Na+/K+-dependent adenosinetriphosphatase activity in mammalian kidney.

Microsomal preparations with Na+/K+-dependent ATPase activity from the outer medulla of rabbit and pig kidney were obtained. Purifications were assaied by centrifugation on sucrose discontinuous gradients and gel-filtration on Sepharose 6B, after detergent incubation. Sodium dodecylsulfate, with ATP as protecting agent, can remove a maximal amount of non specific proteins from the membranes and allows the recovery of a fraction with very high specific activity. The approach to purification by affinity chromatography techniques leads to interesting results, which induce us to pursue the present researchs to applicate an affinity method to Na+/K+-ATPase.     

Molecular identification of the Na+-activated K+ channel

Progress in understanding sodium-activated potassium channels (K(Na)), suggested to function in excitable cells both during physiological conditions and protectively during hypoxia, has been limited by their unknown molecular identity. In this issue of Neuron, Salkoff and coworkers now show that members of the Slo gene family, Slo2.1 and Slo2.2, encode functional K(Na) channels.     

(Na+ + K+)-activated adenosinetriphosphatase: fluorimetric determination of the associated K+ -dependent 3-O-methylfluorescein phosphatase and its use for the assay of enzyme samples with low activities.

Data are presented which prove that 3-O-methylfluorescein phosphate is a substrate for the K⁺-dependent phosphatase that is associated with Na⁺,K⁺-ATPase. Conditions for the continuous fluorimetric assay of 3-O-methylfluorescein phosphatase are described. Enzyme preparations from three different tissues with widely different specific activities exhibit similar K_m values for 3-O-methylfluorescein phosphate. Correlation between Na⁺,K⁺-ATPase activity and K⁺-dependent 3-O-methylfluorescein phosphatase activity is demonstrated in several partially purified enzyme preparations and crude tissue fractions. When the K⁺-dependent 3-O-methylfluorescein phosphatase of a crude rat-brain homogenate is assayed, the activity is a linear function of the amount of homogenate added to the assay mixture. The equivalent of 10 μg of brain tissue may be assayed under the conditions used. The potential value of this highly sensitive fluorimetric method for the assay of enzyme in small samples of various tissues is suggested.     

Stimulation of rat renal Na+, K+-ATPase activity by thyroid hormones

The effect of thyroid hormones (T4, T3 and reverse T3) on rat renal Na+,K+-ATPase activity was investigated by a cytochemical technique. T3 caused stimulation of Na+,K+-ATPase activity in the renal medulla but not in the renal cortex. There was a peak in enzyme activity after cultured renal segments had been exposed to T3 for 11 min and this time of maximal stimulation did not vary with the concentration of T3. A rectilinear response in Na+,K+-ATPase activity was observed over T3 concentration range 10 pmol l-1 to 100 nmol l-1; at higher T3 concentrations, Na+,K+-ATPase activity was inhibited. The enzyme response was totally blocked by specific T3 antiserum. Addition of T4 and reverse T3 (100 fmol l-1 -1 mmol l-1) failed to stimulate Na+,K+-ATPase activity in any part of the kidney. Plasma (neat and diluted 1:10) stimulated the enzyme in parallel with the dose response curve and the stimulatory effect was abolished by prior addition of specific T3 antiserum.     

Cryptosin induces backbone structural changes in cardiac Na+ and K+ dependent adenosinetriphosphatase

Cryptosin, a new cardenolide, was found to preferentially bind to Na,K-ATPase enzyme (7), which is believed to be the ouabain binding site on cardiac sarcolemmal membrane. CD spectral studies revealed that cryptosin, in the presence of Na+ and Mg++ ions, bind to Na,K-ATPase and induce a dose-dependent change in the backbone structure of cardiac Na,K-ATPase.     

ADP binding to (Na + K)-activated ATPase

[¹⁴C]ADP binding to EDTA-washed ox brain cell membranes was increased by Na⁺, but decreased by K⁺, Mg²⁺ and Ca²⁺. Na⁺ abolished the effect of K⁺ on ADP binding by a competitive mechanism, but could not reverse the inhibitory action of Mg²⁺ and Ca²⁺. It is concluded that the cation-induced changes in ADP binding reflect properties of (Na⁺ + K⁺)-activated ATPase.     

