Temperature effects on sodium pump phosphoenzyme distribution in human red blood cells

Phosphorylation of red cell membranes at ambient temperatures with micromolar [32P]ATP in the presence of Na ions produced phosphoenzyme that was dephosphorylated rapidly upon the addition of ADP or K ions. However, as first observed by Blostein (1968, J. Biol. Chem., 243:1957), the phosphoenzyme formed at 0 degrees C under otherwise identical conditions was insensitive to the addition of K ions but was dephosphorylated rapidly by ADP. This suggested that the conformational transition from ADP-sensitive, K-insensitive Na pump phosphoenzyme (E1 approximately P) to K-sensitive, ADP-insensitive phosphoenzyme (E2P) is blocked at 0 degrees C. Since the ATP:ADP exchange reaction is a partial reaction of the overall enzyme cycle dependent upon the steady state level of E1 approximately P that is regulated by [Na], we examined the effects of temperature on the curve relating [Na] to ouabain-sensitive ATP:ADP exchange. The characteristic triphasic curve seen at higher temperatures when [Na] was between 0.5 and 100 mM was not obtained at 0 degrees C. Simple saturation was observed instead with a K0.5 for Na of approximately 1 mM. The effect of increasing temperature on the ATP:ADP exchange at fixed (150 mM) Na was compared with the effect of increasing temperature on (Na + K)-ATPase activity of the same membrane preparation. It was observed that (a) at 0 degrees C, there was significant ouabain-sensitive ATP:ADP exchange activity, (b) at 0 degrees C, ouabain-sensitive (Na + K)-ATPase activity was virtually absent, and (c) in the temperature range 5-37 degrees C, there was an approximately 300-fold increase in (Na + K)-ATPase activity with only a 9-fold increase in the ATP:ADP exchange. These observations are in keeping with the suggestion that the E1 approximately P----E2P transition of the Na pump in human red cell membranes is blocked at 0 degrees C. Previous work has shown that the inhibitory effect of Na ions and the low-affinity stimulation by Na of the rate of ATP:ADP exchange occur at the extracellular surface of the Na pump. The absence of both of these effects at 0 degrees C, where E1 approximately P is maximal, supports the idea that external Na acts through sites on the E2P form of the phosphoenzyme.     

Effects of ATP on the intermediary steps of the reaction of the (Na+ + K+)-ATPase. IV. Effect of ATP on K0.5 for Na+ and on hydrolysis at different pH and temperature.

The pH optimum for (Na+ + K+)-ATPase (ATP phosphohydrolase, EC 3.6.1.3) depends on the combination of monovalent cations, on the ATP concentration and on temperature. ATP decreases the Na+ concentration necessary for half maximum activation, K0.5 for Na+ (Na+ + K+ = 150 mM), and the effect is pH and temperature dependent. At a low ATP concentration a decrease in pH leads to an increase in K0.5 for Na+, while at the high ATP concentration it leads to a decrease. K0.5 for ATP for hydrolysis decreases with an increase in pH. The fractional stimulation by K+ in the presence of Na+ decreases with the ATP concentration, and at a low ATP concentration K+ becomes inhibitory, this being most pronounced at 0 degrees C. The results suggest that (a) ATP at a given pH has two different effects: it increases the Na+ relative to K+ affinity on the internal site (K0.5 for ATP at pH 7.4, 37 degrees C, is less than 10 microM); it increases the molar activity in the presence of Na+ + K+ (K0.5 for ATP at pH 7.4, 37 degrees , is 127 microM), (b) binding of the cations to the external as well as the internal sites leads to pK changes (Bohr effect) which are different for Na+ and for K+, i.e. the selectivity for Na+ relative to K+ depends both on ATP and on the degree of protonation of certain groups on the system, (c) ATP involves an extra dissociable group in the determination of the selectivity of the internal site, and thereby changes the effect of an increase in protonation of the system from a decrease to an increase in selectivity for Na+ relative to K+.     

