(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.     

trans-1,2-Diaminocyclohexane-N,N,N',N'-tetraacetic acid is superior to ethylenediamine-N,N,N',N'-tetraacetic acid for sequestering Mg2+ in 31P NMR experiments involving ATP spectra at neutral and acidic pH

The use of ethylenediamine-N,N,N',N'-tetraacetic acid (EDTA) to sequester Mg2+ from samples containing ATP at acidic or neutral pH prior to 31P NMR spectroscopic analysis leads to significant broadening of the gamma- and beta-phosphorus resonances of ATP as compared to ATP alone. It was found that the use of trans-1,2-diaminocyclohexane-N,N,N',N'-tetraacetic acid (CDTA) reduces the broadening of the ATP resonances. At pH 7.0, 30 mM EDTA in the presence of 5 mM ATP and 7 mM Mg2+ leads to a threefold increase in the peak width of the gamma phosphorus of ATP as compared to 5 mM ATP alone. When 30 mM CDTA is used in the place of EDTA, the peak width decreased to about 80% of the peak width of ATP alone. When the experiment is repeated at pH 8.5, both EDTA and CDTA lead to narrow peak widths with no significant difference between the two spectra. At pH 6.0, the use of EDTA leads to a spectrum that is very noisy, with a 10-fold increase in the peak width as compared to ATP in the absence of Mg2+ at this pH, whereas the increase with CDTA is only 50%. These results do not reflect the difference in chelating strength between EDTA and CDTA: The free Mg2+ concentration in the presence of each chelator, as calculated by the computer program given in the Appendix, was nearly equal at each pH. The results, however, reflect a difference in the lability of the metal-ligand bond between EDTA and CDTA.     

Presence of a low-affinity nucleotide binding site on the (K+ + H+)-ATPase phosphoenzyme

The effects of Mg2+ and nucleotides on the dephosphorylation process of the (K+ + H+)-ATPase phosphoenzyme have been studied. Phosphorylation with [gamma-32P]ATP is stopped either by addition of non-radioactive ATP or by complexing of Mg2+ with EDTA. The dephosphorylation process is slow and monoexponential when dephosphorylation is initiated with ATP. When phosphorylation is stopped by complexing of Mg2+ the dephosphorylation process is fast and biexponential. The discrepancy could be explained by a nucleotide mediated inhibition of the dephosphorylation process. The I0.5 for ATP for this inhibition is 0.1 mM and that for ADP is 0.7 mM, suggesting that a low-affinity binding site is involved. When Mg2+ is present in millimolar concentrations in addition to the nucleotides the dephosphorylation process is enhanced. Evidence has been obtained that Mg2+ acts through lowering the affinity for ATP. In contrast to K+, Mg2+ does not stimulate dephosphorylation in the absence of nucleotides. Mg2+ and nucleotides show the same interaction in the dephosphorylation process of a phosphoenzyme generated from inorganic phosphate. These findings suggest the presence of a low-affinity nucleotide binding site on the phosphoenzyme, as is found in the (Na+ + K+)-ATPase phosphoenzyme. This low-affinity binding site may function as a feed-back mechanism in proton transport.     

