Immunogold localization of adenosine 5'-monophosphate-specific cytosolic 5'-nucleotidase in dog heart.

Adenosine has a major regulatory function in the heart and many tissues. Our previous work showed that a cytosolic (not a membrane, as previously hypothesized) 5'-nucleotidase from dog heart has the kinetic properties consistent with it being the enzyme responsible for adenosine formation from adenosine 5'-monophosphate (AMP) in response to hypoxia or ischemia. In the present study, we evaluated the spatial distribution of AMP-specific cytosolic 5'-nucleotidase in dog heart using electron microscopic immunogold localization. Polyclonal antibodies raised against purified cytosolic 5'-nucleotidase recognized the 43-kd subunit of the enzyme on Western blots of both purified enzyme and the soluble fraction of dog heart homogenates but did not react with proteins extracted from the membrane fraction. Purified cytosolic 5'-nucleotidase and 5'-nucleotidase activity present in the soluble fraction of heart homogenates were inhibited by anti-cytosolic 5'-nucleotidase, but the membrane fraction was not. The monospecific antibodies against the cytosolic 5'-nucleotidase were used for electron microscopic immunogold localization of cytosolic 5'-nucleotidase in dog heart tissue sections. Cytosolic 5'-nucleotidase was found in the cytoplasm of red blood cells, cardiac myocytes, and endothelium; the plasma membrane and interstitium were devoid of gold label. These results are the first to document the presence cytosolic 5'-nucleotidase in specific cell types in the heart and demonstrate the potential for these cell types to produce adenosine via cytosolic 5'-nucleotidase.