The effects of temperature and ouabain on steady-state Na and K exchanges in human lymphocytes

The temperature-dependence of steady-state exchanges of K and Na were determined under conditions in which cell viability, ATP, water, Ca, and Mg were not confounding variables. Steady-state ion contents are near-normal between 37° and 10° C, Below 10° C K is replaced by Na in a mole-for-mole fashion with significant net K retention and Na exclusion occurring even below 3°C. The rates of steady-state Kand Na exchanges have markedly different temperature-dependences; between 37° and 10° C, for example, that of K decreases markedly while that of Na remains near-normal, and there is no consistent correlation between the steady-state exchanges and the ionic contents. Ouabain increases steady-state Na flux at 37° C and induces a more marked temperature-dependence over the entire temperature range. This effect is not due simply to inhibition of some processes and unmasking others; it mirrors a pronounced effect of ouabain on the intrinsic properties of Na self-exchange. These results are compatible with a model based on two simple concepts: (1) partial ionic exclusion from cellular water that is ordered by interaction with proteins; and (2) ionic accumulation mediated by adsorption onto and desorption from fixed macromolecular anionic sites, the majority of which interact with one another in a cooperative fashion. In this view, the sharp temperature transition in the net replacement of K by Na below 10° C is due to a critical transition in the selectivity of the cooperatively interacting adsorption sites. The rates of steady-state self-exchanges of K and Na are determined by parameters of ion-site interaction, and the major set of ion-adsorbing sites that interact cooperatively have a steep thermal activation energy of ionic self-exchange. When they are in the K-preferring state above 10° C, exchange of K has a steep temperature-dependence. When they are in the Na-preferring state below 10° C, exchange of Na has a steep temperature-dependence. When these sites are forced into a Na-preferring state at all temperatures by treatment with ouabain, exchange of Na acquires a steep temperature-dependence over the entire temperature range 37° to 0° C.     

ATP and integrity of human red blood cells

In spite of the well known significance of ATP in the energy dependent life processes, the role of ATP in maintaining cellular integrity is poorly understood. A possible model for studying ATP dependent life processes is to monitor the kinetics of changes seen intra/extracellularly during ATP depletion. In our model system anticoagulated human whole blood was incubated at different temperatures to reduce intracellular ATP without addition of any chemicals. The red blood cells in their own plasma were incubated for several days at 4 degrees C or at 37 degrees C, and ATP, glucose, K+, Na+, hemoglobin, water content, mean corpuscular volume (MCV), pH and Ca2+ were analyzed in time-sequences. All the examined parameters remained practically unchanged at 4 degrees C, while at 37 degrees C total ATP and glucose decreased parallel and after a transient increase of MCV, the water content of red blood cells decreased. As the actual ATP fell below 10% of the initial ATP content (at 48 h), the release of potassium sharply increased. Release of hemoglobin started only after 96 hours of incubation. Maximums of changes of the examined parameters were found at different time intervals. The maximal speed of concentration changes for glucose was found at 12-24 hours of incubation and at 24-36 hours for ATP, at 48-60 hours for K+(-)Na+ and after 96 hours for hemoglobin.     

Effect of temperature on the reversal of the calcium ion pump in sarcoplasmic reticulum.

Sarcoplasmic-reticulum vesicles were actively loaded with Ca2+ in the presence of phosphate, and the ADP-induced Ca2+ efflux and ATP synthesis were measured as a function of temperature. Arrhenius plots show break points for both processes at about 18 and 37 degrees C. Between 18 and 37 degrees C, Ca2+ efflux and ATP synthesis occur with an activation energy of 67.2-71.4 kJ/mol, whereas it is about 189-210 kJ/mol for temperatures below 18 degrees C. Above 37 degrees C, the rates of ADP-induced Ca2+ release and of ATP synthesis sharply decline until the temperature reaches about 42 degrees C. Above this temperature, the Ca2+ efflux increases again even in absence of ADP, although the synthesis of ATP is inhibited, which reflects leakiness of the vesicles. The results show that the transition temperatures for ADP-induced Ca2+ efflux and for ATP synthesis resemble those for active Ca2+ uptake, which indicates that the same coupling mechanism is involved during the inward and outward Ca2+ translocations across the membrane.     

