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PubMed1988

Metabolism of pyrimidine bases and nucleosides by Pseudomonas fluorescens biotype F.

Pyrimidine metabolism in Pseudomonas fluorescens biotype F, and its ability to grow in liquid culture on pyrimidines and related compounds was investigated. It was found that uracil, uridine, cytosine, cytidine, deoxycytidine, dihydrouracil, dihydrothymine, beta-alanine or beta-aminoisobutyric acid could be utilized by this pseudomonad as a sole nitrogen source. Only uridine, cytidine, beta-alanine, beta-aminoisobutyric acid or ribose were capable of supporting its growth as a sole source of carbon. In solid medium, the pyrimidine analogue 5-fluorouracil or 5-fluorouridine could prevent P. fluorescens biotype F growth at a low concentration while a 20-fold higher concentration of 5-fluorocytosine, 5-fluorodeoxyuridine or 6-azauracil was necessary to block its growth. The pyrimidine salvage enzymes cytosine deaminase, nucleoside hydrolase, uridine phosphorylase, thymidine phosphorylase and cytidine deaminase were assayed. Only cytosine deaminase and nucleoside hydrolase activities could be detected under the assay conditions used. The effect of growth conditions on cytosine deaminase and nucleoside hydrolase levels in the micro-organism was explored. Cytosine deaminase activity was shown to increase if glycerol was substituted for glucose as the sole carbon source or if asparagine replaced (NH4)2SO4 as the sole nitrogen source in each respective medium. In contrast, nucleoside hydrolase activity remained virtually unchanged whether the carbon source in the medium was glucose or glycerol. A decrease in nucleoside hydrolase activity was witnessed when asparagine was present in the medium instead of (NH4)2SO4 as the sole source of nitrogen.

Cytosine Deaminase↗
PubMed1978

Amino acid transport in thymic- and spleen-derived lymphocytes.

We have examined the transport of amino acids by the sodium-dependent "A" and "ASC" system in thymic- and splenic-derived lymphocytes from the Long-Evans rat. Lymphocytes derived from the thymus transport amino acids by both the "A" and "ASC" systems, whereas lymphocytes from the spleen transport amino acids by the "ASC" system only. Thymic lymphocytes are capable of establishing a steady state distribution ratio of 7.9 for 2-aminoisobutyric acid, but splenic lymphocytes can attain only 3.5. The steady state distribution ratio of alanine was the same in both cell types. Sodium-independent transport is also different in splenic and thymic lymphocytes. But both cells move amino acids by a Na+-independent system for mediated exchange-diffusion. The studies show that lymphocytes derived from the spleen and thymus transport amino acids differently, and that the "T" lymphocytes from the spleen have membrane transport systems different from "T" lymphocytes from the thymus.

Alanine↗
PubMed1986

Stimulative effect of glia maturation factor and acetylcholine on active amino acid uptake and Ca2+,Mg2+-ATPase activity in nodose ganglia excised from adult rats.

During aerobic incubation at 37 degrees C, active uptake of labeled non-metabolizable alpha-aminoisobutyric acid (AIB) into isolated nodose ganglia (NG) excised from adult rats was accelerated to nearly twice that of the control, by the addition of glia maturation factor (GMF, 5 micrograms/ml) in a dose-dependent manner. A similar but moderate stimulative effect on ganglionic AIB uptake was caused by the addition of acetylcholine (ACh, 1 mM) plus eserine (0.1 mM). This effect, however, was not antagonized by nicotinic (hexamethonium, C6, 0.1 mM) or muscarinic (atropine, 0.1 mM) blockers. The GMF-induced amino acid uptake seemed to be inhibited by further addition of ACh. On the other hand, ganglionic Ca2+,Mg2+-ATPase activity was greatly stimulated by either GMF or ACh. These results suggest that the increase in AIB uptake induced by GMF or ACh is possibly linked to Ca2+,Mg2+-ATPase activity in NG cell membranes.

