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The reaction cycle of the Na+/K+-ATPase includes several intermediate stages, which are produced by the binding of different physiological ligands accompanied by conformational changes of the enzyme (2). Recently we have shown that extensive proteolytic digestion in the presence of various physiological ligands could be a useful tool in studies of rearrangements of membrane-associated segments of Na+/K+ -ATPase (10). Distinctive conformational changes were revealed by N-terminal amino acid sequence analysis of the digests following SDS PAGE (10). The changes in cleavage patterns result from alterations in domain-domain interactions of the protein. Here, we exploit this approach to shed light on events which occur following ouabain binding. We addressed this question by a comparative study of the posttryptic products obtained after extensive degradation of Na+/K+-ATPase after phosphorylation, in the presence of potassium or Mg,Pi,ouabain.

The chemical modification of purified renal Na+/K+-ATPase with the fluorescent carboxyl-selective reagent, 4-(diazomethyl)-7-(diethylamino)-coumarin (DEAC), results in enzyme inactivation via disruption of the monovalent cation binding sites and loss of K+ and Na+ binding capacity. Modification of 1 or 2 carboxyl residues in the α-subunit in a K+ or Na+-preventable manner leaves the ATP binding unaltered and the enzyme is still able to undergo E1 ↔ E2 transitions. These characteristics of the DEAC-modified enzyme are evidence that the modified residues are located at the cation binding site (1, 2). Furthermore, the specificity of DEAC to react with carboxyl groups supports the early idea that carboxyl residues, in or near the membrane, would be involved in the cation oclusion and transport by the Na+/K+-ATPase.

The sodium pump or Na,K-ATPase, maintains the Na + and K + gradients across eukaryotic cell membranes at the expense of ATP. Incubation of purified canine renal Na,K-ATPase with 4-acetamido-4′-isothiocyanatostilbene-2,2′-disulfonic acid (SITS) inhibited the ATPase activity. Both the labeling of the protein and the loss or ATPase activity were prevented by co-incubation with ADP (acting as an ATP analog) or KCI. Only the α-subunit was labeled by SITS. The α-subunit from the inhibited enzyme was extensively digested with trypsin, and SITS-labeled peptides were purified by reverse-phase HPLC and sequenced. The amino acid sequence determined, His-Leu-Leu-Val-Met-X-Gly-Ala-Pro-Glu, indicated that SITS modifies Lys-501 (X) on the α-subunit of Na,K-ATPase.

We have recently reported that the Na pump alpha-subunit has cytosolic-oriented oligosaccharides which were sensitive to cleavage by an enzyme specific for hydrolysis of N-linked glycans [Pedemonte et al. (1990) Proc. Natl. Acad. Sci. U.S.A. 87, 9789-9793]. We now describe experiments that characterize the saccharides and further substantiate our previous findings. Bovine milk galactosyltransferase has been used in conjunction with radiolabeled UDP-galactose to label N-acetylglucosamine residues on the protein. The Na pump alpha-subunit contains some O-linked carbohydrates; however, the bulk (> 80%) of the radioactivity was found in oligosaccharides sensitive to peptide:N-glycosidase F degradation but not to alkaline hydrolysis. Alkaline hydrolysis produced degradation of the protein, and the [3H]Gal radiolabeled carbohydrates remained bound to peptides and were released by subsequent peptide N-glycosidase F treatment. The exogenously galactosylated sugars cleaved by the glycosidase were analyzed by liquid chromatography and had elution volumes identical to a galactose-N-acetylglucosamine disaccharide standard. Since the galactose was exogenously added, we propose that the N-linked glycans on the alpha-subunit of the Na pump are composed of a single sugar residue, which is probably N-acetylglucosamine.

Chemical modification of specific residues in enzymes, with the characterization of the type of inhibition and properties of the modified activity, is an established approach in structure-function studies of proteins. This strategy has become more productive in recent years with the advances made in obtaining primary sequence information from gene-cloning technologies. This article discusses the application of chemical modification procedures to the study of the Na(+)-K(+)-ATPase protein. A wide array of information has become available about the kinetics, enzyme structure, and various conformational states as a result of the combined use of inhibitors, ligands, modifiers, and proteolytic enzymes. We will review a variety of reagents and approaches that have been employed to arrive at structure-function correlates and discuss critically the limits and ambiguities in the type of information obtained from these methodologies. Chemical modification of the Na(+)-pump protein has already provided a body of data and will, we anticipate, guide the efforts of mutagenesis studies in the future when suitable expression systems become available.