Pb2+ and imidazole-activated phosphorylation by ATP of (Na+ + K+)-ATPase

In order to study whether Pb2+ and imidazole increase the ATP phosphorylation level of (Na+ + K+)-ATPase by the same mechanism, the effects of both compounds on phosphorylation and dephosphorylation reactions of the enzyme have been studied. Imidazole in the presence of Mg2+ increases steady-state phosphorylation of (Na+ + K+)-ATPase by decreasing, in a competitive way, the K+-sensitivity of the formed phospho-enzyme (E-P . Mg). If Pb2+ is present during phosphorylation, the rate of phosphorylation increases and a K+- and ADP-insensitive phosphointermediate (E-P . Pb) is formed. Pb2+ has no effect on the K+-sensitivity of E-P . Mg and EDTA is unable to affect the K+-insensitivity of E-P . Pb. These effects indicate that Pb2+ acts before or during phosphorylation with the enzyme. Binding of Na+ to E-P . Pb does not restore K+-sensitivity either. However, increasing Na+ during phosphorylation in the presence of Pb2+ leads to formation of the K+-sensitive intermediate (E-P . Mg), indicating that E-P . Pb is formed via a side path of the Albers-Post scheme. ATP and ADP decrease the dephosphorylation rate of both E-P . Mg and E-P . Pb. Above optimal concentration, Pb2+ also decreases the steady-state phosphorylation level both in the absence and in the presence of Na+. This inhibitory effect of Pb2+ is antagonized by Mg2+.     

The substitution of calcium for magnesium in H+,K+-ATPase catalytic cycle. Evidence for two actions of divalent cations

In order to determine the role of divalent cations in the reaction mechanism of the H+,K+-ATPase, we have substituted calcium for magnesium, which is required by the H+,K+-ATPase for phosphorylation from ATP and from PO4. Calcium was chosen over other divalent cations assayed (barium and manganese) because in the absence of magnesium, calcium activated ATP hydrolysis, generated sufficiently high levels of phosphoenzyme (573 +/- 51 pmol.mg-1) from [gamma-32P]ATP to study dephosphorylation, and inhibited K+-stimulated ATP hydrolysis. The Ca2+-ATPase activity of the H+,K+-ATPase was 40% of the basal Mg2+-ATPase activity. However, the Ca2+,K+-ATPase activity (minus the Ca2+ basal activity) was only 0.7% of the Mg2+,K+-ATPase, indicating that calcium could partially substitute for Mg2+ in activating ATP hydrolysis but not in K+ stimulation of ATP hydrolysis. Approximately 0.1 mM calcium inhibited 50% of the Mg2+-ATPase or Mg2+,K+-ATPase activities. Inhibition of Mg2+,K+-ATPase activity was not competitive with respect to K+. Inhibition by calcium of Mg2+,K+ activity p-nitrophenyl phosphatase activity was competitive with respect to Mg2+ with an apparent Ki of 0.27 mM. Proton transport measured by acridine orange uptake was not detected in the presence of Ca2+ and K+. In the presence of Mg2+ and K+, Ca2+ inhibited proton transport with an apparent affinity similar to the inhibition of the Mg2+, K+-ATPase activity. The site of calcium inhibition was on the exterior of the vesicle. These results suggest that calcium activates basal turnover and inhibits K+ stimulation of the H+,K+-ATPase by binding at a cytosolic divalent cation site. The pseudo-first order rate constant for phosphoenzyme formation from 5 microM [gamma-32P]ATP was at least 22 times slower in the presence of calcium (0.015 s-1) than magnesium (greater than 0.310 s-1). The Ca.EP (phosphoenzyme formed in the presence of Ca2+) formed dephosphorylated four to five times more slowly that the Mg.EP (phosphoenzyme formed in the presence of Mg2+) in the presence of 8 mm trans-1,2-diaminocyclohexane-N,N,N',N'-tetraacetic acid (CDTA) or 250 microM ATP. Approximately 10% of the Ca.EP formed was sensitive to a 100 mM KCl chase compared with greater than 85% of the Mg.EP. By comparing the transient kinetics of the phosphoenzyme formed in the presence of magnesium (Mg.EP) and calcium (Ca.EP), we found two actions of divalent cations on dephosphorylation.(ABSTRACT TRUNCATED AT 400 WORDS)     

The effect of Mg2+ and chelating agents on intermediary steps of the reaction ofNa+,K+-activated ATPase.

