Physicochemical and Kinetic Properties of E-NTPDase Activity in Lymphocytes Isolated from Bone Marrow, Thymus and Mesenteric Lymph Nodes of Wistar Rats

Abstract

Abstract Plasma membrane anchored nucleotidases (E-ATPDases), as the E-NTPDase family, could hydrolyse 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 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 (0 to 120 min) and protein (1 to 6 μg protein) intervals. The optimal activity was attained at 37ºC in a pH value of 8.0. The essence of cofactor was shown by availability of Ca2+ or Mg2+ within the concentration range of 0 to 1000 µM in the reaction medium, with both ions showing similar effect. 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 actions of E-NTPDase isoforms -1 and -2 in the isolated cells. Furthermore, different kinetic behaviour of the nucleotide hydrolysis in each resident 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 observed variation in enzyme activity of each resident lymphocyte may account for the different physiological signalling of nucleotides in each immune organ.