PARTIAL PURIFICATION OF SOLUBLE 5′-NUCLEOTIDASE FROM RABBIT HEART: 39

Abstract

Cytosolic 5′-nucleotidase from rabbit heart has been purified approximately 40-fold to a specific activity of 0.6 μmol/mg protein/min. At this stage of purification, the enzyme is free of other phosphatases and is stable to repeated cycles of freezing and thawing over a 3 months period. Soluble 5′-nucleotidase has an absolute requirement for divalent metal ions and is activated by ATP, GTP, and ADP. Activation by these nucleotides causes a shift from a sigmoidal to a hyperbolic substrate saturation curve. ATP (1 mM) increases the Kapp of the enzyme for AMP about 5-fold. Activation by GTP is similar. Activation by ADP also increases Kapp, but high concentrations of ADP cause a decrease in Vmax. AMP is the preferred substrate in terms of Vmax/Kapp, with AMP>UMP>GMP>IMP. Nucleoside monophosphates other than AMP are inhibitory at high substrate concentrations. The fully activated enzyme exhibits a Kapp for AMP of 2.5 mM. The enzyme purified by us has properties different from the soluble liver enzyme described by Van Den Berghe et al. (Biochem. J. (1977) 162, 611) and from the plasma membrane enzyme from rat heart described by Naito & Lowenstein (Biochem. (1981) 20, 5188) with respect to both substrate specificity and regulatory properties.