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Radiation inactivation was used to estimate the molecular size of a Na(+)-dependent amino acid transport system in Ehrlich ascites cell plasma membrane vesicles. Na(+)-dependent alpha-aminoisobutyric acid uptake was measured after membranes were irradiated at -78.5 degrees C in a cryoprotective medium. Twenty-five percent of the transport activity was lost at low radiation doses (less than 0.5 Mrad), suggesting the presence of a high molecular weight transport complex. The remaining activity (approximately 75% of total) decreased exponentially with increasing radiation dose, and a molecular size of 347 kDa was calculated for the latter carrier system. Vesicle permeability and intravesicular volume were measured to verify that losses in transport activity were due to a direct effect of radiation on the transporter and not through indirect effects on the structural integrity of membrane vesicles. Radiation doses 2-3-fold higher than those required to inactivate amino acid transport were needed to cause significant volume changes (greater than 15%). Vesicle permeability was unchanged by the irradiation. The structural integrity of plasma membrane vesicles was therefore maintained at radiation doses where there was a dramatic decrease in amino acid transport. The relationship between the fragmentation of a 120-130-kDa peptide, a putative component of the Na(+)-dependent amino acid carrier [McCormick, J. I., & Johnstone, R. M. (1988) Proc. Natl. Acad. Sci. U.S.A. 85, 7877-7881], and loss of transport activity in irradiated membranes was also examined. Peptide loss was quantitated by Western blot analysis.(ABSTRACT TRUNCATED AT 250 WORDS)

A reconstitution assay was used to measure transport activity during purification of a Na+-dependent amino acid transporter from Ehrlich cell plasma membrane. Cholate/urea-solubilized membranes were fractionated on a Sepharose 6B column and transport activity was recovered in the column void volume. Centrifugation of the void volume fraction at 105,000 X g and reextraction of the pellet with 1% octyl glucoside led to recovery of an extract whose specific transport activity was nearly 30-fold higher than that of the original solubilized extract with a recovery of 38% of the original activity. The properties of amino acid uptake in the purified reconstituted transporter were identical to those in native plasma membrane vesicles. The major component present in the purified fraction had a molecular mass of 120-130 kDa. Strong evidence that this 120- to 130-kDa peptide contains a component of the amino acid transporter was obtained by immunoprecipitation of transport activity from solubilized membranes with an antibody against the 120- to 130-kDa peptide. This study tentatively identifies a component of the Na+-dependent amino acid transporter as a peptide with an apparent molecular mass of 120-130 kDa.

Inside-out membrane vesicles have been prepared from sheep reticulocytes. With these vesicles, Na+-dependent glycine uptake and net accumulation have been demonstrated to occur in reverse, i.e., from extravesicular (normal cytoplasmic) to intravesicular (normal extravesicular) surface. Uptake and accumulation are inhibited by energization of the sodium pump by ATP whereby the Na+ electrochemical gradient is dissipated. Glycine-dependent Na+ uptake was also observed, providing evidence that Na+-dependent glycine influx into these vesicles, equivalent to normal efflux, is characterized by Na+-glycine co-transport.

Na+-dependent glycine transport has been studied in reticulocyte-enriched fractions of blood obtained after massive bleeding of sheep. The activity is dependent on the sodium electrochemical potential and the membrane potential. The sodium chemical gradient was varied by changing either external or internal Na+ and the membrane potential, by addition of valinomycin. Similar results were obtained with resealed reticulocyte ghosts. Under conditions optimal for sodium pumping (intracellular Na+ greater than 50mM), ouabain inhibited glycine uptake prior to any measurable change in the cellular Na+ suggesting that in these cells an electrogenic sodium pump is sufficiently active to contribute to the membrane potential. Na+-dependent glycine transport undergoes a marked decrease during long-term incubation at 37 degrees C. During this time, the cells maintain their integrity and ATP content but undergo maturation as evidenced in the decrease in cells with reticulocyte morphology.

