Computed energy profiles for ion transport in the gramicidin A channel

Energy profiles have been established for the transport of Na⁺, K⁺ and Cs⁺ through the gramicidin A channel. In Urry’s head-to-headβ _3.3 ^6.3 left-handed helical dimer structure, using a refined methodology for the calculation of intra and intermolecular interactions. The computations show the important role, for the energy profile and the position of the possible binding site, of the flexible ethanolamine chain, whose significance was till now overlooked. The calculations indicate that the barriers at the entrance and at the center of the channel should be in the order Na⁺ XXX K⁺ XXX Cs⁺ but predict also that the energies of the binding sites should be the greatest for Na⁺ and, then, comparable for K⁺ and Cs⁺. The indications concerning the barriers are confirmed by experiment.     

Nigericin forms highly stable complexes with lithium and cesium

Nigericin is a monocarboxylic polyether molecule described as a mobile K⁺ ionophore unable to transport Li⁺ and Cs⁺ across natural or artificial membranes. This paper shows that the ion carrier molecule forms complexes of equivalent energy demands with Li⁺, Cs⁺, Na⁺, Rb⁺, and K⁺. This is in accordance with the similar values of the complex stability constants obtained from nigericin with the five alkali metal cations assayed. On the other hand, nigericinalkali metal cation binding isotherms show faster rates for Li⁺ and Cs⁺ than for Na⁺, K⁺, and Rb⁺, in conditions where the carboxylic proton does not dissociate. Furthermore, proton NMR spectra of nigericin-Li⁺ and nigericin-Cs⁺ complexes show wide broadenings, suggesting strong cation interaction with the ionophore; in contrast, the complexes with Na⁺, K⁺, and Rb⁺ show only clear-cut chemical shifts. These latter results support the view that nigericin forms highly stable complexes with Li⁺ and Cs⁺ and contribute to the explanation for the inability of this ionophore to transport the former cations in conditions where it catalyzes a fast transport of K⁺>Rb⁺>Na⁺.Part of the results of this paper were presented at the 14th International Congress of Biochemistry in Prague, Czechoslovakia.     

Ion Selectivity of the KcsA Channel: A Perspective from Multi-Ion Free Energy Landscapes

Potassium (K⁺) channels are specialized membrane proteins that are able to facilitate and regulate the conduction of K⁺ through cell membranes. Comprising five specific cation binding sites (S₀-S₄) formed by the backbone carbonyl groups of conserved residues common to all K⁺ channels, the narrow selectivity filter allows fast conduction of K⁺ while being highly selective for K⁺ over Na⁺. To extend our knowledge of the microscopic mechanism underlying selectivity in K⁺ channels, we characterize the free energy landscapes governing the entry and translocation of a Na⁺ or a K⁺ from the extracellular side into the selectivity filter of KcsA. The entry process of an extracellular ion is examined in the presence of two additional K⁺ in the pore, and the three-ion potential of mean force is computed using extensive all-atom umbrella sampling molecular dynamics simulations. A comparison of the potentials of mean force yields a number of important results. First, the free energy minima corresponding to configurations with extracellular K⁺ or Na⁺ in binding site S₀ or S₁ are similar in depth, suggesting that the thermodynamic selectivity governed by the free energy minima for those two binding sites is insignificant. Second, the free energy barriers between stable multi-ion configurations are generally higher for Na⁺ than for K⁺, implying that the kinetics of ion conduction is slower when a Na⁺ enters the pore. Third, the region corresponding to binding site S₂ near the center of the narrow pore emerges as the most selective for K⁺ over Na⁺. In particular, while there is a stable minimum for K⁺ in site S₂, Na⁺ faces a steep free energy increase with no local free energy well in this region. Lastly, analysis shows that selectivity is not correlated with the overall coordination number of the ion entering the pore, but is predominantly affected by changes in the type of coordinating ligands (carbonyls versus water molecules). These results further highlight the importance of the central region near binding site S₂ in the selectivity filter of K⁺ channels.     