The occurrence of thermotolerance in non-proliferating rat hepatocytes in primary culture

The occurrence of thermotolerance, induced by an initial heat treatment at 42 degrees C for 30 min, was studied in adult non-proliferating rat hepatocytes in primary culture. Heat treatment at 42 degrees C for 30 min did not affect cell morphology, cell attachment, Na+, K+ pump activity, K+ content and lactate dehydrogenase accumulation into the medium. In contrast, after exposure to 44 degrees C for 30 min a dramatic change in all these parameters was observed. However, of the cells, which remained attached to the substratum 24 h after treatment, Na+, K+ pump activity and K+ content appeared to be normal compared with untreated cells. Cells, pre-treated at 42 degrees C for 30 min, followed by incubation at 37 degrees C for 16 h, were found to be completely thermal resistant against heat treatment at 44 degrees C, as judged by cell morphology, detachment from the substratum, lactate dehydrogenase accumulation, Na+, K+ pump activity and K+ content. These results show that induction and development of thermotolerance can be studied in non-proliferating cells in primary culture.     

Active ion transport in the renal proximal tubule. I. Transport and metabolic studies

Various aspects of the interrelationship between ion transport and cellular metabolism were investigated using a suspension of rabbit cortical tubules that were mainly proximal in nature. Using the intact tubules, the compartmentation of K within the renal cell was studied by performing 42K uptake studies. The oxygen consumption (QO2) of the tubules was measured under similar conditions, as well as when the Na pump was stimulated by increasing Na+ entry with nystatin. In addition, the state 3 rate of respiration was measured when the mitochondria of digitonin-permeabilized tubules were stimulated by ADP. At 37 and 25 degrees C, a single-compartmental uptake of 42K was observed, which suggests that extracellular K+ communicates with a single compartment within the renal cell. Between 37 and 15 degrees C, the ouabain- sensitive QO2 and the initial 42K uptake rate were parallel in an Arrhenius-type plot, which indicated that active ion transport and oxidative phosphorylation remain tightly coupled within this temperature range. At all temperatures between 37 and 15 degrees C, nystatin stimulated the QO2, which demonstrates that the entry of Na+ into the renal cells was rate limiting for active Na+ transport throughout this temperature range. Between 37 and 20 degrees C, the nystatin-stimulated QO2 was nearly equal to the state 3 rate of respiration, which suggests that active ion transport may be limited by ATP availability under these conditions. At 15 degrees C, nystatin addition stimulated the QO2 well below the state 3 respiratory rate.     

Effect of amiodarone on membrane fluidity and Na+/K+ ATPase activity in rat-brain synaptic membranes

In rat-brain synaptic membranes at a fixed temperature (37 degrees C), amiodarone dose-dependently inhibits the Na+/K+ ATPase activity (IC50 approximately equal to 2.10(-5)M) and produces a linear increase in the degree of fluorescence depolarization (P) of 1,6-diphenylhexatriene embedded in the lipid matrix. Amiodarone has no effect on Mg++ ATPase and K+PNPase activity up to 3.10(-4)M. Studies carried out at different temperatures indicate that 10(-5)M amiodarone inhibits the Na+/K+ ATPase and decreases the lipid fluidity at all the temperatures studied (9 - 40 degrees C). The compound significantly displaces the temperature of transition observed around 20 degrees C in both Na+/K+ ATPase activity and lipid fluidity to 24 degrees C with no changes in slopes. The results suggest that part of the selective inhibition of Na+/K+ ATPase activity by amiodarone could be due to the effects of the drug on lipid dynamics.     