Modification of ligand binding to the Na+/K+-activated ATPase

Interactions between the ligands Mg2+, K+, and substrate and the Na+/K+-activated ATPase were examined in terms of a rapid-equilibrium, random-order, terreactant kinetic scheme for the K+-nitrophenyl phosphatase reaction that is catalyzed by this enzyme. At 37 degrees C and pH 7.5 the derived values for the dissociation constants from the free enzyme were 0.2, 0.08, and 1.4 mM for Mg2+, K+, and substrate, respectively. For Mg2+ interactions, the presence of 20% (v/v) dimethyl sulfoxide (Me2SO) increased the calculated affinity 25-fold; higher concentrations increased affinity still further. Neither reducing the temperature to 20 degrees C nor altering the pH from 6.5 to 8.3 appreciably changed the affinity for Mg2+ in the absence or presence of Me2SO. The Mg2+ sites are thus characterized by an absence of functional groups ionizable in the pH range 6.5-8.3, with binding driven by entropy changes, and with Me2SO, probably through solvation effects on the protein, increasing affinity for Mg2+ close to that for Ca2+ and Mn2+. By contrast, for K+ interactions, the presence of 20% Me2SO increased the calculated affinity only by half; moreover, reducing the temperature to 20 degrees C and the pH to 6.5 both increased affinity and diminished the response to Me2SO. The K+ sites are thus characterized by a marked sensitivity to pH and temperature, presumably through alterations in enzyme conformational equilibria that in turn are modifiable by Me2SO. Inhibition by higher concentrations of Mg2+, which varies inversely with the K+ concentration, was decreased by Me2SO. Finally, for substrate interactions, the presence of 20% Me2SO increased the calculated affinity 4-fold, and, as for Mg2+-binding, neither reducing the temperature nor varying the pH over the range 6.5-8.3 appreciably altered the affinity in the absence or presence of Me2SO. Thus, the substrate sites, like the Mg2+ sites, are characterized by an absence of functional groups ionizable in this range, with binding driven by entropy changes, and with Me2SO increasing affinity for substrate, in this case probably through favoring the partitioning of substrate from the medium into the hydrophobic active site.     

Vanadate selectively inhibits the K0+-activated Na+ efflux in squid axons

The effects of internally applied 1 mM vanadate on the Na⁺ efflux in dialysed squid axons were found to depend on the presence of external K⁺. In K⁺-free artificial sea water, vanadate did not produce any change in the rate of Na⁺ efflux, whereas in the presence of 10 mM K⁺ the Na⁺ efflux was reduced to values even lower than those observed in the absence of K⁺ (inversion of the K⁺-free effect). In vanadate-poisoned axons, K⁺ and NH₄⁺ at low concentrations activated Na⁺ efflux, but at high concentrations both cations were inhibitory. However, NH₄⁺ was always a better activator and a poorer inhibitor than K⁺.     

Hydrolysis of adenylyl imidodiphosphate in the presence of Na+ + Mg+ by (Na+ + K+)-activated ATPase

(1) Contrary to what has usually been assumed, (Na⁺ + K⁺)-ATPase slowly hydrolyses AdoPP[NH]P in the presence of Na⁺ + Mg²⁺ to ADP-NH₂ and P_i. The activity is ouabain-sensitive and is not detected in the absence of either Mg²⁺ or Na²⁺. The specific activity of the Na⁺ + Mg²⁺ dependent AdoPP[NH]P hydrolysis at 37°C and pH 7.0 is 4% of that for ATP under identical conditions and only 0.07% of that for ATP in the presence of K⁺. The activity is not stimulated by K⁺, nor can K⁺ replace Na⁺ in its stimulatory action. This suggests that phosphorylation is rate-limiting. Stimulation by Na⁺ is positively cooperative with a Hill coefficient of 2.4; half-maximal stimulation occurs at 5–9 mM. The K_m value for AdoPP[NH]P is 17 μM. At 0°C and 21°C the specific activity is 2 and 14%, respectively, of that at 37°C. AMP, ADP and AdoPP[CH₂]P are not detectably hydrolysed by (Na⁺ + K⁺)-ATPase in the presence of Na⁺ + Mg²⁺. (2) In addition, AdoPP[NH]P undergoes spontaneous, non-enzymatic hydrolysis at pH 7.0 with rate constants at 0, 21 and 37°C of 0.0006, 0.006 and 0.07 h^?1, respectively. This effect is small compared to the effect of enzymatic hydrolysis under comparable conditions. Mg²⁺ present in excess of AdoPP[NH]P reduces the rate constant of the spontaneous hydrolysis to 0.005 h^?1 at 37°C, indicating that the MgAdoPP[NH]P complex is virtually stable to spontaneous hydrolysis, as is also the case for its enzymatic hydrolysis. (3) A practical consequence of these findings is that AdoPP[NH]P binding studies in the presence of Na⁺ + Mg²⁺ with enzyme concentrations in the mg/ml range are not possible at temperatures above 0°C. On the other hand, determination of affinity in the (Na⁺ + K⁺)-ATPase reaction by competition with ATP at low protein concentrations (μg/ml range) remains possible without significant hydrolysis of AdoPP[NH]P even at 37°C.     

Evidence against Na+-pump mediation of Ca2+-activated K+ transport and diuretic-sensitive (Na+/K+)-cotransport

Proteoliposomes reconstituted from purified Na⁺ pumps show neither Ca²⁺ activation nor bumetanide inhibition of Rb⁺ uptake, suggesting that the Na⁺ pump does not mediate these passive fluxes.     

The effect of chelators on Mg2+, Na+-dependent phosphorylation of (Na+ + K+)-activated ATPase.

