MURINE ECTO-5'-NUCLEOTIDASE (CD73) - CDNA CLONING AND TISSUE DISTRIBUTION

The murine cDNA, encoding the purine catabolic enzyme, ecto-5'-nucleot idase (NT), was cloned and the tissue-specific distribution, of both t he mRNA and enzyme activity was examined. Starting with kidney RNA and primers based on the known rat sequence, reverse transcriptase-polyme rase chain reaction (RT-PCR) was utilized to obtain the complete seque nce for the translated portion of the murine cDNA. Murine NT is 94% id entical to human NT at the amino acid (aa) level and 86% identical at the nucleotide (nt) level. NT enzyme assays revealed greater than tenf old more NT activity in mature vs. immature murine T- and B-lymphocyte s. A similar increase in NT activity was also found when the pre-B-cel l line, 70Z/3, was induced to produce surface kappa light chains with lipopolysaccharide (LPS) and gamma-interferon (gamma-IFN). Thus, cultu re systems in which murine lymphocytes mature may be useful for examin ing the mechanisms of NT gene regulation, as well as the function of N T in the immune system. In tissues, enzyme activity varied over 30-fol d, from the lowest levels in skeletal muscle, thymus and spleen to hig hest in placenta, kidney and forestomach. Levels of mRNA, as determine d by RNase protection assay, showed increased NT expression in the ear ly gestation site, as compared to non-pregnant uterus, and in day-19.5 placenta, as compared to day-13 chorioallantoic placenta. Messenger R NA levels were in general proportional to enzyme activity, except in t he lung and glandular stomach where mRNA levels were higher than expec ted, based on enzyme activity. Western blotting suggested that these t issues may contain a form of NT with lower enzymatic activity than in other tissues. NT activity was inversely related to reported adenosine deaminase (ADA) activity in several organs in the gastrointestinal tr act. Thus, there appear to be multiple components of NT regulation, in cluding coordinate regulation with ADA, as well as developmental and t issue-specific regulation.