(1) It has been investigated how varying concentrations of free magnesium with and without EDTA influence the properties of the phospho-enzyme formed in the presence of sodium by the (Na+ plus K+)-activated enzyme system. (2) The phospho-enzyme formed in the presence of sodium and a high concentration of free magnesium has the same rate of (a) spontaneous dephosphorylation, (b) dephosphorylation after addition of potassium, and (c) dephosphorylation after addition of ADP, as a phospho-enzyme formed in the presence of sodium and a low concentration of magnesium. (3) With sodium and a given concentration of free magnesium, high or low, EDTA present during formation of the phospho-enzyme leads to a decrease in the rate of (a) spontaneous dephosphorylation, and (b) dephosphorylation after addition of potassium to the phospho-enzyme. (4) The rate of dephosphorylation after addition of ADP to phospho-enzyme formed without and with EDTA is the same. But as the rate of spontaneous dephosphorylation is lower with EDTA than without, ADP gives a higher increase in the rate of dephosphorylation of phospho-enzyme formed with EDTA than without. (5) The experiments thus show that the reported different sensitivity towards potassium and ADP of phospho-enzyme formed in the presence of a low and high concentration of free magnesium, respectively, is due to the EDTA used to decrease the free magnesium concentration and not to the decrease in the free magnesium as such.     

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.     

Bovine brain Na+,K+-stimulated ATP phosphohydrolase studied by a rapid-mixing technique. K+-stimulated liberation of [32P] orthophosphate from [32P] phosphoenzyme and resolution of the dephosphorylation into two phases.

Dephosphorylation of [32P]phosphoenzyme of bovine brain Na+,K+-stimulated ATP phosphohydrolase (EC 3.6.1.3), labelled by [gamma-32P]ATP, was investigated at 21 degrees C by means of a rapid-mixing technique. On addition of a high concentration of KCl (10 mM) to [32P]phosphoenzyme at steady state in the presence of Mg2+ and Na+, very rapid dephosphorylation was obtained. Simultaneously, the amount of [32P]orthophosphate increased at about the same rate. It was concluded that this K+-stimulated dephosphorylation and liberation of [32P]orthophosphate from the [32P]phosphoenzyme was rapid enough to participate in the Na+,K+-stimulated hydrolysis of ATP. In order to study the dephosphorylation in absence of continuing 32P-labelling, excess unlabelled ATP or a chelator of Mg2+ was added. Simultaneous addition of a high concentration of KCl to the [32P]phosphoenzyme formed in the presence of Mg2+ and Na+ but in the absence of K+, resulted in an initial very rapid phase and a subsequent slower phase of dephosphorylation. With KCl also initially present in the incubation medium, only the slow phase was observed. The slow phase of dephosphorylation also seemed to be sufficiently rapid to participate in the Na+, K+-stimulated ATPase reaction. On addition of a high concentration of ADP (5 mM) to [32P]phosphoenzyme formed in the presence of Mg2+ and Na+, an initial comparatively rapid, and later slow phase of dephosphorylation were detected. This gave further support for different forms of phosphoenzyme. Approximate concentrations of these forms, in the absence and presence of KCl, were estimated by extrapolation and the turnover of these forms was calculated. The nature of the kinetically different components of phosphoenzyme and their role in the Na+, K+-stimulated ATPase reaction is discussed.     

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.     

The reaction of Mg2+ with the Ca2+-ATPase from human red cell membranes and its modification by Ca2+

