Properties and Regulation of Adenylate Kinase From Zea mays Leaf Operating in C₄ Pathway Photosynthesis

Abstract

Adenylate kinase from the mesophyll cells of Zea mays was partially purified and its kinetic and regulatory properties determined. Fractionation of mesophyll cell extracts by (NH₄)₂SO₄ precipitation, filtration on Sephacryl S-200 and diethylaminoethylcellulose chromatography gave a fraction purified about 90-fold. For the reaction in the direction of ADP synthesis (forward reaction), activity was influenced by a complex interaction between Mg²⁺, ATP and pH. Optimal activity was observed with a Mg²⁺ to ATP ratio of about unity and the pH optimum under these conditions was about 8.0. With an excess of Mg²⁺, activity was reduced and the pH optimum was shifted to lower values. Varying ATP or AMP in a lower range of concentrations gave simple Michaelis- Menten responses. However, at higher concentrations there was a complex response to increasing ATP, and AMP became inhibitory. For the reverse reaction the K_m (ADP) was about 70 µM and the pH optimum was between 8.0 and 8.5. AMP was a strong inhibitor of the reverse reaction (competitive with respect to ADP), the K_I being about 40 µM. Several other metabolites tested had no effect on adenylate kinase activity. Adenylate kinase extracted from washed mesophyll chloroplasts had essentially identical properties.