Identification and immunolocalization of two isoforms of ATP-diphosphohydrolase (ATPDase) in the pig immune system

The occurrence of a variety of purine receptors in the immune system indica tes that extracellular purines play important functional roles. Extracellul ar purine concentrations are, in great part, determined by ectonucleotidase s, namely, the ATP diphosphohydrolase, also identified as CD39, a lymphocyt e cell surface marker. The latter enzyme converts triphospho- and diphospho nucleosides to nucleoside monophosphates. In this study, high levels of ATP ase and ADPase activities have been found in homogenates of the different p ig lymphoid organs. Specific activities decreased in the following order: s pleen > bone marrow > thymus > lymph glands. The parallel decrease in ATPas e and ADPase activities, in the presence of sodium azide, indicated that an ATP diphosphohydrolase (ATPDase) was responsible for these activities. Par ticulate fractions, prepared from the different lymphoid organs by ultracen trifugation on a sucrose cushion, showed about a 10-fold enrichment of ATPD ase activity. Identity of ATPDase was confirmed by electrophoretograms of t he particulate fractions and Western immunoblots, with an antibody that rec ognizes ATPDases from different sources. Two isoforms of ATPDase were found (I and II), corresponding to molecular masses of 78,000 and 54,000, respec tively, as estimated by SDS-PAGE. Immunohistochemical localization was carr ied out on these different organs: In spleen, reaction was found in both wh ite and red pulps. A particularly intense reaction was put in evidence in n ervous fibers of this organ. Immunolocalization also showed positive reacti ons with tonsilar lymphoid structures, diffuse lymphoid tissues, and nodule s associated with stomach, duodenum, jejunum, and ileum. In addition, our o bservations establish the presence of ATPDase in lymphocytes and macrophage s of the pig immune system. (C) 1999 Academic Press.