Robert Henry (Rob) Costa, an authority on liver biology and cancer, died on September 1, 2006 at his home in Oak Park, IL. ![Graphic][1] Rob was born in Galesburg, IL and grew up in Bethesda, MD. He attended the University of California at Irvine, where he received his doctorate in Molecular

Treatment of purified renal Na,K-ATPase with dihydro-4,4'-diisothiocyanatostilbene-2,2'-disulfonate (H2DIDS) produces both reversible and irreversible inhibition of the enzyme activity. The reversible inhibition is unaffected by the presence of saturating concentrations of the sodium pump ligands Na+,K+, Mg2+, and ATP, while the inactivation is prevented by either ATP or K+. The kinetics of protection against inactivation indicate that K+ binds to two sites on the enzyme with very different affinities. Na+ ions with high affinity facilitate the inactivation by H2DIDS and prevent the protective effect of K+ ions. The H2DIDS-inactivated enzyme no longer exhibits a high-affinity nucleotide binding site, and the covalent binding of fluorescein isothiocyanate is also greatly reduced, but phosphorylation by Pi is unaffected. The kinetics of inactivation by H2DIDS were first order with respect to time and H2DIDS concentration. The enzyme is completely inactivated by the covalent binding of one H2DIDS molecule at pH 9 per enzyme phosphorylation site, or two H2DIDS molecules at pH 7.2. H2DIDS binds exclusively to the alpha-subunit of the Na,K-ATPase, locking the enzyme in an E2-like conformation. The profile of radioactivity, following trypsinolysis and SDS-PAGE, showed H2DIDS attachment to a 52-kDa fragment which also contains the ATP binding site. These results suggest that H2DIDS treatment modifies a specific conformationally sensitive amino acid residue on the alpha-subunit of the Na,K-ATPase, resulting in the loss of nucleotide binding and enzymatic activity.

We have synthesized 2-nitro-5-azidobenzoyl (NAB) derivatives of ouabain as photoaffinity labels of the cardiac glyocoside binding site of Na, K-ATPase. [3HzNAB-ouabain was found to bind to the same number of sites on Na, K-ATPase (purified from pig kidney outer medulla) as ouabain (1.9 nmol/mg), with approximately the same affinity (Kk(ouabain)/Kd(NAB-ouabain) congruent to 1.6), and ouabain was fully competitive uith NAB-ouabain at these sites. NAB-ouabain binding and inhibition were reversible in the dark, but on exposure to ultraviolet light (310-370 nm) 30-40% of the binding and ihibition became irreversible; this binding was shown to be covalent by stability to trichloroacetic acid, organic solvents, and heat denaturation. Covalent labeling was prevented by photolysis of NAB-ouabain prior to the experiment, or by prior incubation of the enzyme with ouabain. On sodium dodecyl suffate-polyacrylamide gels of labeled Na,K-ATPase, about half of the covalently bound [3H]NAB-ouabain migrated with the large polypeptide (molecular weight congruent to 95 000), and half migrated with a small polypeptide (molecular weight congruent to 12 000); noncovalently bound NAB-ouabain (60-70% of total label) ran with the tracking dye. A similar labeling pattern was obtained utilizing NaI microsomes prepared from pig kidney outer medulla. The small polypeptide was characterized as an acidic proteolipid by extractability into acid chloroform/methanol; labeling of this component by NAB-ouabain is the first demonstration that it is directly associated with the Na,K-ATPase. The results of our characterization of NAB-ouabain show that it has the required specificity, covalency, and efficiency of labeling for application in structural studies of Na,K-ATPase subunit interactions.

The properties of a recently synthesized photolabile chelator for divalent cations are described, the affinity of which for Ca2+ changes by some 5 orders of magnitude on illumination. The compound 1-(2-nitro-4,5-dimethoxyphenyl)-N,N,N',N'-tetrakis[(oxycarbonyl)me thyl]-1,2-ethanediamine (DM-nitrophen) binds Ca2+ (Kd approximately 5.0 x 10(-9) M) and Mg2+ (Kd approximately 2.5 x 10(-6) M) with relatively high affinities. On exposure of the DM-nitrophen-Ca2+ complex to UV light in the 350-nm range, the chelator is cleaved yielding iminodiacetic products with a much lower affinity for Ca (Kd approximately 3 x 10(-3) M) and the free [Ca2+] increases. The quantum yield for Ca2+ release is 0.18. In experiments with chemically skinned skeletal muscle fibers, a fully relaxed fiber equilibrated with DM-nitrophen-Ca2+ complex produced maximal contraction after a single flash from a frequency-doubled ruby laser (347 nm). Half-maximal tension was achieved in approximately 40 ms, some 5 times faster than that obtained after a rapid solution change from a Ca2+-free to a Ca2+-containing solution. In experiments with resealed human erythrocyte ghosts, irradiation of ghosts containing the DM-nitrophen-Ca2+ complex activates a Ca2+-dependent K+ efflux pathway, which is not observed in the absence of illumination. DM-nitrophen is sufficiently stable and photolabile to be used as a caged Ca (or caged Mg) for the rapid photoinitiation of divalent cation-dependent processes over a wide concentration range with a significant increase in temporal resolution over conventional mixing methods.

