A simple and efficient method for reconstitution of amino acid and glucose transport systems from Ehrlich ascites cells

Solubilized Ehrlich cell plasma membrane proteins were incorporated into lipid vesicles in the presence of added phospholipid, using Sephadex G-50 chromatography combined with a freeze-thaw step. Liposomes formed in K+ exhibited high levels of Na+-dependent, alpha-aminoisobutyric acid uptake which was electrogenic and inhibited by other amino acids. The transport activity reconstituted was similar to that observed in native plasma membrane vesicles. In addition to transport by system A, leucine exchange activity (system L), Na+-dependent serine exchange activity (system ASC), and stereospecific glucose transport activity were also reconstituted. The latter was inhibited by D-glucose, D-galactose, cytochalasin B, and mercuric chloride. The medium used for reconstitution was critical for the recovery of Na+-dependent amino acid transport. The use of Na+ in the reconstitution procedure led to formation of liposomes which displayed little Na+-dependent and gradient-stimulated amino acid uptake. In contrast, all transport activities studied were efficiently reconstituted in K+ medium.     

Identification of proximal tubular transport functions in the established kidney cell line, OK

OK cells, derived from an American opossum kidney, were analyzed for proximal tubular transport functions. In monolayers, L-glutamate, L-proline, L-alanine, and alpha-methyl-glucopyranoside (alpha-methyl D-glucoside) were accumulated through Na+-dependent and Na+-independent transport pathways. D-Glucose and inorganic sulfate were accumulated equally well in the presence or absence of Na+. Influx of inorganic phosphate was only observed in the presence of Na+. Na+/alpha-methyl D-glucoside uptake was preferentially inhibited by phlorizin and D-glucose uptake by cytochalasin B. An amiloride-sensitive Na+-transport was also identified. In isolated apical vesicles (enriched 8-fold in gamma-glutamyltransferase), L-glutamate, L-proline, L-alanine, alpha-methyl D-glucoside and inorganic phosphate transport were stimulated by an inwardly directed Na+-gradient as compared to an inwardly directed K+-gradient. L-Glutamate transport required additionally intravesicular K+. D-Glucose transport was similar in the presence of a Na+- and a K+-gradient. Na+/alpha-methyl D-glucoside uptake was inhibited by phlorizin whereas cytochalasin B had no effect on Na+/D-glucose transport. An amiloride-sensitive Na+/H+ exchange mechanism was also found in the apical vesicle preparation. It is concluded that the apical membrane of OK cells contains Na+-coupled transport systems for amino acids, hexoses, protons and inorganic phosphate. D-Glucose appears a poor substrate for the Na+/hexose transport system.     

Characterization of tryptophan transport in human placental brush-border membrane vesicles.

The characteristics of tryptophan uptake in isolated human placental brush-border membrane vesicles were investigated. Tryptophan uptake in these vesicles was predominantly Na+-independent. Uptake of tryptophan as measured with short incubations occurred exclusively by a carrier-mediated process, but significant binding of this amino acid to the membrane vesicles was observed with longer incubations. The carrier-mediated system obeyed Michaelis-Menten kinetics, with an apparent affinity constant of 12.7 +/- 1.0 microM and a maximal velocity of 91 +/- 5 pmol/15 s per mg of protein. The kinetic constants were similar in the presence and absence of a Na+ gradient. Competition experiments showed that tryptophan uptake was effectively inhibited by many neutral amino acids except proline, hydroxyproline and 2-(methylamino)isobutyric acid. The inhibitory amino acids included aromatic amino acids as well as other system-1-specific amino acids (system 1 refers to the classical L system, according to the most recent nomenclature of amino acid transport systems). The transport system showed very low affinity for D-isomers, was not affected by phloretin or glucose but was inhibited by p-azidophenylalanine and N-ethylmaleimide. The uptake rates were only minimally affected by change in pH over the range 4.5-8.0. Tryptophan uptake markedly responded to trans-stimulation, and the amino acids capable of causing trans-stimulation included all amino acids with system-1-specificity. The patterns of inhibition of uptake of tryptophan and leucine by various amino acids were very similar. We conclude that system t, which is specific for aromatic amino acids, is absent from human placenta and that tryptophan transport in this tissue occurs via system 1, which has very broad specificity.     