Dependence of the optical absorption and Na+ binding energies of coumarin-crown ethers on the size and attachment position of ether ring: density functional investigation

The crowned coumarin complexes are well known compounds for their ion recognition abilities. They undergo photophysical changes upon cation binding. On the basis of density functional theory calculations, we examined the sodium cation (Na⁺) binding energies of coumarin-crown ethers based on 15-Crown-5 (15 C5) and 18-Crown-6 (18 C6) as well as the optical absorptions of coumarin-crown ethers based on 12-Crown-4 (12 C4), 15 C5 and 18 C6. We explored why the attachment of crown ether ring to coumarin affects the Na⁺ binding energies of coumarin-crown ethers and also why the optical absorption of coumarin is modified by the crown ethers. Our study reveals that the Na⁺ ion binding energies of coumarin-crown ethers depend strongly on the size of the crown ether ring and also on the attachment position of the ether ring on coumarin. These factors affect the intramolecular charge transfer and overall stability of the complexes. The absorptions of the coumarin and ether ring parts of coumarin-crown ether are red shifted from those of isolated coumarin and crown ether, respectively. The red-shift of the coumarin ester group absorption is much stronger depending on the attachment position of the ether ring to coumarin. The absorption intensity of the coumarin part in coumarin-crown ethers is reduced for the benzene group absorption, but is enhanced for the ester group absorption.

Enniatin B and Valinomycin as Ion Carriers: An Empirical Force Field Analysis

Abstract

The alkali-ion binding properties of two natural depsipeptide ion carriers, enniatin B (EnB) and valinomycin (VM), are examined and compared by the empirical force field method. While VM has been shown to bind preferentially K⁺, Rb⁺, and Cs⁺ over Na⁺ in most solvents, EnB is considerably less specific.

We find that EnB forms two kinds of complexes, internal and external. In internal complexes, the ion binds to all six carbonyl oxygens, while in external ones, only three oxygens, preferentially those of the D-hydroxy-isovaleryl residues, are bound. The size of the internal cavity is best suited for Na⁺, while K⁺ and Rb⁺ squeeze in asymmetrically by distorting the molecule, and Cs⁺ not at all. External binding is much less specific. Since internal complexes possess much higher strain energies than external ones, the latter may be at least as stable as the former, even in fairly non-polar solvents.

VM is calculated to bind only internally, and with much less strain energy than EnB. The size of its internal cavity is well suited for binding the ions K⁺, Rb⁺, and Cs⁺, but is too big for Na⁺. The difference between the binding energies of Na⁺ and K⁺ is much smaller than that between the corresponding hydration enthalpies, thus explaining the binding preference for the latter ion.     

Molecular analysis of the mechanism of potassium uptake through the TRK1 transporter of Saccharomyces cerevisiae

The TRK-HKT family of K⁺ transporters mediates K⁺ and Na⁺ uptake in fungi and plants. In this study, we have investigated the molecular mechanism involved in the movement of alkali cations through the TRK1 transporter of Saccharomyces cerevisiae. The model that best explains the activity of ScTRK1 is a cotransport of two K⁺ or Rb⁺, both of which bind the two binding sites of ScTRK1 with very high affinities in K⁺-starved cells. Na⁺ can be transported in the same way but it exhibits a much lower affinity for the second binding site. Therefore, only at critical concentration ratios between K⁺ and Na⁺, or Rb⁺ and Na⁺, the transporter takes up Na⁺ together with K⁺ or Rb⁺. Mutation analyses suggest that the two binding sites are located in the P fragment of the first MPM motif of the transporter, and that Gln⁹⁰ is involved in these binding sites. ScTRK1 can be in two states, medium or high affinity, and we have found that Leu⁹⁴⁹ is involved in the oscillation of the transporter between these two states. ScTRK1 mediates active K⁺ uptake. This is not Na⁺-coupled and direct coupling of ScTRK1 to a source of chemical energy seems more probable than K⁺-H⁺ cotransport.     

Effect of metal ions (Li+, Na+, K+, Mg2+, Ca2+, Ni2+, Cu2+ and Zn2+) and water coordination on the structure and properties of l-histidine and zwitterionic l-histidine

Interactions between metal ions and amino acids are common both in solution and in the gas phase. The effect of metal ions and water on the structure of l-histidine is examined. The effect of metal ions (Li⁺, Na⁺, K⁺, Mg²⁺, Ca²⁺, Ni²⁺, Cu²⁺ and Zn²⁺) and water on structures of His·M(H₂O)m, m = 0.1 complexes have been determined theoretically employing density functional theories using extended basis sets. Of the five stable complexes investigated the relative stability of the gas-phase complexes computed with DFT methods (with one exception of K⁺ systems) suggest metallic complexes of the neutral l-histidine to be the most stable species. The calculations of monohydrated systems show that even one water molecule has a profound effect on the relative stability of individual complexes. Proton dissociation enthalpies and Gibbs energies of l-histidine in the presence of the metal cations Li⁺, Na⁺, K⁺, Mg²⁺, Ca²⁺, Ni²⁺, Cu²⁺ and Zn²⁺ were also computed. Its gas-phase acidity considerably increases upon chelation. Of the Lewis acids investigated, the strongest affinity to l-histidine is exhibited by the Cu²⁺ cation. The computed Gibbs energies ΔG are negative, span a rather broad energy interval (from ?130 to ?1,300 kJ/mol), and upon hydration are appreciably lowered.     