Purification of Na+,K+-ATPase expressed in Pichia pastoris reveals an essential role of phospholipid-protein interactions

Na+,K+-ATPase (porcine alpha/his10-beta) has been expressed in Pichia Pastoris, solubilized in n-dodecyl-beta-maltoside and purified to 70-80% purity by nickel-nitrilotriacetic acid chromatography combined with size exclusion chromatography. The recombinant protein is inactive if the purification is done without added phospholipids. The neutral phospholipid, dioleoylphosphatidylcholine, preserves Na+,K+-ATPase activity of protein prepared in a Na+-containing medium, but activity is lost in a K+-containing medium. By contrast, the acid phospholipid, dioleoylphosphatidylserine, preserves activity in either Na+- or K+-containing media. In optimal conditions activity is preserved for about 2 weeks at 0 degrees C. Both recombinant Na+,K+-ATPase and native pig kidney Na+,K+-ATPase, dissolved in n-dodecyl-beta-maltoside, appear to be mainly stable monomers (alpha/beta) as judged by size exclusion chromatography and sedimentation velocity. Na+,K+-ATPase activities at 37 degrees C of the size exclusion chromatography-purified recombinant and renal Na+,K+-ATPase are comparable but are lower than that of membrane-bound renal Na+,K+-ATPase. The beta subunit is expressed in Pichia Pastoris as two lightly glycosylated polypeptides and is quantitatively deglycosylated by endoglycosidase-H at 0 degrees C, to a single polypeptide. Deglycosylation inactivates Na+,K+-ATPase prepared with dioleoylphosphatidylcholine, whereas dioleoylphosphatidylserine protects after deglycosylation, and Na+,K+-ATPase activity is preserved. This work demonstrates an essential role of phospholipid interactions with Na+,K+-ATPase, including a direct interaction of dioleoylphosphatidylserine, and possibly another interaction of either the neutral or acid phospholipid. Additional lipid effects are likely. A role for the beta subunit in stabilizing conformations of Na+,K+-ATPase (or H+,K+-ATPase) with occluded K+ ions can also be inferred. Purified recombinant Na+,K+-ATPase could become an important experimental tool for various purposes, including, hopefully, structural work.     

Increased permeability and subsequent resealing of the host cell membrane early after infection of Escherichia coli with bacteriophage T1.

The addition of T1 to cells growing at 37 degrees C in a minimal medium at 0.4 mM Mg2+ rapidly induced an irreversible loss of K+ and Mg2+ and uptake of Na+ by the cells. Both the ATP pool of the cells and the transmembrane proton motive force were reduced. These cells did not lyse from within, since viral DNA replication and the maturation of the 36,000-molecular-weight phage head protein were inhibited. By contrast, cells lysed when infected at 5.4 mM Mg2+. In these cells, T1 initially induced K+ efflux and Na+ influx and lowered the cytoplasmic ATP concentration. After a few minutes, the cation gradients and ATP pool were restored to levels close to that of control cells. At 5.4 mM Mg2+, the shutoff of host protein synthesis was delayed and coincided with the restoration of the ATP pool. In an ATP synthase-negative mutant, infection with T1 did not affect the cytoplasmic ATP concentration but inhibited host protein synthesis with the same rate as it did in wild-type cells.     

Inhibition of gill (Na+ + K+)-ATPase in rainbow trout (Salmo gairdneri) by a purinedisulfide analog of adenosine triphosphate.

The effect of the adenosine triphosphate analog, 6,6'-dithiobis(inosinyl imidodiphosphate), (sIMP-PNP)2, was tested on the ouabain-sensitive (Na+ + K+)-ATPase (ATP phosphohydrolase, EC 3.6.1.3) and the ouabain-insensitive Mg2+ - ATPase in microsomes prepared from gill tissue of sea water-adapted rainbow trout, Salmo gairdneri. The (Na+ + K+)-ATPase was completely inhibited by low concentrations of (sIMP-PNP)2 (6 micrometer) but the Mg2+ - ATPase was unaffected by the inhibitor at concentrations as high as 28 micrometer, supporting the suggestion that the two activities represent separate enzymes. The specificity of inactivation could be demonstrated both at a physiological temperature (13 degrees C) and at 37 degrees C. The rates of inactivation were similar at both temperatures. Inactivation of the (Na+ + K+)-ATPase by (sIMP-PNP)2 was reversed by dithiothreitol, suggesting that the inhibitor forms a mixed disulfide with sulfhydryl groups on the enzyme. The inability of substrate (either ATP or its analog, adenyl-5'-yl imidodiphosphate) to protect against inactivation suggests that (sIMP-PNP)2 is reacting with sulfhydryl groups which are not associated with the active site.     

