Reconstitution experiments provide no evidence for a role for the 53-kDa glycoprotein in coupling Ca2+ transport to ATP hydrolysis by the (Ca(2+)-Mg2+)-ATPase in sarcoplasmic reticulum.

The sarcoplasmic reticulum (SR) of skeletal muscle contains a 53 kDa glycoprotein of unknown function, as well as the (Ca(2+)-Mg2+)-ATPase. It has been suggested that the glycoprotein couples the hydrolysis of ATP by the ATPase to the transport of calcium. It has been shown that if SR vesicles are solubilized in cholate in media containing low K+ concentrations followed by reconstitution, then vesicles are formed containing the glycoprotein and with ATP hydrolysis coupled to Ca2+ accumulation, as shown by a large stimulation of ATPase activity by addition of A23187. In contrast, if SR vesicles are solubilized in media containing a high concentration of K+, then the vesicles that are produced following reconstitution lack the glycoprotein and show low stimulation by A23187 (Leonards, K.S. and Kutchai, H. (1985) Biochemistry 24, 4876-4884). We show that the effect of K+ on reconstitution does not follow from any changes in the amount of glycoprotein but rather from an effect of K+ on the detergent properties of cholate. In low K+ media, the cmc of cholate is high, cholate is a relatively poor detergent and incomplete solubilization results in 'reconstitution' of vesicles with the correct orientation of ATPase molecules. In high K+ media, the cmc of cholate is reduced and more complete solubilization of the SR leads to a true reconstitution with the formation of vesicles with a random orientation of ATPase molecules. The experiments provide no evidence for an effect of the glycoprotein on the (Ca(2+)-Mg2+)-ATPase.     

A mitochondrial protein fraction catalyzing transport of the K+ analog T1+.

A protein fraction has been obtained from detergent-solubilized mitochondrial membranes by its affinity for quinine, an inhibitor of K+ transport. A peptide derived from the predominant 53 kDa protein in this fraction is found to be identical in sequence to a portion of aldehyde dehydrogenase. Antigenically unrelated bands at 97, 77, 57, and 31 kDa are also seen on polyacrylamide gels. Observations utilizing a fluorescent probe entrapped in the lumen of membrane vesicles indicate that the reconstituted protein fraction imparts permeability to the K+ analog Tl+. These and other findings suggest that the affinity purified fraction includes a cation transport catalyst.     

Formations of electrochemical proton gradient and adenosine triphosphate in proteoliposomes containing purified adenosine triphosphatase and bacteriorhodopsin.

Proteoliposome vesicles containing both bacteriorhodopsin of Halobacterium halobium and H+-translocating ATPase [EC 3.6,1.3] of a thermophilic bacterium, PS3, (TF0-F1) were reconstituted by either the dialysis method or the sonication method. Generation of the electrochemical proton gradient (deltamuH+) in these vesicles was measured using 9-aminoacridine for estimation of the chemical (deltapH) component and 8-anilinonaphthalene sulfonate for the electrical (deltaphi) component). In illuminated bacteriorhodopsin-vesicles the deltamuH+ reached 180-190 mV when reconstituted by the dialysis method and 210-220 mV when reconstituted by the sonication method. Vesicles reconstituted from both TF0-F1 and bacteriorhodopsin by the dialysis method generated a deltapH+ of about 200 mV on addition of ATP, while vesicles prepared by the sonication method generated very little deltamuH+, if any. These vesicles generated similar deltamuH+ on illumination to that found in bacteriorhodopsin-vesicles. Using vesicles reconstituted from both TF0-F1 and bacteriorhodopsin by the dialysis method, light dependent ATP synthesis was measured in relation to deltamuH+ formation. It was necessary to generate a deltamuH+ of above 170 mV for demonstration of appreciable formation of ATP and the greater the deltamuH+, the faster the rate of ATP synthesis.     