1. The effect of free Mg2+, MgEDTA and MgCDTA on the phofphorylation of the (Na+ + K+)-activated ATPase (ATP phosphohydrolase, EC 3.6.1.3) has been studied. 2. 10 mM trans-1,2-diaminocyclohexane-N,N,N',N'-tetraacetic acid (CDTA) added simultaneously with [gamma-32P]ATP to a solution containing the enzyme, 1 mM Mg2+ and 150 mM Na+ does not prevent formation of phospho-enzyme. When [gamma-32P]ATP is added after CDTA the level of phospho-enzyme obtained decreases with increase in the time interval between addition of CDTA and ATP. The inability of CDTA to prevent the formation of phospho-enzyme becomes more pronounced when the medium contains MgEDTA. In the presence of CDTA the maximum amount of phospho-enzyme formed increases with the MgEDTA concentration. 3. Without CDTA the steady-state level of phospho-enzyme is directly proportional to the logarithm of free Mg2+ concentration. Neither with suboptimal nor with optimal concentrations of free Mg2+ does MgEDTA have an effect on the level of phospho-enzyme formed. 4. Using the phospho-enzyme level as a measure of free Mg2+ the experiments show that CDTA reacts slower with Mg2+ than does EDTA, but the stability constant of MgCDTA complex is higher than of MgCDTA, complex. 5. Due to the higher stability constant, of MgCDTA, as compared to MgEDTA, addition of CDTA to a medium containing free Mg2+ and MgEDTA will not only chelate the free Mg2+, but it will also shift the equilibrium from MgEDTA towards MgCDTA, i.e. MgEDTA acts as a source of free Mg2+ which is then chelated by CDTA. The experiments show that it takes minutes before Mg2+, EDTA and CDTA come to equilibrium. Provided the dissociation of MgEDTA is faster than the formation of the MgCDTA complex, the medium will contain a concentration of free Mg2+ which at any given instant is near in equilibrium with a slowly decreasing concentration of MgEDTA; this free Mg2+ can support phosphorylation. This can explain why the rate with which CDTA stops phosphorylation decreases with an increase in the MgEDTA concentration. 6. When phosphorylation is stopped by addition of unlabelled ATP, the rate of dephosphorylation is faster than when it is stopped by addition of CDTA both with and without EDTA in the medium. CDTA reacts too slowly with Mg2+ to be used as a chelator in studies where a fast removal of Mg2+ is required. 7. A previous finding has been verified, namely that the rate of spontaneous, of K+-stimulated and of ADP-stimulated dephosphorylation is independent of the Mg2+ concentration during formation of phospho-enzyme.     

(Na+, K+)-activated adenosinetriphosphatase of axonal membranes, cooperativity and control. Steady-state analysis.

1. The ATP sites. Homotropic interactions between ATP sites have been studied in a very large range of Na+ and K+ concentrations. The ( Na+, K+)-activated ATPase displays Michaelis-Menten kinetics for ATP under standard concentration conditions of Na+ (100 mM) and K+ (10 mM). The steady-state kinetics behavior changes at very low concentrations of K+ where negative cooperativity is observed. The existence of a high affinity and a low affinity site for ATP was clearly demonstrated from the study of the ATP stimulated hydrolysis of p-nitrophenylphosphate in the presence of Na+ and K+. The ratio of apparent affinities of high and low affinity sites for ATP is 86 at pH 7.5. 2. The Na+ sites. The binding of Na+ to its specific stimulatory sites (internal sites) is characterized by positive cooperativity with a Hill coefficient n(H(Na+))=2.0. Homotropic interactions between Na+ sites are unaffected by variations of the K+ concentration. 3. The K+ sites. (a) Binding of K+ to the (external) stimulatory site of the ATPase has been analyzed by following the (Na+, K+)-ATPase activity as well as the p-nitrophenylphosphatase activity in the presence of Na+ and K+ (with or without ATP). Binding is characterized by a Hill coefficient of 1.0 and a K(0.5(K+))=0.1 to 0.8 mM. The absence of positive or negative cooperativity persists between 5 mM and 100 mM Na+. (b) The analysis of the p-nitrophenylphosphatase or of the 2, 4 dinitrophenylphosphatase activity in the presence of K+ alone indicates the existence of low affinity sites for K+ with positive homotropic interactions. The characteristics of stimulation in that case are, K(0.5)=5 mM, n(H)=1.9. The properties of this family of site(s) are the following: firstly, saturation of the low affinity site(s) by K+ prevents ATP binding to its high affinity internal site. Secondly, saturation of the low affinity sites for K+ prevents binding of Na+ to its internal sites. Thirdly, this family of sites disappears in the presence of ATP, p-nitrophenylphosphate or of both substrates, when Na+ binds to its internal sites. Na+ binding to its specific stimulatory sites provokes the formation of the high affinity type of site for K+. 4. Mg2+ stimulation of the (Na+, K+)-ATPase is characterized by a Hill coefficient n(H(Mg2+))=1.0 and a K(0.5(Mg2+))=1 mM stimulation is essentially a V effect. Heterotropic effects between binding of Mg2+ and substrate to their respective sites are small. Heterotropic interactions between the Ms2+, Na+ and K+ sites are also small. 5. The fluidity of membrane lipids also controls the (Na+, K+)-ATPase activity. Phase transitions or separations in the membrane hardly affect recognition properties of substrates, Na+, K+ and Mg2+ for their respective sites on both sides of the membrane. Only the rate of the catalytic transformation is affected.