High intensity pulsed light sources are currently being used in two major applications in muscle research. The first of these involves rapid photolytic release of metabolites, e.g. adenosine triphosphate or calcium ions, from inert, photosensitive precursors. This allows rapid kinetic experiments in muscle fibres which would otherwise be impossible because of the long diffusion times even of small molecules into the lattice of thick and thin filaments. Examples of the use of lasers and flash lamps for this purpose will be given, with particular emphasis on dynamic structural investigations using synchrotron radiation as a high intensity X-ray source for monitoring structural changes by low angle diffraction. The second application involves the use of solid state lasers to produce powerful light pulses in the infrared region to achieve temperature jumps in muscle fibres. This, in conjunction with the use of synchrotron radiation, allows the kinetics of interesting structural transitions in muscle fibres to be studied.

Irreversible inhibition of Na,K-ATPase and K+-dependent p-nitrophenylphosphatase activities was produced by incubation of purified Na,K-ATPase enzyme with 1-ethyl-3(3-dimethylaminopropyl)carbodiimide (EPC). Inhibition was time and [EPC] dependent and displayed first order kinetics with respect to time. The [EPC] to reduce the enzyme velocity by 50% for Na,K-ATPase and phosphatase activities was 1.6 and 2.2 mM, respectively. Analysis of the kinetics of inhibition by EPC indicated that reaction at one site was sufficient to produce inhibition. Inhibition was greatly reduced by the presence of Mg2+, Na+, K+, choline, or Tris (decreasing order of effectiveness); ATP was without effect. This suggests that cation-bound enzyme forms were less reactive with the carbodiimide than free enzyme; ATP-bound enzyme was as reactive. Apparently the cations Na+, Mg2+, Tris, and choline stabilize E1 forms of the enzyme which are different from the E1 form stabilized by ATP. Addition of [14C]glycine ethyl ester (Gly-OEt) resulted in incorporation of radioactivity into both alpha and beta subunits that was dependent upon the presence of EPC, and the incorporation was reduced by the cations which reduced the inhibition due to EPC. Simultaneous addition of Gly-OEt with EPC prevented inhibition, although 14C incorporation still took place. If Gly-OEt addition was delayed the initial inactivation was not affected, but little subsequent inactivation occurred. The protection against inactivation by EPC occurs on the addition of other exogenous nucleophiles, such as aminoethane or ethylenediamine. Dicyclohexylcarbodiimide, a more potent hydrophobic carbodiimide inhibitor, shows similar effects; the inhibition due to dicyclohexylcarbodiimide is also prevented by the simultaneous presence of a nucleophile. After treatment with a carbodiimide and exogenous nucleophile the Na,K-ATPase has modified carboxyl residues but is not inhibited. Thus, modification of the cation-protectable carboxyl groups does not by itself cause inhibition. It seems likely that the inhibition of activity due to carbodiimide alone is not due to the modification of a carboxyl group per se but to the formation of an intramolecular bond between the carbodiimide-activated carboxylic acid and an endogenous nucleophile. The formation of such bonds suggests the close juxtaposition of amine and carboxyl groups in the secondary structure of the enzyme.

The rate of valinomycin-induced KCl efflux from human red cells and ghosts was measured in the presence and absence of phlorizin. Extracellular phlorizin accelerated the KCl efflux. The effect depended on the phlorizin concentration and showed half saturation at about 0.4mM phlorizin. Hunter's procedure was used to calculate Cl permeabilities (PCl) by means of the Goldman equation from rate constants of K+ loss in valinomycin-treated ghosts. For saturating phlorizin concentrations a 20-fold increase, approximately, ofPCl was calculated. The observed increase inPCl is in contrast to the almost total inhibition of Cl− equilibrium exchange. Similar to the effects on anion exchange permeability the effect onPCl is only observed when phlorizin is present at the outer surface of the erythrocyte membrane while internal phlorizin is without effect. A similar asymmetry was observed in the stimulation of valinomycin-induced K+ exchange at identical K+ concentrations on both sides of the membrane. The effects of phlorizin were only observed if net KCl flow was out of the cells but not if it was in the opposite direction. The effect of phlorizin on net KCl movements and sugar transfer were unaltered when the phlorizin was subjected to several consecutive purifications. This indicates that the observed effects are due to the glycoside and not to contaminations with its aglycone.

