Identification and purification of the calcium-regulated Ca²⁺-ATPase from the endoplasmic reticulum of a higher plant mechanoreceptor organ

Erythrosin b, a potent inhibitor of the Ca²⁺-ATPases and the Ca²⁺-release channel (BCCl) in mechanosensitive tissue of Bryonica dioica Jacq., effectively suppresses of tendril's reaction to touch, suggesting that Ca²⁺-transporters are involved in signal transduction in this organ. The Ca²⁺-ATPase located in the endoplasmic reticulum (ER) represents a multiregulated enzyme that is stimulated by calmodulin (CaM). KCl and lysophospholipids. Limited proteolysis of ER-membranes by trypsin results in an irreversible activation of the Ca²⁺-ATPase and loss of the CaM sensitivity, presumably through removal of an autoinhibitory domain where CaM binds. Mild trypsination mimics the effects of CaM on V(max) and the affinity for Ca²⁺ and ATP. Irrespective of a trypsin treatment, the enzyme can be additionally stimulated by KCl and lysolipids, indicating that the sites of interaction for these effectors are not located in the domain removed by the protease. CaM-stimulated ATPase activity was purified from microsomal and ER fractions using a combination of CaM-affinity and anion-exchange chromatography. The isolated polypeptide was enzymatically active, showed a calcium-dependent mobility-shift in SDS-PAGE from 109 kDa in the absence of Ca²⁺ to 104 kDa in the presence of 10 mM CaCl₂ and could be radiolabeled with [³⁵S]-CaM. The characteristics of the purified enzyme remained closely similar to those of the ER-bound Ca²⁺-transporting activity, including the enzymatic data, CaM stimulation, and the sensitivity towards a range of inhibitors.