5'-Nucleotidase from rat heart

Abstract

5'-Nucleotidase has been 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 molecular weight 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, AMP, isoAMP, 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 second optimum being observed under some conditions. Magnesium ions activate the enzyme above pH 7.5 in the presence of some buffer ions but not of others. Magnesium ions show only a slight activation when the reaction is run in diethanolamine buffer, pH 9.5, at 30 degrees C; the activation in this buffer is considerably greater when the reaction is run at 37 degrees 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 pH9. 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.

Partial characterization of 5′-nucleotidase from giant squid (Dosidicus gigas) mantle Caracterización parcial de la enzima 5′-nucleotidasa del manto de calamar gigante (Dosidicus gigas)

“…Activator or inhibitor effect depends on 5 0 -nucleotidase source, even though MgCl 2 generally acts as an activator. Similar results were reported by Naito and Lowenstein (1981), who evaluated MgCl 2 effect on 5 0 -nucleotidase from rat heart at concentration range from 0 to 50 mM, getting up to 140% of activity with 20 mM of MgCl 2 . Moreover, Marseno et al (1993a) evaluated the effect of the same salt (from 0 to 10 mM) on 5 0 -nucleotidase purified of rock fish, noticing an increase of 200% at concentration of 10 mM.…”

“…On the other hand, when Skladanowski et al (1996) were working with 5 0 -nucleotidase from rat they observed an ADP activator effect in range from 0 to 1 mM, in which its specific activity increased from 0.1 to 0.3 U/ mg. Nevertheless, both ATP and ADP may also act as inhibitors depending on the enzyme source (Naito & Lowenstein, 1981). Our results assume that 5 0 -nucleotidase enzyme from squid mantle was activated when squid declined the ATP content; therefore ADP and AMP levels started to accumulate inside the cell.…”

Expression of ecto-5'-nucleotidase (CD73) in normal mammary gland and in breast carcinoma

“…Ecto-5'-NT catalyses the extracellular dephosphorylation of purine and pyrimidine ribo-and deoxyribonucleotide monophosphates to the corresponding ribo-and deoxyribo-nucleosides (Naito & Lowenstein, 1981). These compounds then may be transported inside the cell and reconverted to nucleotides via the purine salvage pathway.…”

Purification and properties of bovine liver plasma membrane 5′ nucleotidase

“…The apparent molecular weight of the native enzyme is about 140000. The dimeric form of the 5'-nucleotidase has been described in rat liver [29], mouse liver [32], rat heart [33]. In contrast, a monomeric 5'-nucleotidase was obtained in pig lymphocytes [28].…”