Like phlorizin, two glycosidic esters of phlorizin, the 4-azido-2-nitrobenzoate (ANB-phlorizin) and the 2-nitrobenzoate (NB-phlorizin) were found to be effective inhibitors of SO 4 2− equilibrium exchange at the outer but not at the inner membrane surface of the human erythrocyte ghost. After photolysis of ghost suspensions in the presence of extracellular ANB-phlorizin an irreversible inhibition of SO 4 2− exchange was observed, while photolysis of intracellular ANB-phlorizin was without effect. After photolysis in the presence of extracellular or intracellular triatated ANB-phlorizin gel electrophoresis of the labelled membranes revealed similar locations of binding. These findings suggest that the sidedness of action of ANB-phlorizin could not be related to inaccesibility of the inner membrane surface for the agent but that inhibition occurs via binding to fixed sites at the outer membrane surface that are not associated with a mobile carrier which crosses the membrane.

Resealed human red cell ghosts containing caged ATP (Kaplan et al., 1978) and [3H]ADP were irradiated at 340 nm. The photochemical release of free ATP initiated a rapid transphosphorylation reaction (ATP:ADP exchange), a component of which is inhibited by ouabain. The reaction rate was measured by following the rate of appearance of [3H]ATP. The sodium pump-mediated ATP:ADP exchange reaction showed high-affinity stimulation by Mg ions (less than 10 microM) and was inhibited at higher levels. At optimal [Mg], extracellular Na (Nao) had a biphasic effect. Nao progressively inhibited the reaction rate between 0 and 10 mM and stimulated at higher levels. Intracellular Na (Nai) activated the reaction; the rate was maximal when Nai was 1 mM and remained unaltered up to 115 mM Nai at constant Nao. Extracellular K ions (Ko) inhibited the reaction; at high Nao, half-maximal inhibition was observed with 0.9 mM Ko. Lio inhibited the exchange rate with a lower affinity than Ko; half-maximal inhibition was produced by approximately 50 mM Lio. Intracellular K ions were without dramatic effect on the reaction rate in the concentration range where Ko inhibited completely. The relationship between these observations and previous studies on porous preparations is discussed, as well as the extent to which these observations support the hypothesis that the sodium pump-mediated ATP:ADP exchange reaction accompanies the Na:Na exchange transport mode of the sodium pump.

We have recently shown that inactivation of renal Na,K-ATPase by 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide occurs via an intramolecular cross-link formed between an activated carboxyl group and an endogenous nucleophile (Pedemonte, C.H., and Kaplan, J.H. (1986) J. Biol. Chem. 261, 3632-3639). The modified enzyme shows the same level of Rb+ binding as untreated enzyme: 3.16 and 2.93 ATP-sensitive mumol of Rb+ binding/mumol of phosphoenzyme, respectively. Thus, the Rb+ binding site and the transition accomplished by low affinity nucleotide binding which accelerates de-occlusion are not greatly affected by the carbodiimide inactivation. 1 mM K+ reduces the ADP binding to the high affinity nucleotide binding site to the same extent in normal and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide-treated enzyme and Na+ counteracts this effect. Thus, the competition between Na+ and K+ ions for binding to the free enzyme are also largely unaltered by the modification. Phosphorylation from ATP (microM) in the presence of Na+ and Mg2+ ions and from inorganic phosphate in the presence of Mg2+ ions (in the absence or presence of ouabain) is greatly inhibited (85%) following carbodiimide treatment. The extent of inhibition of phosphorylation quantitatively correlates with the residual Na,K-ATPase activity (15%). Consequently, the rate of inactivation by carbodiimide is reduced when a greater proportion of the enzyme is in the phosphorylated form. Fluoroscein isothiocyanate, which inhibits the Na,K-ATPase by covalently modifying a lysine residue close to the high affinity binding site for ATP in the alpha-subunit does not bind to the carbodiimide-inactivated enzyme. Since high affinity nucleotide binding is only partially inhibited by the modification produced by the carbodiimide this suggests that the lysine residue to which fluoroscein isothiocyanate binds is not specifically required for competent nucleotide binding.

