PARTIAL PURIFICATION AND PROPERTIES OF ATP - GMP PHOSPHOTRANSFERASE FROM RAT LIVER

A nucleoside monophosphate kinase has been purified 250-fold from normal adult rat liver by centrifugation, isoelectric precipitation, ammonium sulfate fractionation, alumina Cγ adsorption, DEAE-cellulose chromatography, and hydroxylapatite adsorption. The enzyme preparation is highly specific for (d)GMP as the phosphate acceptor and for (d)ATP as the phosphate donor. XMP, IMP, TMP, (d)AMP, (d)CMP, UMP, 6-thio(d)GMP, guanosine, and deoxyguanosine will not serve as substrates. 6-thio(-d)GMP, 8-azaGMP, and 8-bromoGMP can replace (d)GMP to some extent. The enzyme prepared from the Morris 7793 or Dunning hepatomas has the same substrate specificity with respect to 6-thio analogs of (d)GMP. The enzyme preparation is free of ATP-ase and nucleoside diphosphokinase activity. The ratio of GMP to dGMP kinase activity remains constant throughout the purification. dGMP kinase activity is stimulated 3-fold by potassium ion and to a lesser extent by ammonium and rubidium and is inhibited by cesium and lithium. Sodium has no effect. GMP kinase activity is relatively unaffected by these metal ions. The enzyme requires magnesium ion for activity. The optimum ratio of ATP to magnesium is 1. This ratio is unaffected by the presence of potassium. The enzyme has a molecular weight of about 20,500, as determined by gel filtration. The molecular weight is unaffected by potassium. The GMP and dGMP kinase activities are (1) nonadditive, (2) inhibited by 8-aza, 6-thio and 8-bromoGMP, (3) unaffected by 6-thioGMP, (4) inhibited by iodoacetate, iodoacetamide, p-hydroxymercuribenzoate, N-ethylmaleimide and 5,5′-dithiobis-(2-nitrobenzoic acid), and (5) unaffected by mercaptoethanol and dithiothreitol. Potassium has no effect on the relative rate of inactivation by iodoacetate or iodoacetamide or on the ability of GMP and ATP to protect the enzyme from iodoacetamide. © 1969.