Trans-stimulation of glycine uptake by cellular glycine in Ehrlich cells is a Na+-dependent phenomenon. In contrast trans-stimulated methionine or leucine uptake is Na+-independent. Trans-stimulated uptake of glycine does not show any characteristics of an ex change process but rather appears to be due to changes in membrane potential which occur as a result of a net Na+-dependent loss of cellular amino acids. Trans-stimulated influx of glycine occurs during the time of net loss of cellular glycine and is absent when the cellular amino acid level is at steady or when the cell is depolarized. Exchange of leucine or methionine occurs when the amino acid level is at steady state and it is not directly affected by depolarizing agents such as gramicidin.

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.

Na+-dependent amino acid transport can be reconstituted from solubilized Ehrlich cell plasma membranes by addition of asolectin vesicles, gel filtration, and a freeze-thaw cycle. Removal of phosphatidic acid (approximately 10% of the total lipid) by Ba2+ precipitation reduces the efficiency of reconstitution of Na+-dependent amino acid transport by approximately 73% and decreases intravesicular volume of the proteoliposomes by approximately 43%. The loss of transport activity is not due to exclusion of specific proteins during reconstitution. The phosphatidic acid-free liposomes are less permeable and require more time to attain an equilibrium distribution of solute. Transport activity and intravesicular volume can be restored to Ba2+-precipitated asolectin proteoliposomes by addition of egg-phosphatidic acid during reconstitution. The extent of recovery of transport activity is proportional to the change in intravesicular volume and depends on the amount of phosphatidic acid present. Replacement of phosphatidic acid with 20% phosphatidylserine or phosphatidylglycerol leads to increases in intravesicular volume with little or no increase in amino acid transport. Generation of phosphatidic acid in situ by treatment of Ba2+-precipitated proteoliposomes with phospholipase D also restored transport. The observed increase in transport activity (9-fold) is accompanied by a 46% increase in intravesicular volume, presumably caused by vesicle fusion. Phosphatidic acid is also required for successful reconstitution of Na+-dependent amino acid transport from pure phosphatidylcholine:phosphatidylethanolamine (1:1) mixtures with only a small change (approximately 16%) in intravesicular volume. The results provide evidence for both indirect and direct effects of phosphatidic acid on reconstitution of Na+-dependent amino acid transport. The indirect effects occur through enlargement of intravesicular volume, large vesicles showing higher rates of transport. However, there is also evidence to indicate a specific effect of phosphatidic acid on the Na+-dependent amino acid transporter, since other acidic lipids may change intravesicular volume without a commensurate change in transport activity.

Cells depleted of amino acids show lower rates of glycine or aminoisobutyric acid uptake than do freshly isolated cells. In the amino acid-depleted cells, addition of valinomycin stimulates amino acid influx at least to the level observed in freshly isolated cells. In cells containing high levels of cellular amino acids, valinomycin has little effect on influx of amino acids. It is concluded that the transport of amino acids in freshly isolated cells is elevated compared to depleted cells because the cells are hyperpolarized by the continuous loss of cellular amino acids during the transport assay. During this hyperpolarization by amino acid loss, transport of amino acids is not further stimulated by valinomycin at low external [K+] (10 mM +/- 5 mM). With the exception of preloading with glycine, cells preloaded with a single amino acid to a concentration greater than 20 mM show reduced rates of glycine and aminoisobutyric acid influx at early times (less than 15 min) compared to amino acid-depleted cells. The reduction of influx is transient and by 30 min, influx is greater in preloaded than in amino acid-depleted cells. Knowing that increases and decreases in the membrane potential are achieved by using varying external [K+] in the presence of valinomycin and propranolol, and using amino acid-depleted cells, it can be shown that an increased membrane potential increases the V for glycine and aminoisobutyric acid influx. A decrease in the potential difference results in a decreased V. Changes in Km also occur when the membrane potential is varied.