Sodium gradient-dependent L-glutamate transport is localized to the canalicular domain of liver plasma membranes. Studies in rat liver sinusoidal and canalicular membrane vesicles

The driving forces for L-glutamate transport were determined in purified canalicular (cLPM) and basolateral (i.e. sinusoidal and lateral; blLPM) rat liver plasma membrane vesicles. Initial rates of L-glutamate uptake in cLPM vesicles were stimulated by a Na+ gradient (Na+o greater than Na+i), but not by a K+ gradient. Stimulation of L-glutamate uptake was specific for Na+, temperature sensitive, and independent of nonspecific binding. Sodium-dependent L-glutamate uptake into cLPM vesicles exhibited saturation kinetics with an apparent Km of 24 microM, and a Vmax of 21 pmol/mg X min at an extravesicular sodium concentration of 100 mM. Specific anionic amino acids inhibited L-[3H]glutamate uptake and accelerated the exchange diffusion of L-[3H]glutamate. An outwardly directed K+ gradient (K+i greater than K+o) further increased the Na+ gradient (Na+o greater than Na+i)-dependent uptake of L-glutamate in cLPM vesicles, resulting in a transient accumulation of L-glutamate above equilibrium values (overshoot). The K+ effect had an absolute requirement for Na+. In contrast, in blLPM the initial rates of L-glutamate uptake were only minimally stimulated by a Na+ gradient, an effect that could be accounted for by contamination of the blLPM vesicles with cLPM vesicles. These results indicate that hepatic Na+ gradient-dependent transport of L-glutamate occurs at the canalicular domain of the plasma membrane, whereas transport of L-glutamate across sinusoidal membranes results mainly from passive diffusion. These findings provide an explanation for the apparent discrepancy between the ability of various in vitro liver preparations to transport glutamate and suggest that a canalicular glutamate transport system may serve to reabsorb this amino acid from bile.     

Leucine-proton cotransport system in Chang liver cell

The stimulatory effect of an inward H+ gradient on the Na+-independent L-leucine uptake by the plasma membrane vesicles from Chang liver cells (Mohri, T., Mitsumoto, Y., and Ohyashiki, T. (1983) Biochem. Int. 7, 159-167) has been shown to be due to the increase of the Km value without changing the Vmax value in the transport kinetics. The uptake of leucine by the vesicles is accompanied by intravesicular acidification, and a stimulated uptake of leucine by the countertransport with a high concentration of leucine in the vesicles enhances the acidification. All of these uptakes of leucine and proton and their stimulations are amplified by imposing an inward proton gradient. These results suggest appreciably different affinities of proton for the leucine transport carrier in the inner and outer sides of the plasma membrane. A rapid decrease in the cytoplasmic pH was observed only in the first minute of incubation of intact cells with leucine in Na+-containing medium. But the leucine-dependent decrease of the cytoplasmic pH persisted longer when either Na+ in the medium was replaced by choline or amiloride was present along with Na+. Addition of amiloride to Na+-containing medium was inhibitory on the leucine uptake of cells, without effect on the early phase of glycine uptake. We conclude that Chang liver cells are provided in their plasma membrane with an amino acid-H+ cotransport system, and this is coupled to the amiloride-sensitive Na+/H+ exchange system.     

Reconstitution into liposomes of the B degrees -like glutamine-neutral amino acid transporter from renal cell plasma membrane