Acetylcholine↗
PubMed1978

Cyclic AMP mediated increased amino acid transport: effect of isoproterenol administration.

A single injection of isoproterenol (IPR) stimulates cell proliferation in rodent salivary glands after a lag period of about 24 hrs. Among the many events occurring prior to stimulated DNA synthesis, there is an early increase in cAMP levels and elevated transport of amino acids into the parotid gland. Amino acid transport is also elevated in liver and pancreas, tissues not induced to proliferate by IPR. IPR-stimulated alpha-aminoisobutyric acid (AIB) transport in parotid, pancreas and liver is augmented by prior injection of theophylline and is mimicked by dibutyryl cAMP. In all three tissues, changes in cAMP levels and subsequent increases in AIB transport appear to be closely related events. Since only the parotid gland is stimulated to grow after IPR injection, amino acid transport and growth would not appear to be directly related.

Amino Acids↗
PubMed1992

Amino acid transport by the cultured human placental trophoblast: effect of insulin on AIB transport.

Insulin responsiveness in the human placenta is controversial. This study evaluated insulin stimulation of alpha-aminoisobutyric acid (AIB) uptake in cultured human placental trophoblasts. Both Na(+)-dependent and -independent components of AIB uptake were present in cultured trophoblasts. Na(+)-dependent AIB uptake was significantly stimulated by insulin in a time-dependent manner, as early as 2 h, with a maximum at 12 h of continuous exposure to hormone. Insulin treatment for 4 h increased both the initial uptake rate and the final intracellular concentration. Stimulation was dependent on insulin concentration, with significant stimulation beginning at 10(-9) M. Insulin treatment increased maximum velocity but not the Michaelis constant. Approximately 75% of basal (unstimulated) AIB uptake was inhibited by 10 mM alpha-methylaminoisobutyric acid (MeAIB). The insulin-stimulated increment above basal AIB uptake was completely inhibited by 10 mM MeAIB. Cycloheximide treatment significantly reduced basal and stimulated AIB uptake, although a significant response to insulin persisted. Na(+)-dependent AIB uptake was also stimulated by glucagon, dexamethasone, and 8-bromoadenosine 3',5'-cyclic monophosphate, but not by vasopressin. This study further characterizes amino acid uptake by the human placenta and demonstrates that the Na(+)-dependent component of AIB uptake by the cultured trophoblasts is stimulated by physiological concentrations of insulin.

Amino Acids↗
PubMed1975

Neutral amino acid transport pathways in uptake of L-thyroxine by Ehrlich ascites cells.

Neutral amino acid pathways were investigated as possible mediators of L-thyroxine (T4) entry into ascites cells. 14C-Labeled T4, alpha-aminoisobutyric acid (AIB), L-phenylalanine (Phe), and cycloleucine (cLeu) uptakes were measured at initial extracellular amino acid concentrations of 1 muM in a modified Krebs-Ringer phosphate buffer, pH 7.4. T4 uptake was markedly slower than that of AIB, Phe, or cLeu over a 25-min period. T4 uptake was neither competitively stimulated nor inhibited by either AIB or Phe when these latter amino acids were varied over the concentration range 1.0 x 10(-6) to 1.0 x 10(-2) M. Preloading cells with 30 mM L-methionine did not enhance the uptake of T4. TJ efflux from preloaded cells was not affected by either AIB or Phe in the extracellular fluid (ECF). The uptakes of AIB, Phe, and cLeu were markedly altered by replacement of ECF Na+ with choline+, whereas T4 uptake was unchanged. It was concluded that neural amino acid transport pathways (specifically A, L, ASC, and beta) do not participate in the transmembrane transfer of L-thyroxine into Ehrlich ascites cells.

Amino Acids↗
PubMed1999

beta-alanine and alpha-fluoro-beta-alanine concentrative transport in rat hepatocytes is mediated by GABA transporter GAT-2.