The effect of hyperthermia on the sodium-potassium pump in Chinese hamster ovary cells

The effect of hyperthermia on the Na+-K+ pump was determined by measuring influx and efflux of 86Rb+ in Chinese hamster ovary cells from 31 to 50 degrees C. The maximum initial rate of ouabain-sensitive influx increased with temperature between 31 and 45 degrees C although Km increased significantly above 37 degrees C, implying a diminished affinity of the transport protein for its substrate. The changes in the kinetics of influx at temperatures up to 45 degrees C were rapidly reversible on return to 37 degrees C. Above 45 degrees C an irreversible decrease in 86Rb+ uptake was observed. Efflux of 86Rb+ increased from 31 to 40 degrees C but above 43 degrees C showed a small but significant decrease. The study of 86Rb+ influx after varying times of exposure to elevated temperatures showed that the Na+-K+ pump remains functional in cells which are reproductively dead. We have shown that although the kinetics of K+ transport are sensitive to temperature changes in the range used in clinical hyperthermia, the inactivation of the Na+-K+ pump is not a primary event in cell killing.     

Effect of temperature on nucleotide pools and monoclonal antibody production in a mouse hybridoma

The specific monoclonal antibody productivity (q(Mab)) of a murine hybridoma (CC9C10) increased with incubation temperature in the range 33 degrees C to 39 degrees C. q(Mab) was constant at each temperature and was independent of the phase of culture. The q(Mab) increased 97% at 39 degrees C and decreased by 21% at 33 degrees C compared with controls at 37 degrees C. Specific rates of substrate (glucose and glutamine) utilization and byproduct (lactate and ammonia) formation also increased with temperature but the yield coefficient, Y(Lac/Llc') was constant for 33 degrees C to 39 degrees C and Y(Amm/Gin) was constant for 37 degrees C to 39 degrees C. Y(Amm/Gin) at 33 degrees C was lower than the control. Changes in specific nucleotide concentrations and ratios were monitored by analysis of intracellular nucleotide pools. The NTP ratio, (ATP + GTP)/(UTP + CTP), increased and the U-ratio (UTP/UDP-GNac) decreased during the course of each culture, whereas the adenylate energy charge, (ATP + 0.5ADP)/(ATP + ADP + AMP), remained relatively constant at a value 0.8. The relative content of UDP-/N acetyl galactosamine, UDP-N acetyl glucosamine, and NAD increased with incubation temperature, whereas the relative ATP content, SA(ATP + ADP + AMP)/SU (UTP + UDP-sugars) ratio, purine/pyrimidine, ATP/GTP, and U-ratio decreased at higher incubation temperatures. It is possible that these nucleotide parameters may have a regulatory role in the changes of q(Mab) observed at the higher temperatures. (c) 1994 John Wiley & Sons, Inc.     

The regulation of ATPase-ATPase interactions in sarcoplasmic reticulum membrane. I. The effects of Ca2+, ATP, and inorganic phosphate

Two-dimensional crystalline arrays of Ca2+-ATPase molecules develop after treatment of sarcoplasmic reticulum vesicles with Na3VO4 in calcium-free medium (Dux, L., and Martonosi, A. (1983) J. Biol. Chem. 258, 2599-2603). The formation of Ca2+-ATPase crystals is inhibited by Ca2+ (2 microM), or ATP (5 mM), but not by ADP, 5'-adenylylimidodiphosphate, or adenylylmethylenediphosphonate. ATPase crystals did not form at 37 degrees C and exposure of preformed crystals to 37 degrees C for 1 h caused the disappearance of crystal lattice. Inorganic orthophosphate (1 mM at pH 6.0) promoted the formation of a distinct crystal form of Ca2+-ATPase, which was different from that produced by Na3VO4. These observations indicate that Ca2+, ATP, inorganic phosphate, pH, and temperature influence the interactions between ATPase molecules in the sarcoplasmic reticulum membrane.     