Media prepared with CDTA and low concentrations of Ca2+, as judged by the lack of Na+-dependent phosphorylation and ATPase activity of (Na+ +K+)-ATPase preparations are free of contaminant Mg2+. In these media, the Ca2+-ATPase from human red cell membranes is phosphorylated by ATP, and a low Ca2+-ATPase activity is present. In the absence of Mg2+ the rate of phosphorylation in the presence of 1 microM Ca2+ is very low but it approaches the rate measured in Mg2+-containing media if the concentration of Ca2+ is increased to 5 mM. The KCa for phosphorylation is 2 microM in the presence and 60 microM in the absence of Mg2+. Results are consistent with the idea that for catalysis of phosphorylation the Ca2+-ATPase needs Ca2+ at the transport site and Mg2+ at an activating site and that Ca2+ replaces Mg2+ at this site. Under conditions in which it increases the rate of phosphorylation, Ca2+ is without effect on the Ca2+-ATPase activity in the absence of Mg2+ suggesting that to stimulate ATP hydrolysis Mg2+ accelerates a reaction other than phosphorylation. Activation of the E1P----E2P reaction by Mg2+ is prevented by Ca2+ after but not before the synthesis of E1P from E1 and ATP, suggesting that Mg2+ stabilizes E1 in a state from which Mg2+ cannot be removed by Ca2+ and that Ca2+ stabilizes E1P in a state insensitive to Mg2+. The response of the Ca2+-ATPase activity to Mg2+ concentration is biphasic, activation with a KMg = 88 microM is followed by inhibition with a Ki = 9.2 mM. Ca2+ at concentration up to 1 mM acts as a dead-end inhibitor of the activation by Mg2+, and Mg2+ at concentrations up to 0.5 mM acts as a dead-end inhibitor of the effects of Ca2+ at the transport site of the Ca2+-ATPase.     

Specific effects of spermine on Na+,K+-adenosine triphosphatase

Specific effects of spermine on Na+,K+-ATPase were observed using an enzyme partially purified from rabbit kidney microsomes by extraction with deoxycholate. 1. Spermine competed with K+ for K+-dependent, ouabain-sensitive nitrophenylphosphatase. The K1 for spermine was 0.075 mm in the presence of 1 mM Mg2+ and 5 mM p-nitrophenylphosphate at pH 7.5. 2. spermine activated Na+,K+-ATPase over limited concentration ranges of K+ and Na+ in the presence of 0.05 mM ATP. The spermine concentration required for half maximal activation was 0.055 mM in the presence of 1 mM K+, 10 mM Na+, 1 mM Mg2+, and 0.05 mM ATP. 3. The activation of Na+,K4-ATPase was not due to substitution of spermine for K+, Na+, or Mg2+. 4. When the concentration of K+ or Na+ was extremely low, or in excess, spermine did not activate Na+,K+-ATPase, but inhibited it slightly. 5. Plots of 1/v vs. 1/[ATP] at various concentrations of spermine showed that spermine decreased the Km for ATP without changing the Vmax. 6. Plots of 1/v vs. 1/[ATP] at concentrations of K+ from 0.05 mM to 0.5 mM showed that K+ increased the Km for ATP with increase in the Vmax in the presence of 0.2 mM spermine similarly to that in the absence of spermine. The contradictory effects of spermine on this enzyme system suggest that the K+-dependent monophosphatase activity does not reflect the second half (the dephosphorylation step) of the Na+,K+-ATPase catalytic cycle.     

The pre-steady-state hydrolysis of ATP by porcine brain (Na+ + K+)-dependent ATPase.

The hydrolysis of [gamma-32P]ATP by porcine brain (Na+ + K+)-stimulated ATP phosphohydrolase (EC 3.6.1.3) has been studied at 28 degree C in a rapid mixing quenched-flow apparatus. An "early burst" in the release of Pi from ATP has been observed when the enzyme is mixed with ATP, Na+ and a relatively high concentration of K+ (10 mM) but the burst is less pronounced with 0.5 mM K+. This "early burst" of Pi release is suppressed when the enzyme is pre-mixed with 10 mM K+ or 20% (v/v) dimethylsulphoxide before mixing with ATP and Na+, and premixing of enzyme with Na+ antagonizes this effect of dimethylsulphoxide. The results have been analysed by a non-linear least squares regression treatment and are consistent with a mechanism involving three steps, one of which may be a relatively slow change in enzyme conformation following release of Pi from its covalent linkage with the enzyme, in addition to formation of the enzyme-substrate complex. Rate constants (and S.E.) for these steps have been calculated and the roles of phospho-enzyme and other intermediates in the reaction mechanism of the transport ATPase are dicussed.     

