Jens G. Nørby

Binding of ATP to brain microsomal ATPase Determination of the ATP-binding capacity and the dissociation constant of the enzyme-ATP complex as a function of K+ concentration

Abstract 1. 1. The binding of the ATP molecule to brain microsomal (Na+ + K+)-activated ATPase was studied by a rapid dialysis rate technique. 2. 2. The experiments were performed at 2° in the presence of 10 mM EDTA to minimize hydrolysis of ATP. The ionic strength was 0.073 M, pH 7.4. 3. 3. The specific activity of the preparations and the Na+ + K+ activity/Mg2+ activity ratio were changed by heat denaturation. 4. 4. The dissociation constant of the enzyme-ATP complex ( E- ATP ) was 0.12 μM. 5. 5. The Mg2+ requirement for binding of ATP to the enzyme, if any, was much lower than that for hydrolysis of ATP. The binding seemed to be independent of Na+. 6. 6. Increasing the concentration of K+ up to 3 mM led to an increase in the apparent dissociation constant of E- ATP towards a maximum (0.7 μM). The effect of K+ could be described by a model involving the formation of the following complexes: E- ATP , K + -E and K + -E- ATP . 7. 7. Proportionality was found between the ATP-binding capacity (nmoles/mg protein) and (Na+ + K+)-ATPase activity (μmoles P1 released per mg protein per h), suggesting that binding to (Na+ + K+)-ATPase was measured. The catalytic center activity based on this assumption was 7000 ± 470 min −1 .

Scatchard plot: Common misinterpretation of binding experiments

Abstract The widespread misinterpretation in the literature of ligand-protein binding experiments which show upward curvature in Scatchard plots is emphasized. The most commonly encountered errors are discussed and references to the correct methods of resolution of upward-curved Scatchard plots are given.

On the specificity of the ATP-binding site of (Na+ + K+)-activated ATPase from brain microsomes

Abstract In order to obtain information on the specificity of the ATP-binding site of ATPase from brain microsomes, the influence of pyrophosphate, adenine, adenosine, AMP, ADP, CTP, GTP, and ITP on ATP binding was investigated by a rapid dialysis rate technique previously described. 1. 1. Pyrophosphate, adenine, adenosine and AMP in concentrations up to about 3 orders of magnitude higher than that of free ATP did not affect the binding of ATP. 2. 2. Less ATP was bound in the presence than in the absence of ADP. Based on a model for competitive binding the affinity of the ATP-binding site for ADP was found to be 5 times smaller than for ATP. 3. 3. CTP, GTP and ITP were only able to displace ATP from the ATP-binding site in concentrations very high relative to that of free ATP. The affinity for these triphosphates was at least 250–850 times lower than for ATP, CTP having the highest affinity of the three compounds examined. 4. 4. The fact that the nucleotide specificity of the ATP-binding site is similar to the substrate specificity of (Na + + K + )-activated ATPase strongly suggests that this binding site is identical with the substrate site of (Na + + K + )-activated ATPase. 5. 5. It is concluded that the 6-amino group of the purine and pyrimidine ring as well as the β-phosphate group are essential for binding to the substrate site of (Na + + K + )-activated ATPase, and that binding of ATP also involves a link between the γ-phosphate group and the enzyme.

(Na+ + K+)-ATPase: confirmation of the three-pool model for the phosphointermediates of Na+-ATPase activity. Estimation of the enzyme-ATP dissociation rate constant

The dephosphorylation kinetics of acid-stable phosphointermediates of (Na+ + K+)-ATPase from ox brain, ox kidney and pig kidney was studied at 0 degree C. Experiments performed on brain enzyme phosphorylated at 0 degree C in the presence of 20-600 mM Na+, 1 mM Mg2+ and 25 microM [gamma-32P]ATP show that irrespectively of the EP-pool composition, which is determined by Na+ concentration, all phosphoenzyme is either ADP- or K+-sensitive. After phosphorylation of kidney enzymes at 0 degree C with 1 mM Mg2+, 25 microM [gamma-32P]ATP and 150-1000 mM Na+ the amounts of ADP- and K+-sensitive phosphoenzymes were determined by addition of 1 mM ATP + 2.5 mM ADP or 1 mM ATP + 20 mM K+. Similarly to the previously reported results on brain enzyme, both types of dephosphorylation curves have a fast and a slow phase, so that also for kidney enzymes a slow decay of a part of the phosphoenzyme, up to 80% at 1000 mM Na+, after addition of 1 mM ATP + 20 mM K+ is observed. The results obtained with the kidney enzymes seem therefore to reinforce previous doubts about the role played by E1 approximately P(Na3) as intermediate of (Na+ + K+)-ATPase activity. Furthermore, for both kidney enzymes the sum of ADP- and K+-sensitive phosphoenzymes is greater than E tot. In experiments on brain enzyme an estimate of dissociation rate constant for the enzyme-ATP complex, k-1, is obtained. k-1 varies between 1 and 4 s-1 and seems to depend on the ligands present during formation of the complex. The highest values are found for enzyme-ATP complex formed in the presence of Na+ or Tris+. The results confirm the validity of the three-pool model in describing dephosphorylation kinetics of phosphointermediates of Na+-ATPase activity.

