Comparative study on recombinant chloroplastic and cytosolic ascorbate peroxidase isozymes of spinach

The spinach stromal, thylakoid-bound, and cytosolic ascorbate peroxidase isozymes (EC 1.11.1.11) were overexpressed in Escherichia coli, and their enzymatic properties were compared with the respective native isozymes. The purification of the recombinant stromal and cytosolic ascorbate peroxidases using the conventional column chromatography yielded 0.73 and 2.2 mg of protein/liter of bacteria culture with enzyme activities of 800 and 486 micromol min-1 mg protein-1, respectively. In every respect, the recombinant stromal, thylakoid-bound, and cytosolic ascorbate peroxidase isozymes exhibited identical enzymatic properties with each native isozyme. Specifically, the recombinant stromal and thylakoid-bound ascorbate peroxidase isozymes showed high utilization of ascorbate as an electron donor and had a very short lifetime in ascorbate-depleted medium. Polyclonal antibodies raised against both purified recombinant stromal and cytosolic ascorbate peroxidase isozymes were prepared. Both antibodies showed a cross-reaction with the recombinant and native ascorbate peroxidase isozymes.