Conformational transitions in fluorescein-labeled (Na,K)ATPase reconstituted into phospholipid vesicles

Fluorescein-labeled (Na,K)ATPase reconstituted into phospholipid vesicles has been used to study conformational transitions. Addition of K+ or Na+ to the vesicle medium induces fluorescence changes characteristic of the E2(K) or E1Na states of fluorescein-labeled (Na,K)ATPase (Karlish, S.J.D. (1980) J. Bioenerg. Biomembr. 12, 111-136). The cation effects are exerted from the cytoplasmic surface of inside-out-oriented pumps. Equilibrium cation titrations and measurements of rates of conformational transitions have led to the following observations. 1) The rate of E2(K)----E1Na or E2(T1)----E1Na is 4-6-fold faster and E1K----E2(K) is about 2-fold slower in vesicles compared to enzyme. In equilibrium titrations the K0.5 for K+ is higher and that for Na+ is lower for vesicles compared to enzyme. The conformational equilibrium E(1)2K----E2(2K) is apparently shifted toward E(1)2K in vesicles compared to enzyme. 2) Diffusion potentials, positive-outside, induced with valinomycin or Li+ ionophore AS701, do not affect the rates of E2(T1)----E1Na or E1K----E2(K), or equilibrium cation titrations. This demonstrates that the conformational transitions E(1)2K----E2(2K) are voltage-insensitive steps, confirming a prediction based on transport experiments. 3) In vesicles containing choline, K+, Na+, or Li+, the rate of E2(T1)----E1Na increases in the order given. Vesicles with reconstituted fluorescein-labeled (Na,K)ATPase provide a convenient system for correlating directly properties of conformational transitions with cation transport.     

Role of Ca2+-activated K+ channel in regulation of NaCl reabsorption in thick ascending limb of Henle's loop

1. Reabsorption of NaCl in the thick ascending limb of Henle's loop involves the integrated function of the Na+,K+,Cl- -cotransport system and a Ca2+-activated K+ channel in the luminal membrane with the Na+,K+-pump and a net Cl- conductance in the basolateral membrane. 2. Assay of K+ channel activity after reconstitution into phospholipid vesicles shows that the K+ channel is stimulated by Ca2+ in physiological concentrations and that its activity is regulated by calmodulin and phosphorylation from cAMP dependent protein kinase. 3. For purification luminal plasma membrane vesicles are isolated and solubilized in CHAPS. K+ channel protein is isolated by affinity chromatography on calmodulin columns. The purified protein has high Ca2+-activated K+ channel activity after reconstitution into vesicles. 4. The purified K+ channel consists of two proteins of 51 and 36 kDa. Phosphorylation from cAMP dependent protein kinase stimulates K+ channel activity and labels the 51 kDa band. The 36 kDa band is rapidly cleaved by trypsin and may be involved in Ca2+ stimulation. 5. Opening of the K+ channel by Ca2+ in physiological concentrations and regulation by calmodulin and phosphorylation by protein kinase may mediate kinetic and hormonal regulation of NaCl transport across the tubule cells in TAL.     

The C-terminally truncated NuoL subunit (ND5 homologue) of the Na+-dependent complex I from Escherichia coli transports Na+

The NADH:quinone oxidoreductase (complex I) from Escherichia coli acts as a primary Na+ pump. Expression of a C-terminally truncated version of the hydrophobic NuoL subunit (ND5 homologue) from E. coli complex I resulted in Na+-dependent growth inhibition of the E. coli host cells. Membrane vesicles containing the truncated NuoL subunit (NuoLN) exhibited 2-4-fold higher Na+ uptake activity than control vesicles without NuoLN. Respiratory proton transport into inverted vesicles containing NuoLN decreased upon addition of Na+, but was not affected by K+, indicating a Na+-dependent increase of proton permeability of membranes in the presence of NuoLN. The His-tagged NuoLN protein was solubilized, enriched by affinity chromatography, and reconstituted into proteoliposomes. Reconstituted His6-NuoLN facilitated the uptake of Na+ into the proteoliposomes along a concentration gradient. This Na+ uptake was prevented by EIPA (5-(N-ethyl-N-isopropyl)-amiloride), which acts as inhibitor against Na+/H+ antiporters.     

