Asymmetric incorporation of Na+, K+-ATPase into phospholipid vesicles

Purified lamb kidney Na⁺, K⁺-ATPase, consisting solely of the Mτ = 95,000 catalytic subunit and the M_τ～- 44,000 glycoprotein, was solubilized with Triton X-100 and incorporated into unilamellar phospholipid vesicles. Freeze-fracture electron microscopy of the vesicles showed intramembranous particles of approximately 90–100 Å in diameter, which are similar to those seen in the native Na⁺,K⁺-ATPase fraction. Digestion of the reconstituted proteins with neuraminidase indicated that the glycoprotein moiety of the Na⁺,K⁺-ATPase was asymmetrically oriented in the reconstituted vesicles, with greater than 85% of the total sialic acid directed toward the outside of the vesicles. In contrast, in the native Na⁺,K⁺-ATPase fraction, the glycoprotein was symmetrically distributed. Purified glycoprotein was also asymmetrically incorporated into phospholipid vesicles using Triton X-100 and without detergents as described by R. I. MacDonald and R. L. MacDonald (1975, J. Biol. Chem.250, 9206–9214). The glycoprotein-containing vesicles were 500–1000 Å in diameter, unilamellar, and, in contrast to the vesicles containing the Na⁺,K⁺-ATPase, did not contain the 90- to 100-Å intramembranous particles. These results indicate that the intramembranous particles observed in the native Na⁺,K⁺-ATPase and in the reconstituted Na⁺,K⁺-ATPase are not due to the glycoprotein alone, but represent either the catalytic subunit, or the catalytic plus the glycoprotein subunit.     

Electron microscopy of phospholipid vesicles reconstituted with purified renal Na,K-ATPase

Purified Na,K-ATPase after reconstitution into phospholipid vesicles catalyzed an active coupled transport with a ratio close to 3Na/2K. A uniform population of closed vesicles with average diameters close to 900 A are observed after freeze-fracture and thin sectioning. After freeze-fracture intramembranous particles with diameters of 80-100 A are observed. The data suggest that these particles correspond to Na,K-ATPase molecules.     

Influence of voltage and ATP on ion-channel of (Na,K)ATPase incorporated into solvent-free phospholipid planar bilayers

Purified (Na,K)ATPase was incorporated into solvent free phospholipid bilayers made on patch-clamp pipettes. In the absence of ATP, the incorporated enzyme acted as an ion-channel which underwent opening and closing (switching) upon application of transmembrane potential gradient of more than 40 mV. The minimum conductance was about 40 pS. It was inhibited by ouabain from one side. ATP added to the opposite side shifted the threshold potential for switching of the channel to 80 mV. Furthermore the magnitude of minimum conductance decreased to 6-10 pS in the presence of ATP.     

Electrogenic and electroneutral transport modes of renal Na/K ATPase reconstituted into proteoliposomes

Summary This paper describes measurements of electrical potentials generated by renal Na/K-ATPase reconstituted into proteoliposomes, utilizing the anionic dye, oxonol VI. Calibration of absorption changes with imposed diffusion potentials allows estimation of absolute values of electrogenic potentials.ATP-dependent Na_cyt/K_exc exchange in K-loaded vesicles generates large potentials, up to 250 mV. By comparing initial rates or steady-state potentials with ATP-dependent²²Na fluxes in different conditions, it is possible to infer whether coupling ratios are constant or variable. For concentrations of Na_cyt (2–50mm) and ATP (1–1000 m) and pH's (6.5–8.5), the classical 3Na_cyt/2K_exc coupling ratio is maintained. However, at low Na_cyt concentrations (<0.8mm), the coupling ratio is apparently less than 3Na_cyt/2K_exc.ATP-dependent Na_cyt/congener_exc exchange in vesicles loaded with Rb, Cs, Li and Na is electrogenic. In this mode congeners, including Na_exc, act as K_exc surrogates in an electrogenic 3Na_cyt/2congener_exc exchange. (ATP+Pi)-dependent K_cyt/K_exc exchange in K-loaded vesicles is electroneutral.ATP-dependent uncoupled Na flux into Na- and K-free vesicles is electroneutral at pH 6.5–7.0 but becomes progressively electrogenic as the pH is raised to 8.5. The²²Na flux shows no anion specificity. We propose that uncoupled Na flux is an electroneutral 3Na_cyt/3H_exc exchange at pH 6.5–7.0 but at higher pH's the coupling ratio changes progressively, reaching 3Na/no ions at pH 8.5. Slow passive pump-mediated net K uptake into Na- and K-free vesicles is electroneutral, and may also involve K_cyt/H_exc exchange.We propose the general hypothesis that coupling ratios are fixed when cation transport sites are saturated, but at low concentrations of transported cations, e.g., Na_cyt in Na/K exchange and H_exc in uncoupled Na flux, coupling ratios may change.     