Computational Flux Balance Analysis Predicts that Stimulation of Energy Metabolism in Astrocytes and their Metabolic Interactions with Neurons Depend on Uptake of K<Superscript>+</Superscript> Rather than Glutamate

Brain activity involves essential functional and metabolic interactions between neurons and astrocytes. The importance of astrocytic functions to neuronal signaling is supported by many experiments reporting high rates of energy consumption and oxidative metabolism in these glial cells. In the brain, almost all energy is consumed by the Na⁺/K⁺ ATPase, which hydrolyzes 1 ATP to move 3 Na⁺ outside and 2 K⁺ inside the cells. Astrocytes are commonly thought to be primarily involved in transmitter glutamate cycling, a mechanism that however only accounts for few % of brain energy utilization. In order to examine the participation of astrocytic energy metabolism in brain ion homeostasis, here we attempted to devise a simple stoichiometric relation linking glutamatergic neurotransmission to Na⁺ and K⁺ ionic currents. To this end, we took into account ion pumps and voltage/ligand-gated channels using the stoichiometry derived from available energy budget for neocortical signaling and incorporated this stoichiometric relation into a computational metabolic model of neuron-astrocyte interactions. We aimed at reproducing the experimental observations about rates of metabolic pathways obtained by ¹³C-NMR spectroscopy in rodent brain. When simulated data matched experiments as well as biophysical calculations, the stoichiometry for voltage/ligand-gated Na⁺ and K⁺ fluxes generated by neuronal activity was close to a 1:1 relationship, and specifically 63/58 Na⁺/K⁺ ions per glutamate released. We found that astrocytes are stimulated by the extracellular K⁺ exiting neurons in excess of the 3/2 Na⁺/K⁺ ratio underlying Na⁺/K⁺ ATPase-catalyzed reaction. Analysis of correlations between neuronal and astrocytic processes indicated that astrocytic K⁺ uptake, but not astrocytic Na⁺-coupled glutamate uptake, is instrumental for the establishment of neuron-astrocytic metabolic partnership. Our results emphasize the importance of K⁺ in stimulating the activation of astrocytes, which is relevant to the understanding of brain activity and energy metabolism at the cellular level.     

DFT study of the interaction of cytidine and 2′-deoxycytidine with Li, Na, and K: effects of metal cationization on sugar puckering and stability of the N-glycosidic bond

Density functional theory (DFT) calculations were performed at the B3LYP level with a 6-311++G(d,p) basis set to systematically explore the geometrical multiplicity and binding strength for complexes formed by Li⁺, Na⁺, and K⁺ with cytidine and 2′-deoxycytidine. All computational studies indicate that the metal ion affinity (MIA) decreases from Li⁺ to Na⁺ and K⁺ for cytosine nucleosides. For example, for cytidine the affinity for the above metal ions are 79.5, 55.2, and 41.8 and for 2′-deoxycytidine, 82.8, 57.4, and 42.2 kcal/mol, respectively. It is also interesting to mention that linear correlations between calculated MIA values and the atomic numbers (Z) of the above metal ions were found. The influence of metal cationization on the coordination modes and the strength of the N-glycosidic bond in cytosine nucleosides have been studied. In all cases, the N1-C1′ bond distance changes upon introducing a positive charge in the nucleosides. It has been found that metal binding significantly changes the values of the phase angle of pseudorotation P in the sugar unit of these nucleosides. With respect to the sugar ring, metal binding changes the values of the glycosyl torsion angle and sugar ring conformation. The present calculations in the gas phase provide the first clues on the intrinsic chemistry of these systems and may be of value for studies of the influence of metal cations on the conformational behavior and function of nucleic acids.     