Sodium and buffer cations inhibit dephosphorylation of (Na+ + K+)-ATPase

Effects of various cations on the dephosphorylation of (Na+ + K+)-ATPase, phosphorylated by ATP in 50 mM imidazole buffer (pH 7.0) at 22 degrees C without added Na+, have been studied. The dephosphorylation in imidazole buffer without added K+ is extremely sensitive to K+-activation (Km K+ = 1 microM), less sensitive to Mg2+-activation (Km Mg2+ = 0.1 mM) and Na+-activation (Km Na+ = 63 mM). Imidazole and Na+ effectively inhibit K+-activated dephosphorylation in linear competitive fashion (Ki imidazole 7.5 mM, Ki Na+ 4.6 mM). The Ki for Na+ is independent of the imidazole concentration, indicating different and non-interacting inhibitory sites for Na+ and imidazole. Imidazole inhibits Mg2+-activated dephosphorylation just as effective as K+-activated dephosphorylation, as judged from the Ki values for imidazole in the two processes. Tris buffer and choline chloride, like imidazole, inhibit dephosphorylation in the presence of residual K+ (less than 1 microM), but less effectively in terms of I50 values and extent of inhibition. Tris inhibits to the same extent as choline. This indicates different inhibitory sites for Tris or choline and for imidazole. These findings indicate that high steady-state phosphorylation levels in Na+-free imidazole buffer are due to the induction of a phosphorylating enzyme conformation and to the inhibition of (K+ + Mg2+)-stimulated dephosphorylation.     

Inhibition of oxidative phosphorylation by Ca2+ or Sr2+: a competition with Mg2+ for the formation of adenine nucleotide complexes

Intramitochondrial Sr2+, similar to Ca2+, inhibits oxidative phosphorylation in intact rat-liver mitochondria. Both Ca2+ and Sr2+ also inhibit the hydrolytic activity of the ATPase in submitochondrial particles. Half-maximal inhibition of ATPase activity was attained at a concentration of 2.5 mM Ca2+ or 5.0 mM Sr2+ when the concentration of Mg2+ in the medium was 1.0 mM. The inhibition of ATPase activity by both cations was strongly decreased by increasing the Mg2+ concentration in the reaction medium. In addition, kinetical data and the determination of the concentration of MgATP, the substrate of the ATPase, in the presence of different concentrations of Ca2+ or Sr2+ strongly indicate that these cations inhibit ATP hydrolysis by competing with Mg2+ for the formation of MgATP. On the basis of a good agreement between these results with submitochondrial particles and the results of titrations of oxidative phosphorylation with carboxyatractyloside or oligomycin in mitochondria loaded with Sr2+ it can be concluded that intramitochondrial Ca2+ or Sr2+ inhibits oxidative phosphorylation in intact mitochondria by decreasing the availability of adenine nucleotides to both the ADP/ATP carrier and the ATP synthase.     

Phosphorylation of Cl-, HCO3--stimulated Mg2+-ATPase of plasma membranes of carp (Cyprinus carpio L.) brain sensitive to GABA(A)-ergic ligands

Phosphorylation of the sensitive to GABA(A)-ergic ligands Cl-, HCO3--stimulated Mg2+-ATPase of the plasma membranes from fish brain by [gamma-32P]ATP was investigated in the presence of Mg2+. It was established, that formation of the phosphoprotein at 0-1 degrees C is dependent on time incubation and concentration of Mg2+ in the incubation medium. Hydroxylamine (50 mM) and pH (10) completely inhibited formation of phosphorylated intermediate. Ions of Cl- (10 mM)+HCO3- (2 mM) and also GABA (1-100 microM) dephosphorylated the enzyme. The dephosphorylating effect of GABA on the membrane samples did not appear in the presence of bicuculline. o-Vanadate (10 microM) eliminates the dephosphorylating effect of anions and GABA on the phosphoprotein. It was established by SDS-PAAG electrophoresis and autoradiographia that investigated phosphorylation and GABA(A)-induced dephosphorylation is performed by the protein with molecular weight aproximately 56 kDa. Such molecular weight has a subunit which forms oligomer composition of the sensitive to GABA(A)-ergic ligands Cl-, HCO3--ATPase from fish brain. The obtained data demonstrated that Cl, HCO3- ATPase from fish brain can be directly phosphorylated by [gamma-32P]ATP in the presence of Mg2+ and forms the phosphorylation intermediate.     

