Unconventional Secretion of Fibroblast Growth Factor 2 Is Mediated by Direct Translocation across the Plasma Membrane of Mammalian Cells

Human platelets contain an ATP diphosphohydrolase activity (apyrase, EC 3.6.1.5) that is Ca2+ dependent, hydrolyses ATP and ADP and also GTP, ITP, CTP, GDP, IDP, CDP. The enzyme does not hydrolyse AMP, p-nitrophenylphosphate, inorganic phosphate or glucose-6-phosphate. Contaminant activities were ruled out because the enzyme was not inhibited by 2 μg/d ouabain, 1.0 μM levamisole, 10 μM ApSA or 1.0 mM azide. The enzyme was sensitive to 100 μM orthovanadate, 100μMApSA and 10 mM azide, reagents that have been described as inhibitors of some other apyrases. A strong inhibition by 1.0 mM NEM was observed, indicating that sulphydryl groups are involved in the enzyme activity. The parallel behaviour of ATPase and ADPase activities and the competition plot presented suggest that ATP and ADP hydrolysis occurs at the same active site. ATP diphosphohydrolase from human platelets may be involved in the modulation of nucleotide concentration in the circulation and thus in vascular tonus.

ATP diphosphohydrolase activity (apyrase, EC 3.6.1.5) in human blood platelets.

Purification of the blood vessel atp diphosphohydrolase, identification and localisation by immunological techniques

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.

https://febs.onlinelibrary.wiley.com/doi/pdf/10.1111/j.1432-1033.1995.066_c.x

A. Banda

Papers

Human placental ATP-diphosphohydrolase : biochemical characterization, regulation and function