CHARACTERIZATION, FUNCTIONAL RECONSTITUTION AND ACTIVATION BY FUSICOCCIN OF A CA-2+-ATPASE FROM CORYDALIS-SEMPERVIRENS PERS CELL-SUSPENSION CULTURES
Cell suspension cultures of Corydalis sempervirens have proven ideal for the study of fusicoccin action [Schulz et al. (1990) Planta 183: 83] and express the fusicoccin-binding protein as well as a plasma membrane H+-ATPase which is activated by the fungal toxin. Microsomal vesicles prepared from these cells accumulate Ca2+ in the presence of Mg-ATP. The protonophore carbonylcyanide m-chlorophenylhydrazone did not inhibit the Mg-ATP dependent Ca2+-transport into the vesicles. This process is thus due to the activity of at least one primary active, ATP-driven, Ca2+-pump. The enzyme was characterized in deta It has a pH optimum of 7.2, an apparent K(m) of 0.3 mM (ATP), 12-mu-m (Ca2+), accepts ATP > ITP almost-equal-to GTP > CTP almost-equal-to UTP, and is strongly (K(i), app 0.75-mu-M) inhibited by erythrosine B but less so (K(i), app 95-mu-M) by orthovanadate. These characteristics are typical for the plasma membrane Ca2+-ATPase characterized from differentiated tissues [Graf and Weiler (1990) Physiol. Plant. 75: 634]. Fusicoccin activates the erythrosine-sensitive Ca2+-pump by lowering its K(m) for ATP, when added to living cells prior to tissue homogenization. Thus, fusicoccin appears to activate at least two ion-translocating ATPases in one and the same tissue, suggesting that the toxin's mechanism of action is complex and not restricted to activation of the H+-ATPase. FC has no effect when administered to microsomes. The microsomal enzyme was solubilized and reconstituted into asolectin liposomes in functional form. The reconstituted, erythrosine sensitive Ca2+-ATPase was insensitive to fusicoccin. Thus, components essential for toxin action are either lost or inactivated during subcellular fractionation. It is likely that FC action requires soluble components.
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