The effect of sodium on inorganic phosphate- and p-nitrophenyl phosphate-facilitated ouabain binding to (Na+ + K+)-activated ATPase

The effect of the hydrolysis product P_i and the artificial substrate p-nitrophenyl phosphate (p-nitrophenyl-P) on ouabain binding to (Na⁺ + K⁺)-activated ATPase was investigated.The hypothesis that (Mg²⁺ + p-nitrophenyl-P)-supported ouabain binding might be due to P_i release and thus (Mg²⁺ + P_i)-supported could not be confirmed.The enzyme · ouabain complexes obtained with different substrates were characterized according to their dissociation rates after removal of the ligands facilitating binding. The character of the enzyme · ouabain complex is determined primarily by the monovalent ion present during ouabain binding, but, qualitatively at least, it is immaterial whether binding was obtained with p-nitrophenyl phosphate or P_i.The presence or absence of Na⁺ during binding has a special influence upon the character of the enzyme · ouabain complex. Without Na⁺ and in the presence of Tris ions the complex obtained with (Mg²⁺ + P_i) and that obtained with (Mg²⁺ + p-nitrophenyl-P) behaved in a nearly identical manner, both exhibiting a slow decay. High Na⁺ concentration diminished the level of P_i-supported ouabain binding, having almost no effect on p-nitrophenyl phosphate-supported binding. Both enzyme · ouabain complexes, however, now resembled the form obtained with (Na⁺ + ATP), as judged from their dissociation rates and the K⁺ sensitivity of their decay. The complexes obtained at a high Na⁺ concentration underwent a very fast decay which could be slowed considerably after adding a low concentration of K⁺ to the resuspension medium. The most stable enzyme · ouabain complex was obtained in the presence of Tris ions only, irrespective of whether p-nitrophenyl phosphate or P_i facilitated complex formation. The presence of K⁺ gave rise to a complex whose dissociation rate was intermediate between those of the complexes obtained in the presence of Tris and a high Na⁺ concentration.It is proposed that the different ouabain dissociation rates reflect different reactive state of the enzyme. The resemblance between the observations obtained in phosphorylation and ouabain binding experiments is pointed out.     

The action of androgens on Na+,K+-ATPase activity of erythrocyte membranes

The effect of androgens (testosterone, androsterone, dehydroepiandrosterone and dehydroepiandrosterone sulfate) on erythrocyte membrane during their nonspecific binding was investigated. The change in erythrocyte membrane Na⁺,K⁺-ATPase activity was measured at different hormone concentration in a suspension. It is shown that the dependence has dome-shaped character: at the elevated hormone concentration Na⁺,K⁺-ATPase activity starts to increase, reaches its maximum, and then decreases. The hypothesis is put forward that an increase in microscopists of erythrocyte membrane first intensifies Na⁺,K⁺-ATPase activity due to the growth of the maximum energy of membrane phonons, and then decreases it due to hindering conformational transitions in the enzyme molecule.     

Interaction of some Pd(II) complexes with Na+/K+-ATPase: Inhibition,kinetics, prevention and recovery

The aim of this work was to investigate the influence of [PdCl₄]^{2 ?} , [PdCl(dien)]⁺ and [PdCl(Me₄dien)]⁺ complexes on Na⁺/K⁺-ATPase activity. The dose-dependent inhibition curves were obtained in all cases. IC₅₀ values determined by Hill analysis were 2.25 × 10^{? 5} M, 1.21 × 10^{? 4} M and 2.36 × 10^{? 4} M, respectively. Na⁺/K⁺-ATPase exhibited typical Michelis-Menten kinetics in the presence of Pd(II) complexes. Kinetic parameters (V_max, K_m) derived using Eadie–Hofstee transformation indicated a noncompetitive type of Na⁺/K⁺-ATPase inhibition. The inhibitor constants (K_i) were determined from Dixon plots. The order of complex affinity for binding with Na⁺/K⁺-ATPase, deducted from K_i values, was [PdCl₄]^{2 ?} >[PdCl(dien)]⁺>[PdCl(Me₄dien)]⁺. The results indicated that the potency of Pd(II) complexes to inhibit Na⁺/K⁺-ATPase activity depended strongly on ligands of the related compound. Furthermore, the ability of SH-donor ligands, l-cysteine and glutathione, to prevent and recover the Pd(II) complexes-induced inhibition of Na⁺/K⁺-ATPase was examined. The addition of 1 mM l-cysteine or glutathione to the reaction mixture before exposure to Pd(II) complexes prevented the inhibition by increasing the IC₅₀ values by one order of magnitude. Moreover, the inhibited enzymatic activity was recovered by addition of SH-donor ligands in a concentration-dependent manner.     