ATP synthesis catalyzed by the ATPase complex from Rhodospirillum rubrum reconstituted into phospholipid vesicles together with bacteriorhodopsin

The coupling factor ATPase complex extracted by Triton X-100 from the photosynthetic bacterium Rhodospirillum rubrum could be incorporated into phospholipid vesicles after removal of the Triton. Vesicles reconstituted with this F₀ · F₁-type ATPase together with bacteriorhodopsin were found to catalyze, in the light, net ATP synthesis which was inhibited by the energy transfer inhibitors oligomycin and N,N-dicyclohexylcarbodiimide as well as by uncouplers. In vesicles reconstituted with the crude ATPase up to 50% of the observed rate of phosphorylation was independent on light and bacteriorhodopsin and insensitive to the above-listed inhibitors. This dark activity was, however, completely blocked by the adenylate kinase inhibitor, p¹,p⁵-di(adenosine-5′)pentaphosphate, which did not affect at all the net light-dependent phosphorylation nor the ATP-³²P_i exchange reaction. Vesicles reconstituted with the purified ATPase catalyzed only the light- and bacteriorhodopsin-dependent diadenosine pentaphosphate-insensitive phosphorylation. The rate of this photophosphorylation was found to be proportional to the amount of ATPase and bacteriorhodopsin, and linear for at least 20 min of illumination. These results indicate that the purified ATPase contains the complete assembly of subunits required to transduce electrochemical gradient energy into chemical energy.     

Ratio of Na:K transport in reconstituted sodium pump vesicles

The effect of ATP on the kinetics of Na and K fluxes across the membranes of reconstituted sodium pump vesicles was examined. In the absence of ATP, the active vesicles equilibrated with ⁴²K or ⁸⁶Rb within 6 hours. In contrast, the equilibration of intravesicular Na with external ²²Na was about 4 times slower. In the presence of ATP, the intravesicular K was replaced within 3 min by Na $\text{via}$ a Na:K exchange process. The total intravesicular Na pool was then labeled to the same specific radioactivity as the Na of the medium $\text{via}$ a Na:Na exchange process. The Na:K transport ratio varied with the intravesicular concentrations of Na and K.     

Phosphorylation of purified rat brain Na+ channel reconstituted into phospholipid vesicles by protein kinase C.

