Properties of the conversion of an enzyme-ATP complex to a phosphorylated intermediate in the reaction of Na+-K+-dependent ATPase1.

Previously, we proposed the following reaction machanism for the transport ATPase (EC 3.6.1.3) reaction in the presence of high concentrations of Mg2+ and Na+:(see article). Some kinetic and thermodynamic properties of steps 3 and 4 were investigated, and the following results were obtained. 1. When the reaction was started by adding ATP to the enzyme in the presence of 50 mM Na+ and 0.5 mM K+ or in the presence of 50mM Na+ and 0.5mM Rb+, the amount of E ADP P increased with time and maintained a constant level after reaching a maximum. We could not observe the initial burst of EP formation, which was observed by Post er al. in the presence of 8 mM Na+ and 0.01 mM Rb+. 2. The existence of quasi-equilibrium between E2ATP and E ADP P in the presence of low concentrations of Na+ was suggested by the fact that the values of the reciprocal of the equilibrium constant, K3 of step 3 obtained by the following three methods were almost the same. a) The value of 1+K3 was estimated from the ratio of vo/[EP] to kd, where vo is the rate of ATP hydrolysis in the steady state, [EP] the concentration of EP, and kd the first-order rate constant of EP disappearance after stopping EP formation. b) This value was also calculated from the ratio of the amount of P1 liberated to that of decrease in EP after stopping EP formation. c) The value of K3 was also calculated from the initial rapid decrease in EP on adding K+ and EDTA, assuming that the rapid decrease was due to a shift of the equilibrium toward E2ATP on adding K+. For example, the value of K3 with 10mM NaCL and 0.5mM KCL was 7--11. Although ATP formation due to a shift of the equilibrium toward E2ATP by a K+ jump in the presence of a low concentration of Na+ was observed at 0 degrees, the amount of ATP formed by a K+ jump at 15 degrees was less than the value expected from the shift of the equilibrium. 3. The values of delta H degrees and delta S degrees of step 3 were estimated in the presence of a sufficient amount of Na+ and in the absence of K+. They were +4--+5 kcal mole minus 1 and +15--+16 entropy units mole minus1, respectively. On the basis of kinetic studies of the elementary steps and the overall reaction of Na+-K+-dependent ATPase [EC 3.6.1.3], we (1--4) showed that a phosphorylated intermediate, EP, is formed via two kinds of enzyme-substrate complex, E1ATP and E2ATP, that the EP is in K+-dependent quasi-equilibrium with E2ATP, and that in the presence of high concentration of Mg2+, EP is in a high-energy state and contains bound ADP, E ADP P.(see article).     

Effects of sodium and potassium ions on the elementary steps in the reaction of Na+-K+-dependent ATPase1.

The effects of Na+ and K+ ions on the elementary steps in the reaction of Na+-K+-dependent ATPase (EC 3.6.1.3) were investigated in 0.5-600mM NaCL and 0-10mM KCL, at a fixed concentration (1mM) OF MgCL2, AT PH 8.5 and at 15 degrees. The data were analyzed on the basis of the reaction mechanism in which a phosphorylated intermediate, E ADP P (abbreviated as EP), is formed via two kinds of enzyme-substrate comples, E1ATP and E2ATP, and EP is in equilibrium with E2ATP, and is hydrolyzed to produce P1 and ADP. The following results were obtained: 1. The rate od E2ATP-formation, vf, increased with increase in the Na+ concentration, reached a maximum level, and then decreased with further increase in the Na+ concentration at various K+ concentrations. The value of vf was given as (see article). 2. The reciprocal of the equilibrium constants, K2, of the step E1ATPEQUILIBRIUM E ADP P in the presence of low concentrations of Na+ was larger than that in the presence of high concrntrations of Na+, indicating that the equilibrium shifted markedly toward E2ATP at low concentrations of Na+. The relation of K3 with Na concentration was rather complicated on varying the concentration of K+. However, generally speaking, it increased with increase in the K+ concentration. 3. The decomposition of EP was markedly activated by even low concentrations of K+, and inhibited by high concentrations of Na+. The inhibition by Na+ was partially suppressed by K+. The rate constant of EP-decomposition, vo/(EP), was given by (see article) where (vo/(EP) K+EQUALS0 was the value of vo/[EP] in the absence of K+.     

Transport ATPase: thimerosal inhibits the Na+ +K+-dependent ATPase activity without diminishing the Na+-dependent ATPase activity.

A guinea pig kidney membrane preparation was incubated with thimerosal and then thoroughly washed. Comparison of the properties of the native and the modified membranes showed that (a) Na⁺+K⁺-dependent activity is substantially inhibited by thimerosal; (b) thimerosal does not diminish Na⁺-dependent ATPase activity; and (c) the thimerosal treated enzyme, like the native enzyme, is phosphorylated in the presence of Na⁺ and ATP, and dephosphorylated upon the addition of K⁺. It is suggested that thimerosal does not affect the binding of ATP to the high-affinity catalytic site, but that it blocks the binding of ATP to a low affinity modifying site the occupation of which is essential for the dissociation of the stable K⁺-dephosphoenzyme and the recycling of the enzyme.     

Responses of Na+-K+ ATPase activities from dog olfactory tissue to selected odorants.

Na⁺-K⁺ ATPase activity from nerve ending particle (NEP) fractions of dog olfactory tissue homogenates showed different patterns of response to odorants. Similar turbinal groupings were removed from the right and left sides of the septum in the nasal cavity and NEP preparations were tested with eight different odor compounds, including 2-keto alkane homologs and the optical isomers d- and l-carvone. Odorant stimulation of Na⁺-K⁺ ATPase activity from paired turbinal groupings did not show bilateral symmetry. Different patterns of stimulation were observed for each turbinal grouping and for each odorant. A stimulation of over 200% was observed in one preparation in response to 2-nonanone.A study of the response of Na⁺-K⁺ ATPase activity from individual turbinals showed that the enzyme in each turbinal had a different response pattern to six different odorants. Inhibitory and stimulatory responses were observed for the individual turbinal NEP preparations. These results support the proposal that odor sensing initiation may occur through odorant perturbation of the Na⁺-K⁺ ATPase activity.     

Ouabain-insensitive Na+ stimulation of an Mg-2+ -dependent ATPase in kidney tissue.

1. Freshly prepared microsomal fractions of the outermost cortex of guinea pig kidney show an Mg-2+-dependent ATPase activity which is partially inhibited by 100 mM NaCl, LiCl, KCl, RbCl, CsCl, NH4Cl or choline chloride. 2. If the microsomal preparation is aged by storage at 4 degrees C for 10-15 days, the Mg-2+-dependent activity shows stimulation by Na-+ and Li-+ but not by K-+, Rb-+, Cs-+, NH4-+ or choline. 3. Stimulation is similar with sodium salts of Cl-minus, HCO3-minus, CH3COO-minus, BR-minus, SO4-2-minus or methylsulphonate. 4. Stimulation is insensitive to 1 mM and 10 mM ouabain. 5. Stimulation is unaltered by the presence of 0.5 mM ethyleneglycol-bis-(beta-aminoethyl ether)N,N'-tetracetic acid. 6. Stimulation is 100% inhibited by 2 mM ethacrynic acid, a concentration which inhibits only 30% of the Mg-2+-dependent ATPase and 50% of the (Na-++K-+)-stimulated ATPase. 7. Some of these characteristics coincide with those of an ouabain-resistant, K-+-independent, ethacrynic acid-sensitive mode of Na-+ extrusion out of guinea pig kidney cortex cells.