Asymmetric reconstitution of the glucose transporter from Ehrlich ascites cell plasma membrane: role of alkali cations

Gel chromatography of solubilized Ehrlich cell plasma membranes and preformed asolectin vesicles coupled to a freeze-thaw cycle results in the reconstitution of 3-O-methyl-D-glucose transport. The transport activity of the liposomes formed is critically dependent on the cation present during reconstitution. Liposomes formed in K+ show high levels of carrier-mediated 3-O-methyl-D-glucose uptake (495 pmol/min/mg protein) while those formed in Na+ do not (33 pmol/min/mg protein). The inactivity in Na+ is not due to a diminished incorporation of glucose transporter nor is it due to carrier molecules reconstituted with a different orientation from those in K+ liposomes. Instead, the low glucose transport level in Na+ liposomes is related to the small size of vesicles formed with Na+. A second freeze-thaw cycle in K+ causes a two- to threefold increase in the available intravesicular volume of Na+ liposomes and results in an eightfold increase in carrier-mediated 3-O-methyl-D-glucose uptake. K+ liposomes, treated in an identical manner, show only a twofold increase in uptake. The glucose transporter was identified as a protein with a molecular mass range of 44.7 to 66.8 kDa, by the D-glucose-inhibitable photoincorporation of [3H]cytochalasin B. The carrier protein is inserted in reconstituted vesicles in a nonrandom manner with at least 80% of the molecules oriented with the cytoplasmic domain accessible to the external medium. In contrast, the neutral Na+-dependent amino acid transport system appears to be randomly reconstituted.     

Sidedness of K+ activation of calcium transport in the reconstituted sarcoplasmic reticulum calcium pump

Sidedness of the effect of K+ on Ca transport by the sarcoplasmic reticulum Ca pump reconstituted into soybean phospholipid vesicles was investigated. The reconstituted vesicles which sustained a high rate of Ca transport even in the absence of Ca-precipitating anions exhibited low passive permeabilities to 42K+, 86Rb+, or 45Ca2+. Evidence was presented that K+ activated the Ca pump on the external surface of the vesicles and that it was not taken up by the vesicles during the pump activity. In the presence of high externally added K+, the reconstituted vesicles preloaded with K+ exhibited a significantly higher Ca transport activity than the vesicles preloaded with Tris+ but not the ones preloaded with Li+. Ca transport by the K+-loaded vesicles was accompanied by a small amount of K+ efflux, which corresponded to about 20% of the amount of Ca+ taken up. Since the intravesicular K+ did not affect the turnover of the ADP-insensitive component (E2P) of the phosphoenzyme intermediate formed during the pump cycle, it was concluded that the intravesicular K+ stimulated the Ca pump activity indirectly by compensating the charge imbalance caused by the electrogenic Ca2+ movement. These results thus indicate that K+ activates the Ca pump only on the cytoplasmic side of the sarcoplasmic reticulum membrane, but it is not obligately transported across the membrane under conditions where K+ fully activates the Ca pump.     

The reconstituted isolated uncoupling protein is a membrane potential driven H+ translocator.

The isolated uncoupling protein (UCP) from brown fat adipose tissue mitochondria has been reconstituted into artificial phospholipid vesicles. Because of the high lability of H+ transport, several new steps have been introduced in the reconstitution; the detergent octyl-POE, the addition of phospholipids to mitochondria prior to solubilization and purification, the vesicle formation by rapid removal of detergent with polystyrene beads and of external salts by a mixed ion exchange. In the K+-loaded proteoliposomes, H+ influx can be induced by a diffusion potential on addition of valinomycin. H+ influx is inhibited to more than 90% by GTP addition, in the assay for UCP activity. By reversing delta psi with external K+, H+ efflux is measured, however, at a four times lower rate. In vesicles loaded with internal GTP, H+ influx is fully inhibited but can be activated by Dowex-OH treatment to an even higher rate than that found in the GTP-free vesicles. Binding studies with GTP show that most of the active UCP are oriented with the binding site outside as in mitochondria, and that in GTP-loaded vesicles GTP is also bound at the outside. The rate of H+ transport is linearly dependent on the membrane potential. Despite the ordered orientation, there is no 'valve' mechanism, since there is H+ efflux with a reversed potential. pH dependency is only small between pH 6.5 and 7.5, indicating that the H+-translocating site differs from the highly pH-dependent nucleotide-binding site. The turnover number of reconstituted UCP is commensurate with mitochondrial function and indicates a carrier instead of a channel-type H+ transport.(ABSTRACT TRUNCATED AT 250 WORDS)     