Na+ dependent [3H]glutamine uptake was found in liposomes reconstituted with solubilized rat kidney brush border in the presence of intraliposomal K+. The reconstituted system was optimised with respect to the critical parameters of the cyclic detergent removal procedure, i.e., the detergent used for the solubilization, the protein concentration, the detergent/phospholipid ratio and the number of passages through a single Amberlite column. Time dependent [3H]glutamine accumulation in proteoliposomes occurred only in the presence of external Na+ and internal K+. The transporter showed low if there is any tolerance towards the substitution of Na+ or K+ for other cations. Valinomycin strongly stimulated the transport indicating that it is electrogenic. Intraliposomal glutamine had no effect. From the dependence of the transport rate on the Na+ concentration cooperativity index close to 1 was derived, indicating that 1 Na+ should be involved in the cotransport with glutamine. The electrogenicity of the transport originated from the Na+ transport. Optimal rate of 0.1 mM [3H]glutamine uptake was found in the presence of 50 mM intraliposomal K-gluconate. At higher K-gluconate concentrations the transport rate decreased. The activity of the reconstituted transporter was pH dependent with optimal function in the range pH 6.5-7.0. [3H]glutamine (and [3H]leucine) uptake was inhibited by all the neutral but not by the positively or negatively charged amino acids. The sulfhydryl reagents HgCl2, mersalyl, p-hydroxymercuribenzoate and the substrate analogue 2-aminobicyclo[2,2,1]heptane-2-carboxylate strongly inhibited the transporter, whereas the amino acid analogue alpha-(methylamino)isobutyrate had no effect. The inhibition by mersalyl was protected by the presence of the substrate. On the basis of the Na+ dependence, the electrogenic transport mode and the specificity towards the amino acids, the reconstituted transporter was classified as B degrees-like.     

Active amino acid transport in plasma membrane vesicles from Simian virus 40-transformed mouse fibroblasts. Characteristics of electrochemical Na+ gradient-stimulated uptake.

Selectively permeable membrane vesicles isolated from Simian virus 40-transformed mouse fibroblasts catalyzed Na+ gradient-coupled active transport of several neutral amino acids dissociated from intracellular metabolism. Na+-stimulated alanine transport activity accompanied plasma membrane material during centrifugation in discontinuous dextran 110 gradients. Carrier-mediated transport into the vesicle was demonstrated. When Na+ was equilibrated across the membrane, countertransport stimulation of L-[3H]alanine uptake occurred in the presence of accumulated unlabeled L-alanine, 2-aminoisobutyric acid, or L-methionine. Competitive interactions among neutral amino acids, pH profiles, and apparent Km values for Na+ gradient-stimulated transport into vesicles were similar to those previously described for amino acid uptake in Ehrlich ascites cells, which suggests that the transport activity assayed in vesicles is a component of the corresponding cellular uptake process. Both the initial rate and quasi-steady state of uptake were stimulated as a function of a Na+ gradient (external Na+ greater than internal Na+) applied artificially across the membrane and were independent of endogenous (Na+ + K+)-ATPase activity. Stimulation by Na+ was decreased when the Na+ gradient was dissipated by monensin, gramicidin D or Na+ preincubation. Na+ decreased the apparent Km for alanine, 2-aminoisobutyric acid, and glutamine transport. Na+ gradient-stimulated amino acid transport was electrogenic, stimulated by conditions expected to generate an interior-negative membrane potential, such as the presence of the permeant anions NO3- and SCN-. Na+-stimulated L-alanine transport was also stimulated by an electrogenic potassium diffusion potential (K+ internal greater than K+ external) catalyzed by valinomycin; this stimulation was blocked by nigericin. These observations provide support for a mechanism of active neutral amino acid transport via the "A system" of the plasma membrane in which both a Na+ gradient and membrane potential contribute to the total driving force.     

Sodium-dependent amino acid transport in reconstituted membrane vesicles from Ehrlich ascites cell plasma membranes

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.     

Sodium-ion stimulated amino acid uptake in membrane vesicles of alkalophilic Bacillus no. 8-1

Membrane vesicles were isolated from alkalophilic Bacillus No. 8-1, and the active transport of amino acids was studied. The transport of amino acids was dependent upon substrate oxidation and the presence of Na+. Concentrative uptake of amino acids was stimulated by the addition of an artificial electron donor system, ascorbate-phenazine methosulfate (PMS), and to a lesser extent by NADH, while succinate, L-lactate, and alpha-glycerol-phosphate did not stimulate the uptake. N,N,N',N'-Tetramethyl-p-phenylenediamine (TMPD) and cytochrome c were able to replace PMS, and reduced forms of these compounds were also very efficient electron donors. Amino acid transport was dependent on electron transfer, and inhibition of NADH oxidation by cyanide, 2-heptyl-4-hydroxyquinoline-N-oxide (HOQNO), and sodium azide directly prohibited serine transport. The pH optima for serine transport lay between pH 8 and 9 for all energy sources. Sodium ion stimulated serine transport in the presence of NADH, NADH plus cytochrome c or succinate plus PMS, but had no stimulatory effect on the corresponding dehydrogenase activities. Sodium ion was also required for accumulation of serine in response to an artificial membrane potential where the respiratory chain was not operative. These results indicated that the stimulatory effect of Na+ on amino acid uptake was on the transport process itself.     

