183 Purification and regulation of cytosolic 5′-nucleotldaae I from rabbit heart

Rabbit heart contains two cytosolic 5′-nucleotidases, termed N-I and II, which can be separated using phosphocellulose chromatography. N-I prefers AMP over IMP as a substrate, in contrast to N-II which prefers IMP over AMP. N-II closely resembles the soluble enzyme found in heart and liver of chicken and rat with respect to substrate specificity (Itoh and coworkers, Comp. Biochem. Biophys. 81B, 159-163 (1985); Biochim. Biophys. Acta 657, 402-410 (1981); Biochem. J., 235, 847-851 (1986); and Naito. Biochim. Biophys. Acta 438, 159-168 (1976)). N-I has been purified approximately 1000-fold to a specific activity of 90 μmol/mg protein/min. The enzyae is strongly activated by ADP; it is not activated by ATP. Gel filtration indicates a molecular weight for N-I of about 255,000. At low [AMP], the activity of N-I is stimulated by a regulatory protein (R) in the presence of ATP. R was also purified to homogeneity. Gel electrophoresis in the presence of sodium dodecyl sulfate shows a subunit molecular weight for R of about 25,000. N-I is most likely the enzyme responsible for the release of adenosine from AMP under conditions of hypoxia or increased work load.