In vitro protein translocation into inverted membrane vesicles prepared from Vibrio alginolyticus is stimulated by the electrochemical potential of Na+ in the presence of Escherichia coli SecA

A protein translocation system was reconstituted from inverted membrane vesicles prepared from Na+ pump-possessing Vibrio alginolyticus and purified Escherichia coli SecA. The translocation required ATP and was stimulated by the functioning of the Na+ pump, suggesting that the electrochemical potential of Na+, but not that of H+, is important for protein translocation in Vibrio.     

Reconstitution of the mitochondrial non-selective Na+/H+ (K+/H+) antiporter into proteoliposomes

Mitochondria contain two Na+/H+ antiporters, one of which transports K+ as well as Na+. The physiological role of this non-selective Na+/H+ (K+/H+) antiporter is to provide mitochondrial volume homeostasis. The properties of this carrier have been well documented in intact mitochondria, and it has been identified as an 82,000-dalton inner membrane protein. The present studies were designed to solubilize and reconstitute this antiporter in order to permit its isolation and molecular characterization. Proteins from mitoplasts made from rat liver mitochondria were extracted with Triton X-100 in the presence of cardiolipin and reconstituted into phospholipid vesicles. The reconstituted proteoliposomes exhibited electroneutral 86Rb+ transport which was reversibly inhibited by Mg2+ and quinine with K0.5 values of approximately 150 and 300 microM, respectively. Incubation of reconstituted vesicles with dicyclohexylcarbodiimide resulted in irreversible inhibition of 86Rb+ uptake into proteoliposomes. Incubation of vesicles with [14C]dicyclohexylcarbodiimide resulted in labeling of an 82,000-dalton protein. These properties, which are also characteristic of the native Na+/H+ (K+/H+) antiporter, lead us to conclude that this mitochondrial carrier has been reconstituted into proteoliposomes with its known native properties intact.     

Cation selectivity characteristics of the reconstituted voltage-dependent sodium channel purified from rat skeletal muscle sarcolemma

In this report, the alkali metal cation selectivity of the purified, voltage-dependent sodium channel from rat skeletal muscle is described. Isolated sodium channel protein (980-2840 pmol of saxitoxin binding/mg of protein) was reconstituted into egg phosphatidylcholine vesicles, and channels were subsequently activated by either batrachotoxin (5 X 10(-6) M) or veratridine (5 X 10(-4) M). Activation of the reconstituted sodium channel by batrachotoxin permitted rapid specific influx of cations into channel-containing vesicles. Quenched flow kinetic techniques were adapted to allow resolution of the kinetics of cation movement. Uptake rates for 42K+, 86Rb+, and 137Cs+ were measured directly and half-times for equilibration at 18 degrees C were determined to be 350 ms, 2.5 s, and 10 s, respectively, in this vesicle population. 22Na+ equilibration occurred within the mimimum quenching time of the apparatus (90 ms) but an upper limit of 50 ms at 18 degrees C could be assigned to its half-time. Based on this upper estimate for Na+, cation selectivity ratios of the batrachotoxin-activated channel were Na+ (1):K+ (0.14):Rb+ (0.02):Cs+ (0.005). Toxin-stimulated influx could be blocked by saxitoxin with a Ki of approximately 5 X 10(-9) M at 18 degrees C. Rates of cation movement through veratridine-activated channels were much slower, with half-times of 1.0, 1.2, 2.0, and 2.6 min at 36 degrees C for Na+, K+, Rb+, and Cs+, respectively. The temperature dependences of batrachotoxin and veratridine-stimulated cation uptake were markedly different. The activation energies for 86Rb+ and 137Cs+ movement into batrachotoxin-activated vesicles were 7.6 and 6.1 kcal/mol, respectively, while comparable measurements for these two cations in veratridine-activated vesicles yielded activation energies of 31 kcal/mol. Measurements of cation exchange with batrachotoxin-activated channels may reflect characteristics of an open sodium channel while the process of channel opening itself may be rate-limiting when veratridine is used for activation.     

