Purification and Properties of Human Placental ATP Diphosphohydrolase

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Abstract
ATP diphosphohydrolase activity (ATP-DPH) has been previously identified in the paniculate fraction of human term placenta [Papamarcaki, T. & Tsolas, O. (1990) Mol. Cell. Biochem. 97, 1–8]. In the present study we have purified to homogeneity and characterized this activity. A 260-fold purification has been obtained by solubilization of the particulate fraction and subsequent chromatography on DEAE Sepharose CL-6B and 5′-AMP Sepharose 4B. The preparation has been shown to be free of alkaline phosphatase even though the placental extract is rich in this activity. The purified enzyme is a glycoprotein and migrates as a single broad band of 82 kDa on SDS/PAGE. The same band is obtained after photoaffinity labeling of the enzyme with 8–azido-[α-32P]ATP. The enzyme has a broad substrate specificity, hydrolyzing triphosphonucleosides and diphosphonucleosides but not monophosphonucleosides or other phosphate esters. The activity is dependent on the addition of divalent cations Ca2+ or Mg21. The Km values for ATP and ADP were determined to be 10μM and 20 μM, respectively. Maximum activity was found at pH 7.0–7.5 with ATP as substrate, and pH 7.5–8.0 with ADP. The enzymic activity is inhibited by NaN3 NaF3 adenosine 5′-[β,γ-imido]triphosphate and adenosine 5′-[α,β-methylene]triphosphate. Protein sequence analysis showed ATP-DPH to be N-terminally blocked. Partial internal amino acid sequence information was obtained after chymotryptic cleavage and identified a unique sequence with no significant similarity to known proteins. ATP-DPH activity has been reported to be implicated in the prevention of platelet aggregation, hydrolysing ADP to AMP and thus preventing blood clotting.

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