ABSTRACT
The mechanism of Na,K-ATPase function is reviewed. The peculiarities of hydrolysis of various substrates are described. The experimental results testify to the effect of substrate structure on the E2----E1 transition, rate of Na+ transport, K-dependent phosphatase activation and the quaternary structure of Na,K-ATPase. A conclusion is drawn that the proton-acceptor properties of the substrate play a role in the regulation of ion transport by Na,K-ATPase.
Subject(s)
Sodium-Potassium-Exchanging ATPase/metabolism , Chemical Phenomena , Chemistry , Structure-Activity Relationship , Substrate SpecificityABSTRACT
The radiation inactivation analysis of Na+, K+-ATPase, (EC 3.6.1.37) from two different sources was carried out using ATP, CTP, GTP and p-NPP as substrates. In the case of Na+, K+-ATPase from the bovine brain the relation between the logarithm of the residual activity and the radiation dose is strictly linear, which permits calculating 75-90 kDa (for 3 mM GTP and 10 mM p-NPP). Duck salt glands Na+, K+-ATPase reveals larger target sizes: 350 kDa for ATP hydrolysis in saturating concentrations and 145-190 kDa in the case of GTP and p-NPP or low concentration of ATP (30 microM). A conclusion is drawn that while hydrolyzing substrates with complex kinetics (ATP and CTP) the enzyme functions like oligomer. The interaction of nucleotide with substrate-binding site of low affinity induces the aggregation of monomers.
Subject(s)
Sodium-Potassium-Exchanging ATPase/analysis , Affinity Labels , Animals , Brain/enzymology , Catalysis , Cattle , Ducks , Hydrolysis , Kinetics , Substrate SpecificityABSTRACT
Specific activity of Na,K-ATPase from kidney outer medulla microsomes of spontaneously hypertensive rats (SHR) and of normotensive Kyoto-Wistar rats (WKY) at different stages of ontogenesis was studied. A positive correlation was observed between the blood pressure level and specific Na,K-ATPase activity in SHR kidney medulla. At the malignant stage of the disease, the specific activity of Na,K-ATPase exceeded that of control by 67%. The inhibiting effect of blood serum from SHR on purified preparations of Na,K-ATPase was far more pronounced than that of WKY.
Subject(s)
Hypertension/enzymology , Kidney Medulla/enzymology , Sodium-Potassium-Exchanging ATPase/antagonists & inhibitors , Aging , Animals , Female , Rats , Rats, Inbred SHR , Rats, Inbred WKY , Sodium-Potassium-Exchanging ATPase/blood , Time FactorsABSTRACT
The ability of ATP, CTP, ITP, GTP, UTP and two synthetic ATP analogs to provide for ouabain-sensitive Na+ accumulation into proteoliposomes with a reconstituted Na+,K+-ATPase (ATP phosphohydrolase, EC 3.6.1.37) was investigated. A correlation between the proton-accepting properties of the nucleotides and their ability to provide for active transport was found. The proton-accepting properties of the substrate seem to be a necessary condition for the shift from the K-form of Na+,K+-ATPase--an immutable step in the active translocation of Na+ and K+ through the Na+ pump.
Subject(s)
Ribonucleotides/metabolism , Sodium-Potassium-Exchanging ATPase/metabolism , Sodium/metabolism , Animals , Biological Transport, Active , In Vitro Techniques , Ion Channels/metabolism , Potassium/metabolism , Proteolipids , Protons , Structure-Activity RelationshipABSTRACT
The ability of ATP, CTP, ITP, GTP and UTP to induce ouabain-sensitive accumulation of Na+ by proteoliposomes with a reconstituted Na/K-pump was studied. At low Na+/K+ ratio (20 mM/50 mM), a correlation was observed between the proton-accepting capacity of the nucleotide and its efficiency as an active transport substrate. In order to test the hypothesis on the role of the negative charge in position 1 of the purine (3-pyrimidine) base of the nucleotide in the reversible transitions from the Na- to the K-conformations of Na,K-ATPase, two ATP analogs (N1-hydroxy-ATP possessing a proton-accepting ability and N1-methoxy-ATP whose molecule carries a negative charge quenched by a methyl group) were used. The first substrate provides for active accumulation of Na+ by proteoliposomes at a rate similar to that of ATP, whereas the second substrate is fairly ineffective.
Subject(s)
Proteolipids/metabolism , Sodium-Potassium-Exchanging ATPase/metabolism , Sodium/metabolism , Adenosine Triphosphate/metabolism , Animals , Biological Transport, Active/drug effects , Ducks , In Vitro Techniques , Kinetics , Ouabain/pharmacology , Substrate SpecificitySubject(s)
Adenosine Triphosphatases/analysis , Membrane Lipids/analysis , Animals , Biological Transport , Cattle , Cell Membrane/enzymology , Chemical Phenomena , Chemistry, Physical , Ducks , Electron Spin Resonance Spectroscopy , Male , Membrane Lipids/isolation & purification , Rabbits , Salt Gland/enzymologyABSTRACT
Hydrolysis of umbelliferone phosphate is inhibited by sodium ions and ATP added to the incubation medium together with Na+ activates it, CTP, ITP, UTP and GTP produce the same effect. Acetyl phosphate, p-nitrophenyl phosphate, ADP and adenylyl imidodiphosphate beta, gamma-NH-ATP, ATP nonhydrolyzable analog, have no activating effect. ATP produces a mixed inhibition of umbelliferone phosphate hydrolysis both in the presence and absence of Na+. A scheme is suggested which explains the mechanism of the nucleotide activating effect.
Subject(s)
Adenosine Triphosphatases/metabolism , Adenosine Triphosphate/metabolism , Cytidine Triphosphate/metabolism , Cytosine Nucleotides/metabolism , Sodium-Potassium-Exchanging ATPase/metabolism , Allosteric Site , Animals , Cation Transport Proteins , Ducks , Enzyme Activation , Hydrolysis , In Vitro Techniques , Kinetics , Molecular Conformation , Phosphates , Sodium , Substrate Specificity , Umbelliferones/metabolismABSTRACT
Kinetic analysis of hydrolytic activity of (Na+, K+)-ATPase purified from duck salt glands shows that several substrates are hydrolysed in different manners. UTP, GTP and ITP are hydrolysed in accordance with usual Michaelis kinetics with the single Km value and with no cooperatively (Hill coefficient, nH = 1), while CTP is hydrolysed, like ATP, in accordance with non-Michaelis kinetics with two Km values. Hydrolysis of the last two substrates in the range of the second Km is characterised by positive cooperativity with nH greater than 1.
Subject(s)
Salt Gland/enzymology , Sodium-Potassium-Exchanging ATPase/metabolism , Animals , Ducks , Kinetics , Ribonucleotides , Substrate SpecificityABSTRACT
The protein and lipid composition of Na,K-ATPase from duck salt glands were characterized. A kinetic analysis of hydrolysis of two substrates, one of which (ATP) provides and the other (ITP) does not provide for cation active transport was carried out. In both cases two Km values were obtained and were found equal to 10 and 330 microM for ATP and 35 and 710 microM for ITP, respectively. This suggests the existence of substrate sites with high and low affinities. The Hill coefficient for the ATP hydrolysis was equal to 1.4-1.6; the ITP hydrolysis was non-cooperative. It was assumed that positive cooperative interactions between Na,K-ATPase protomers are necessary for active translocation of Na+ and K+.
