Solubilization and Reconstitution of Ca<sup>2+</sup> Pump from Corn Leaf Plasma Membrane

Kasai, Minobu; Muto, Shoshi

doi:10.1104/pp.96.2.565

Cited by 14 publications

(6 citation statements)

References 17 publications

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“…The analogy with animals would suggest that the activity should be present in the plasma membrane only, and both this and several other investigations indicate that the plant plasma membrane may contain a CaM-stimulated Ca2" pump (Table III; 5,12,30,31). In other studies, no CaM stimulation of plasma membrane ATPase activity or Ca2" uptake has been found (15,16,21). In the present work, however, we clearly show that the major part of the ATP-dependent CaM-stimulated Ca2" uptake is not in the plasma membrane, because only about 6% of the total activity was associated with the plasma membrane fraction and the remaining part with the intracellular membrane fraction (Table III).…”

Section: Further Characterization Of the Reconstituted Ca2+-atpasementioning

confidence: 56%

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

Askerlund¹,

Evans²

1992

Plant Physiol.

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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

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Section: Further Characterization Of the Reconstituted Ca2+-atpasementioning

confidence: 56%

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

Askerlund¹,

Evans²

1992

Plant Physiol.

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“…266. 2 Abbreviations: CaM, calmodulin; PM, plasma membrane; BTP, bis-tris propane, (1 ,3-bis(tris(hydroxymethyl)methylamino)-propane; EB, erythrosin B; W7, (N-(6-aminohexyl)-5-chloro-l-naphthalenesulfonamide; W5, N-(6-aminohexyl)-l-naphthalenesulfonamide; Brij 58, polyoxyethylene 20 cetyl ether. been proposed to represent the PM Ca-ATPase on the basis ofits structural and immunological similarities with the erythrocyte Ca-ATPase (3,4,16).…”

mentioning

confidence: 99%

Plasma Membrane Ca-ATPase of Radish Seedlings

Rasi‐Caldogno¹,

Carnelli²,

Michelis³

1992

Plant Physiol.

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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...

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“…A prolonged increase in the concentration of free Ca 2+ in the cytosol after cell response is dangerous for cells (Rasmussen and Barret 1984). Ca 2 § activated ATPase, with its high affinity for Ca 2 § and low pumping capacity, is known to remove Ca 2 § from plant cells (Kasai and Muto 1991). In addition, calmodulin and other Ca2+-binding proteins can bind Ca 2 § within some intracellular compartments (Marme 1985, Navazio et al 1995.…”

Section: Introductionmentioning

confidence: 99%

The oppositely directed Ca2+ and Na+ transmembrane transport in algal cells

et al. 2000

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Summary. The countertransport of Ca 2+ and Na + across the membranes of the unicellular fresh-water alga Chlamydornonas reinhardtii CW-15 and two Dunaliella species differing in salt tolerance was studied. All algae used are devoid of cell walls. The calcium uptake by two Dunaliella species depended markedly on the intracellular sodium concentration. This calcium uptake was accompanied by Na + release. For 15 and 30 s after artificial gradient formation (Na+int greater than Na+ext), the ratio of released Na + to absorbed Ca 2+ was 3 : 1 and 4 : 1, respectively. For the extremely halotolerant D. salina, the apparent Michaelis constant of the Ca 2+ uptake was 33 gM, and for the marine halotolerant alga D. maritima, it was equal to 400 gM, presuming more efficient Na+-for-Ca 2+ exchange in 19. salina cells. Ouabain, an inhibitor of Na+/K+-ATPase, suppressed Na + transfer by 25%, whereas the agents blocking Ca >-Channels did not affect the transport of Ca 2+ and Na +. The oppositely directed transmembrane Ca 2+ and Na + transfer was shown to depend on the external concentrations of Na + and H +. In the fresh-water alga C. reinhardtii CW-15 (Na+ext greater than Na+in0, the direction of Ca 2+ and Na + fluxes across the plasma membrane was opposite to those described for Dunaliella cells. The results obtained point to the ability of the Na+-Ca 2+ exchanger function in plasma membranes of algal ceils.

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Solubilization and Reconstitution of Ca²⁺ Pump from Corn Leaf Plasma Membrane

Cited by 14 publications

References 17 publications

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

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

Plasma Membrane Ca-ATPase of Radish Seedlings

The oppositely directed Ca2+ and Na+ transmembrane transport in algal cells

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Solubilization and Reconstitution of Ca2+ Pump from Corn Leaf Plasma Membrane

Cited by 14 publications

References 17 publications

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

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

Plasma Membrane Ca-ATPase of Radish Seedlings

The oppositely directed Ca2+ and Na+ transmembrane transport in algal cells

Contact Info

Product

Resources

About

Solubilization and Reconstitution of Ca²⁺ Pump from Corn Leaf Plasma Membrane

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

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