Regulation of the microtubule-lysosome interaction: activation by Mg2+ and inhibition by ATP

We developed a sedimentation assay to characterize and quantify the association of purified lysosomes to reconstituted microtubules (Mithieux, G., Audebet, C. and Rousset. B. (1988) Biochim. Biophys. Acta 969, 121-130). In the present work, we have examined the potential regulatory role of ATP and Mg2+ on the microtubule-lysosome interaction. The formation of microtubule-lysosome complexes takes place in the absence of Mg2+, but is activated by the addition of Mg2+; both the rate of the interaction and the amount of complexes formed are increased. The maximal effect is observed between 1.5 and 3.5 mM free Mg2+. Measured at the plateau of the interaction, the proportion of microtubules bound to lysosomes increases as a function of free Mg2+ concentration; at optimal concentration of free Mg2+, 90% of the microtubules present in the incubation mixture are bound to lysosomes. ATP induces a concentration-dependent inhibition of the formation of microtubule-lysosome complexes. The half-maximal effect is obtained at an ATP concentration of 0.83 +/- 0.11 mM (n = 7). The effect of ATP is not related to ATP hydrolysis, since ATP exerts its inhibitory action in the presence of EDTA. The ATP effect is mimicked by GTP, p[NH]ppA and tripolyphosphate, ADP and pyrophosphate, but not by AMP or phosphate. In the presence of 1 mM ATP, a Mg2+ concentration of 3 mM (corresponding to 2 mM free Mg2+) is required to overcome the inhibition caused by ATP; above 3 mM, Mg2+ exerts its activating effect. Since the modulating effects of ATP and Mg2+ are obtained at concentrations closed to those occurring in intact cells, we conclude that the regulation of the microtubule-lysosome interaction reported in this paper could be of physiological significance.     

Role of Mg2+ in the Ca2+-Ca2+ exchange mediated by the membrane-bound (Ca2+, Mg2+)-ATPase of sarcoplasmic reticulum vesicles

Sarcoplasmic reticulum vesicles were preloaded with either 45Ca2+ or unlabeled Ca2+. The unidirectional Ca2+ efflux and influx, together with Ca2+-dependent ATP hydrolysis and phosphorylation of the membrane-bound (Ca2+, Mg2+)-ATPase, were determined in the presence of ATP and ADP. The Ca2+ efflux depended on ATP (or ADP or both). It also required the external Ca2+. The Ca2+ concentration dependence of the efflux was similar to the Ca2+ concentration dependences of Ca2+ influx, Ca2+-dependent ATP hydrolysis, and phosphoenzyme formation. The rate of the efflux was approximately in proportion to the concentration of the phosphoenzyme up to 10 microM Ca2+. These results and other findings indicate that the Ca2+ efflux represents the Ca2+-Ca2+ exchange (between the external medium and the internal medium) mediated by the phosphoenzyme. In the range of 0.6-5.2 microM Mg2+, no appreciable Ca2+-Ca2+ exchange was detected although phosphoenzyme formation occurred to a large extent. Elevation of Mg2+ in the range 5.2 microM-4.8 mM caused a remarkable activation of the exchange, whereas the amount of the phosphoenzyme only approximately doubled. The kinetic analysis shows that this activation results largely from the Mg2+-induced acceleration of an exchange between the bound Ca2+ of the phosphoenzyme and the free Ca2+ in the internal medium. It is concluded that Mg2+ is essential for the exposure of the bound Ca2+ of the phosphoenzyme to the internal medium.     

