Molecular and biochemical comparison of two different apyrases from Arabidopsis thaliana

Recent findings indicate that extracellular ATP (xATP) plays an important r egulatory role in both animals and plants. The turnover of xATP is controll ed largely by the activity of ecto-phosphatases, including ecto-apyrases. T wo apyrases, termed Atapy1 (Arabidoysis thaliana apyrase 1) and Atapy2, wer e cloned and sequenced. The transcripts of Atapy1 and Atapy2 are widely dis tributed; however, the expression patterns are not identical. In roots, for example, the mRNA level of Atapy1 is greater than that of Atapy2. Atapy1 a nd Atapy2 are 87% identical at the amino acid sequence level. Both contain four apyrase conserved regions (ACRs), an ATP-binding motif and a hydrophob ic segment at the N-terminus. However, only Atapy1 demonstrates a calmoduli n (CaM)-binding domain. The two cDNAs were overexpressed in bacteria, and t he resulting proteins were biochemically analyzed. The purified proteins di splayed enzymatic properties characteristic of apyrases, such as the hydrol ysis of ATP and ADP, but not AMP, and an insensitivity to inhibitors of ATP ases. In a CaM-binding assay, only the protein of Atapy1 bound to CaM under the conditions tested. To date Atapyl represents the only other apyrase, b esides pea NTPase, shown to contain a functional CaM-binding domain. (C) 20 00 Editions scientifiques et medicales Elsevier SAS.