Na+, K+, H+ and Cl- permeability properties of rabbit skeletal muscle sarcolemmal vesicles

The ion permeability properties of rabbit skeletal muscle sarcolemmal vesicles were investigated by means of radioisotope flux, membrane potential, and light-scattering measurements. An enriched sarcolemmal fraction was obtained from the 22-27% region of sucrose gradients after isopycnic centrifugation. The presence of contaminating sarcoplasmic reticulum was assessed with the use of a purified sarcoplasmic reticulum vesicle fraction. 22Na+, 86Rb+, 36Cl-, and [3H]sucrose flux measurements indicated that the sarcolemmal fraction possessed isotope spaces ranging between 1.5 and 4 microliters/mg protein. Membrane potential measurements using the voltage-sensitive fluorescent probe 3,3'-dipentyl-2,2'-oxadicarbocyanine iodide (diO-C5-(3)) indicated that sarcolemmal vesicles were impermeable to H+ and Na+ but that 10-15% of the vesicles were permeable to K+. Light-scattering measurements indicated a small fraction of sarcolemmal vesicles were permeable to both K+ and Cl-. Whether the low permeability of sarcolemmal vesicles to Na+, K+, and Cl- is the result of a low concentration of ion channels or the inactivation of these channels during isolation is at present uncertain.     

Properties of light and heavy vesicles simultaneously prepared from hog gastric mucosae

We obtained two kinds of vesicle preparations which were of different density from the same gastric mucosae of hogs stimulated with food before slaughter. Both kinds contained H+,K+-ATPase. The light vesicle preparation differed from the heavy vesicle preparation as follows: the KCl permeability across the membrane of heavy vesicles was larger than that of light vesicles, the actin (46-kDa peptide on SDS-polyacrylamide gel) content of heavy vesicles was much higher than that of light vesicles, and the H+,K+-ATPase activity of heavy vesicles was less sensitive to a monoclonal antibody raised against light vesicles (HK2032) than that of light vesicles. Furthermore, there was a drastic difference in reactivity to SCH 28080, which is an H+,K+-ATPase-specific inhibitor and reacts competitively with the K+-high affinity site. SCH 28080 is more potent in light vesicles than in heavy vesicles. These results suggest that the conformation of H+,K+-ATPase changed during the translocation from tubulovesicles to the apical plasma membrane. On the other hand, H+,K+-ATPase activities in both vesicles had similar pH and [K+] dependences.     

Purification and partial characterization of the (H+,K+)-transporting adenosinetriphosphatase from fundic mucosa

The microsomal (H+,K+)-ATPase systems from dog and pig fundic mucosa were purified to homogeneity and partially characterized. The method involves sodium dodecyl sulfate (SDS) (0.033% w/v) extraction of the microsomal non-ATPase proteins under appropriate conditions followed by sucrose density gradient centrifugation. Two distinct membrane bands of low (buoyant density = 1.08 g/mL) and high (buoyant density = 1.114 g/mL) densities having distinct enzymatic and chemical composition were harvested. The low-density membrane was highly enriched in Mg2+- or Ca2+-stimulated ATPase and 5'-nucleotidase activities but totally devoid of (H+,K+)-ATPase and K+-p-nitrophenylphosphatase activities. The latter two activities were found exclusively in the high-density membrane. SDS-polyacrylamide gel electrophoresis revealed the high-density membranes to consist primarily of a major 100-kilodalton (kDa) protein and a minor 85-kDa glycoprotein, the former being the catalytic subunit of the (H+,K+)-ATPase. The amino acid composition of the pure dog (H+,K+)-ATPase revealed close similarities with that from pig. The N-terminal amino acid was identified to be lysine as the sole residue. Similar to the high-density membrane-associated pure (H+,K+)-ATPase, the low-density membranes containing high Mg2+-ATPase activity also contained a 100-kDa peptide and a 85-kDa glycopeptide in addition to numerous low molecular weight peptides. Also, similar to the pure (H+,K+)-ATPase, the Mg2+-ATPase-rich fraction produced an E approximately P unstable to hydroxylamine and partially (about 25%) sensitive to K+ but having a slow turnover. The levels of E approximately P produced by the pure (H+,K+)-ATPase- and Mg2+-ATPase-rich fractions were 1400 and 178 pmol/mg of protein, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)     

