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+-rele ase channel (BCCl) in mechanosensitive tissue of Bryonia dioica Jacq., effectively suppresses a 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 res ults in an irreversible activation of the Ca2+-ATPase and loss of the CaM sensitivity, presumably through removal of an autoinhibitory domai n where CaM binds. Mild trypsination mimics the effects of CaM on V-ma x and the affinity for Ca2+ and ATP. Irrespective of a trypsin treatme nt, the enzyme can be additionally stimulated by KCl and lysolipids, i ndicating that the sites of interaction for these effecters are not lo cated in the domain removed by the protease. CaM-stimulated ATPase act ivity was purified from microsomal and ER fractions using a combinatio n of CaM-affinity and anion-exchange chromatography. The isolated poly peptide was enzymatically active, showed a calcium-dependent mobility- shift in SDS-PAGE from 109 kDa in the absence of Ca2+ to 104 kDa in th e presence of 10 mM CaCl2 and could be radiolabeled with [S-35]-CaM. T he characteristics of the purified enzyme remained closely similar to those of the ER-bound Ca2+-transporting activity, including the enzyma tic data, CaM stimulation, and the sensitivity towards a range of inhi bitors.