The mechanism of adenosine formation in cells - Cloning of cytosolic 5 '-nucleotidase-I

Adenosine increases blood flow and decreases excitatory nerve firing. In th e heart, it reduces rate and force of contraction and preconditions the hea rt against injury by prolonged ischemia. Based on indirect kinetic argument s, an AMP-selective cytosolic 5'-nucleotidase designated cN-I has been impl icated in adenosine formation during ATP breakdown. The molecular identity of cN-I is unknown, although an IMP/GMP-selective cytosolic 5'-nucleotidase (cN-II) and an ecto-5'-nucleotidase (e-N) have been cloned. We utilized th e high abundance of cN-I in pigeon heart to purify a 40-kDa subunit for par tial protein sequencing and subsequent cDNA cloning. We obtained a full-len gth clone encoding a novel 40-kDa peptide, unrelated to cN-II or e-N, that was most abundant in heart, brain, and breast muscle. Immunolocalization in heart showed a striated cytoplasmic location, suggesting association with contractile elements. Transient expression in COS-7 cells, generated a 5'-n ucleotidase that catalyzed adenosine formation from AMP, which was increase d during ATP catabolism, In conclusion, the cloning and expression of cN-I provides definitive evidence of its ability to produce adenosine during ATP breakdown.