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.     

The role of potassium and chloride ions on the Na+/acidic amino acid cotransport system in rat intestinal brush-border membrane vesicles

The Na+/L-glutamate (L-aspartate) cotransport system present at the level of rat intestinal brush-border membrane vesicles is specifically activated by the ions K+ and Cl-. The presence of 100 mM K+ inside the vesicles drastically enhances the uptake rate and the transient intravesicular accumulation (overshoot) of the two acidic amino acids. It has been demonstrated that the activation of the transport system depended only in the intravesicular K+ concentration and that in the absence of any sodium gradient, an outward K+ gradient was unable to influence the Na+/acidic amino acid transport system. It was also found that Cl- could specifically activate the Na+-dependent L-glutamate (L-aspartate) uptake either in the presence or in the absence of K+. Also the effect of Cl- was observed only in the presence of an inward Na+ gradient and it was noted to be higher when chloride ion was present on both sides of the membrane vesicles. No influence (activation or accumulation) was observed in the absence of the Na+ gradient and in the presence of chloride gradient. L-Glutamate uptake measured in the presence of an imposed diffusion potential and in the presence of K+ or Cl- did not show any translocation of net charge.     

Sidedness of (sodium, potassium)-adenosine triphosphate of inside-out red cell membrane vesicles. Interactions with potassium.

Inside-out membrane vesicles of human red cells, prepared according to the method of Steck et al. (1970) Science 168, 255-257) have sufficiently low cation permeability to allow the examination of the side-specific interactions of ligands with the asymmetric sodium pump complex. In accordance with the known properties of the pump in intact cells the following results were observed: (a) ATP-dependent sodium influx and (b) maximal (sodium, potassium)-ATPase with K+ present inside the vesicles with larger than or equal to 20 micronM ATP. With much lower [ATP], K+ inhibited sodium-activated ATPase. K+ was inhibitory at either surface. Inhibition was different on the two sides since cytoplasmic (extravesicular) Na+ counteracted inhibition by cytoplasmic (extravesicular) K+ but not inhibition by K+ at the plasma or external membrane surface, i.e. intravesicular K+. A decrease in the steady state level of the phosphenzyme intermediate of sodium-activated ATPase was caused also by K+ at either surface. The effect of cytoplasmic K+ is compatible with its competitive inhibition of activation of phosphorylation of the enzyme by cytoplasmic Na+. At 37 degrees, the inhibitory effect of external K+ is due to interaction with the phosphoenzyme to form a stable complex of K+ with the dephosphenzyme resulting in a decreased overall reaction rate but increased turnover of the phosphoenzyme (E-P + K leads to EK + Pi). At 0 degree, external K+ inhibits by interacting with the unphosphorylated enzyme to form an occluded enzyme-K complex. This results in a decreased overall rate but relatively small change in apparent turnover of the phosphoenzyme. At 0 degree, but not at 37 degrees, external Na+ counteracted the inhibitory effects of external K+.     

Protons as substitutes for sodium and potassium in the sodium pump reaction

The role of protons as substitutes for Na+ and/or K+ in the sodium pump reaction was examined using inside-out membrane vesicles derived from human red cells. Na+-like effects of protons suggested previously (Blostein, R. (1985) J. Biol. Chem. 260, 829-833) were substantiated by the following observations: (i) in the absence of extravesicular (cytoplasmic) Na+, an increase in cytoplasmic [H+] increased both strophanthidin-sensitive ATP hydrolysis (nu) and the steady-state level of phosphoenzyme, EP, and (ii) as [H+] is increased, the Na+/ATP coupling ratio is decreased. K+-like effects of protons were evidenced in the following results: (i) an increase in nu, decrease in EP, and hence increase in EP turnover (nu/EP) occur when intravesicular (extracellular) [H+] is increased; (ii) an increase in the rate of Na+ influx into K+(Rb+)-free inside-out vesicles and (iii) a decrease in Rb+/ATP coupling occur when [H+] is increased. Direct evidence for H+ being translocated in place of cytoplasmic Na+ and extracellular K+ was obtained by monitoring pH changes using fluorescein isothiocyanate-dextran-filled vesicles derived from 4',4-diisothiocyano-2',2-stilbene disulfonate-treated cells. With the initial pHi = pHo = pH 6.2, a strophanthidin-sensitive decrease in pHi was observed following addition of ATP provided the vesicles contained K+. This pH gradient was abolished following addition of Na+. With alkali cation-free inside-out vesicles, a strophanthidin-sensitive increase in pH was observed upon addition of both ATP and Na+. The foregoing changes in pHi were not affected by the addition of tetrabutylammonium to dissipate any membrane potential and were not observed at pH 6.8. These ATP-dependent cardiac glycoside-sensitive proton movements indicate Na,K-ATPase mediated Na+/H+ exchange in the absence of extracellular K+ as well as H+/K+ exchange in the absence of cytoplasmic Na+.     

