Kinetic properties of E-NTPDase activity in lymphocytes isolated from bone marrow, thymus and mesenteric lymph nodes of Wistar rats

Plasma membrane anchored nucleotidases (E-ATPDases), as the E-NTPDase family, could hydrolyze and regulate the pericellular levels of nucleotides in lymphocytes. Each immune organ has a different microenvironment and display lymphocytes with different functions and phenotypes. Considering the different functions of each resident subpopulations of lymphocytes, the E-ATPDases activities in bone marrow (BML), thymus (TL) and mesenteric lymph node (MLL) lymphocytes of Wistar rats were characterized. The hydrolysis of extracellular nucleotides (eATP and eADP) showed linearity in time of reaction between 0 and 120 min, and concentration of lymphocytes expressed in proteins between 1 and 6 mu g protein in the reaction medium. The optimal activity was attained at 37 degree celsius in a pH value of 8.0. The necessity of the cofactors Ca2+ and Mg2+ for the enzymatic activity was confirmed through a curve of concentration of 0-1000 mu M in the reaction medium, with both cofactors showing similar effects in the enzymatic activity. The Chevillard plot revealed that the hydrolysis of eATP and eADP occurred at the same active site of the enzyme. The analyses of E-ATPDases inhibitor and enzyme specificity showed possible involvement of E-NTPDase isoforms - 1 and - 2 in the isolated cells. Furthermore, different kinetic behavior of the nucleotide hydrolysis in each resident subpopulation lymphocyte was observed in this study, as MLL showed the higher Vmax with the lowest km values, while TL had the lowest Vmax and high km values. The kinetic values for E-NTPDase activity of each immune tissue lymphocytes can be an important therapeutic target for numeral immune-related diseases.

Automated summary

Plasma membrane anchored nucleotidases (E-ATPDases) play an important role in the hydrolysis and regulation of nucleotides in lymphocytes. The activities of E-ATPDases vary in different immune organs and resident subpopulations of lymphocytes. This study characterizes the E-ATPDases activities in bone marrow, thymus, and mesenteric lymph node lymphocytes of Wistar rats. The results suggest that the kinetic values of E-NTPDase activity in immune tissue lymphocytes could be an important therapeutic target for immune-related diseases.