APPLICATION OF HPLC TO THE STUDY OF THE CHLOROPLAST ATPASE MG2+ DEPENDENT MECHANISM

The determination by HPLC of released ADP from ATP by chloroplast ATPase (CF1) is described. The enzymatic rate measured by this method is well defined, over several minutes. In the case of epsilon subunit-depleted CF1 or activated CF1, the rate is proportional to the enzyme concentration, the steady state theory is followed and the K(m) and V(m) constants have been calculated. The enzymatic activity of CF1 is inhibited by endogenous epsilon subunit and the inhibition constant has been measured. The influences of ionic strength, pH, magnesium ion, phosphate and ADP concentrations have been studied and the results obtained by this method have been compared to previously reported data based on rate determination of released phosphate.