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.

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The effect of calmodulin on the activity of the plasma membrane Ca-ATPase was investigated on plasma membranes purified from radish (Raphanus satlvus L.) seedlings. Calmodulin stimulated the hydrolytic activity and the transport activity of the plasma membrane Ca-ATPase to comparable extents in a manner dependent on the free Ca2+ concentration. Stimulation was marked at low, nonsaturating Ca2 concentrations and decreased increasing Ca2+, so that the effect of calmodulin resulted in an increase of the apparent affinity of the enzyme for free Ca2+. The pattem of calmodulin stimulation of the plasma membrane CaATPase activity was substantially the same at pH 6.9 and 7.5, in the presence of ATP or ITP, and when calmodulin from radish seeds was used rather than that from bovine brain. At pH 6.9 in the presence of 5 micromolar free Ca2+, stimulation of the plasma membrane Ca-ATPase was saturated by 30 to 50 micrograms per milliliter bovine brain calmodulin. The calmodulin antagonist calmidazolium inhibited both basal and calmodulin-stimulated plasma membrane Ca-ATPase activity to comparable extents.CaM2 is a ubiquitous regulatory protein involved in regulatory events in animal and plant cells (22). The hypothesis that CaM regulated the activity ofthe PM Ca-ATPase ofplant cells, similarly to that observed for the erythrocyte Ca-ATPase (8), has been proposed since 1980 (13). However, the available data on the effect of CaM on the activity of the PM CaATPase of plants have been until now fragmentary and sometimes contradictory (reviewed in refs. 5, 12, 15).The data on crude microsomes or on nonwell identified membrane fractions should be taken with caution, because CaM has been reported to stimulate also the active Ca2" transport systems of the tonoplast (1, 21, 29) and of the endoplasmic reticulum (2).A CaM-stimulated Ca-ATPase purified from maize microsomes by CaM-affinity chromatography (3,4,14,16) been proposed to represent the PM Ca-ATPase on the basis ofits structural and immunological similarities with the erythrocyte Ca-ATPase (3, 4, 16). However, to our knowledge, its localization at the PM has not been directly demonstrated, nor has its involvement in the active transport of Ca2" been shown by reconstitution in proteoliposomes. Moreover, CaMstimulation of this purified enzyme could be observed only when ATP was supplied as a substrate (15), whereas the plant PM Ca-ATPase is able to utilize also ITP or GTP as substrates (5,12,15) and stimulation of its transport activity by CaM in PM from red beet has been observed also in the presence of GTP (28).The response to CaM of the PM Ca-ATPase in PM isolated from different materials is quite variable, and in some instances no significant effect ofCaM could be observed (7, 18-21, 24-26, 28, 29). A detailed analysis of the effect of CaM on the plant PM Ca-ATPase is lacking, because the only attempt to characterize the CaM-stimulated activity (26) was severely limited by the difficulties of analyzing PM Ca2`-dependent ATPase activity in the presence ofthe much hi...

In this work, we exploited the capability of the plasma membrane Ca-ATPase to utilize ITP as a substrate to study its characteristics in plasma membrane vesicles purified from radish (Raphanus sativus L.) seedlings. The Demonstrating the hydrolytic activity of the PM Ca-ATPase in native PM vesicles has proven difficult, due to the simultaneous operation of the much more active PM H+-ATPase, which is inhibited by Ca2", already in the micromolar range (5). Taking advantage of the different pH optima of the PM H+-ATPase (approximately 6.5) and of the PM CaATPase (7.0-7.5), we have shown that a Ca2e-dependent ATPase activity inhibited by submicromolar EB is localized at the PM ofradish seeds (21). The biochemical characteristics ofthis activity so far investigated, as well as its functional mol wt determined by radiation-inactivation, are very similar to those determined for ATP-dependent Ca2`uptake into PM vesicles, indicating that it represents the hydrolytic activity of the PM Ca-ATPase (21-23). However, the simultaneous operation of the Ca2t-inhibited PM H+-ATPase is an important source of error in these measurements because any treatment can be expected to influence both activities in the same or in the opposite sense. Because the PM H+-ATPase is highly specific for ATP as a substrate (5), it should be possible to use ITP or GTP as a substrate to measure the hydrolytic activity of the PM Ca-ATPase in native PM vesicles, minimizing complications arising from the simultaneous operation of the H+-ATPase. Analysis of GTP-dependent Ca2" uptake into PM vesicles from red beet has shown that GTP is a useful means to probe the transport function of the PM Ca-ATPase (27), but no data are available on its suitability to measure the hydrolytic activity of this enzyme. Because the presence of regulatory GTP-binding

The effects of calmodulin and of controlled trypsin treatments on the activity of the Caz+ pump were investigated in plasma membrane purified from radish (Raphanus sativus 1.) seedlings. Treatment of the plasma membrane with ethylenediaminetetraacetate (EDTA), which removed about two-thirds of the plasma membrane-associated calmodulin, markedly increased the stimulation of the Caz+ pump by calmodulin. In EDTA-treated plasma membrane, stimulation by calmodulin of the Caz+ pump activity was maximal at low free Caz+ (2-5 p~) and decreased with the increase of free CaZ+ concentration. l h e CaZ+ pump activity was stimulated also by a controlled treatment of the plasma membrane with trypsin: the effect of trypsin treatment depended on the concentration of both trypsin and plasma membrane proteins and on the duration of incubation. Stimulation of the CaZ+ pump activity by trypsin treatment of the plasma membrane was similar to that induced by calmodulin both in extent and in dependence on the free CaZ+ concentration in the assay medium. Moreover, the Caz+ pump of trypsin-treated plasma membrane was insensitive to further stimulation by calmodulin, suggesting that limited proteolysis preferentially cleaves a regulatory domain of the enzyme that is involved in its activation by calmodulin.In plant cells, the extrusion of Ca2+ from the cytoplasm to the apoplast is catalyzed by a Mg-ATP-dependent Ca2+ pump. During the last few years, the transport and hydrolytic activities of the plant PM Ca2+ pump have been characterized in some detail both in native PM vesicles and in proteoliposomes reconstituted with the solubilized and partially purified enzyme (reviewed in Briskin, 1990; Evans et al., 1991;De Michelis et al., 1992a). The most striking characteristics of the plant PM Ca2+ pump are its ability to use ITP or GTP besides ATP as a substrate (Williams et al., 1990; Camelli et al., 1992) and its high sensitivity to inhibition by fluorescein derivatives such as erythrosin B (Rasi-Caldogno et al., 1987, This work was supported by a grant of the Italian Ministry for University and Scientific and Technologic Research (40% quote) and by the Italian Ministry of Agriculture in the framework of the project 'Resistenze genetiche delle piante agrarie agli stress biotici ed abiotici."