Effect of alkali ions on the active transport of neutral amino acids into Streptomyces hydrogenans.

The active transport of neutral amino acids into Streptomyces hydrogenans is inhibited by external Na+. There is no indication that in these cells amino acid accumulation is driven by an inward gradient of Na+. The extent of transport inhibition by Na+ depends on the nature of the amino acid. It decreases with increasing chain length of the amino acid molecules i.e. with increasing non-polar properties of the side chain. Kinetic studies show that Na+ competes with the amino acid for a binding site at the amino acid carrier. There is a close relation between the Ki values for Na+ and the number of C atoms of the amino acids. Other cations also inhibit neutral amino acid uptake competitively; the effectiveness decreases in the order Li+ greater than Na+ greater than K+ greater than Rb+ greater than Cs+. Anions do not have a significant effect on the uptake of neutral amino acids. After prolonged incubation of the cells with 150 mM Na+, in addition to the competitive inhibition of transport Na+ induces an increase in membrane permeability for amino acids.     

Na+-Independent Transporters, LAT-2 and b0,+, Exchange L-DOPA with Neutral and Basic Amino Acids in Two Clonal Renal Cell Lines

The present study examined the functional characteristics of L-DOPA transporters in two functionally different clonal subpopulations of opossum kidney (OKLC and OKHC) cells. The uptake of L-DOPA was largely Na+-independent, though in OKHC cells a minor component (approximately 15%) required extracellular Na+. At least two Na+-independent transporters appear to be involved in L-DOPA uptake. One of these transporters has a broad specificity for small and large neutral amino acids, is stimulated by acid pH and inhibited by 2-aminobicyclo(2,2,l)-heptane-2-carboxylic acid (BCH; OKLC, Ki = 291 mM; OKHC, Ki = 380 mM). The other Na+-independent transporter binds neutral and basic amino acids and also recognizes the di-amino acid cystine. [14C]-L-DOPA efflux from OKLC and OKHC cells over 12 min corresponded to a small amount of intracellular [14C]-L-DOPA. L-Leucine, nonlabelled L-DOPA, BCH and L-arginine, stimulated the efflux of [14C]-L-DOPA in a Na+-independent manner. It is suggested that L-DOPA uses at least two major transporters, systems LAT-2 and b0,+. The transport of L-DOPA by LAT-2 corresponds to a Na+-independent transporter with a broad specificity for small and large neutral amino acids, stimulated by acid pH and inhibited by BCH. The transport of L-DOPA by system b0,+ is a Na+-independent transporter for neutral and basic amino acids that also recognizes cystine. LAT-2 was found equally important at the apical and basolateral membranes, whereas system b0,+ had a predominant distribution in apical membranes.     

Distinct effects of various amino acids on 45Ca2+ fluxes in rat pancreatic islets.

