Characterization of NTPDase (NTPDase1; ecto-apyrase; ecto-diphosphohydrolase; CD39; EC 3.6.1.5) activity in human lymphocytes

• Published in: Biochimica et biophysica acta Vol. 1721; no. 1; pp. 9 - 15
• Main Authors: Leal, Daniela B.R., Streher, Cristiane A., Neu, Tiago N., Bittencourt, Fábio P., Leal, Cláudio A.M., da Silva, José E.P., Morsch, Vera M., Schetinger, Maria R.C.
• Format: Journal Article
• Language: English
• Published: Netherlands Elsevier B.V 18.01.2005
• Subjects: Adenosine Diphosphate - metabolism; Adenosine Triphosphatases - metabolism; Adenosine Triphosphate - metabolism; Antigens, CD - metabolism; Apyrase - metabolism; CD39; Human lymphocyte; Humans; Kinetics; Lymphocytes - enzymology; NTPDase; Substrate Specificity; CD39; NTPDase; Human lymphocyte
• ISSN: 0304-4165; 0006-3002; 1872-8006
• DOI: 10.1016/j.bbagen.2004.09.006

Human lymphocytes contain NTPDase (NTPDase-1; ecto-apyrase; ecto-diphosphohydrolase; CD39; EC 3.6.1.5), a cation-dependent enzyme that hydrolyzes ATP and ADP and also other di- and triphosphate nucleosides, acting at an optimum pH of 8.0. A significant inhibition of ATP and ADP hydrolysis ( P<0.05) was observed in the presence of 20 mM sodium azide. NTPDase inhibitors, 20 mM sodium fluoride, 0.2 mM trifluoperazine and 0.3 mM suramin, significantly decreased ATP and ADP hydrolysis ( P<0.05) and ADP hydrolysis was only inhibited by 0.5 mM orthovanadate ( P<0.05). ATP and ADP hydrolysis was not inhibited in the presence of 0.01 mM Ap5A (P 1,P 5-di(adenosine-5′)pentaphosphate), 0.1 mM ouabain, 1 mM levamisole, 2 μg/mL oligomycin, 0.1 mM N-ethylmaleimide (NEM), or 5 mM sodium azide. With respect to kinetic behavior, apparent K m values of 77.6±10.2 and 106.8±21.0 μM, and V max values of 68.9±8.1 and 99.4±8.5 (mean±S.E., n=3) nmol Pi/min/mg protein were obtained for ATP and ADP, respectively. A Chevilard plot demonstrated that only one enzymatic site is responsible for the hydrolysis of ATP and ADP. The presence of CD39 was determined by flow cytometry, showing a low density of 2.72±0.24% (mean±S.E.; n=30) in human peripheral lymphocytes. The study of NTPDase activity in human lymphocytes may be important to determine the immune response status against infectious agents related to ATP and ADP hydrolysis.