inosine-triphosphate and fructose-1-6-diphosphate

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.

Topics: Acetyl Coenzyme A; Adenosine Triphosphate; Animals; Apyrase; Brain; Cations; Cattle; Cytidine Triphosphate; Cytosol; Fructosediphosphates; Guanosine Triphosphate; Inosine Triphosphate; Kidney; Kinetics; Liver; Nucleoside-Triphosphatase; Propionates; Substrate Specificity; Uridine Triphosphate

The steady-state kinetics of the reaction catalysed by the bloodstream form of Trypanosoma brucei were studied at pH 6.7. In the presence of 50 mM-potassium phosphate buffer, the apparent co-operativity with respect to fructose 6-phosphate and the non-linear relationship between initial velocity and enzyme concentration, which were found when the enzyme was assayed in 50 mM-imidazole buffer [Cronin & Tipton (1985) Biochem. J. 227, 113-124], are not evident. Studies on the variations of the initial rate with changing concentrations of MgATP and fructose 6-phosphate, the product inhibition by fructose 1,6-bisphosphate and the effects of the alternative substrate ITP were consistent with an ordered reaction pathway, in which MgATP binds to the enzyme before fructose 6-phosphate, and fructose 1,6-bisphosphate is the first product to dissociate from the ternary complex.

Topics: Adenosine Triphosphate; Animals; Fructosediphosphates; Guanosine Triphosphate; Inosine Triphosphate; Kinetics; Phosphofructokinase-1; Substrate Specificity; Trypanosoma brucei brucei