Heterogeneity of the vacuolar pyrophosphatase protein fromChenopodium rubrum |
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Authors: | W Kranewitter R Gehwolf M Nagl W Pfeiffer and F -W Bentrup |
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Institution: | (1) Institut für Pflanzenphysiologie, Universit?t Salzburg, Hellbrunnerstrasse 34, A-5020 Salzburg, Austria |
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Abstract: | Summary Activities of the tonoplast ATPase (V-ATPase EC 3.6.1.3) and PPase (V-PPase EC 3.6.1.1) provide the proton gradient driving
the accumulation of various metabolites, organic and inorganic ions in the plant vacuole. We used anion exchange chromatography,
liquid-phase isoelectric focusing (IEF), and continuous-elution native polyacrylamide gel electrophoresis (preparative PAGE)
to enrich the V-PPase from solubilized tonoplast proteins from suspension cultured cells ofChenopodium rubrum L.The fractions were identified by their enzymatic activity, sensitivity towards the specific PPase inhibitor aminomethylenediphosphonate,
apparent molecular weight, and immunological reactivity with an antibody raised against mung bean V-PPase. All these different
methods used for the separation of solubilized tonoplast proteins revealed the existence of two physically separable V-PPase
proteins exhibiting substrate specific enzymatic activity and 66 kDa apparent molecular weight after sodium dodecyl sulfate(SDS)-PAGE.
The isoelectric points of the active V-PPase forms were 5.05 and 5.48 (V-ATPase 6.1). On the basis of the observation of high
recoveries of enzymatic activity after different preparations we suggest that the V-PPase proteins separated may represent
physiologically occurring forms of the enzyme which cannot be distinguished by SDS-PAGE and Western blot. |
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Keywords: | Anion exchange chromatography Chenopodium rubrum Continuous-elution native-polyacrylamide-gel electrophoresis Liquid-phase isoelectric focusing Vacuolar pyrophosphatase |
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