Side-specific effects of sodium on (Na,K)-ATPase. Studies with inside-out red cell membrane vesicles.

Using inside-out vesicles of human red cell membranes, the side-specific effects of Na+ on phosphorylation of (Na,K)-ATPase have been studied using low concentrations of [gamma-32P]ATP (less than or equal to 0.1 microM). Phosphorylation is stimulated by Na+ at the cytoplasmic membrane surface (extravesicular Na+) alone and not by Na+ at the external surface (intravesicular Na+). At 37 degrees C, external Na+ (less than or equal to 10 mM) does, however, increase the steady state level (approximately 2 1/2-fold) of phosphoenzyme above that observed with cytoplasmic Na+ alone; hydrolysis is increased to only a small extent. Little stimulation by external Na+ is observed at 0 degrees C. As Na+ at the cytoplasmic side is decreased to very low levels (less than or equal to 0.2 mM) several kinetic changes are observed: (i) the apparent turnover of phosphoenzyme (ratio Na+-ATP-ase/phosphoenzyme level) is markedly increased (approximately 3-fold, (ii) Rbext sensitivity (inhibition of (Na)-ATPase at low ATP levels) is reduced, and (iii) the ratio of Na+ ions transported per molecule of ATP hydrolyzed is decreased. These results are compatible with a reaction pathway involving a transition from one form of phosphoenzyme, E1-P, to another, E2-P of which the hydrolysis is decreased by moderate levels of external Na+. It is suggested also that an alternate reaction pathway for Na+-ATPase occurs at very low cytoplasmic Na+, one via hydrolysis of E1-P and not associated with Na+ translocation.     

Some total and partial reactions of Na+/K+-ATPase using ATP and acetyl phosphate as a substrate

Acetyl phosphate, as a substrate of (Na+ + K+)-ATPase, was further characterized by comparing its effects with those of ATP on some total and partial reactions carried out by the enzyme. In the absence of Mg2+ acetyl phosphate could not induce disocclusion (release) of Rb+ from E2(Rb); nor did it affect the acceleration of Rb+ release by non-limiting concentrations of ADP. In K+-free solutions and at pH 7.4 sodium ions were essential for ATP hydrolysis by (Na+ + K+)-ATPase; when acetyl phosphate was the substrate a hydrolysis (inhibited by ouabain) was observed in the presence and absence of Na+. In liposomes with (Na+ + K+)-ATPase incorporated and exposed to extravesicular (intracellular) Na+, acetyl phosphate could sustain a ouabain-sensitive Rb+ efflux; the levels of that flux were similar to those obtained with micromolar concentrations of ATP. When the liposomes were incubated in the absence of extravesicular Na+ a ouabain-sensitive Rb+ efflux could not be detected with either substrate. Native (Na+ + K+)-ATPase was phosphorylated at 0 degrees C in the presence of NaCl (50 mM for ATP and 10 mM for acetyl phosphate); after phosphorylation had been stopped by simultaneous addition of excess trans-1,2-diaminocyclohexane-N,N,N',N' tetraacetic acid and 1 M NaCl net synthesis of ATP by addition of ADP was obtained with both phosphoenzymes. The present results show that acetyl phosphate can fuel the overall cycle of cation translocation by (Na+ + K+)-ATPase acting only at the catalytic substrate site; this takes place via the formation of phosphorylated intermediates which can lead to ATP synthesis in a way which is indistinguishable from that obtained with ATP.     