Interactions of K+ with (Na,K)-ATPase orientation of K+-phosphatase sites studied with inside-out red cell membrane vesicles

Inside-out membrane vesicles from human red cells were used to investigate the side specificity of K+ interactions with the K+-activated phosphatase, a partial reaction of the (Na, K)-ATPase. In the absence of Na+ and ATP, K+ at moderate affinity sites at the extravesicular surface (cytoplasmic K+) stimulates activity, whereas intravesicular K+ (K+ normally at the extracellular surface) is without effect. In contrast, under conditions of phosphorylation of (Na, K)-ATPase (Na+ and ATP present), K+ ions acting at high affinity sites at both surfaces are required. It is concluded that an enzyme x K complex is involved in K+-activated phosphatase activity and that it is formed either by interaction of cytoplasmic K+ with the dephosphoenzyme, or as a consequence of extracellular K+ binding and dephosphorylation of the phosphoenzyme formed in the presence of Na+ plus ATP.     

The influence of cytoplasmic sodium concentration on the stoichiometry of the sodium pump

Using inside-out vesicles of human red cell membranes, the effects of cytoplasmic Na+ in the range 0-5 mM on ATP-dependent 22Na+ influx (normal efflux) and 86Rb+ efflux (normal influx) were tested. The sodium pump stoichiometry, i.e. the ratio of net 22Na+ influx:86Rb+ efflux was reduced markedly when the cytoplasmic Na+ was reduced to less than 1 mM. Reduction in cytoplasmic Na+ concentration was associated also with a decreased sensitivity of the pump to effects of extracellular Rb+. Thus, extracellular (intravesicular) Rb+ stimulation observed at high ATP concentration and inhibition observed at low ATP concentration were not observed when the cytoplasmic (extravesicular) Na+ concentration was reduced to less than or equal to 0.2 mM. It is suggested that at low cytoplasmic Na+, the pump can operate with less than maximal sites filled with Na+ ions. Under this condition, it is likely that an enzymic step associated with either the ion translocation step or the enzyme's conformational transition becomes rate-limiting.     

Sodium pump-catalyzed sodium-sodium exchange associated with ATP hydrolysis

Inside-out red cell membrane vesicles have been used to study sodium interactions with the ATP-dependent sodium pump at sites accessible to both membrane surfaces. ATP-dependent 22Na+ influx (equivalent to efflux from cells) shows sigmoid dependence on extravesicular Na+ concentration. This is observed both in the absence of intravesicular cations and in the presence of intravesicular K or Rb ions. The kinetic behavior is similar to that observed earlier with intact cells, (Garay, R. P., and Garrahan, P. J. (1973) J. Physiol. (Lond.) 231, 297-325) and is consistent with a ratio of close to three Na ions transported per molecule of ATP hydrolyzed. With vesicles having relatively high intravesicular sodium concentration, (approximately 50 mM NaCl), the sodium pump effects an ATP-dependent sodium efflux coupled to sodium influx and to strophanthidin-sensitive ATP hydrolysis. The influx:efflux stoichiometry is approximately 1:1, and the influx:ATP hydrolysis ratio is close to 3. This ATP-dependent exchange has a higher affinity for vanadate than ATP plus ADP-dependent sodium exchange. It is concluded that this sodium-sodium exchange mode resembles sodium-potassium exchange whereby intravesicular sodium, i.e. sodium at the extracellular surface, at relatively high concentration, behaves like potassium.     

Tightness and orientation of vesicles from guinea-pig kidney estimated from reactions of adenosine triphosphatase dependent on sodium and potassium ions.

