Characterisation of a salt-stimulated ATPase activity associated with vacuoles isolated from storage roots of red beet (Beta vulgaris L.) |
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Authors: | Robert R Walker Roger A Leigh |
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Institution: | (1) Botany School, University of Cambridge, Downing Street, CB2 3EA Cambridge, UK;(2) Present address: Division of Horticultural Research, C.S.I.R.O., 3505 Merbein, Vic., Australia;(3) Present address: Soils and Plant Nutrition Department, Rothamsted Experimental Station, AL5 2JQ Harpenden, Hertfs, UK |
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Abstract: | Ion stimulation and some other properties of an ATPase activity associated with vacuoles isolated from storage roots of red beet (Beta vulgaris L.) have been determined. The ATPase had a specific requirement for Mg2+ and in the presence of Mg2+ it was stimulated by salts of monovalent cations. The degree of stimulation by monovalent salts was influenced mainly by the anion and the order of effectiveness of the anions tested was Cl->HCO
3
-
>Br->malate>acetate>SO
4
2-
. For any given series of anions the magnitude of the stimulation obtained was influenced by the accompanying cation (NH
4
+
Na+>K+). This cation effect was abolished by 0.01% (v/v) Triton X-100 and it is suggested that it is the result of different permeabilities of membrane vesicles to the cations. There was no evidence of synergistic stimulation of the ATPase by mixtures of Na+ and K+. KCl- and NaCl-stimulation was maximal with salt concentrations in the range 60–150 mM. The true substrate of the enzyme was shown to be MgATP. It was shown that KCl stimulation was the result of an increase in Vmax rather than a change in the affinity of the enzyme for MgATP. The ATPase was inhibited by N,N -dicyclohexylcarbodiimide, diethylstilbestrol, mersalyl and KNO3 but other inhibitors tested (azide, oligomycin, orthovanadate, K3Cr(oxalate)6] and ethyl-3-3-dimethylaminopropyl]carbodiimide) were without effect or caused only partial inhibition at the highest concentration tested. The ATPase activity was equally distributed between pellet and supernatant fractions obtained after the subfractionation of vacuoles but the properties of the ATPase in each fraction were the same. It is suggested that beet vacuoles possess only one ATPase. The properties of the ATPase are compared with those of ATPases associated with other plant membranes and organelles and its possible role in transport at the tonoplast is discussed.Abbreviations ATPF
free ATP
- ATPT
total ATP
- BSA
bovine serum albumen
- DCCD
N,N -dicyclohexylcarbodiimide
- DES
diethylstilbestrol
- DNP
2,4-dinitrophenol
- EDAC
ethyl-3-(3-dimethylaminopropyl)carbodiimide
- Km
apparent Michaelis constant
- MgATP
complex of Mg2+ and ATP
- Mg
F
2+
free Mg2+
- Mg
T
2
total Mg2+
- MES
2-(N-Morpholino)ethanesulphonic acid
- Na2EDTA
disodium ethylenediaminetetraacetic acid
- NEM
N-ethylmaleimide
- Pi
inorganic phosphate
- TCA
trichloroacetic acid
- Tris
tris(hydroxymethyl)methylamine
- Vmax
maximum velocity |
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Keywords: | ATPase Beta Vacuole |
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