Ouabain-binding and phosphorylation of (Na+ + K+) ATPase treated with N-ethylmaleimide or oligomycin.

Ouabain-binding and phosphorylation of (Na+ mk+)-ATPase (EC 3.6.1.3) of the plasma membranes from kidney were investigated after treatment with N-ethylmaleimide or oligomycin. Either of these inhibitors brought about the following changes: the phosphoenzyme, formed in the presence of Na+, Mg2+ and ATP became essentially insensitive to splitting by K+ but was split by ADP. One mole of this ADP-sensitive phosphoenzyme bound one mole of ouabain but the enzyme-ouabain complex was less stable than in the native enzyme primarily because the rate of its dissociation increased. Ouabain was bound to the ADP-sensitive phosphoenzyme in the presence of Mg2+ alone and addition of inorganic phosphate enhanced both the rate of formation and the steady-state level of the enzyme-ouabain complex. The inhibitors did not affect the properties of this second type of complex. Both in the native enzyme and in the enzyme treated with the two inhibitors inorganic phosphate enhanced ouabain binding by phosphorylating the active center of the enzyme as shown (a) by mapping the labeled peptides from the enzyme after peptic digestion, (b) by inhibition of this phosphorylation with Na+ and (c) by the 1:1 stoichiometric relation between this phosphorylation and the amount of bound ouabain. Unlike the phosphoenzyme, the binding of ouabain remained sensitive to K+ in the enzyme treated with the inhibitors. K+ slowed ouabain-binding either in the presence of Na+, Mg2+ and ATP or of Mg2+ and inorganic phosphate. A higher concentration of K+ was needed to slow ouabain-binding either in the presence of Na+, Mg2+ and ATP or of Mg2+ and inorganic phosphate. A higher concentration of K+ was needed to slow ouabain-binding than to stimulate dephosphorylation. This finding is interpreted as being an indication of separate sites for K+ on the enzyme: a site(s) with high K+-affinity which stimulates dephosphorylation, another site(s) with moderate K+-affinity which inhibits ouabain-binding. Inhibitors may enhance formation of the ADP-sensitive phosphoenzyme by blocking interaction between K+ and the site(s) with high affinity.     

Reaction of (Na+ + K+)-dependent adenosine triphosphatase with inorganic phosphate. Regulation by Na+, K+, and nucleotides

Effects of Na+, K+, and nucleotides on Mg2+-dependent phosphorylation of (Na+ + K+)-dependent adenosine triphosphatase by Pi were studied under equilibrium conditions. Na+ was a linear competitive inhibitor with respect to Mg2+ and a mixed inhibitor with respect to Pi. K+ was a partial inhibitor; it interacted with positive cooperativity and induced negative cooperativities in the interactions of Mg2+ and Pi with the enzyme. Adenyl-5'-yl (beta, gamma-methylene)diphosphonate, a nonhydrolyzable analog of ATP, interacted with negative cooperativity to inhibit phosphorylation in competition with Pi. ATP was also a competitive inhibitor. Na+ and K+ acted antagonistically, Na+ and nucleotides inhibited synergistically, and K+ and nucleotides were mutually exclusive. In the presence of ouabain, when nucleotides were excluded from the site inhibiting phosphorylation, a low affinity regulatory site for nucleotides became apparent, the occupation of which reduced the rate of dephosphorylation and the initial rate of phosphorylation of the enzyme without affecting the equilibrium constant of the reaction of Pi with the ouabain-complexed enzyme. The regulatory site was also detected in the absence of ouabain. The data suggest that catalytic and transport functions of the oligomeric enzyme may be regulated by homotropic and heterotropic site-site interactions, ligand-induced slow isomerizations, and distinct catalytic and regulatory sites for ATP.