The effects of three types of amino acids on 45Ca2+ fluxes in rat pancreatic islets have been compared. Alanine, a non-insulinotropic neutral amino acid, transported with Na+, increased 45Ca2+ efflux in the presence or in the absence of extracellular Ca2+, but not in the absence of Na+. Its effects in Na+-solutions were practically abolished by 7 mM-glucose. Alanine slightly stimulated 45Ca2+ influx (5 min uptake) only when Na+ was present. Two insulinotropic cationic amino acids (arginine and lysine) triggered similar changes in 45Ca2+ efflux. They accelerated the efflux in the presence of Ca2+ and inhibited the efflux in a Ca2+-free medium, whether glucose was present or not. In an Na+-free Ca2+-medium, arginine and lysine markedly accelerated 45Ca2+ efflux, but this effect was suppressed by 7 mM-glucose. Arginine stimulated 45Ca2+ influx irrespective of the presence or absence of glucose and Na+. Leucine, a neutral insulinotropic amino acid well metabolized by islet cells, inhibited 45Ca2+ efflux from the islets in a Ca2+-free medium; this effect was potentiated by glutamine. In the presence of Ca2+ and Na+, leucine was ineffective alone, but triggered a marked increase in 45Ca2+ efflux when combined with glutamine. In an Na+-free Ca2+-medium, leucine accelerated 45Ca2+ efflux to the same extent with or without glutamine. Leucine also stimulated 45Ca2+ influx in the presence or in the absence of Na+, but its effects were potentiated by glutamine only in the presence of Na+. The results show that amino acids of various types cause distinct changes in 45Ca2+ fluxes in pancreatic islets. Certain of these changes involve an Na+-mediated mobilization of cellular Ca2+ from sequestering sites where glucose appears to exert an opposite effect.     

Estimation of apparent L-amino acid diffusion in porcine jejunal enterocyte brush border membrane vesicles

There is an overlap of carrier-mediated L-amino acid transport and apparent simple diffusion when measured in intestinal brush border membrane vesicles. Using L-threonine and L-glutamine as representative amino acids, this study was undertaken to estimate apparent simple diffusion of L-amino acids and to establish the effective dosage of HgCl2 for completely blocking carrier-mediated L-amino acid transport in porcine jejunal enterocyte brush border membrane vesicles. Jejunal mucosa was scraped from three pigs weighing 26 kg. Enterocyte brush border membrane vesicles, with an average enrichment of 24-fold in sucrase specific activity, were prepared by Mg2+-precipitation and differential centrifugation. In vitro uptake was measured by the fast filtration manual procedure. HgCl2 blocked the carrier-mediated initial transport of L-threonine and L-glutamine under Na+-gradient condition in a dose-dependent manner. At the minimal concentration of 0.165 micromol HgCl2 mg(-1) protein, carrier-mediated L-threonine and L-glutamine transport was completely inhibited. The apparent L-threonine and L-glutamine diffusion was estimated to be 8.6+/-0.7 and 12.4+/-1.0% of the total uptake at the substrate concentrations of 5 microM (L-threonine) and 50 microM (L-glutamine). Therefore, the treatment of porcine brush border membrane vesicles with a minimum of 0.165 micromol HgCl2 mg(-1) protein completely blocks carrier-mediated L-amino acid transport and enables the direct estimation of apparent L-amino acid diffusion in enterocyte brush border membrane vesicles.     

Postnatal amino acid uptake by the rat small intestine. Energetics of membrane transport systems for amino acids in the developing jejunum

The energetics of amino acid uptake by the developing small intestine was investigated in vitro. L-valine, L-leucine, L-phenylalanine, L-methionine, L-lysine and L-arginine were all actively transported by the newborn rat jejunum. Metabolic inhibitors (e.g. 2,4-dinitrophenol) significantly reduced uptake of all amino acids but uptake against a concentration gradient was not totally abolished. Uptake of all amino acids was reduced at low[Na+]. Inhibition of transport of neutral amino acids by reduced luminal [Na+] was greater than that of basic amino acids, and the tissue was barely able to concentrate the neutral amino acids. [Na+] affected the Michaelis constant (Km) of neutral transport systems for their substrates; for the basic amino acids Km values were unaffected by the presence or absence of Na+. Ouabain significantly inhibited neutral amino acid uptake but had no effect on L-lysine or L-arginine uptake. These results are discussed in terms of the Na+ gradient hypothesis for amino acid transport, and the site of energy input to active transport. The role of glycolysis in providing energy for intestinal transport in the neonatal rat and the efficiency of Na+ dependent and independent transport mechanisms are considered. It is concluded that the energetics of amino acid transport systems in neonatal and adult rats are essentially similar.     