First-principles study of water desorption from montmorillonite surface

Knowledge about water desorption is important to give a full picture of water diffusion in montmorillonites (MMT), which is a driving factor in MMT swelling. The desorption paths and energetics of water molecules from the surface of MMT with trapped Li⁺, Na⁺ or K⁺ counterions were studied using periodic density functional theory calculations. Two paths—surface and vacuum desorption—were designed for water desorption starting from a stationary structure in which water bonds with both the counterion and the MMT surface. Surface desorption is energetically more favorable than vacuum desorption due to water–surface hydrogen bonds that help stabilize the intermediate structure of water released from the counterion. The energy barriers of water desorption are in the order of Li⁺?>?Na⁺?>?K⁺, which can be attributed to the short ionic radius of Li⁺, which favors strong binding with the water molecule. The temperature dependence of water adsorption and desorption rates were compared based on the computed activation energies. Our calculations reveal that the water desorption on the MMT surface has a different mechanism from water adsorption, which results from surface effects favoring stabilization of water conformers during the desorption process.     

Pulsed NMR studies on Na + binding to simple carbohydrates

Pulsed NMR spectroscopy has been used to study Na⁺ binding to several simple carbohydrates in aqueous solution. Changes in the ²³Na spin-lattice relaxation time (T₁) were monitored to indicate complex formation between sodium ions and a ligand. It was found that Na⁺ interacts with these hydroxy-compounds in a manner similar to other metal cations, but very weakly. Among the sugars investigated, $\text{c i s}$-inositol forms the strongest complexes with the stability constant about 1.2 M^?1 (if 1:1 complexes are assumed). A qualitative study of competition between Na⁺ and Ca²⁺ was done, indicating that both cations have the same binding sites.     

Studies on (Na++K+)-activated ATPase: XXXVII. Stabilization by cations of the enzyme-ouabain complex formed with Mg2+ and inorganic phosphate

Dissociation of the (Na⁺+K⁺)-ATPase ouabain complex, formed presence of Mg²⁺ and inorganic phosphate (Complex II), is inhibited by Mg²⁺ (21–45%) and the alkali cations Na⁺ (25–59%) and K⁺ (27–75%) when kidney cortex tissue (bovine, rabbit, guinea pig) is the enzyme source. Choline chloride at 200 mM, equivalent to the highest concentration of NaCl tested, does not inhibit. Dissociation of Complex II from brain cortex (bovine, rat, rabbit) or heart muscle (rabbit) is much less inhibited: 0–11% by Na⁺ and 11–19% by K⁺. The degree of inhibition is not directly related to the size of the dissociation rate constant ($\text{k}{}^{\mathrm{?}}$) of the various complexes, but rather to the extent of interaction between the cation and ouabain binding sites for these tissues.Inhibition curves for Na⁺ and K⁺ are sigmoidal. Half-maximal inhibition for rabbit brain and kidney cortex is at 30–40 mM Na⁺ and 6–10 mM K⁺, and the maximally inhibitory concentrations are 50–150 and 15–20 mM, respectively. Maximal inhibition by Na⁺ or K⁺ for these tissues is the same. For guinea pig kidney cortex Na⁺ and K⁺ are almost equally effective, but 150 mM K⁺ or 200 mM Na⁺ are still not saturating, and inhibition curves indicate high- and low-affinity binding sites for the alkali cations.The inhibition curve for Mg²⁺ is not sigmoidal. In the kidney preparations Mg²⁺ inhibits half-maximally at 0.4-0.5 mM, maximally at 1–3 mM. Maximal inhibition by Mg²⁺ is higher than by Na⁺ or K⁺ for rabbit cortex and lower for guinea pig kidney cortex.There is no competition or additivity among the cations, indicating the existence of different binding sites for Mg²⁺ and the alkali cations.Complex II differs in stability, in the extent of inhibition, in the dependence of inhibition on the cation concentration and in the absence of antagonism between Na⁺ and K⁺, from the ouabain complex formed via phosphorylation by ATP (Complex I). This indicates that the phosphorylation states for the complexes are clearly different.     