Ouabain binding and coupled sodium, potassium, and chloride transport in isolated transverse tubules of skeletal muscle

The affinity and number of binding sites of [3H]ouabain to isolated transverse (T) tubules were determined in the absence and presence of deoxycholate. In both conditions the KD was approximately 53 nM while deoxycholate increased the number of binding sites from 3.5 to 37 pmol/mg protein. We concluded that the ouabain binding sites were located primarily on the inside of the isolated vesicle and that the vesicles were impermeable to ouabain. ATP induced a highly active Na+ accumulation by the T tubules which increased Na+ in the T tubular lumen by almost 200 nmol/mg protein. The accumulation had an initial fast phase lasting 2-3 min and a subsequent slow phase which continued for at least 40 min. The rate of the initial fast phase indicated a turnover number of 20 Na+/s. The Na+ accumulation was prevented by monensin but was unaffected by valinomycin. Ouabain did not influence Na+ uptake, but digitoxin inhibited it. At low K+ the accumulation of Na+ was reduced 3.7-fold below the value at 50 mM K+. 86Rb, employed as a tracer to detect K+, showed a first phase of K+ release while Na+ was accumulated. After 2-3 min, K+ was reaccumulated while Na+ continued to increase in the lumen. T tubules accumulated Cl- on addition of ATP. This suggested that ATP initiated an exchange of Na+ for K+ followed by uptake of Na+ and K+ accompanied by Cl-.     

Demonstration of cooperating alpha subunits in working (Na+ + K+)-ATPase by the use of the MgATP complex analogue cobalt tetrammine ATP

The MgATP complex analogue cobalt-tetrammine-ATP [Co(NH3)4ATP] inactivates (Na+ + K+)-ATPase at 37 degrees C slowly in the absence of univalent cations. This inactivation occurs concomitantly with incorporation of radioactivity from [alpha-32P]Co(NH3)4ATP and from [gamma-32P]Co(NH3)4ATP into the alpha subunit. The kinetics of inactivation are consistent with the formation of a dissociable complex of Co(NH3)4ATP with the enzyme (E) followed by the phosphorylation of the enzyme: (Formula: see text). The dissociation constant of the enzyme-MgATP analogue complex at 37 degrees C is Kd = 500 microM, the inactivation rate constant k2 = 0.05 min-1. ATP protects the enzyme against the inactivation by Co(NH3)4ATP due to binding at a site from which it dissociates with a Kd of 360 microM. It is concluded, therefore, that Co(NH3)4ATP binds to the low-affinity ATP binding site of the E2 conformational state. K+, Na+ and Mg2+ protect the enzyme against the inactivation by Co(NH3)4ATP. Whilst Na+ or Mg2+ decrease the inactivation rate constant k2, K+ exerts its protective effect by increasing the dissociation constant of the enzyme.Co(NH3)4ATP complex. The Co(NH3)4ATP-inactivated (Na+ + K+)-ATPase, in contrast to the non-inactivated enzyme, incorporates [3H]ouabain. This indicates that the Co(NH3)4ATP-inactivated enzyme is stabilized in the E2 conformational state. Despite the inactivation of (Na+ + K+)-ATPase by Co(NH3)4ATP from the low-affinity ATP binding site, there is no change in the capacity of the high-affinity ATP binding site (Kd = 0.9 microM) nor of its capability to phosphorylate the enzyme Na+-dependently. Since (Na+ + K+)-ATPase is phosphorylated Na+-dependently from the high-affinity ATP binding site although the catalytic cycle is arrested in the E2 conformational state by specific modification of the low-affinity ATP binding site, it is concluded that both ATP binding sites coexist at the same time in the working sodium pump. This demonstration of interacting catalytic subunits in the E1 and E2 conformational states excludes the proposal that a single catalytic subunit catalyzes (Na+ + K+)-transport.     

