PURIFICATION AND PROPERTIES OF HUMAN PLACENTAL ATP-DIPHOSPHOHYDROLASE

ATP diphosphohydrolase activity (ATP-DPH) has been previously identifi ed in the particulate fraction of human term placenta [Papamarcaki, T. & Tsolas, O. (1990) Mel. Cell. Biochem. 97, 1-8]. In the present stud y we have purified to homogeneity and characterized this activity. A 2 60-fold purification has been obtained by solubilization of the partic ulate fraction and subsequent chromatography on DEAE Sepharose CL-6B a nd 5'-AMP Sepharose 4B. The preparation has been shown to be free of a lkaline phosphatase even though the placental extract is rich in this activity. The purified enzyme is a glycoprotein and migrates as a sing le broad band of 82 kDa on SDS/PAGE. The same band is obtained after p hotoaffinity labeling of the enzyme with 8-azido-[alpha-P-32]ATP. The enzyme has a broad substrate specificity, hydrolyzing triphosphonucleo sides and diphosphonucleosides but not monophosphonucleosides or other phosphate esters. The activity is dependent on the addition of divale nt cations Ca2+ or Mg2+. The K-m values for ATP and ADP were determine d to be 10 mu M and 20 mu M, respectively. Maximum activity was found at pH 7.0-7.5 with ATP as substrate, and pH 7.5-8.0 with ADP. The enzy mic activity is inhibited by NaN3, NaF, adenosine 5'-[beta,gamma-imido ]triphosphate and adenosine 5'-[alpha,beta methylene]triphosphate. Pro tein sequence analysis showed ATP-DPH to be N-terminally blocked. Part ial internal amino acid sequence information was obtained after chymot ryptic cleavage and identified a unique sequence with no significant s imilarity 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.