Characterisation of a Ca2+-dependent ATP hydrolysis associated with the vacuolar membrane of wheat roots |
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Authors: | Gethyn J Allen Francisco Morais Shelagh Muir R Gareth Wyn Jones Roger A Leigh |
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Institution: | The Plant Laboratory, Dept of Biology, Univ. of York, P.O. Box 373, York YOI 5YW, UK;Depto de Biologia Vegetal, Blaca C2 Campo Grande, Univ. de Lishoa, Portugal;School of Biological Sciences, Univ. of Wales, Bangor, Gwynedd LL57 2UW, UK;Biochemistry and Physiology Dept. Inst. of Arable Crrops Research, Rothamsted Experimental Station, Harpenden, Hertfordshire AL5 2JQ, UK |
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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. |
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Keywords: | Adenosine triphosphatase (ATPase) calcium ion transport Triticum aestivum (wheat) vacuolar membrane (tonoplast) |
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