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Abstract
The advantages of the use of HPLC for the purification of the activated chloroplast F1 ATPase, for the measurement of enzymatic activity (by ADP-ATP separation on anion exchange column) and for ATP binding measurement (by the Hummel and Dreyer chromatographic method on gel filtration columns) are pointed out, by comparison with conventional methods such as ATP regenerating method or centrifugation on Sephadex columns.
The enzymatic data obtained at various ATP and Mg2+ concentrations and with different Mg2+ chelators and the ATP binding data led to the conclusion that metal free ATP is the true substrate of the enzyme and that Mg2+ is the activator. MgATP also binds to the enzymatic sites but with a weaker affinity (KD = 180 M) than ATP (KD = 14 M) and is not hydrolysed. Moreover, no cooperativity between the sites is needed to account for the results.
A schema is proposed in which the protonated form of the lysine of the enzymatic site interacts with the negatively charged γ phosphate of ATP. Complexation with Mg2+ would prevent this interaction.
These results and their interpretation are discussed and compared with those of the literature.