Caged compounds are molecules or ions of physiological interest, e.g. ATP, IP3, cAMP, cGMP, GTP and Ca2+ rendered inactive by chemical modification. The modification introduces a photochemically labile bond, which on exposure to ultraviolet light cleaves rapidly, releasing the active compound. This article reviews some of the major advances and applications of the photorelease approach, and illustrates its potential in several areas of interest to cellular neuroscientists.

2-Nitrobenzyl phosphate and 1-(2-nitro)phenylethyl phosphate have been synthesized and demonstrated to be suitable as photolabile sources of inorganic phosphate. The same protecting groups were attached to the terminal phosphate of adenosine 5'-triphosphate. These caged ATP compounds released adenosine 5'-triphosphate on illumination at 340 nm in aqueous solution and P/sup 3/-1-(2-nitro)phenylethyl-ATP gave about a 70 percent yield in under 30 s. The unphotolyzed caged ATP was neither a substrate nor inhibitor of purified renal Na,K-ATPase (EC 3.61.3). Following photolysis in the presence of the enzyme, the liberated ATP was hydrolyzed but at an inhibited rate. The photo-dependent inhibition could be eliminated by prior addition of glutathione or bisulfite to the irradiated solution. Caged ATP was incorporated into resealed human erythrocyte ghosts prepared from red blood cells depleted of internal energy stores. While the NA : K pump was unable to use incorporated caged ATP as a substrate, the ATP liberated by photolysis activated the pump as evidenced by measurements of K-dependent, ouabain-sensitive Na efflux. Thus the caged ATP can be used as a stable source of ATP unmetabolizable by intracellular ATPases until the ATP is released following photolytic irradiation.

The sodium pump of human red blood cells mediates a Rb:Rb exchange that is dependent for maximal rates upon the simultaneous presence of intracellular ATP (or ADP) and phosphate. We have measured ouabain-sensitive 86Rb uptake into resealed ghosts of human red cells containing ADP and show that arsenate will substitute for phosphate in supporting the Rb:Rb exchange transport mode. The concentration dependence of arsenate-supported Rb:Rb exchange in ghosts containing 2 mM ADP shows both activating and inhibiting phases; the dependence upon phosphate shows similar characteristics. Elevation of the external [Rb] lowers the apparent affinity for arsenate since there is a shift to higher concentrations of arsenate in the activating and inhibiting phases of the arsenate concentration dependence curve. Similarly, elevation of [ADP] substantially reduces the inhibition of Rb:Rb exchange observed at higher [arsenate]. These effects are also observed in phosphate-supported Rb:Rb exchange. The phosphate requirement for Rb:Rb exchange involves phosphorylation of the sodium pump protein; the close agreement between the effects of arsenate and phosphate in supporting Rb:Rb exchange makes it likely that arsenylation of the sodium pump occurs during Rb:Rb exchange. Arsenate efflux from red blood cell ghosts into arsenate-free chloride medium is partially inhibited (77-80%) by DNDS (4,4'-dinitro-2,2'-stilbenedisulfonic acid), this compares with 82-87% inhibition by DNDS of phosphate efflux under the same conditions. It appears that Band III, the red cell anion transport system, accepts arsenate in a similar fashion to phosphate and that a fraction of the flux of both anions may occur through pathways other than Band III. Thus, in human red blood cells, both the sodium pump and the anion exchange transport system will accept arsenate as a phosphate congener and the protein-arsenate interactions are very similar to those with phosphate.

Synthese de derives nitro-2 dimethoxy-4,5 phenyl de l'EDTA et de l'EGTA et complexation par action de CaCl 2 ou MgCl 2

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.

Lysozyme has been shown to increase the rate at which 22 Na + diffuses out of liposomes composed of varying amounts of phosphatidylserine and phosphatidylcholine. As the proportion of phosphatidylserine is increased, the effectiveness with which the protein increases the 22 Na + -diffusion rate becomes greater. The increase in effect with increasing proportions of phosphatidylserine can be related to the amount of protein bound to the vesicles. The simultaneous rate of CrO 4 2− diffusion from the liposomes was not affected by the addition of lysozyme. A tentative model is proposed for this discrimination.

