CHARACTERIZATION OF A CA2-DEPENDENT ATP HYDROLYSIS ASSOCIATED WITH THE VACUOLAR MEMBRANE OF WHEAT ROOTS()

Microsomal fractions from wheat tissues exhibit a higher level of ATP hydrolytic activity in the presence of Ca2+ than Mg2+. Here we charact erise the Ca2+-dependent activity from roots of Triticum aestivum (cv. Troy) and investigate its possible function. Ca2+-dependent ATP hydro lysis 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+, C-l--ATPase on continu ous sucrose gradients indicating that it is associated with the vacuol ar membrane. Ca2+ -dependent ATP hydrolysis can be distinguished from an inhibitory effect of Ca2+ on the plasma membrane K+, Mg2+-ATPase fo llowing microsomal membrane separation using aqueous polymer two phase partitioning. The Ca2+-dependent activity is stimulated by free Ca2with a K-m of 8.1 mu M in the absence of Mg2+ ([CaATP] = 0.8 mM). Vacu olar membrane vesicle preparations contain a higher Ca2+-dependent tha n Mg2+-dependent ATP hydrolysis, although the two activities are not d irectly additive. The nucleotide specificity of the divalent ion-depen dent activities in vacuolar membrane-enriched fractions was low, hydro lysis of CTP and UTP being greater than ATP hydrolysis with both Ca2and Mg2+. The Ca2+-dependent activity did discriminate against dinucle otides, and mononucleotides, and failed to hydrolyse phosphatase subst rates. Despite low nucleotide specificity the Mg2+-dependent activity functioned as a bafilomycin sensitive H+-pump in vacuolar membrane ves icles. Ca2+-dependent ATP hydrolysis was not inhibited by the V-, P-, or F-type ATPase inhibitors bafilomycin, vanadate and azide, respectiv ely, 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 vesic les. No H+ or Ca2+ translocating activity was observed under condition s when the Ca2+-dependent ATP hydrolysis was active.