Inhibition of (Na+,K+)-ATPase by dicyclohexylcarbodiimide. Evidence for two carboxyl groups that are essential for enzymatic activity

N,N'-Dicyclohexylcarbodiimide (DCCD), a reagent that reacts with carboxyl groups under mild conditions, irreversibly inhibits (Na+,K+)-ATPase activity (measured by using 1 mM ATP) with a pseudo-first-order rate constant of 0.084 min-1 (0.25 mM DCCD and 37 degrees C). The partial activities of the enzyme, including (Na+,K+)-ATPase at 1 microM ATP, Na+-ATPase, and the formation of enzyme-acyl phosphate (E-P), decayed at about one-third the rate at which (Na+,K+)-ATPase at 1 mM ATP was lost. The formation of E-P from inorganic phosphate was unaffected by DCCD while K+-phosphatase activity decayed at the same rate as (Na+,K+)-ATPase measured at 1 mM ATP. The enzyme's substrates (i.e., sodium, potassium, magnesium, and ATP) all decreased the rate of DCCD inactivation of (Na+,K+)-ATPase activity measured at either 1 mM or 1 microM ATP. The concentration dependence of the protection afforded by each substrate is consistent with its binding at a catalytically relevant site. DCCD also causes cross-linking of the enzyme into species of very high molecular weight. This process occurs at about one-tenth the rate at which (Na+,K+)-ATPase activity measured at 1 mM ATP is lost, too slowly to be related to the loss of enzymatic activity. Labeling of the enzyme with [14C]DCCD shows the incorporation of approximately 1 mol of DCCD per mole of large subunit; however, the incorporation is independent of the loss of enzymatic activity. The results presented here suggest that (Na+,K+)-ATPase contains two carboxyl groups that are essential for catalytic activity, in addition to the previously known aspartate residue which is involved in formation of E-P.(ABSTRACT TRUNCATED AT 250 WORDS)     

Chromium(III)ATP inactivating (Na+ + K+)-ATPase supports Na+-Na+ and Rb+-Rb+ exchanges in everted red blood cells but not Na+,K+ transport

The chromium(III) complex of ATP, an MgATP complex analogue, inactivates (Na+ + K+)-ATPase by forming a stable chromo-phosphointermediate. The rate constant k2 of inactivation at 37 degrees C of the beta, gamma-bidentate of CrATP is enhanced by Na+ (K0.5 = 1.08 mM), imidazole (K0.5 = 15 mM) and Mg2+ (K0.5 = 0.7 mM). These cations did not affect the dissociation constant of the enzyme-chromium-ATP complex. The inactive chromophosphoenzyme is reactivated slowly by high concentrations of Na+ at 37 degrees C. The half-maximal effect on the reactivation was reached at 40 mM NaCl, when the maximally observable reactivation was studied. However, 126 mM NaCl was necessary to see the half-maximal effect on the apparent reactivation velocity constant. K+ ions hindered the reactivation with a Ki of 70 microM. Formation of the chromophosphoenzyme led to a reduction of the Rb+ binding sites and of the capacity to occlude Rb+. The beta, gamma-bidentate of chromium(III)ATP (Kd = 8 microM) had a higher than the alpha, beta, gamma-tridentate of chromium(III)ATP (Kd = 44 microM) or the cobalt tetramine complex of ATP (Kd = 500 microM). The beta, gamma-bidentate of the chromium(III) complex of adenosine 5'-[beta, gamma-methylene]triphosphate also inactivated (Na+ + K+)ATPase. Although CrATP could not support Na+, K+ exchange in everted vesicles prepared from human red blood cells, it supported the Na+-Na+ and Rb+-Rb+ exchange. It is concluded that CrATP opens up Na+ and K+ channels by forming a relatively stable modified enzyme-CrATP complex. This stable complex is also formed in the presence of the chromium complex of adenosine 5'-[beta, gamma-methylene]triphosphate. Because the beta, gamma-bidentate of chromium ATP is recognized better than the alpha, beta, gamma-tridentate, it is concluded that the triphosphate site recognizes MgATP with a straight polyphosphate chain and that the Mg2+ resides between the beta- and the gamma-phosphorus. The enhancement of inactivation by Mg2+ and Na+ may be caused by conformational changes at the triphosphate site.