Biochemical characterization of purified apyrases in Arabidopsis thaliana

Apyrases (nucleoside triphosphate-diphosphohydrolases) are enzymes that regulate the concentration of NTP and NDP nucleotides in cells by removing their terminal phosphate. Two of the 7 apyrases in Arabidopsis, APY1 and APY2, are 87% identical in primary structure and play important roles in regulating auxin transport and plant growth. To clarify how these apyrases function, this report confirms their localization in purified nuclei and characterizes their enzymatic properties. Immunolocalization and immunoblot assays using a polyclonal antibody (2556) raised to a 24-mer peptide unique to APY1 showed that this calmodulin-binding APY was highly expressed in nuclei purified from etiolated Arabidopsis seedlings, just as a previously characterized calmodulin-binding apyrase, psNTP9, is highly expressed in purified nuclei of etiolated pea seedlings. Crude nuclear extracts assayed by mass spectroscopy identified the presence of APY1. Because nuclei purified from seedlings expressing APY1-GFP and APY2-GFP both showed the GFP signal, both APY1 and APY2 were likely present in the final preparation of APY, which eluted as a 48 kDa monomer from a molecular sieve column. As estimated by silver-staining after its separation on SDS-PAGE, the purified APY was >85% pure. It had a specific activity toward ATP and ADP substrates similar to that of other apyrases purified from plant and animal sources, but did not hydrolyze AMP substrates. These results favor those of prior reports that APY1/2 are nucleoside triphosphate-diphosphohydrolases, but differ from those of an earlier report that found HA-tagged APY1 extracted from light-grown Arabidopsis tissue did not hydrolyze ATP.