Evolution of the sodium pump

Eukaryotic plasma membranes (PMs) are energized by electrogenic P-type ATPases that generate either Na⁺ or H⁺ motive forces to drive Na⁺ and H⁺ dependent transport processes, respectively. For this purpose, animal rely on Na⁺/K⁺-ATPases whereas fungi and plants employ PM H⁺-ATPases. Prokaryotes, on the other hand, depend on H⁺ or Na⁺-motive electron transport complexes to energize their cell membranes. This raises the question as to why and when electrogenic Na⁺ and H⁺ pumps evolved? Here it is shown that prokaryotic Na⁺/K⁺-ATPases have near perfect conservation of binding sites involved in coordination of three Na⁺ and two K⁺ ions. Such pumps are rare in Eubacteria but are common in methanogenic Archaea where they often are found together with P-type putative PM H⁺-ATPases. With some exceptions, Na⁺/K⁺-ATPases and PM H⁺-ATPases are found everywhere in the eukaryotic tree of life, but never together in animals, fungi and land plants. It is hypothesized that Na⁺/K⁺-ATPases and PM H⁺-ATPases evolved in methanogenic Archaea to support the bioenergetics of these ancestral organisms, which can utilize both H⁺ and Na⁺ as energy currencies. Both pumps must have been simultaneously present in the first eukaryotic cell, but during diversification of the major eukaryotic kingdoms, and at the time animals diverged from fungi, animals kept Na⁺/K⁺-ATPases but lost PM H⁺-ATPases. At the same evolutionary branch point, fungi did loose Na⁺/K⁺-ATPases, and their role was taken over by PM H⁺-ATPases. An independent but similar scenery emerged during terrestrialization of plants: they lost Na⁺/K⁺-ATPases but kept PM H⁺-ATPases.     

Neurochemical Evidence that Pristanic Acid Impairs Energy Production and Inhibits Synaptic Na<Superscript>+</Superscript>, K<Superscript>+</Superscript>-ATPase Activity in Brain of Young Rats

Pristanic acid (Prist) accumulates in some peroxisomal disorders characterized by neurologic dysfunction and brain abnormalities. The present work investigated the in vitro effects of Prist on important parameters of energy metabolism in brain cortex of young rats. CO₂ production from labeled acetate and the activities of the respiratory chain complexes I–IV, creatine kinase and synaptic Na⁺, K⁺-ATPase were measured. Prist decreased CO₂ production and the activities of complexes I, II and II–III. Prist also reduced Na⁺, K⁺-ATPase activity, but did not affect the activity of creatine kinase. Considering the importance of the citric acid cycle and the electron flow through the respiratory chain for brain energy production and of Na⁺, K⁺-ATPase for the maintenance of membrane potential, the present data indicate that Prist compromises brain bioenergetics and neurotransmission. It is presumed that these pathomechanisms may be involved in the neurological damage found in patients affected by disorders in which Prist accumulates.     

Solvation free energies of glutamate and its metal complexes: A computer simulation study

Most biological ion channels demonstrate a high degree of selectivity for one type of ion more than others, and in many cases, how they control attaining this is still not clear. So we have studied on some metal ion compounds of glutamate. The Glutamate and its meal ion compounds (Ca²⁺, Na⁺, K⁺ and Li⁺) were first modeled by ab initio calculations and then Monte Carlo simulation was used to calculate solvation free energies and also the complexes free energies for the related structures. The results indicated that Glutamate-Ca²⁺ have more stability in water than other metal ion. Also, it was found out that the more movement in ions; less stability of the structure would result. This trend can be seen both in gas and liquid phase.     

Energy metabolism of reticulocytes: Two different sources of energy for Na+K+-ATPase activity

Total energy production in rabbit reticulocytes amounted to 136·52 ± 6·50μmol ATP h^?1ml^?1 of reticulocytes: 88·3 per cent was provided by oxidative phosphorylation, whereas only 11·7 per cent by aerobic glycolysis. Na⁺K⁺-ATPase accounted for 23 per cent, i.e. 27·65 ± 2·55μmol ATP h^?1ml^?1 of reticulocytes, in the overall energy consumption in reticulocytes of rabbits. Under basal conditions ATP for Na⁺K⁺-ATPase activity was derived exclusively from oxidative phosphorylation. However, when the activity of Na⁺K⁺-ATPase was increased due to the stimulation of adenylate cyclase by (?)-isoprenaline, the additional energy required was provided by aerobic glycolysis. These results indicate that two different compartments, one cytosolic and the other mitochondrial, provide energy for Na⁺K⁺-ATPase activity in reticulocytes.