The characterization of membrane transport systems requires a study of the sidedness of the effects of modifying agents. Previous work with phlorizin showed that although this agent inhibits sugar transport at either surface of the red cell membrane, the effects on anion transport and acceleration of Clnet movements [l] and an inhibition of Clor SO;equilibrium exchange [2,3] can only be observed if the agent is added to the external medium. Intracellular phlorizin was without effect except for a slight acceleration of Cl-/Clequilibrium exchange at low pH [4]. Similar studies on the sidedness of the effects of two disulfonic acids, 4,4’-diacetamido stilbene-2,2’-disulfonic acid (DAS) and 2-(4’-aminophenyl)d-methylbenzene-thiazole-3’,7-disulfonic acid (APMB), revealed that the former compound inhibits SO:equilibriufixchange only at the outer membrane surface while APMB produced a strong inhibition at both surfaces [5]. The present work characterizes the actions of these two inhibitors on Cltransport as measured under conditions of Clequilibrium exchange, and net flow. The sidedness was the same under both experimental conditions. This indicates that, although Clequilibrium exchange proceeds about 10’ times faster than SOiequilib

Coupled active transport of Na+ and K+ across cellular plasma membranes is mediated by (Na+ + K+)-stimulated Mg2+-dependent ATPase. Active cation transport by this Na pump involves a cyclic Na-dependent phosphorylation of the enzyme by intracellular ATP and hydrolytic dephosphorylation of the phosphoenzyme, stimulated by K+ (ref. 1). In human red blood cells, skeletal muscle and squid axons, replacement of extracellular K by Na results in a ouabain-sensitive efflux of Na coupled to an influx of extracellular Na. There is apparently no net Na movement nor net hydrolysis of ATP. The rate of Na:Na exchange is stimulated by increased levels of ADP and exchange transport is not observed in cells totally depleted of intracellular ATP. These characteristics suggest that the biochemical mechanism underlying the Na exchange mode of the Na pump involves phosphorylation of the enzyme by ATP (which requires intracellular Na) followed by its dephosphorylation by ADP. Such a reaction has been observed in partially purified (Na+ + K+) ATPase from a variety of sources and its dependence on Na concentration has been described (although not previously for the red cell enzyme). In the present work, intracellular ATP:ADP exchange reaction was initiated by photoreleased ATP following brief irradiation at 350 nm of ghosts containing caged-ATP. The ouabain-sensitive component of the ensuing ATP:ADP exchange reaction shows a biphasic response to extracellular Na. External Na in the range 0--10 mM has an inhibitory effect whilst increasing concentrations beyond this range stimulate the rate of exchange in a roughly linear fashion up to 100 mM Na. These results represent the first direct demonstration of the sidedness of the effects of Na on this partial sequence in the overall enzyme cycle and bear a qualitative resemblance to the Na effects on the Na-ATPase which occur in the absence of intracellular ADP in human red blood cells.

In the last decade, the burgeoning interest in membrane proteins and the development of new techniques for their isolation and characterization have resulted in an enormous increase in our knowledge of the sodium pump as a membrane protein. At the same time, more extensive studies on the transport characteristics of the system have enhanced our understanding of the sodium pump as an ion-transporting system. In the last several years, the sodium pump has been the subject of many reviews. These include: the reaction mechanism (6, 10, 18,44); Na activation of enzymatic activity and transport (27); charac­ teristics of cardiac glycoside binding (15); ligand interactions (39); and enzyme distribution in the renal tubule (25). The papers presented at three meetings dedicated to the sodium pump are also an excellent source of detailed studies of the system during the last decade (2, 22, 53).

Publisher Summary Extracellular Na in the range 0– 5mM has an inhibitory effect on the rate of ATP : ADP exchange, while increasing concentrations in the range 10mM–100mM stimulate the rate in a roughly linear fashion. These studies enable to identify the inhibitory and low affinity stimulatory portions of the curve with sites for extracellular Na. It has been speculated that the activation of the ATP : ADP exchange reaction in the range 10–100mM Na is because of a shift in the equilibrium from ADP-insensitive to ADP-sensitive phosphoenzyme; if this model is correct, it is evident that extracellular Na is responsible for the regulation. The pattern of the external Na dependence of the Na : Na exchange seen in human red blood cells is very similar to the dependence observed for ATP : ADP exchange, an initial inhibition followed by stimulation. To relate the biochemical properties of the Na pump with the associated transport modes, it is important to be able to measure the respective rates under identical experimental conditions. The measurement of the rate of isotopic Na : Na exchange in the resealed ghost system using caged-ATP make such measurements feasible. Such studies enable to describe more precisely the relationship between elementary steps in the biochemical sequence and events in the transport process.