Phosphorylation of voltage-sensitive Na+ channels in neurons by protein kinase C slows Na+ channel inactivation and reduces peak Na+ currents. Na+ channels purified from rat brain and reconstituted into phospholipid vesicles under conditions that restore Na+ channel function were rapidly phosphorylated by protein kinase C on their 260-kDa alpha subunit. The phosphorylation reaction required Ca2+, diolein, and phosphatidylserine for activation of protein kinase C, and the rate of phosphorylation of reconstituted Na+ channels was 3- to 4-fold faster than for Na+ channels in detergent solution. Phosphorylation was on serine residues in three distinct tryptic phosphopeptides designated A, B, and C. Up to 2.5 mol of phosphate were incorporated per mol of Na+ channel. Following maximum phosphorylation by protein kinase C, cAMP-dependent protein kinase was able to incorporate more than 2.25 mol of phosphate per mol of Na+ channel indicating that these two kinases phosphorylate distinct sites. However, prior phosphorylation by cAMP-dependent protein kinase prevented phosphorylation of phosphopeptide B indicating that both kinases phosphorylate the site in this peptide. Phosphopeptide B shown here to be phosphorylated by protein kinase C and phosphopeptide 7 previously shown to be phosphorylated by cAMP-dependent protein kinase co-migrate on two-dimensional phosphopeptide maps and evidently are identical. The reduction in peak Na+ currents caused by both protein kinase C and cAMP-dependent protein kinase may result from phosphorylation of this single common site.     

Electrical potential accelerates the E1P(Na)----E2P conformational transition of (Na,K)-ATPase in reconstituted vesicles

We have used renal (Na,K)-ATPase, covalently labeled with fluorescein, and phospholipid vesicles reconstituted with labeled enzyme, to detect conformational transitions induced by acetyl phosphate in the presence of Mg2+ and Na+ ions. Equilibrium fluorescence measurements show quenching of the fluorescein fluorescence, which is thought to reflect conversion of the initial E1 form to the phosphorylated E2P form. These fluorescence changes occur on inside-out-oriented pumps. The rates of acetyl phosphate-induced fluorescence changes have been measured using a stopped-flow fluorimeter. The rate of fluorescence quenching (1.5-3 s-1) is a measure of the rate of the E1P(Na)----E2P transition. The quenching is preceded by a fast fluorescence increase (12.3 +/- 4 s-1) associated with phosphorylation of E1 to E1P(Na), shown clearly in experiments with enzyme treated with oligomycin. Oligomycin greatly reduces the rate of the fluorescence quenching (0.044 +/- 0.01 s-1). Using potassium-loaded vesicles treated with valinomycin or lithium-loaded vesicles treated with Li+ ionophore N,N'-diheptyl-N,N'-didiethyl ether, 5,5-dimethyl-3,7-dioxanonanediamide in order to induce electrical diffusion potentials, negative inside, the rates of the fluorescence quenching are accelerated by up to 4-fold. The experiments demonstrate that the conformational transition E1P(Na)----E2P, associated with transport of 3 Na+ ions, is a voltage-sensitive reaction, carrying a net positive charge. This confirms a prediction based on transport experiments. In experiments with fluorescein-labeled (Na,K)-ATPase, the use of acetyl phosphate rather than ATP, which does not bind, provides a valuable tool to detect fluorescence signals accompanying steps in the turnover cycle.     

A new method for making phospholipid vesicles and the partial reconstitution of the (Na+, K+-activated ATPase

It has been found that vesicles of phospholipid (96% (w/w) phosphatidylcholine; 4% (w/W) phosphatidylserine) can be formed by dialysis of a solution of the phospholipid in the detergent, sodium deoxycholate. Depending upon the composition of the dialysis medium, small closed vesicles apparently bounded by one or two membranes or large multi-walled structures are produced. The former are predomiant if only univalent ions are present in the dialysis buffer. As the Mg²⁺ concentration is raised above about 0.1 mM multiwalled structures are found.The (Na⁺,K⁺)-ATPase (EC 3.6.1.3) from cattle brain microsomes has been solubilized with deoxycholate. Dialysis of this material after the addition of the above phospholipid mixture in detergent also produces membrane-bound vesicles. Sucrose density gradient centrifugation has been used to demonstrate that the phospholipid, (Na⁺,K⁺)-ATPase and protein reaggregate together only if the phospholipid and solubilized protein are mixed before dialysis. This method of forming artificial membranes may be a useful way of studying transport proteins in isolation as the vesicles appear to be small and closed.     

pH-dependent fusion induced by vesicular stomatitis virus glycoprotein reconstituted into phospholipid vesicles