The charge stoichiometry of cytochrome c oxidase in the reconstituted system.

Purified cytochrome c oxidase was reconstituted into phospholipid vesicles having high internal pH buffering capacity. In the presence of valinomycin, 2 K+ ions were taken up by the vesicles per electron transferred from cytochrome c to oxygen. The charge stoichiometry of 2 was obtained from simultaneous measurement of changes of K+, H+, and oxygen in the medium after addition of the reductant ascorbate/TMPD (N,N,N',N'-tetramethyl-p-phenylenediamine). The changes in oxygen concentration were measured with a fast responding oxygen electrode (90% response time, 0.4 s). The existence of a proton pump in cytochrome c oxidase could thus be confirmed, and its charge stoichiometry measured, in a reconstituted system uncomplicated by other respiratory chain components.     

Reconstitution of (Na+ + K+)-ATPase proteoliposomes having the same turnover rate as the membranous enzyme

Membranous (Na+ + K+)-ATPase from the electric eel was solubilized with 3-[3-cholamidopropyl)-dimethylammonio)-1-propanesulfonate (Chaps). 50 to 70% of the solubilized enzyme was reconstituted in egg phospholipid liposomes containing cholesterol by using Chaps. The obtained proteoliposomes consisted of large vesicles with a diameter of 134 +/- 24 nm as the major component, and their protein/lipid ratio was 1.25 +/- 0.07 g protein/mol phospholipid. The intravesicular volume of these proteoliposomes is too small to consistently sustain the intravesicular concentrations of ligands, especially K+, during the assay. The decrease in K+ concentration was cancelled by the addition of 20 microM valinomycin in the assay medium. The low value of the protein/lipid ratio suggests that these proteoliposomes contain one Na+/K+-pump particle with a molecular mass of 280 kDa per one vesicle as the major component. In these proteoliposomes, the specific activity of the (Na+ + K+)-ATPase reaction was 10 mumol Pi/mg protein per min, and the turnover rate of the ATP-hydrolysis was 3500 min-1, the same as the original enzyme under the same assay condition. The ratio of transported Na+ to hydrolyzed ATP was 3, the same as that in the red cell. The proteoliposomes could be disintegrated by 40-50 mM Chaps without any significant inactivation. This disintegration of proteoliposomes nearly tripled the ATPase activity compared to the original ones and doubled the specific ATPase activity compared to the membranous enzyme, but the turnover rate was the same as the original proteoliposomes and the membranous enzyme. This disintegration of proteoliposomes by Chaps suggests the selective incorporation of the (Na+ + K+)-ATPase particle into the liposomes and the asymmetric orientation of the (Na+ + K+)-ATPase particle in the vesicle.     

