5'-Nucleotidase from rat heart

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Abstract
5''-Nucleotidase was extracted from rat heart and purified to apparent homogeneity. The enzyme is a glycoprotein. Gel electrophoresis in the presence of sodium dodecyl sulfate indicates that the apparent MW of the subunit is 74,000 at several different gel concentrations. Cross-linking of the native enzyme with dimethylpimelimidate followed by gel electrophoresis shows that the enzyme is a dimer. The enzyme hydrolyzes all nucleoside 5''-monophosphates tested. A comparison of Vmax/Km for 14 different substrates shows that AMP is the best substrate. The enzyme shows lowest Km values for AMPS [adenosine 5''-O-thiophosphate], AMP, isoAMP [3-.beta.-ribofuranosyladenine 5''-monophosphate], GMP and IMP. It shows no activity with nucleoside 2''- and 3''-monophosphates, sugar phosphates and p-nitrophenyl phosphate, even when tested at high enzyme concentrations. The optimum activity of the enzyme occurs at pH 7.5 with AMP as substrate. Above this pH, buffer ions affect the activity in a complex manner, a 2nd optimum being observed under some conditions. Mg activate the enzyme above pH 7.5 in the presence of some buffer ions but not of others. Mg2+ show only a slight activation when the reaction is run in diethanolamine buffer, pH 9.5, at 30.degree. C; the activation in this buffer is considerably greater when the reaction is run at 37.degree. C. The enzyme is strongly inhibited by free ADP, maximum inhibition occurring below pH 6. The ADP inhibition is diminished as the pH is raised above 6, becoming negligible above pH 9. The enzyme is inhibited by EDTA. The inhibition is partially reversed when the EDTA is removed from the enzyme by gel filtration. This as well as other evidence indicates that the enzyme contains a tightly bound metal ion.