Plasma Membrane Ca-ATPase of Radish Seedlings

Abstract

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...

A Single Gene May Encode Differentially Localized Ca2+-ATPases in Tomato.

“…These data are consistent with the lack of a calmodulin binding domain in the deduced LCA amino acid sequence (Wimmers et al, 1992). In previous studies, calmodulin sensitivity was reported for the PM Ca2+-ATPases from maize (Robinson et al, 1988;Briars and Evans, 1989), red beet roots (Williams et al, 1990), carrot cell cultures (Hsieh et al, 1991), and radish seedlings (Rasi-Caldogno et al, 1992. In contrast, calmodulin had no effect on the PM Ca2+-ATPases from pea roots (Butcher and Evans, 1987) and Commelina communis leaves (Siebers et al, 1990).…”

Plasma Membrane Ca-ATPase of Radish Seedlings

“…The pH sensitivity of the PM CaATPase affinity for Ca2", and thus of its activity at physiological Ca2+ concentrations, might constitute a linkage between Ca2" and pH homeostasis in the plant cell (2,12). These points will be discussed further in the accompanying paper with respect to the role of calmodulin in regulating the activity of the PM Ca-ATPase (19).…”

Identification of the Plasma Membrane Ca2+-ATPase and of Its Autoinhibitory Domain

“…In fact, it is half-saturated by Ca2+ in the micromolar range and has a very high affinity for MgATP (Rasi-Caldogno et al, 1989;Carnelli et al, 1992;De Michelis et al, 1992. Finally, the PM Ca2+-ATPase activity is regulated by CaM, which strongly increases the V,,, of the reaction, thus making the enzyme up-regulated by an increase of cytosolic Ca2+ and Ca2+-CaM concentrations (Malatialy et al, 1988;Robinson et al, 1988;Williams et al, 1990;Erdei and Matsumoto, 1991;Carnelli et al, 1992;Rasi-Caldogno et al, 1992;De Michelis et al, 1993).…”