1. The effects of the addition of Ca2+ on ouabain-resistant 22Na+ efflux from Na+-loaded strips of rabbit portal anterior mesenteric vein in Ca2+-free media have been studied. 2. Na+ efflux into Li+ media containing 5 mM-KCl is rapidly and transiently stimulated some 4- to 5-fold on the addition of Ca2+ (1.2 mM). No stimulation is observed if the Li+ medium is K+ free or if Na+ replaces Li+ ions. This Ca2+-activated Na+ efflux is not obligatorily coupled to Na+ influx. 3. The stimulation of Na+ efflux could also be triggered by the addition of 5 mM-K+ to a Ca2+-containing K+-free medium. The Ca2+-activated increase in Na+ efflux also occurred when K+ was the sole monovalent extracellular cation. Rb+ could substitute for the K+ requirement. Thus the Na+ efflux is not mediated by a system which has a specific requirement for counter-transport of Li+ or one in which Li+ but not K+ are counter-transported such as the familiar Na+-H+ exchange system. Acidification of the external medium reduced the Ca2+-stimulated Na+ efflux, in keeping with the conclusion that this efflux was not due to Na+-H+ exchange. 4. Progressive reduction of external [Ca2+] increased the time-lag to peak activation of Na+ efflux, suggesting that the effects of added Ca2+ were mediated by a rise in intracellular Ca2+. Under experimental conditions which did not result in activation of the Na+ efflux by the addition of extracellular Ca2+ alone (e.g. in Na+ media), addition of Ca2+ plus the Ca2+ ionophore, ionomycin, stimulated Na+ efflux. This further confirms that intracellular sites for Ca2+ are critical for the activation of Na+ efflux. In the absence of ionophore, in Na+ media, intracellular Ca2+ is not sufficiently increased when extracellular Ca2+ is added. A partial (40%) block of Ca2+-activated Na+ efflux by amiloride (2 X 10(-3) M) could also be overcome by the addition of ionomycin. 5. The lack of effect of a variety of inhibitors suggests that the Ca2+-stimulated Na+ efflux mechanism is not mediated via a Na+-K+-Cl- co-transport system or a Na+-H+ counter-transport system, or Na+-Ca2+ exchange. 6. The activation of Na+ efflux in smooth muscle by Ca2+ ions seems to involve Ca2+ entry partially via an extracellular Ca2+-intracellular Na+ exchange and also through other parallel pathway(s), followed by a rise in intracellular Ca2+ that activates Na+ efflux through a Ca2+-sensitive Na+ channel or other transport pathway.

Treatment of the Na,K-ATPase enzyme, isolated from canine renal outer medulla, with thimerosal (ethylmercurithiosalicylate) resulted in significant inhibition of the overall Na,K-ATPase with only slight, if any, inhibition of the Na-ATPase and ATP:ADP exchange activities. The K-stimulated PNPPase activity was stimulated [see G. R. Henderson and A. Askari (1977) Arch. Biochem. Biophys. 182, 221-226]. Examination of the Na dependence of the ATPase and ATP:ADP exchange activities revealed an Na-independent, ouabain-sensitive activity that was inhibited by Na in the range 0-10 mM. At greater than or equal to 10 mM concentration of Na the treated and modified enzymes showed similar activities. The apparent affinity of the modified enzyme for ATP in the presence of 100 mM Na was the same as that of the untreated enzyme (0.2-0.3 microM). In the absence of Na, the modified enzyme hydrolyzed ATP with a relatively low affinity (about 120 microM). The enhancement of p-nitrophenylphosphatase (PNPPase) activity measured in the presence of K ions was due to the appearance of K-independent, ouabain-sensitive PNPP activity. The modification was without major affect on the apparent affinity of the enzyme for K ions in the PNPPase activity. Treatment of the thimerosal-modified enzyme with dithiothreitol removed (or greatly reduced) the cation-independent, ouabain-sensitive activities and the Na,K-ATPase activity returned. Modification of a set of enzyme -SH groups in the Na,K-ATPase enzyme made it able to hydrolyze ATP in the absence of Na ions and PNPP in the absence of K ions. The -SH groups modified by thimerosal are evidently critical to the major dephosphoenzyme conformational changes but are not involved in the major transport conformational change between phosphoenzymes.

The band 3 protein-mediated, valinomycin-induced KCl efflux continues to increase with increasing [KCl] when the Cl−/Cl− equilibrium exchange becomes saturated. This suggests the existence of a band 3-mediated component of Cl− flux that contributes to the electrical conductance without being related to slippage; i.e., equilibration of the unloaded transport protein between the two membrane surfaces.