Many hydrophilic amino acids are transported via exchange by the Na+-independent L system in the Ehrlich cells. However, this exchange is measurable only at relative high concentrations of the small hydrophilic amino acids. Whereas leucine saturated this transport system at 0.2 mM, with threonine, serine, or alanine the system was below saturation at 30 mM. All amino acids capable of exchanging showed similar EA values (congruent to 20 X 10(3) cal X mol-1, 1 cal = 4.1868 J) and maximum velocities. Depending on the temperature, the apparent Km values for different amino acids differed by one or two orders of magnitude. These data suggest that for the small hydrophilic amino acids, the relatively slow rate of transport is due to the unfavorable Km values.

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.

Plasma membranes, isolated from Ehrlich ascites tumor cells, were dissolved in 2% cholate, 4 M urea and then reformed into liposomes upon dialysis at 4 degrees with exogenous phospholipids. Reconstituted vesicles regain the ability to transport amino acids. Na+ was shown to accelerate the uptake of alpha-aminoisobutyrate, phenylalanine, and methionine, but not leucine or epsilon-aminohexanoic acid. With the reconstituted vesicles, methionine, but not leucine, inhibited the uptake of alpha-aminoisobutyrate. An apparent Km value for alpha-aminoisobutyrate uptake of 3.0 mM was obtained. This value is close to that observed with the intact cells and the native membrane vesicles. A Na+ gradient (high Na+ outside) increased alpha-aminoisobutyrate uptake, whereas a reversed gradient (high Na+ inside) increased alpha-aminoisobutyrate efflux. The latter flux was increased by valinomycin, suggesting electrogenic transport. A modest extent of coupling between a Na+ gradient and uphill flow of alpha-aminoisobutyrate was observed.

Light-induced Na+ efflux was observed in sub-bacterial particles of Halobacterium halobium loaded and suspended in 4 M NaCl solution. The Na+ efflux was not ATP driven, since ATPase inhibitors were without effect or even enhanced efflux at low light intensity. Uncouplers, on the other hand, inhibited Na+ efflux, the inhibition being complete at low light intensity. The Na+ efflux was accompanied by proton influx. Both processes were dependent on light intensity, unaffected or enhanced by ATPase inhibitors and similarly affected by uncouplers. Proton influx was not observed in particles loaded with 4 M KCl instead of 4 M NaCl. Na+ transport in the dark could be induced by artificial formation of a pH difference across the membrane; changing the sign of the pH difference reversed the direction of the Na+ transport. Proton influx in the dark followed the artificial formation of a sodium gradient [Na+]in less than [Na+]out). These results may be explained by a Na+/H+ antiport mechanism. The fluxes of Na+ and H+ were of comparable magnitude, but the initial rate of Cl- efflux in the same experiment was one-third of the initial rate of Na+ efflux. Consequently Cl- is not regarded as a participant in the Na+ efflux mechanism.

Influx and efflux of glycine have been examined as a function of external and internal Na+ concentrations, respectively, when : formula: (see text) = O. With : formula: (see text) = O it was found that at comparable external and cellular Na+ levels, the Km for efflux was larger by an order of magnitude than the value for influx and the V for efflux was several times greater than the V for influx. For both fluxes the major effect of Na+ was to decrease the Km value. The observations are consistent with the conclusion that the Na+-dependent transport system is asymmetric per se. Influx and efflux of glycine were increased in a near linear manner by increasing the Na+ concentration from 13 to 100 mM, the half-time for glycine equilibration being a funcion of the Na+ concentration in absence of an electrochemical potential difference for Na+. In Na+-free media ([Na+] less than 5 mM) equilibration of glycine between cells and medium was not achieved after 60 min at 25 degrees C. With : formula: (see text) = O, efflux (or uptake) of glycine was not affected by internal (or external) K+ between 20 and 120 mM suggesting that K+ plays no direct role in Na+-dependent transport of glycine in Ehrlich cells.