Proton gradient-dependent transport of glycine in rabbit renal brush-border membrane vesicles

This study describes evidence for the existence of a H+/glycine symport system in rabbit renal brush-border membrane vesicles. An inward proton gradient stimulates glycine transport across the brush-border membrane, and this H+-driven glycine uptake is attenuated by the protonophore carbonyl cyanide p-trifluoromethoxyphenylhydrazone. It is a positive rheogenic process, i.e. the H+-dependent glycine uptake is further enhanced by an intravesicular negative potential. Glycine uptake is stimulated to a lesser degree by an inward Na+ gradient. H+-dependent glycine uptake is inhibited by sarcosine (69%), an analog amino acid, imino acids (proline 81%, hydroxy proline 67%), and beta-alanine (31%), but not by neutral (L-leucine) or basic (L-lysine) amino acids. The results demonstrate that H+ glycine co-transport system in rabbit renal brush-border membrane vesicles is a carrier-mediated electrogenic process and that transport is shared by imino acids and partially by beta-alanine.     

Sodium-dependent transport of branched-chain amino acids by a monensin-sensitive ruminal peptostreptococcus.

A recently isolated ruminal peptostreptococcus which produced large amounts of branched-chain volatile fatty acids grew rapidly with leucine as an energy source in the presence but not the absence of Na. Leucine transport could be driven by an artificial membrane potential (delta psi) only when Na was available, and a chemical gradient of Na+ (delta uNa+) also drove uptake. Because Na+ was taken up with leucine and a Z delta pH could not serve as a driving force (with or without Na), it appeared that leucine was transported in symport with Na+. The leucine carrier could use Li as well as Na and had a single binding site for Na+. The Km for Na was 5.2 mM, and the Km and Vmax for leucine were 77 microM and 328 nmol/mg of protein per min, respectively. Since valine and isoleucine competitively inhibited (Kis of 90 and 49 microM, respectively) leucine transport, it appeared that the peptostreptococcus used a common carrier for branched-chain amino acids. Valine or isoleucine was taken up rapidly, but little ammonia was produced if they were provided individually. The lack of ammonia could be explained by an accumulation of reducing equivalents. The ionophore, monensin, inhibited growth, but leucine was taken up and deaminated at a slow rate. Monensin caused a loss of K, an increase in Na, a slight increase in delta psi, and a decrease in intracellular pH. The inhibition of growth was consistent with a large decrease in ATP.(ABSTRACT TRUNCATED AT 250 WORDS)     

Characteristics of D-alanine transport by luminal membrane vesicles from pars convoluta and pars recta of rabbit proximal tubule

Uptake of D-alanine against a concentration gradient has been shown to occur with isolated luminal-membrane vesicles from pars convoluta or pars recta of rabbit proximal tubule. Renal D-alanine transport systems, displaying the following characteristics, were shown: (1) In vesicles from pars convoluta, the uptake of D-alanine was mediated by both Na+-dependent and Na+-independent transport processes. It was found that an inwardly directed H+-gradient could drive the transport of D-alanine into the vesicles both in the presence and absence of Na+. Thus, in addition to Na+, the transport of D-alanine is influenced by the H+-gradient. (2) In vesicles from pars recta, the transient accumulation of D-alanine was strictly dependent on Na+, since no 'overshoot' was ever observed in the absence of Na+. Although the Na+-dependent uptake of D-alanine was stimulated at acid pH, H+ did not substitute for Na+, as it apparently does in pars convoluta, but instead potentiated the Na+ effect. (3) Addition of L-alanine to vesicle preparations, both from pars convoluta and from pars recta, specifically inhibited renal uptake of D-alanine. A comparison between the transport characteristics of D- and L-alanine indicated that these two isomers of alanine probably share common transport systems located along the proximal tubule of rabbit kidney.     