Purified G-protein from vesicular stomatitis virus was reconstituted into egg phosphatidylcholine vesicles by detergent dialysis of octyl glucoside. A homogeneous population of reconstituted vesicles could be obtained, provided the protein to lipid ratio was high (about 0.3 mol % protein) and the detergent removal was slow. The reconstituted vesicles were assayed for fusion activity using electron microscopy and fluorescence energy transfer. The fusion activity mediated by the viral envelope protein was dependent upon pH, temperature, and target membrane lipid composition. Incubation of reconstituted vesicles at low pH with small unilamellar vesicles containing negatively charged lipids resulted in the appearance of large cochleate structures, as shown by electron microscopy using negative stain. This process did not cause leakage of a vesicle-encapsulated aqueous marker. The rate of fusion was pH-dependent with a pK of about 4 and the apparent energy of activation for the fusion was 16 +/- 1 kcal/mol. G-protein-mediated fusion showed a large preference for target membranes which contain phosphatidylserine or phosphatidic acid. Inclusion of 36% cholesterol in any of the lipid compositions had no effect on the rate of fusion. These reconstituted vesicles provide a system to study the mechanism of pH-dependent fusion induced by a viral spike protein.     

Magnetic resonance study of 13C-enriched glycophorin A reconstituted into phospholipid vesicles

$\text{[S-[}{}^{13}\text{C}\text{]}\text{methylmethionine-8 and -81]glycophorin}$ A was reconstituted into l-α-phosphatidyl choline vesicles. Results indicate that the $\text{S-[}{}^{13}\text{C}\text{]}\text{methylmethyionine}\text{-81}$ residue in the phospholipid bilayer has limited mobility and is not susceptible to dealkylation, whereas the opposite effects are indicated for the $\text{S-[}{}^{13}\text{C}\text{]}\text{methylmethionine}\text{-8}$ residue.     

Conformational study of poly(L-lysine) interacting with acidic phospholipid vesicles

Circular dichroism measurements were carried out on poly(L-lysine) in the presence of vesicles of the negatively charged phospholipids, phosphatidylserine (PS; from bovine brain), phosphatidic acid (PA; prepared from egg yolk lecithin) and dimyristoylphosphatidylglycerol (DMPG). PS vesicles induced a conformational change in poly(L-lysine) from random coil to alpha-helix structure in 5 mM Tes (pH 7.0), whereas PA vesicles gave rise to beta-structure in the same buffer. The fraction of alpha-helix, F alpha (or beta-structure, F beta), increased with increasing PS (or PA) concentration, reaching a saturation value of about 0.7 (or about 1). Mixed vesicles comprising PS and dilauroylphosphatidylcholine (DLPC) also induced alpha-helix conformation, however, the saturation value of F alpha diminished with decreasing PS content in mixed vesicles. On the other hand, the spectral patterns for poly(L-lysine) in DMPG vesicle suspensions exhibited the coexistence of alpha-helix and beta-structure. Both F alpha and F beta increased with DMPG concentration and reached saturation values of about 0.5. Mixed vesicles composed of DMPG and dimyristoylphosphatidylcholine (DMPC) led to a reduction in F beta, while F alpha remained almost constant. The diversity in ordered structure induced by different phospholipid vesicles suggests the participation of lipid head groups in determining the secondary structure of poly(L-lysine) adsorbed on the vesicular surface.     

