Characterisation of a Ca²⁺‐dependent ATP hydrolysis associated with the vacuolar membrane of wheat roots

Abstract

Microsomal fractions from wheat tissues exhibit a higher level of ATP hydrolytic activity in the presence of Ca2+ than Mg2+. Here we characterise the Ca2+‐dependent activity from roots of Triticum aestivum lev. Troy) and investigate its possible function. Ca2+‐dependent ATP hydrolysis in the microsomal fraction occurs over a wide pH range with two slight optima at pH 5.5 and 7.5. At these pHs the activity co‐migrates with the major peak of nitrate‐inhibited Mg2+. Cl‐ATPase on continuous sucrose gradients indicating that it is associated with the vacuolar membrane. Ca2+‐dependent ATP hydrolysis can be distinguished from an inhibitory effect of Ca2+ on the plasma membrane K+, Mg2+‐ATPase following microsomal membrane separation using aqueous polymer two phase partitioning. The Ca2+‐dependent activity is stimulated by free Ca2+ with a Km of 8.1 μM in the absence of Mg2+ ([CaATP] = 0.8 mM). Vacuoiar membrane vacuolar preparations contain a higher Ca2+‐dependent than Mg2+‐dependent ATP hydrolysis, although the two activities are not directly additive. The nucleotide specificity of the divalent ion‐dependent activities in vacuolar membrane‐enriched fractions was low. hydrolysis of CTP and UTP being greater than ATP hydrolysis with both Ca2+ and Mg2+ The Ca2+‐dependent activity did discriminate against dinucleotides, and mononucleotides. and failed to hydrolyse phosphatase substrates. Despite low nucleotide specificity the Mg2+‐dependent activity functioned as a bafilomycin sensitive H+‐pump in vacuolar membrane vesicles. Ca2+‐dependent ATP hydrolysis was not inhibited by the V‐, P‐, or F‐type ATPase inhibitors bafilomycin. vanadate and azide, respectively. nor by the phosphatase inhibitor molybdate, but was inhibited 20% at pH 7.5 by K+. Possible functions of Ca2+‐dependent hydrolysis as a H+‐pump or a Ca2+‐pump was investigated using vacuolar membrane vesicles. No H+ or Ca2+ translocating activity was observed under conditions when the Ca2+‐dependent ATP hydrolysis was active.