Molecular and biochemical comparison of two different apyrases from Arabidopsis thaliana

Recent findings indicate that extracellular ATP (xATP) plays an important regulatory role in both animals and plants. The turnover of xATP is controlled largely by the activity of ecto-phosphatases, including ecto-apyrases. Two apyrases, termed Atapy1 (Arabidopsis thaliana apyrase 1) and Atapy2, were cloned and sequenced. The transcripts of Atapy1 and Atapy2 are widely distributed; however, the expression patterns are not identical. In roots, for example, the mRNA level of Atapy1 is greater than that of Atapy2. Atapy1 and Atapy2 are 87 % identical at the amino acid sequence level. Both contain four apyrase conserved regions (ACRs), an ATP-binding motif and a hydrophobic segment at the N-terminus. However, only Atapy1 demonstrates a calmodulin (CaM)-binding domain. The two cDNAs were overexpressed in bacteria, and the resulting proteins were biochemically analyzed. The purified proteins displayed enzymatic properties characteristic of apyrases, such as the hydrolysis of ATP and ADP, but not AMP, and an insensitivity to inhibitors of ATPases. In a CaM-binding assay, only the protein of Atapy1 bound to CaM under the conditions tested. To date Atapy1 represents the only other apyrase, besides pea NTPase, shown to contain a functional CaM-binding domain.