Biomaterial Database

A study of the vanadate-trapped state of the (Na,K)-ATPase. Evidence against interacting nucleotide site models. 1980

R L Smith, and K Zinn, and L C Cantley

The state of the Na+- and K+-stimulated adenosine triphosphatase ((Na,K)-ATPase) which binds vanadate was investigated by taking advantage of the slow rate of vanadate release (koff = 0.32/h at 25 degrees C and 0.045/h at 4 degrees C). Vanadate release from the enzyme could be accelerated approximately 2-fold by addition of K+ and 50-fold by addition of Na+. The K+ effect saturated hyperbolically with a K1/2 of 0.5 mM, while the Na+ effect had a sigmoidal activation curve and K1/2 of 250 mM. These results indicate that either Na+ or K+ can equilibrate with the vanadate-"trapped" enzyme prior to vanadate release. In the presence of vanadate, a saturable Mn2+ binding site could be detected with a dissociation constant of 120 nM. When 54Mn2+ was added during incubation of the enzyme with vanadate, 1 mol of 54Mn2+ could be trapped/mol of vanadate trapped and the two metals dissociated in parallel. This result indicates a single divalent cation site is involved in stabilizing vanadate (and probably phosphate) binding. Addition of 1 to 4 mM of ATP to the vanadate-trapped enzyme had no affect on the rate of vanadate release. Also, the high affinity ATP site could not be detected in equilibrium-binding studies with the vanadate-trapped enzyme. Since kinetic experiments indicate that vanadate binding is competitive with the low affinity ATP site (Cantley, L. C., Jr., Cantley, L. G., and Josephson, L. (1978) J. Biol. Chem. 253, 7361--7368) and the high affinity ATP site is absent on vanadate-trapped enzyme, it appears unlikely that ATP can occupy either a high or low affinity site on the vanadate-trapped enzyme. We show that nonlinear Lineweaver-Burk plots for ATP hydrolysis can be explained by a single hydrolysis site which exhibits a low affinity for ATP prior to the rate-limiting E2 to E1 conformational change, but a high affinity for ATP following the conformational change.

UI	MeSH Term	Description	Entries
D007668	Kidney	Body organ that filters blood for the secretion of URINE and that regulates ion concentrations.	Kidneys
D007700	Kinetics	The rate dynamics in chemical or physical systems.
D008274	Magnesium	A metallic element that has the atomic symbol Mg, atomic number 12, and atomic weight 24.31. It is important for the activity of many enzymes, especially those involved in OXIDATIVE PHOSPHORYLATION.
D008345	Manganese	A trace element with atomic symbol Mn, atomic number 25, and atomic weight 54.94. It is concentrated in cell mitochondria, mostly in the pituitary gland, liver, pancreas, kidney, and bone, influences the synthesis of mucopolysaccharides, stimulates hepatic synthesis of cholesterol and fatty acids, and is a cofactor in many enzymes, including arginase and alkaline phosphatase in the liver. (From AMA Drug Evaluations Annual 1992, p2035)
D011485	Protein Binding	The process in which substances, either endogenous or exogenous, bind to proteins, peptides, enzymes, protein precursors, or allied compounds. Specific protein-binding measures are often used as assays in diagnostic assessments.	Plasma Protein Binding Capacity,Binding, Protein
D004285	Dogs	The domestic dog, Canis familiaris, comprising about 400 breeds, of the carnivore family CANIDAE. They are worldwide in distribution and live in association with people. (Walker's Mammals of the World, 5th ed, p1065)	Canis familiaris,Dog
D000254	Sodium-Potassium-Exchanging ATPase	An enzyme that catalyzes the active transport system of sodium and potassium ions across the cell wall. Sodium and potassium ions are closely coupled with membrane ATPase which undergoes phosphorylation and dephosphorylation, thereby providing energy for transport of these ions against concentration gradients.	ATPase, Sodium, Potassium,Adenosinetriphosphatase, Sodium, Potassium,Na(+)-K(+)-Exchanging ATPase,Na(+)-K(+)-Transporting ATPase,Potassium Pump,Sodium Pump,Sodium, Potassium ATPase,Sodium, Potassium Adenosinetriphosphatase,Sodium-Potassium Pump,Adenosine Triphosphatase, Sodium, Potassium,Na(+) K(+)-Transporting ATPase,Sodium, Potassium Adenosine Triphosphatase,ATPase Sodium, Potassium,ATPase, Sodium-Potassium-Exchanging,Adenosinetriphosphatase Sodium, Potassium,Pump, Potassium,Pump, Sodium,Pump, Sodium-Potassium,Sodium Potassium Exchanging ATPase,Sodium Potassium Pump
D000818	Animals	Unicellular or multicellular, heterotrophic organisms, that have sensation and the power of voluntary movement. Under the older five kingdom paradigm, Animalia was one of the kingdoms. Under the modern three domain model, Animalia represents one of the many groups in the domain EUKARYOTA.	Animal,Metazoa,Animalia
D014638	Vanadates	Oxyvanadium ions in various states of oxidation. They act primarily as ion transport inhibitors due to their inhibition of Na(+)-, K(+)-, and Ca(+)-ATPase transport systems. They also have insulin-like action, positive inotropic action on cardiac ventricular muscle, and other metabolic effects.	Decavanadate,Metavanadate,Orthovanadate,Oxyvanadium,Vanadyl,Monovanadate,Sodium Vanadate,Vanadate,Vanadate, Sodium
D014639	Vanadium	A metallic element with the atomic symbol V, atomic number 23, and atomic weight 50.94. It is used in the manufacture of vanadium steel. Prolonged exposure can lead to chronic intoxication caused by absorption usually via the lungs.	Vanadium-51,Vanadium 51