Pump current and Na+/K+ coupling ratio of Na+/K+-ATPase in reconstituted lipid vesicles

A method is described for studying the coupling ratio of the Na+/K+ pump, i.e., the ratio of pump-mediated fluxes of Na+ and K+, in a reconstituted system. The method is based on the comparison of the pump-generated current with the rate of K+ transport. Na+/K+-ATPase from kidney is incorporated into the membrane of artificial lipid vesicles; ATPase molecules with outward-oriented ATP-binding site are activated by addition of ATP to the medium. Using oxonol VI as a potential-sensitive dye for measuring transmembrane voltage, the pump current is determined from the change of voltage with time t. In a second set of experiments, the membrane is made selectively K+-permeable by addition of valinomycin, so that the membrane voltage U is equal to the Nernst potential of K+. Under this condition, dU/dt reflects the change of intravesicular K+ concentration and thus the flux of K+. Values of the Na+/K+ coupling ratio determined in this way are close to 1.5 in the experimental range (10-75 mM) of extravesicular (cytoplasmic) Na+ concentrations.     

Reconstitution of (Na+ + K+)-ATPase into phospholipid vesicles with full recovery of its specific activity

(Na+ + K+)-ATPase from rectal glands of the spiny dogfish has been reconstituted into phospholipid vesicles. The nonionic detergent octaethyleneglycoldodecyl monoether ( C12E8 ) is used to dissolve both the enzyme and the lipids and reconstitution is accomplished by subsequent removal of the detergent by adsorption to polystyrene beads. About 60% of the enzyme incorporates in the right-side-out orientation (r/o). The fraction of molecules in the inside-out orientation (i/o) increases from about 10% to about 30% with a parallel decrease in the fraction of 'non-oriented' (n-o) molecules (both sides exposed) when the protein/lipid ratio decreases from 1:10 to 1:75. The orientation of enzyme molecules detected from vanadate binding is the same as measured from activity, i.e., the turnover of the enzyme molecule in the different orientations is the same. The recovery of the specific activity of the incorporated enzyme increases with an increase in the protein/lipid ratio and is 100% with a protein/lipid ratio of about 1:20 or higher. Full recovery is only obtained provided a proper lipid composition is chosen which includes both negatively charged phospholipids, preferably phosphatidylinositol, and cholesterol. The ATP-dependent, K+-stimulated Na+-influx is found to be about 35 mumol Na+ per mg (i/o)-protein per min at 22 degrees C in 1:10 protein/lipid liposomes. The specific activity corresponds to 3 Na+ transported per ATP molecule hydrolyzed.     

ATP synthesis catalyzed by purified DCCD-sensitive ATPase incorporated into reconstituted purple membrane vesicles

Purified dicyclohexylcarbodiimide-sensitive ATPase from a thermophilic bacterium (TF₀·F₁) and purple membranes from Halobacterium halobium were incorporated into P-lipid vesicles. The reconstituted vesicles took up protons dependent on either illumination or addition of ATP. Net formation of ATP was observed when the vesicles were illuminated in the presence of ADP and Pi and this was completely abolished by addition of an uncoupler or energy transfer inhibitor. These results indicate that purified DCCD-sensitive ATPase, consisting of 8 kinds of polypeptides, was capable of ATP synthesis coupled with proton translocation.     

Incorporation of bovine enterokinase in reconstituted soybean phospholipid vesicles

Bovine enterokinase was incorporated into vesicles reconstituted from a soybean phospholipid mixture. A thin film hydration procedure (MacDonald, R. I., and MacDonald, R. C. (1975) J. Biol. Chem. 250, 9206-9214) produced vesicles with 40% of the enterokinase activity bound in the membrane. The highest incorporation was observed when cholesterol or dimyristoylphosphatidylethanolamine was added to the soybean phospholipids. Crude and highly purified enterokinase preparations were incorporated to the same extent suggesting that other membrane components were not required for a successful reconstitution. The properties of enterokinase in phospholipid vesicles were compared with those of alkaline phosphatase, which was also added to the reconstitution system, and with the enzyme activities present in vesicles prepared from brush-border membranes. The enzyme activities were not released by solutions of high ionic strength and remained associated with the phospholipid vesicles on gel filtration, ultracentrifugation, and sucrose density centrifugation. Enterokinase and alkaline phosphatase had their active sites exposed to substrate in the brush-border membrane vesicles. In soybean phospholipid vesicles half of the active sites of both enzymes were on the outside, since release of the enzyme with Triton X-100 almost doubled the units of enzyme present. Incubation of the soybean phospholipid and brush-border membrane vesicles with papain released the exposed molecules of enterokinase. The released enzyme molecules were fully active but could not be reincorporated into phospholipid vesicles. This suggests that the structure imbedded in the lipid bilayer was essential for a successful reconstitution. We conclude that the reconstituted soybean phospholipid vesicles are a suitable membrane system for the further study of membrane-bound enterokinase.     