In order to study the "sidedness" of the ligands of the Na+, K+-ATPase in the phosphorylation from [32P]ATP, tight vesicles were prepared from guinea pig kidney and partially purified by a two-stage sucrose and Ficoll gradient centrifugation procedure. These vesicles were derived presumably from plasma membrane fragments resealed after the initial disruption of the cells during homogenization. Tightness of the vesicles was estimated according to activation by the nonionic detergent, Triton X-100. Treatment with Triton X-100 increased both the activity of the Na+, K+-ATPase and its Na+-dependent phosphorylation from [32P]ATP at least three-fold. Activation of both functions also appeared when the vesicles were shocked osmotically. These results suggest that the preparation contains a major population of tight normal vesicles (approximately 75%) in which the phosphorylation site faces the intravesicular solution. In the response to ouabain breakdown of the phosphoenzyme was inhibited in vesicles treated with Triton X-100 but not in intact ones as if ouabain could not get to its binding site. Correspondingly in phosphorylation from ATP pretreatment with ouabain in the presence of inorganic phosphate produced less inhibition in intact vesicles than in those disrupted with Triton X-100 beforehand. These data suggest the presence of an everted vesicle fraction in the preparation (approximately 20%). Apparently only a small fraction of the vesicles was leaky. In the everted vesicles the action of K+ on the phosphoenzyme was slow. In order to accelerate the dephosphorylation in intact vesicles as effectively as in disrupted ones, K+ had to be added before the start of phosphorylation. This supports the view that K+ was acting from the side of the membrane opposite to that where the gamma-phosphoryl group was accepted from ATP.     

Transient state in the phosphorylation of sodium- and potassium- transport adenosine triphosphatase by adenosine triphosphate

The rate of phosphorylation of sodium and potassium ion-transport adenosine triphosphatase by 10 microM [gamma-32P]ATP was much slower with Ca2+ than with Mg2+ (0.13-10 mM) in the presence of 16 to 960 mM Na+ at 0 degrees C and pH 7.4. In the presence of a fixed concentration of Mg2+ or Ca2+, the rate became slower with increasing Na+ concentration. When the Na+ concentration was fixed, the rate became slower with decreasing divalent cation concentration. Sodium ions appear to antagonize the divalent cation in the phosphorylation to slow its rate. In the presence of 1 mM Ca2+ and 126 or 270 mM Na+, the rate was slow enough to permit the manual addition of a chasing solution at various times before the phosphorylation reached the steady state. Therefore, we studied the time-dependent change of the sensitivity to ADP or to K+ of the phosphoenzyme by a chase with unlabeled ATP containing ADP or K+ during the time range from the transient to the steady state of the phosphorylation. The ADP sensitivity decreased and the K+ sensitivity increased with the progress of the phosphorylation. With 270 mM Na+, the phosphoenzyme found at 1 s, when its amount was 5.5% of the maximum level, was virtually completely sensitive to ADP. Under these conditions, it was concluded that the form of the phosphoenzyme initially produced from the enzyme.ATP complex has ADP sensitivity and that the phosphoenzyme acquires K+ sensitivity later. The initially produced ADP-sensitive phosphoenzyme partially lost its normal instability and sensitivity upon adding a chelating agent, probably because of dissociation of a divalent cation from the phosphoenzyme.     

Na+-Ca2+ exchange in squid optic nerve membrane vesicles is activated by internal calcium

The role of intracellular Ca2+ as essential activator of the Na+-Ca2+ exchange carrier was explored in membrane vesicles containing 67% right-side-out and 10% inside-out vesicles, isolated from squid optic nerves. Vesicles containing 100 microM free calcium exhibited a 2-fold increase in the initial rate of Na+i-dependent Ca2+ uptake as compared with vesicles where intravesicular calcium was chelated by 2 mM EGTA or 10 mM HEDTA. The activatory effect exerted by intravesicular Ca2+ on the reverse mode of Na+-Ca2+ exchange (i.e. Na+i-Ca2+o exchange) is saturated at about 100 microM Ca2+i and displays an apparent K 1/2 of 12 microM. Intravesicular Ca2+ produced activation of Na+i-Ca2+i exchange activity rather than an increase in Ca2+ uptake due to Ca2+-Ca2+ exchange. The presence of Ca2+i was essential for the Na+i-dependent Na+ influx, a partial reaction of the Na+-Ca2+ exchanger. In fact, the Na+ influx levels in vesicles loaded with 2 mM EGTA were close to those expected from diffusional leak while in vesicles containing Ca2+i an additional Na+-Na+ exchange was measured. The results suggest that in nerve membrane vesicles Ca2+ at the inner aspect of the membrane acts as an activator of the Na+-Ca2+ exchange system.     

