Simultaneous Kinetic Measurements of Adenine Nucleotides in Isolated Vacuoles by HPLC Technique
Numerous substances are contained in plant vacuoles. Some of them are highly concentrated like sugar and organic acids which may be representative of the accumulation processes occurring in vacuoles. There are many data in the literature suggesting that transport is energy-dependent and linked to protons pumping by a tonoplast ATPase (for a review see Sze, 1985). However, in a complex system like plant vacuoles, the hydrolysis of ATP may be catalyzed not only by ATPases but also by other phosphorolytic enzymes which are not involved in an energization process of the membrane. Thus, the estimation of ATPases activities is an important and difficult point, since the results from many usual titration methods are more or less specific of the ATP hydrolysis into ADP (Fiske and Subbarow, 1925; Strehler, 1965; Adam, 1965). Whether the titration is specific or not for ATP, the data show different aspects of the adenine nucleotide hydrolysis catalyzed by tonoplast enzymes (Dupaix et al., 1986; Hill et al., 1986). It is possible to remove this uncertainty by simultaneously monitoring the evolvement of the adenine nucleotides in the presence of purified tonoplast vesicles by the suitable reverse phase HPLC technique described below.
KeywordsAdenine Nucleotide Ammonium Molybdate Adenylate Kinase Potassium Dihydrogen Phosphate Acer Pseudoplatanus
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