ADP binding to (Na + K)-activated ATPase

[¹⁴C]ADP binding to EDTA-washed ox brain cell membranes was increased by Na⁺, but decreased by K⁺, Mg²⁺ and Ca²⁺. Na⁺ abolished the effect of K⁺ on ADP binding by a competitive mechanism, but could not reverse the inhibitory action of Mg²⁺ and Ca²⁺. It is concluded that the cation-induced changes in ADP binding reflect properties of (Na⁺ + K⁺)-activated ATPase.     

Functional activity of oligosaccharide-deficient (Na,K)ATPase expressed in Xenopus oocytes

(Na,K)ATPase from Torpedo californica was expressed in Xenopus laevis oocytes in the presence of tunicamycin by injecting mRNAs for the alpha- and beta-subunits derived from the cloned cDNAs into the oocytes. The oligosaccharide-deficient ATPase thus synthesized was transported to the oocyte plasma membrane, where it exhibited virtually the same ATPase activity, ouabain-binding capacity and 86Rb+ transport activity as the fully glycosylated enzyme. We conclude that the oligosaccharide chains on the beta-subunit has no effect on the catalytic activities of (Na,K)ATPase.     

Conformational transitions between Na+-bound and K+-bound forms of (Na+ + K+)-ATPase, studied with formycin nucleotides.

1. Fluorescence measurements have shown that formycin triphosphate (FTP) or formycin diphosphate (FDP) bound to (Na+ + K+)-ATPase (ATP phosphohydrolase, EC 3.6.1.3) in Na+-containing media can be displaced by the following ions (listed in order of effectiveness): Tl+, K+, Rb+, NH4+, Cs+. 2. The differences between the nucleotide affinities displayed by the enzyme in predominantly Na+ and predominantly K+ media in the absence of phosphorylation, are thought to reflect changes in enzyme conformation. These changes can therefore be monitored by observing the changes in fluorescence that accompany net binding or net release of formycin nucleotides. 3. The transition from a K+-bound form (E2-(K)) to an Na+-bound form (E1-Na) is remarkably slow at low nucleotide concentrations, but is accelerated if the nucleotide concentration is increased. This suggests that the binding of nucleotide to a low-affinity site on E2-(K) accelerates its conversion to E1-Na; it supports the hypothesis that during the normal working of the pump, ATP, acting at a low affinity site, accelerates the conversion of dephosphoenzyme, newly formed by K+-catalysed hydrolysis of E2P, to a form in which it can be phosphorylated in the presence of Na+. 4. The rate of the reverse transformation, E1-Na to E2-(K), varies roughly linearly with the K+ concentration up to the highest concentration at which the rate can be measured (15 mM). Since much lower concentrations of K+ are sufficient to displace the equilibrium to the K-form, we suggest that the sequence of events is: (i) combination of K+ with low affinity (probably internal) binding sites, followed by (ii) spontaneous conversion of the enzyme to a form, E2-(K), containing occluded K+. 5. Mg2+ or oligomycin slows the rate of conversion of E1-Na to E2-(K) but does not significantly affect the rate of conversion of E2-(K) to E1-Na. 6. In the light of these and previous findings, we propose a model for the sodium pump in which conformational changes alternate with trans-phosphorylations, and the inward and outward fluxes of both Na+ and K+ each involve the transfer of a phosphoryl group as well as a change in conformation between E1 and E2 forms of the enzyme or phosphoenzyme.