Reconstitution and Characterization of a Calmodulin-Stimulated Ca²⁺-Pumping ATPase Purified from Brassica oleracea L.

Abstract

Purification and functional reconstitution of a calmodulin-stimulated Ca2"-ATPase from cauliflower (Brassica oleracea L.) is described. Activity was purified about 120-fold from a microsomal fraction using calmodulin-affinity chromatography. The purified fraction showed a polypeptide at 115 kD, which formed a phosphorylated intermediate in the presence of Ca2 , together with a few polypeptides with lower molecular masses that were not phosphorylated. The ATPase was reconstituted into liposomes by 3-([cholamidopropylJ-dimethylammonio-)1-propanesulfonate (CHAPS) dialysis. The proteoliposomes showed ATP-dependent Ca21 uptake and ATPase activity, both of which were stimulated about 4-fold by calmodulin. Specific ATPase activity was about 5 &mol min-' (mg protein)-', and the Ca21/ATP ratio was 0.1 to 0.5 when the ATPase was reconstituted with entrapped oxalate. The purified, reconstituted Ca2 -ATPase was inhibited by vanadate and erythrosin B, but not by cyclopiazonic acid and thapsigargin. Activity was supported by ATP (100%) and GTP (50%) and had a pH optimum of about 7.0. The effect of monovalent and divalent cations (including Ca21) on activity is described. Assay of membranes purified by two-phase partitioning indicated that approximately 95% of the activity was associated with intracellular membranes, but only about 5% with plasma membranes. Sucrose gradient centrifugation suggests that the endoplasmic reticulum is the major cellular location of calmodulin-stimulated Ca21-pumping

A calmodulin‐stimulated Ca2+‐ATPase from plant vacuolar membranes with a putative regulatory domain at its N‐terminus1

“…Second, a synthetic peptide corresponding to this region bound strongly to CaM. Whereas binding of the native enzyme to CaM is strictly dependent on Ca2+ [6], the synthetic peptide bound to CaM in both the presence and absence of Ca2+ (Fig. 4A).…”

“…The properties of the purified enzyme have been described [5,6]. Trypsin digestion and sequencing of the resulting peptides were carried out by Bo Ek, Uppsala Genetic Center, Swedish University of Agricultural Sciences.…”

Modulation of an Intracellular Calmodulin-Stimulated Ca2+-Pumping ATPase in Cauliflower by Trypsin (The Use of Calcium Green-5N to Measure Ca2+ Transport in Membrane Vesicles)

“…In contrast, CaM-stimulated Ca2+-ATPases in plants have been reported to be present in the ER (Hsieh et al, 1991;Askerlund and Evans, 1992;Gilroy and Jones, 1993;Liss and Weiler, 1994;Logan and Venis, 1995), the vacuolar membrane (Gavin et al, 1993), and the chloroplast envelope (Nguyen and Siegenthaler, 1985), as well as the plasma membrane (Robinson et al, 1988;Rasi-Caldogno et al, 1993, 1995 Olbe and M. Sommarin, unpublished data; for a review, see Askerlund and Sommarin, 1996). CaM-stimulated Ca2+-ATPase in cauliflower (Brassica olevacea L.) was earlier found to be located mainly in a low-density intracellular membrane fraction that was suggested to be the ER, but also in the plasma membrane (Askerlund and Evans, 1992). More recent investigations have shown that the distribution of the intracellular CaM-stimulated Ca2+-ATPase in cauliflower correlates much better with vacuolar membrane markers than with ER markers, suggesting that it may be located in the vacuolar membrane (P. Askerlund, unpublished data).…”

Distinction between Endoplasmic Reticulum-Type and Plasma Membrane-Type Ca2+ Pumps (Partial Purification of a 120-Kilodalton Ca2+-ATPase from Endomembranes)

“…So far only a few CaM-stimulated Ca2+-ATPases (of 120 and 111-115 kD) have been purified from low-density endomembranes of plants (this work; Askerlund and Evans, 1992;Askerlund, 1996); however, numerous reports of CaM-stimulated Ca2+ transport in endomembranes (Brauer et al, 1990;Hsieh at al., 1991;Gilroy and Jones, 1993;Bush and Wang, 1995) illustrate that this is a common feature in plants. Recent studies in corn roots have attributed a low-density, CaM-stimulated Ca2+ pump to the vacuole alone (Gavin et al, 1993;Pfeiffer and Hager, 1993); however, in the tendrils of Bryonia, CaMstimulated Ca'+ transport was found in purified ER after ribosomes had been stripped (Liss and Weiler, 1994).…”

“…The animal ER-type Ca2' pump is distinguished from the animal PM-type Ca2+ pump by its sensitivity to thapsigargin or cyclopiazonic acid, and by its insensitivity to CaM (Schatzmann, 1989;Siedler et al, 1989;Carafoli 1992); however, the results from plants are more ambiguous. For example, Ca2+ pump activity in the ER fraction is insensitive to CaM in wheat aleurone layers (Bush and Wang, 1995) and in garden cress (Buckhout, 1984), yet other studies show high CaM-stimulated Ca2+ transport in endomembranes, including the ER from carrot (Daucus carota) suspension cells (Hsieh et al, 1991), cauliflower florets (Askerlund and Evans, 1992), corn roots (Brauer et al, 1990), and tendrils (Liss and Weiler, 1994).…”