51] ATP-ADP exchange enzyme from spinach chloroplasts

Abstract

Publisher Summary This chapter discusses the determination of ATP–ADP exchange enzyme from spinach chloroplasts. The enzyme catalyzes the transfer of the terminal phosphate from ATP to ADP. When 14 C-labeled ADP is used as the acceptor, the rate of the reaction can be calculated from the distribution of label between ATP and ADP as a function of time, provided the reaction has not proceeded to equilibrium (uniform labeling). The method is a simplification of that involved in mitochondrial exchange enzyme, in that the reaction mixture does not have to be chromatographed quantitatively because only the ratio of label in ATP to that in ADP is required. The use of labeled ADP is preferred over that of labeled ATP because any breakdown products of the former do not interfere with the assay, while the latter gives rise upon storage to labeled ADP. This gives a large zero-time value for ADP- 14 C and requires a correction in the rate calculation. In the course of purification of the ATP–ADP exchange enzyme, spinach chloroplasts from 3 kg of leaves are isolated using a grinding medium containing 0.4 M sucrose, 25 m M Tris·HCl, pH 8.0, and 10 m M NaCl.