Cytosolic 5′-nucleotidase, an ubiquitous IMP preferring nucleotide phosphatase has been purified and characterised from different sources.1 The enzyme acts through the formation of a phosphorylated enzyme intermediate, and catalyses both the hydrolysis of the mononucleotides and the transfer of the phosphate to a nucleoside acceptor, thus operating a monucleotide interconversion.2 The enzyme is specific for the 6-hydroxypurine nucleotides and is able to phosphorylate inosine, deoxyinosine, guanosine and many purine nucleoside analogues including 2′,3′-dideoxyinosine and deoxycoformycin.3 The enzyme is activated by a number of nucleoside diphosphates and triphosphates such as ATP and ADP, and by 2,3-bisphosphoglycerate (BPG), furthermore it is inhibited by phosphate.1'4 The sequence of human, avian and bovine cytosolic 5′-nucleotidase has been determined, showing a degree of homology higher than 90%, among the different species.5'6 A similar homology has been described for proteins such as actin and histones, whose structure and function are of fundamental importance in the living cells. The high conservation of protein sequence, the widespread distribution of the enzyme and its complex regulation indicate that the activity of cytosolic 5′-nucleotidase must be of fundamental importance probably in the regulation of the intracellular IMP concentration. In the course of our work we became aware that our final enzyme preparation contained two polypeptides with different electrophoretic mobil- ity, both cross-reacting with specific antibodies raised against cytosolic 5′-nucleotidase. Furthermore, both polypeptides were able to form the phosphorylated intermediate, indicating that they were active 5′-nucleotidases.2 The two forms were separated by affinity chroma-tography on ADP-Agarose and their regulatory characteristics were studied. Recently we noticed that even though ADP and BPG, separately, act at a too high concentration to be considered physiological regulators, there is a synergistic effect among the two compounds. In fact, the presence of physiological ADP concentration significantly lowers the amount of BPG necessary to exert an activatory effect on the form with the lower apparent molecular weight, and viceversa.7 The other form, on the contrary, was activated at a greater extent by ADP and BPG but at different concentrations with respect to the other form, and the synergistic effect was absent.8 With the aim to collect more information on the nature, origin, and physiological function, if any, of these enzyme forms we studied the distribution of enzyme activity, mRNA and electrophoretic mobility in rat tissues.KeywordsEnzyme PreparationEnzyme Specific ActivityPhosphotransferase ActivityPurine Nucleoside AnalogueSimilar HomologyThese keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.