Coupling of Ca2+ transport to ATP hydrolysis by the Ca2+-ATPase of sarcoplasmic reticulum: potential role of the 53-kilodalton glycoprotein

An essential feature of the function of the Ca2+-ATPase of sarcoplasmic reticulum (SR) is the close coupling between the hydrolysis of ATP and the active transport of Ca2+. The purpose of this study is to investigate the role of other components of the SR membrane in regulating the coupling of Ca2+-ATPase in SR isolated from rabbit skeletal muscle, reconstituted SR, and purified Ca2+-ATPase/phospholipid complexes. Our results suggest that (1) it is possible to systematically alter the degree of coupling obtained in reconstituted SR preparations by varying the [KC1] present during cholate solubilization, (2) the variation in coupling is not due to differences in the permeability of the reconstituted SR vesicles to Ca2+, and (3) vesicles reconstituted with purified Ca2+-ATPase are extensively uncoupled under our experimental conditions regardless of the lipid/protein ratio or phospholipid composition. In reconstituted SR preparations prepared by varying the [KC1] present during cholate treatment, we find a direct correlation between the relative degree of coupling between ATP hydrolysis and Ca2+ transport and the level of the 53-kilodalton (53-kDa) glycoprotein of the SR membrane. These results suggest that the 53-kDa glycoprotein may be involved in regulating the coupling between ATP hydrolysis and Ca2+ transport in the SR.     

Effect of alkali cations on freeze-thaw-dependent reconstitution of amino acid transport from Ehrlich ascites cell plasma membrane

Na+-dependent amino acid transport can be reconstituted by gel filtration of disaggregated plasma membrane and asolectin vesicles coupled to a freeze-thaw cycle. The resultant transport activity is markedly affected by the nature of the reconstitution medium. Reconstitution in K+ permits the formation of active liposomes, whereas reconstitution in Na+, Li+, or choline does not. Electron micrographs of K+ liposomes show a wide variation in liposome sizes. Ficoll density gradient fractionation of K+ liposomes shows that the largest vesicles are lipid rich, have the lowest density, and have the highest level of Na+-dependent amino acid transport. Liposomes formed in Na+ have a 34% smaller trapped volume than K+ liposomes and lack a population of large vesicles. A second freeze-thaw in K+ restores activity to Na+ liposomes which now contain large low density active vesicles. Fluorescence measurements of freeze-thaw-induced mixing of vesicle lipids indicates that the absence of large vesicles in Na+ liposomes is due to inhibition by Na+ of lipid vesicle fusion events during freezing and thawing. The large vesicle fraction is enriched in a 125-kDa peptide. It has not yet been established whether this peptide is part of the transport system for neutral amino acids.     

Influence of sterols and phospholipids on sarcolemmal and sarcoplasmic reticular cation transporters

We have examined the influence of different sterols and phospholipids on the activities of the cardiac sarcolemmal Na+-Ca2+ exchanger and Na+,K+-ATPase and the sarcoplasmic reticular Ca2+-ATPase in reconstituted proteoliposomes. When either the solubilized Na+-Ca2+ exchanger or the Na+,K+-ATPase is reconstituted into phosphatidylcholine (PC):phosphatidylserine (30:50 by weight) vesicles, high cholesterol levels (20% by weight) are required for activity to be expressed. This sterol requirement is highly specific for cholesterol. Several cholesterol analogues with minor structural changes are unable to support Na+-Ca2+ exchange or Na+,K+-ATPase activities. When solubilized sarcolemma is reconstituted into PC:cardiolipin vesicles, however, the requirement for cholesterol is lost. Substantial activity can be obtained in the complete absence of cholesterol or in the presence of several cholesterol analogues. Thus, sterol/protein interactions can be highly dependent on the phospholipid environment. In contrast, the skeletal muscle sarcoplasmic reticular Ca2+-ATPase functions equally well in the presence or absence of cholesterol after reconstitution into either PC:phosphatidylserine or PC:cardiolipin proteoliposomes. Phospholipid requirements of the transporters were also examined. The sarcolemmal Na+-Ca2+ exchanger, Na+,K+-ATPase, and the sarcoplasmic reticular Ca2+-ATPase all function optimally in the presence of phosphatidylserine or cardiolipin after reconstitution. Thus, the sarcolemmal cation transporters have similar sterol and phospholipid requirements and may have structural similarities in their hydrophobic regions. The sarcoplasmic reticular Ca2+ pump evolved in a low cholesterol membrane and has different lipid interactions. These findings may have general applicability to other plasma membrane and endoplasmic reticular enzymes.