homology with the PM Ca²⁺-ATPase of mammals is PEA1, which encodes a putative Ca²⁺-ATPase of the plastid envelope of Arabidopsis thaliana (Huang et al., 1993b). Figure 3 shows the western blot of the major fractions of our purification procedure labeled with an antiserum against a portion of the protein encoded by PEA1 (kindly supplied by N.E. Hoffman). The antiserum labeled two bands of about 133 and 120 kD in all of the fractions tested. The 133-kD band was the more heavily labeled in the EDTA-eluted fraction (lane 4), whereas the fraction that did not bind to CaM-agarose (lane 3) was most enriched for the 120-kD protein.

View larger version (121K): [in this window] [in a new window]   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).

	DISCUSSION

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In this paper we report the first purification, to our knowledge, of the Ca²⁺-ATPase of the PM of plant cells. To achieve this goal we have used PM purified from germinating radish seedlings (Rasi-Caldogno et al., 1987, 1989, 1995).

The PM Ca²⁺-ATPase tightly bound to the CaM-agarose matrix and was not substantially eluted by EGTA (even when the concentration was increased up to 10 mm, data not shown), which has been widely used to elute the plant CaM-stimulated Ca²⁺-ATPases of endomembranes (Askerlund and Evans, 1992; Theodoulou et al., 1994; Askerlund, 1996; Hwang et al., 1997; Malmstrom et al., 1997). The tight binding of the PM Ca²⁺-ATPase to the CaM-agarose matrix suggests a high affinity of the enzyme for CaM. This result is in agreement with the previously reported observation that in native PM the Ca²⁺-ATPase is only slightly stimulated by exogenous CaM unless endogenous CaM is stripped by extensive washings with Ca²⁺ chelators (Williams et al., 1990; Rasi-Caldogno et al., 1993). In contrast, stimulation of the Ca²⁺-ATPase activity by exogenous CaM is easily detected in endomembrane fractions (Hsieh et al., 1991; Askerlund and Evans, 1992; Bush and Wang, 1995; Askerlund, 1996; Askerlund and Sommarin, 1996; Hwang et al., 1997). The affinity for CaM may thus be a crucial difference between the PM Ca²⁺-ATPase and Ca²⁺-ATPases of endomembranes, and is worthy of further investigation.

Washing of the column with 5 mm EDTA eluted about 25% of the loaded activity and as much as 40% of the CaM-dependent activity. The EDTA-eluted fraction, which was about 70-fold enriched in Ca²⁺-ATPase activity, was highly enriched in a 133-kD polypeptide, which was labeled by ¹²⁵I-CaM overlay, as well as by treatment with low concentrations of FITC. All of these characteristics closely match those determined for the PM Ca²⁺-ATPase in native PM (Rasi-Caldogno et al., 1995). A second band of about 120 kD was present in variable amounts in the EDTA-eluted fraction. This band, which was enriched in the fraction that did not bind to CaM-agarose, and which was labeled by FITC but not by ¹²⁵I-CaM overlay, has been previously identified as a proteolytic product of the PM Ca²⁺-ATPase (Rasi-Caldogno et al., 1995). Its presence in the purified Ca²⁺-ATPase fraction probably reflects the activity of a co-purifying protease activity; accordingly, it was most abundant in the fractions in which the Ca²⁺-ATPase activity was less stimulated by CaM (see Fig. 1B). Attempts to minimize proteolysis by means of various protease inhibitors have thus far been unsuccessful.

The yield and specific activity of the partially purified PM Ca²⁺-ATPase agree with the best reports on the endomembrane- localized, CaM-stimulated Ca²⁺-ATPase (Dieter and Marmè, 1981; Briars et al., 1988; Evans et al., 1989, 1992; Askerlund and Evans, 1992; Theodoulou et al., 1994; Askerlund, 1996; Hwang et al., 1997).

The PM Ca²⁺-ATPase cross-reacts with an antiserum against a putative chloroplast envelope Ca²⁺-ATPase of A. thaliana (Huang et al., 1993b), which is highly homologous to the tonoplast Ca²⁺-ATPase of cauliflower (Malmstrom et al., 1997), as well as to mammalian PM Ca²⁺-ATPases (Huang et al., 1993b). This result confirms that the same family of CaM-stimulated Ca²⁺-ATPases is expressed in different membranes of plant cells (Evans, 1994; Askerlund and Sommarin, 1996).

Availability of a purified Ca²⁺-ATPase of the PM is an essential prerequisite for cloning its gene. In fact, despite biochemical evidence indicating that CaM-stimulated Ca²⁺-ATPases are the most abundant in plant cells, screening of cDNA libraries with heterologous probes yielded only clones of putative Ca²⁺-ATPases homologous to the sarcoplasmic reticulum Ca²⁺-ATPase of mammals (Perez-Prat et al., 1992; Wimmers et al., 1992). This is probably due to the fact that heterologous probes mostly used for searching for P-type ATPases are based on the highly conserved nucleotide-binding site, which is likely to be variant in plant CaM-stimulated Ca²⁺-ATPases. In fact, plant CaM-stimulated Ca²⁺-ATPases differ from other P-type Ca²⁺-ATPases for their ability to use GTP or ITP as alternative substrates and for their very high sensitivity to inhibition by fluorescein derivatives (Rasi-Caldogno et al., 1987, 1989; Williams et al., 1990; Olbe and Sommarin, 1991; Carnelli et al., 1992; De Michelis et al., 1993), which act as competitive inhibitors with respect to the nucleoside-triphospate substrate (De Michelis et al., 1993).

Only very recently (Malmstrom et al., 1997) has a gene encoding the cauliflower tonoplast Ca²⁺-ATPase been identified using probes derived from the partial amino acid sequence of the purified enzyme: the deduced amino acid sequence shows high similarity to the mammalian PM Ca²⁺-ATPase, but has the striking characteristic that the putative CaM-binding domain appears localized in the N-terminal rather than in the C-terminal domain (Malmstrom et al., 1997). The putative chloroplast envelope Ca²⁺-ATPase, which is similar to the mammalian PM Ca²⁺-ATPase, lacks the C-terminal CaM-binding domain and has an extended N-terminal domain (Huang et al., 1993b); PM-type Ca²⁺-ATPases lacking the C-terminal CaM-binding domain have also been identified in the tonoplast of Saccharomyces cerevisiae (Cunningham and Fink, 1994) and Dictyostelium discoideum (Moniakis et al., 1995).

It will be interesting to find out whether displacement of the CaM-binding domain is a common feature of nonanimal PM-type Ca²⁺-ATPases, or if it is typical of the endomembrane-localized members of this family. Unfortunately, preliminary attempts to sequence the 133-kD polypeptide in our purified PM Ca²⁺-ATPase from radish seedlings have failed due to N-terminal blockage. Work is in progress to obtain partial amino acid sequences from tryptic fragments that are suitable for designing specific probes to isolate the PM Ca²⁺-ATPase cDNA.

	FOOTNOTES

   Received July 25, 1997; accepted October 27, 1997.

	ABBREVIATIONS

Abbreviations: Brij 58, polyoxyethylene-20-cetyl ether. CaM, calmodulin. FITC, fluorescein isothiocyanate. PM, plasma membrane.

	ACKNOWLEDGMENTS

We wish to thank Dr. N.E. Hoffman (Carnegie Institution of Washington, Stanford, CA) for the generous gift of the antiserum against the putative plastid envelope Ca²⁺-ATPase.

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