ABSTRACT
Soluble NTPase, differing in its properties from known proteins exhibiting NTPase activity, was purified from bovine brain to homogeneity. The enzyme has pH optimum at 7.5 and shows absolute dependence on bivalent cations and broad substrate specificity towards nucleoside-5 -tri- and -diphosphates, characteristics of apyrases. The NTPase follows Michaelis-Menten kinetics in the range of investigated substrate concentrations, the apparent K(m) values for UTP, ITP, GTP, CTP, CDP, and ATP being 86, 25, 41, 150, 500, and 260 microM, respectively. According to gel-filtration and SDS-PAGE data, the molecular mass of the enzyme is 60 kD. The NTPase is localized in the cytosol fraction and expressed in different bovine organs and tissues. Total NTPase activity of extracts of bovine organs and tissues decreases in the following order: liver > heart > skeletal muscle > lung > brain > spleen > kidney ~ small intestine. The enzyme activity can be regulated by acetyl-CoA, alpha-ketoglutarate, and fructose-1,6-diphosphate acting as activators in physiological concentrations, whereas propionate exhibits an inhibitory effect.
Subject(s)
Apyrase/isolation & purification , Apyrase/metabolism , Brain/enzymology , Nucleoside-Triphosphatase/isolation & purification , Nucleoside-Triphosphatase/metabolism , Acetyl Coenzyme A/metabolism , Adenosine Triphosphate/metabolism , Animals , Apyrase/chemistry , Cations/metabolism , Cattle , Cytidine Triphosphate/metabolism , Cytosol/metabolism , Fructosediphosphates/metabolism , Guanosine Triphosphate/metabolism , Inosine Triphosphate/metabolism , Kidney/enzymology , Kinetics , Liver/enzymology , Nucleoside-Triphosphatase/chemistry , Propionates/metabolism , Substrate Specificity , Uridine Triphosphate/metabolismABSTRACT
Soluble nucleoside triphosphatase differing in its properties from all known proteins with NTPase activity was partially purified from bovine kidneys. The enzyme has pH optimum of 7.5, molecular mass of 60 kDa, as estimated by gel chromatography, and shows an absolute dependence on divalent metal ions. NTPase obeyed Michaelis-Menten kinetics in the range of substrate concentration tested from 45 to 440 microM; the apparent Km for inosine-5'-triphosphate was calculated to be 23.3 microM. The enzyme was found to possess a broad substrate specificity, being capable of hydrolyzing various nucleoside-5'-tri- as well as diphosphates.