Sodium ions as substitutes for protons in the gastric H,K-ATPase

In view of the striking homology among various ion-translocating ATPases including Na,K-ATPase, Ca-ATPase, and H,K-ATPase, and the recent evidence that protons can replace cytoplasmic sodium as well as potassium in the reaction mechanism of the Na,K-ATPase (Polvani, C., and Blostein, R. (1988) J. Biol. Chem. 263, 16757-16763), we studied the role of sodium as a substitute for protons in the H,K-ATPase reaction. Using hog gastric H,K-ATPase-rich inside-out membrane vesicles we observed 22Na+ influx which was stimulated by intravesicular potassium ions (K+i) at pH 8.5 but not at pH 7.1. This sodium influx was observed in medium containing ATP and was inhibited by vanadate and SCH28080, a selective inhibitor of the gastric H,K-ATPase. At least 2-fold accumulation of sodium was observed at pH 8.5. Experiments aimed to determine the sidedness of the alkaline pH requirement for K+i-dependent sodium influx showed that K+i-activated sodium influx depends on pHout and is unaffected by changes in pHin. These results support the conclusion that sodium ions substitute for protons in the H,K-ATPase reaction mechanism and provide evidence for a similarity in ion selectivity and/or binding domains of the Na,K-ATPase and the gastric H,K-ATPase enzymes.     

Isolation of plasma membrane vesicles from rabbit skeletal muscle and their use in ion transport studies

A method has been developed for the isolation of sealed plasma membrane vesicles from rabbit white skeletal muscle. The final preparation was highly purified as indicated by enrichment of plasma membrane marker enzymes (i.e. ouabain-sensitive (Na+,K+)-ATPase, adenylate cyclase, and acetylcholinesterase). The absence of sarcoplasmic reticulum and mitochondria as contaminants was indicated by the low specific activity of marker enzymes, i.e. Ca2+-ATPase, succinate-cytochrome c reductase, and monoamine oxidase. Thin section and negative staining electron microscopy confirmed the absence of sarcoplasmic reticulum and mitochondrial contamination. The plasma membrane preparation consisted largely of sealed vesicles as observed by electron microscopy and as also demonstrated by latency of enzymic activities, which were unmasked by preincubation with detergent (sodium dodecyl sulfate). Membrane sidedness was estimated from latency of ouabain-sensitive (Na+,K+)-ATPase activity and acetylcholinesterase activity. The latency studies suggest that most of the vesicles are oriented inside out with respect to the orientation of the sarcolemma membrane in the muscle fiber. The inside-out plasma membrane vesicles actively accumulated sodium ions upon addition of ATP. The sodium ions were concentrated greater than 8-fold inside the vesicles and were released upon addition of the ionophore monensin. The sodium ions were taken up in the presence of K+ or NH4+ but not of choline. Uptake was inhibited by low concentrations of vanadate or digitoxin. The Na+ uptake was concomitant with Rb+ efflux. Therefore, the sodium ion transport and the resulting gradients formed appear to have been generated by the ouabain-sensitive (Na+,K+)-ATPase. Batrachotoxin, which opens Na+ channels in excitable tissues, prevents most of the Na+ uptake, suggesting the presence of toxin-activated Na+ channels in these plasma membrane vesicles.     

Existence of ADP- and KCl-insensitive phosphoenzyme intermediate of Na+,K(+)-ATPase at alkaline Ph

The Na(+)-ATPase activity of Na+,K(+)-ATPase in the absence of K+ was least dependent on the sodium concentration when the pH was 9.5. Around 40% of the phosphoenzyme formed from ATP in the presence of 0.5 mM MgCl2 at alkaline pH was insensitive to both KCl and ADP. High-Na+ chase reversed this insensitivity, i.e., the phosphoenzyme became sensitive to KCl or ADP. On the other hand, phosphorylation at 0.1 mM MgCl2 instead of 0.5 mM showed at least 95% sensitivity to KCl. These observations suggest that ADP- and KCl-insensitive phosphoenzyme was formed when excess Mg++ was present during phosphorylation at alkaline pH. This phosphoenzyme might be an intermediate in the process of ATP hydrolysis.     

Calcium and magnesium regulation of phosphorylation by ATP and ITP in sarcoplasmic reticulum vesicles.