Sodium-dependent transport of branched-chain amino acids by a monensin-sensitive ruminal peptostreptococcus

A recently isolated ruminal peptostreptococcus which produced large amounts of branched-chain volatile fatty acids grew rapidly with leucine as an energy source in the presence but not the absence of Na. Leucine transport could be driven by an artificial membrane potential (delta psi) only when Na was available, and a chemical gradient of Na+ (delta uNa+) also drove uptake. Because Na+ was taken up with leucine and a Z delta pH could not serve as a driving force (with or without Na), it appeared that leucine was transported in symport with Na+. The leucine carrier could use Li as well as Na and had a single binding site for Na+. The Km for Na was 5.2 mM, and the Km and Vmax for leucine were 77 microM and 328 nmol/mg of protein per min, respectively. Since valine and isoleucine competitively inhibited (Kis of 90 and 49 microM, respectively) leucine transport, it appeared that the peptostreptococcus used a common carrier for branched-chain amino acids. Valine or isoleucine was taken up rapidly, but little ammonia was produced if they were provided individually. The lack of ammonia could be explained by an accumulation of reducing equivalents. The ionophore, monensin, inhibited growth, but leucine was taken up and deaminated at a slow rate. Monensin caused a loss of K, an increase in Na, a slight increase in delta psi, and a decrease in intracellular pH. The inhibition of growth was consistent with a large decrease in ATP.(ABSTRACT TRUNCATED AT 250 WORDS)     

Reversed transport of amino acids in Ehrlich cells.

Gramicidin induces a marked Na+-dependent efflux of amino acids from Ehrlich cells. In absence of Na+, gramicidin does not alter the efflux. In presence gramicidin, glycine efflux is inhibited by methionine and less so by leucine. Glycine efflux caused by HgCl2 is neither Na+ dependent nor inhibitable by amino acids. Neither efflux of inositol which is transported by an Na+-dependent route, nor efflux of several other solutes which are transported by Na+-independent routes, is affected by gramicidin. The antibiotic appears to permit a reversal in the direction of of the operation of the Na+-dependent amino acid transport system. The increased efflux is partly, but not entirely, due to an increase in the cellular Na+ concentration and a reduction of the electrochemical potential difference for Na+.     

Characterization and chemical modification of the Na(+)-dependent bile-acid transport system in brush-border membrane vesicles from rabbit ileum.

The Na(+)-dependent uptake system for bile acids in the ileum from rabbit small intestine was characterized using brush-border membrane vesicles. The uptake of [3H]taurocholate into vesicles prepared from the terminal ileum showed an overshoot uptake in the presence of an inwardly-directed Na(+)-gradient ([Na+]out > [Na+]in), in contrast to vesicles prepared from the jejunum. The Na(+)-dependent [3H]taurocholate uptake was cis-inhibited by natural bile acid derivatives, whereas cholephilic organic compounds, such as phalloidin, bromosulphophthalein, bilirubin, indocyanine green or DIDS - all interfering with hepatic bile-acid uptake - did not show a significant inhibitory effect. Photoaffinity labeling of ileal membrane vesicles with 3,3-azo- and 7,7-azo-derivatives of taurocholate resulted in specific labeling of a membrane polypeptide with apparent molecular mass 90 kDa. Bile-acid derivatives inhibiting [3H]taurocholate uptake by ileal vesicles also inhibited labeling of the 90 kDa polypeptide, whereas compounds with no inhibitory effect on ileal bile-acid transport failed to show a significant effect on the labeling of the 90 kDa polypeptide. The involvement of functional amino-acid side-chains in Na(+)-dependent taurocholate uptake was investigated by chemical modification of ileal brush-border membrane vesicles with a variety of group-specific agents. It was found that (vicinal) thiol groups and amino groups are involved in active ileal bile-acid uptake, whereas carboxyl- and hydroxyl-containing amino acids, as well as tyrosine, histidine or arginine are not essential for Na(+)-dependent bile-acid transport activity. The irreversible inhibition of [3H]taurocholate transport by DTNB or NBD-chloride could be partially reversed by thiols like 2-mercaptoethanol or DTT. Furthermore, increasing concentrations of taurocholate during chemical modification with NBD-chloride were able to protect the ileal bile-acid transporter from inactivation. These findings suggest that a membrane polypeptide of apparent M(r) 90,000 is a component of the active Na(+)-dependent bile-acid reabsorption system in the terminal ileum from rabbit small intestine. Vicinal thiol groups and amino groups of the transport system are involved in Na(+)-dependent transport activity, whereas other functional amino acids are not essential for transport activity.