Solubilization and further chromatographic purification of highly purified, membrane-bound Na,K-ATPase

Highly purified membrane-bound Na,K-ATPase from pig kidney outer medulla was dissolved in the non-ionic detergent C12E8. Chromatography of the dissolved material on a DEAE matrix yielded enzymatical material having a ouabain-binding capacity of 6.9 nmoles per mg protein (measured according to Lowry et al., with bovine serum albumin as standard). This material, which after addition of lipids had the same K+-phosphatase turnover as the membrane-bound enzyme, could consist entirely of live molecules with a molecular weight of 145 kDa, a value close to that expected for alpha beta-promoters of Na,K-ATPase.

The role of Mg2+ and K+ in the phosphorylation of Na+,K+-ATPase by ATP in the presence of dimethylsulfoxide but in the absence of Na+

We have previously demonstrated that Na+,K(+)-ATPase can be phosphorylated by 100 microM ATP and 5 mM Mg2+ and in the absence of Na+, provided that 40% dimethylsulfoxide (Me2SO) is present. Phosphorylation was stimulated by K+ up to a steady-state level of about 50% of Etot (Barrabin et al. (1990) Biochim. Biophys. Acta 1023, 266-273). Here we describe the time-course of phosphointermediate (EP) formation and of dephosphorylation of EP at concentrations of Mg2+ from 0.1 to 5000 microM and of K+ from 0.01 to 100 mM. The results were simulated by a simplified version of the commonly accepted Albers-Post model, i.e. a 3-step reaction scheme with a phosphorylation, a dephosphorylation and an isomerization/deocclusion step. Furthermore it was necessary to include an a priori, Mg(2+)- and K(+)-independent, equilibration between two enzyme forms, only one of which (constituting 14% of Etot) reacted directly with ATP. The role of Mg(2+) was two-fold: At low Mg2+, phosphorylation was stimulated by Mg2+ due to formation of the substrate MgATP, whereas at higher concentrations it acted as an inhibitor at all three steps. The affinity for the inhibitory Mg(2+)-binding was increased several-fold, relative to that in aqueous media, by dimethylsulfoxide. K+ stimulated dephosphorylation at all Mg(2+)-concentrations, but at high, inhibitory [Mg2+], K+ also stimulated the phosphorylation reaction, increasing both the rate coefficient and the steady-state level of EP. Generally, the presence of Me2SO seems to inhibit the dephosphorylation step, the isomerization/deocclusion step, and to a lesser extent (if at all) the phosphorylation reaction, and we discuss whether this reflects that Me2SO stabilizes occluded conformations of the enzyme even in the absence of monovalent cations. The results confirm and elucidate the stimulating effect of K+ on EP formation from ATP in the absence of Na+, but they leave open the question of the molecular mechanism by which Me2SO, inhibitory Mg2+ and stimulating K+ interact with the Na+,K(+)-ATPase.

Phosphorylation of Na+,K+-ATPase by ATP in the presence of K+ and dimethylsulfoxide but in the absence of NA+

Purified Na+, K(+)-ATPase was phosphorylated by [gamma-32P]ATP in a medium containing dimethylsulfoxide and 5 mM Mg2+ in the absence of Na+ and K+. Addition of K+ increased the phosphorylation levels from 0.4 nmol phosphoenzyme/mg of protein in the absence of K+ to 1.0 nmol phosphoenzyme/mg of protein in the presence of 0.5 mM K+. Higher velocities of enzyme phosphorylation were observed in the presence of 0.5 mM K+. Increasing K+ concentrations up to 100 mM lead to a progressive decrease in the phosphoenzyme (EP) levels. Control experiments, that were performed to determine the contribution to EP formation from the Pi inevitably present in the assays, showed that this contribution was of minor importance except at high (20-100 mM) KCl concentrations. The pattern of EP formation and its KCl dependence is thus characteristic for the phosphorylation of the enzyme by ATP. In the absence of Na+ and with 0.5 mM K+, optimal levels (1.0 nmol EP/mg of protein) were observed at 20-40% dimethylsulfoxide and pH 6.0 to 7.5. Addition of Na+ up to 5 mM has no effect on the phosphoenzyme level under these conditions. At 100 mM Na+ or higher the full capacity of enzyme phosphorylation (2.2 nmol EP/mg of protein) was reached. Phosphoenzyme formed from ATP in the absence of Na+ is an acylphosphate-type compound as shown by its hydroxylamine sensitivity. The phosphate radioactivity was incorporated into the alpha-subunit of the Na+, K(+)-ATPase as demonstrated by acid polyacrylamide gel electrophoresis followed by autoradiography.