Membrane phosphorylation and nucleoside triphosphatase activity of sarcoplasmic reticulum vesicles isolated from rabbit skeletal muscle were studied using ATP and ITP as substrates. The Ca2+ concentration was varied over a range large enough to saturate either the high affinity Ca2+-binding site or both high and low affinity binding sites. In intact vesicles, which are able to accumulate Ca2+, the steady state level of enzyme phosphorylated by either ATP or ITP is already high in 0.02 mM Ca2+ and does not vary as the Ca2+ concentration is increased to 10 mM. Essentially the same pattern of membrane phosphorylation by ATP is observed when leaky vesicles, which are unable to accumulate Ca2+, are used. However, for leaky vesicles, when ITP is used as substrate, the phosphoenzyme level increases 3- to 4-fold when the Ca2+ concentration is raised from 0.02 to 20 mM. When Mg2+ is omitted from the assay medum, the degree of membrane phosphorylation by ATP varies with Ca2+ in the same way as when ITP is used in the presence of Mg2+. Membrane phosphorylation of leaky vesicles by either ATP or ITP is observed in the absence of added Mg2+. When these vesicles are incubated in media containing ITP and 0.1 mM Ca2+, addition of Mg2+ up to 10 mM simultaneously decreases the steady state level of phosphoenzyme and increases the rate of ITP hydrolysis. When ATP is used, the addition of 10 mM Mg2+ increases both the steady state level of phosphoenzyme and the rate of ATP hydrolysis. When the Ca2+ concentration is raised to 10 or 20 mM, the degree of membrane phosphorylation by either ATP or ITP is maximal even in the absence of added Mg2+ and does not vary with the addition of 10 mM Mg2+. In these conditions the ATPase and ITPase activities are activated by Mg2+, although not to the level observed in 0.1 mM Ca2+. An excess of Mg2+ inhibits both the rate of hydrolysis and membrane phosphorylation by either ATP or ITP.     

Proton-activated rubidium transport catalyzed by the sodium pump

Although the sodium pump normally exchanges three sodium for two potassium ions, experiments with inside-out red cell membrane vesicles show that the stoichiometry is reduced when the cytoplasmic sodium concentration is decreased to less than 1 mM. The present study was designed to gain insight into the question whether other monovalent cations, particularly protons, can act as sodium congeners in effecting pump-mediated potassium transport (ATP-dependent rubidium efflux from inside-out vesicles). The results show that at low cytoplasmic sodium concentration, an increase in proton concentration effects a further reduction in sodium:rubidium stoichiometry, to a value less than the minimal expected (1Na+:3Rb+). Furthermore, when vesicles containing 86RbCl are incubated in nominally sodium-free medium. ATP-dependent net rubidium efflux (normal influx) occurs when the pH is reduced from approximately 7.0 to 6.2 or less. This efflux is inhibited by strophanthidin and vanadate. These experiments support the notion that the sodium pump can operate as an ATP-dependent proton-activated rubidium (potassium) pump without obligatory countertransport of sodium ions.     

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

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

Inhibitory effects of cations on the gastric H+, K+ -ATPase. A potential-sensitive step in the K+ limb of the pump cycle

The presence of a cation inhibitory site on the dephosphoform of the H+, K+ -ATPase was confirmed by comparing the effects of K+ and NH4+ on overall activity and on phosphorylation and dephosphorylation. Inhibition of ATPase activity was pronounced at high cation/ATP ratios, but NH4+ was much less effective. At 60 mM cation, although the ATPase activity was greater in the presence of NH4+ (17.1 mumol/mg.h) as compared to K+ (5.1 mumol/mg.h), dephosphorylation of preformed phosphoenzyme was faster with K+ (2101 min-1) than with NH4+ (1401 min-1). Increasing K+ concentrations at the cytosolic face of the enzyme, at constant ATP, decreased the rate of phosphorylation from 1343 to 360 min-1 at 25 mM K+. Increasing ATP concentrations in the presence of constant K+ concentrations accelerated ATPase activity and increased the steady-state phosphoenzyme level. Therefore, inhibition by cations was due to cation stabilization of a dephospho form of the enzyme at a cytosolically accessible cation-binding site. ATP promoted cation dissociation from this site. In ion-permeable vesicles, increasing K+ concentrations, at constant ATP, activated and then inhibited ATPase activity, with a K0.5(I) of 22 mM. In intact, ion-impermeable inside-out vesicles, in the presence of valinomycin, ATPase activity increased up to 175 mM K+. Collapse of this potential by the addition of the electrogenic protonophore 3,3',4', 5-tetrachlorosalicylanilide restored the K+ inhibition of ATPase activity. Thus, the cation inhibition of the ATPase activity appears to be voltage-sensitive; and hence, its connection to the voltage sensitivity of acid secretion demonstrated in intact gastric mucosa is discussed.     

Dinitrophenyl glutathione efflux from human erythrocytes is primary active ATP-dependent transport.

