Purification and properties of human placental ATP diphosphohydrolase.
ATP diphosphohydrolase activity (ATP-DPH) has been previously identified in the particulate 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-[alpha-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 Mg2+. The Km values for ATP and ADP were determined to be 10 microM and 20 microM, 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, NaF, adenosine 5'-[beta,gamma-imido]triphosphate and adenosine 5'-[alpha,beta-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.
Christoforidis
S
S
Laboratory of Biological Chemistry, University of Ioannina Medical School, Greece.
Papamarcaki
T
T
Galaris
D
D
Kellner
R
R
Tsolas
O
O
D000345:Affinity Labels; D000469:Alkaline Phosphatase / Q000378:metabolism; D000595:Amino Acid Sequence; D001081:Apyrase / Q000037:antagonists & inhibitors / Q000302:isolation & purification* / Q000378:metabolism; D002851:Chromatography, High Pressure Liquid; D002852:Chromatography, Ion Exchange; D004591:Electrophoresis, Polyacrylamide Gel; D006801:Humans; D006863:Hydrogen-Ion Concentration; D008969:Molecular Sequence Data; D010920:Placenta / Q000201:enzymology*; D012995:Solubility; D013045:Species Specificity; D013194:Staining and Labeling
D000345:Affinity Labels; D000469:Alkaline Phosphatase; D001081:Apyrase