Thallium binding to native and radiation-inactivated Na+/K+-ATPase

The number of high-affinity K+-binding sites on purified Na+/K+-ATPase from pig kidney outer medulla has been assessed by measurement of equilibrium binding of thallous thallium, Tl+, under conditions (low ionic strength, absence of Na+ and Tris+) where the enzyme is in the E2-form. Na+/K+-ATPase has two identical Tl+ sites per ADP site, and the dissociation constant varies between 2 and 9 microM. These values are identical to those for Tl+ occlusion found previously by us, indicating that all high-affinity binding leads to occlusion. The specific binding was obtained after subtraction of a separately characterized unspecific adsorption of Tl+ to the enzyme preparations. Radiation inactivation leads to formation of modified peptides having two Tl+-binding sites with positive cooperativity, the second site-dissociation constant approximating that for the native sites. The radiation inactivation size (RIS) for total, specific Tl+ binding is 71 kDa, and the RIS for Tl+ binding with original affinity is approx. 190 kDa, equal to that of Na+/K+-ATPase activity and to that for Tl+ occlusion with native affinity. This latter RIS value confirms our recent theory that in situ the two catalytic peptides of Na+/K+-ATPase are closely associated. The 71 kDa value obtained for total Tl+ sites is equal to that for total binding of ATP and ADP and it is clearly smaller than the molecular mass of one catalytic subunit (112 kDa). The Tl+-binding experiments reported thus supports the notion that radiation inactivation of Na+/K+-ATPase is a stepwise rather than an all or none process.

A kinetic model for N-ethylmaleimide inhibition of the (Na+ + K+)-ATPase from rectal glands of Squalus acanthias

Abstract (1) The time-course of inhibition by N- ethylmaleimide (NEM) of ( Na + + K + )- ATPase and K + -p- nitrophenylphosphatase activity of ( Na + + K + )- ATPase from Squalus acanthias has been followed over a 4000-fold inhibitor concentration range. The ( Na + + K + )- ATPase and the K + -p- nitrophenylphosphatase were inhibited in parallel at all inhibitor concentrations. (2) The data obtained have led to a model with the following features. (a) The enzyme is inactivated via two routes in which the reactivities towards NEM are different. This can explain that about 80% of the activity disappears 40–1000-times faster than the remaining 20%. (b) A primary reaction of SH groups with NEM without inhibition which is required before (c) a subsequent rection with NEM and inhibition can take place (i.e., a lag period). (d) A complicated concentration dependence of the rates of inactivation in the rapid phase which is interpreted in terms of an additional equilibrium between two different enzyme conformations. (3) The results are compatible with our previous finding (Esmann, M. (1982) Biochim. Biophys. Acta 688, 260–270) that more than one SH group per enzyme molecule are modified when the enzyme is inactivated.

The effect of di]methylsulfoxide on the substrate site of Na+/K+-ATPase studied through phosphorylation by inorganic phosphate and ouabain binding

To obtain further information on the role of H2O at the substrate site of Na+/K(+)-ATPase, we have studied the enzymes reaction with P(i) and ouabain in 40% (v/v) Me2SO (dimethylsulfoxide). When the enzyme (E) was incubated with ouabain (O) for 5 min in a 40% (v/v) Me2SO-medium with 5 mM MgCl2 and 0.5 mM KCl (but no phosphate), ouabain was bound (as EO). Subsequent incubation with P(i) showed that E, but not EO, was rapidly phosphorylated (to EP). Long-time phosphorylation revealed that EO is also phosphorylated by P(i) albeit very slowly (t1/2 about 60 min) and that binding of ouabain to EP also is very slow. The EOP complex is stable, i.e., the t1/2 for the loss of P(i) is >> 60 min in contrast to about 1 min in water. These results in 40% Me2SO are distinctly different from what would be obtained in a watery milieu: ouabain would bind slowly and inefficiently in the absence of P(i), and ouabain would catalyse phosphorylation from P(i) rather than retard it. Equilibrium binding of [3H]ouabain to E and EP in water or 40% Me2SO confirmed these observations: Kdiss in water is 11 microM and 12 nM for EO and EOP, respectively, whereas in Me2SO they are 112 nM and 48 nM. It is suggested that the primary effect of the lowered water activity in 40% Me2SO is a rearrangement of the substrate site so that it also in the absence of P(i) attains a transition state configuration corresponding to the phosphorylated conformation. This would be sensed by the ouabain binding site and lead to high affinity ouabain binding in the absence of P(i).