Dinitrophenyl S-glutathione is accumulated by inside-out vesicles made from human erythrocytes in a process totally dependent on ATP and Mg2+. The vesicles were shown to accumulate dinitrophenyl S-glutathione against a concentration gradient. The vesicles were able to concentrate this glutathione derivative even in the absence of membrane potential. This indicated that the ATP-dependent uptake of dinitrophenyl S-glutathione by inside-out vesicles represented an active transport process. Neither extravesicular EGTA nor intravesicular ouabain inhibited the transport process, indicating that neither the Ca2+-ATPase nor the Na+, K+-ATPase were involved. These results indicated that dinitrophenyl S-glutathione uptake by inside-out vesicles probably represented primary active transport. The uptake of dinitrophenyl S-glutathione was a linear function of time (up to 5 h) and vesicle protein. The rate of uptake was optimal between pH 7.0 and 8.0 and at 37 degrees C. The Km values determined for dinitrophenyl S-glutathione and ATP were 0.29 mM and 1 mM, respectively. The transport process was completely inhibited by vanadate and by p-hydroxymercuribenzene sulphonate and inhibited to a lesser extent by N-ethylmaleimide. GTP could efficiently substitute for ATP as an energy source for the transport process, but CTP and UTP were comparatively much less effective.     

The modulation of rat brain Na(+)-Ca2+ exchange by K+

The involvement of potassium ions in the Na(+)-Ca2+ exchange process was studied in rat brain synaptic plasma membrane (SPM) vesicles. Addition of equimolar [K+] to the intravesicular and the extravesicular medium led to a stimulation of the Na+ gradient-dependent Ca2+ influx; this stimulation was noticeable already at 0.5 mM and reached its maximum at 2 mM K+. The magnitude of the K+ stimulation was between 1.3-2.5-fold in different SPM preparations. K+ ions also stimulated the Na(+)-dependent Ca2+ efflux. K+ stimulation of Na(+)-Ca2+ exchange is of considerable specificity, since it is not mimicked by either Li+ or H+. The following lines of evidence suggest that K+ modulation of Na(+)-Ca2+ exchange involves the catalytic moiety of the transporter itself and not an unrelated K+ channel which modulates the membrane potential. 1) K+ stimulation of the transport process was conserved following reconstitution of the transporter into phospholipid-rich liposomes, an experimental condition which presumably separates the native membrane proteins among different vesicular structures. 2) K+ stimulation of Na+ gradient-dependent Ca2+ influx persists also when the build up of negative inside membrane potential is prevented by addition of carbonyl cyanide p-trifluoromethoxy phenylhydrazone which renders the membrane highly permeable to protons both in the native and the reconstituted preparation. 3) K+ stimulation of Na+ gradient-dependent Ca2+ influx is obtained also when tetraethylammonium chloride, 2,3-diaminopyridine and Cs+ are added to the Ca2+ uptake medium. Reconstituted SPM vesicles take up 86Rb+ in response to activation of Na+ gradient-dependent Ca2+ influx. The ratio of Ca2+ taken up by SPM vesicles in a Na+ gradient-dependent manner to the corresponding amounts of Rb+ taken up varies between 8-5 in different SPM preparations. If the stoichiometry of the process is 1 Rb+/1 Ca2+, then Rb+ cotransport is mediated by 10-20% of the transporters present in the preparation.     

Inhibition by vanadate of the reactions catalyzed by the (Na+ + K+)-stimulated ATPase. A transient state kinetic characterization

The interaction of vanadate with the (Na+ + K+)-stimulated ATPase from electric organ was investigated using the acid quench-flow technique. At 21 degrees C, incubation of the enzyme with 1.3 to 1.6 muM vanadate in the presence of 75 mM Na+ and 25 mM K+ strongly inhibits phosphorylation by ATP. Enzyme activity remaining under these conditions shows no change in the apparent rates of phosphorylation or dephosphorylation, although effects were noted which suggest that vanadate increases the reverse rate of dephosphorylation. Ten micromolar vanadate, sufficient to inhibit the (Na+ + K+)-stimulated ATPase by more than 98%, has no effect on phosphorylation in the presence of Na+ alone. Phosphoenzyme formed in the presence of Na+ and K+ consists of rapidly and slowly decaying components which differ in sensitivity to vanadate. Up to 2 muM vanadate suppresses predominantly the rapidly decaying phosphoenzyme, while at higher concentrations vanadate inhibits both the rate and level of formation of the slowly decaying phosphoenzyme. These results indicate that vanadate is a useful reagent for distinguishing between these two phosphorylation reactions.