Publisher Summary The importance of sulfhydryl groups in maintaining the functional integrity of the Na, K-ATPase has been well established. The modification of one set of –SH groups on the enzyme by N -ethylmaleimide leads to specific changes in the functional catalytic properties of the Na, K-ATPase. The treatment with N -ethylmaleimide blocks the usual sequential transition from phosphoenzyme, which is ADP sensitive and K insensitive (E 1 P) to one which is K sensitive and ADP insensitive (E 2 P). The major consequence of this modification is, along with the inhibition of Na, K-ATPase activity, an increase in the partial reaction, which depends upon the level of E 1 P—that is, ATP/ADP exchange. The treatment of Na, K-ATPase enzyme with thimerosal (ethylmercurithiosalicylate) results in a significant inhibition of the overall Na, K-ATPase activity, without significantly inhibiting Na-ATPase, ATP: ADP exchange or K-stimulated p -nitrophenylphosphatase (PNPPase) activities. The normal-complex Na-activation kinetics seen in the Na-ATPase activity became monotonic after mercurial modification. The Na dependence of the ATPase activity of the untreated enzyme is compared with that of the enzyme after modification with thimerosal.

Publisher Summary This chapter discusses sodium pump-catalyzed ATP–ADP exchange in red blood cells together with the effects of intracellular and extracellular Na and K ions. When human red blood cells are incubated in K-free media containing Na ions, an ouabain-sensitive exchange of intracellular and extracellular Na ions is observed. Subsequent studies on this transport mode of Na pump have established that the Na–Na exchange rate is increased by elevated levels of ADP, is independent of ATP in the range 300 μM–1500 μM, and will not take place in resealed ghosts containing ADP but not ATP. Although ATP is required for the Na pump to support Na–Na exchange, there is no net hydrolysis of ATP associated with the transport. These observations support the suggestion that the biochemical events catalyzed by the Na pump protein while mediating Na–Na exchange are the reversible phosphorylation of the protein by ATP and its dephosphorylation by ADP. This chapter characterize the sidedness of the Na-activating (and inhibiting) effects and discusses the validity of the hypothesis that ATP–ADP exchange and Na–Na exchange are coupled simultaneous biochemical and transporting reactions.

1. 1. By incorporating varying amounts of stearylamine or dicetylphosphate with phosphatidylcholine into vesicles, positively charged or negatively charged liposomes, respectively, were produced. Anion (CrO 4 2− efflux rates were shown to be influenced by the prevailing charge on the liposome, the efflux rates being greater with positively charged vesicles. 2. 2. The addition of the soluble basic protein, lysozyme, to suspensions of negatively charged liposomes produced an increase in the diffusion rate of CrO 4 2− across the bilayers. The diffusion rate of CrO 4 2− across positively charged bilayers was unaffected by the addition of lysozyme. A semi-empirical rate equation was derived in order to compare the effectiveness of the protein in altering the efflux rates under varying conditions. 3. 3. The amount of protein bound to the liposomes was measured using an ultrafiltration technique, and was correlated to the increase in anion permeability caused by the addition of protein.

The Na pump is an αβ heterodimer responsible for maintaining fluid and electrolyte homeostasis in mammalian cells. We engineered MDCK cell lines expressing α1flag-, β1flag-, or β2myc-subunits via a tetracycline (tet)-regulated promoter and a cell line expressing both stable β1myc and tet-regulated β1flag in order to examine regulatory mechanisms of Na pump subunit expression. Overexpression of β1flag increases total β-subunit levels by greater than 200% without changes in α-subunit abundance, however, endogenous β1-subunit (β1E) abundance is decreased. β1E down-regulation does not occur during β2 overexpression. The decrease in β-subunit expression is not accompanied by any change in mRNA levels. In addition, the degradation rate of β-subunits is not altered by β1flag overexpression. Cells stably expressing β1myc, when induced to express β1flag-subunits show reduced β1myc- and β1E-subunit abundance, indicating that these effects occur via the coding sequences of the down-regulated polypeptides. Similarly, MDCK cells over-expressing α1flag-subunits exhibit a reduction of endogenous α1 (α1E) protein with no change in α mRNA levels or β-subunits. The reduction in α1E compensates for α1flag-subunit expression, resulting in unchanged total α-subunit abundance. Thus, regulation of α-subunit expression maintains its native level whereas β-subunit is not as tightly regulated and its abundance can increase substantially over native levels. These effects are also seen in HEK cells. This is the first indication that cellular Na pump subunit abundance is modulated by translational repression. We will discuss the mechanism of this novel, potentially important mode of Na pump regulation.