Studies on the compartmentalization of uridine catabolic metabolism in liver have indicated accumulation of beta-alanine as well as alpha-fluoro-beta-alanine (FbetaAL) for 5-fluorouracil in the hepatocytes. Using preparations of rat hepatocytes we were able to identify a Na+-dependent transport with high affinity for beta-alanine and GABA with Michaelis constant (Km) of 35.3 and 22.5 microM, respectively. A second Na+-dependent kinetic component with Km >1 mM was also identified. The sigmoidal profile of beta-alanine uptake with respect to Na+ shows the involvement of multiple ions of sodium in the transport process. A Hill coefficient of 2.6 +/- 0.4 indicates that at least two sodium ions are cotransported with beta-alanine. The flux of beta-alanine was also shown to be chlorine dependent. The substitution of this anion with gluconate, even in the presence of Na+, reduced the intracellular concentrative accumulation of beta-alanine to passive diffusion level, indicating that both Na+ and Cl- are essential for the activity of this transporter. The transport of beta-alanine was inhibited by GABA, hypotaurine, beta-aminoisobutyric acid, and FbetaAL in a competitive manner. However, concentrations up to 1 mM of L- and D-alanine, taurine, and alpha-aminoisobutyric acid did not affect beta-alanine uptake. Considering the similarities in substrate specificity with the rat GAT-2 transporter, extracts of hepatocytes were probed with the anti-GABA transporter antibody R-22. A 80-kDa band corresponding to GAT-2 was present in the hepatocyte and in the GAT-2 transfected Madin-Darby canine kidney cell extract, confirming the extraneural localization of this transporter. In view of these results, the neurotoxic effects related to the administration of uridine and 5-fluorouracil could be explained with the formation of beta-alanine and FbetaAL and their effect on the cellular reuptake of GABA.

Animals↗
PubMed1995

Inhibition of oxidative insult in cultured cells by a novel 6-chromanol-containing antioxidant.

N18-RE-105 neuronal hybridoma cells were used in a cell culture system to evaluate the protective effects of a novel 6-chromanol-containing antioxidant, U78517F. First, the incorporation of the compound into the cells was evaluated, using a serum albumin carrier. Then the cells were exposed to peroxide-generating compounds, and the cell injury was estimated from the loss of alpha-aminoisobutyric acid (AIB) transport. We found that U78517F only protected the cells significantly when the degree of oxidative insult was below a certain limit; the measurable protection of cells by U78517F against either cumene hydroperoxide or H2O2 was limited to a narrow range of concentrations of the reactive oxygen species generator. Additionally, the protection provided by U78517F was largely localized to the cell membrane and did not extend to protection of mitochondrial function. The action of U78517 was fully consistent with a direct radical scavenging in the cells. The results indicate that the following factors must be taken into account for evaluation of antioxidants in cell culture: (a) the delivery of a compound to cells, especially when the compound is lipophilic; (b) the nature and extent of the oxidative insult used to evaluate protection; and (c) the location of the protective agent in the cells.

Amino Acid Transport Systems↗
PubMed1987

Altered peripheral amino acid uptake in obstructive jaundice.

To characterize amino acid metabolism in obstructive jaundice, the amino acid uptake in tissues of bile duct-ligated (BDL) rats was determined. Fischer 344 rats underwent either bile duct ligation or sham laparotomy and were pair fed for 72 hr. Amino acid uptake was determined in peripheral skeletal muscle (quadriceps femoris, soleus, and rectus abdominis), liver, blood, and other tissues by accumulation of alpha-[14C]-aminoisobutyric acid following intracardiac injection. Although total hepatic amino acid uptake was unaltered in the BDL animals compared with the sham-operated controls, amino acid uptake in peripheral skeletal muscle was significantly decreased in all muscle groups studied in the BDL rats. The relative concentration (percentage dose per gram normalized to the animal mass) for quadriceps femoris was 0.16 +/- 0.02 for BDL and 0.32 +/- 0.04 for sham-operated rats, P less than 0.005. Muscle protein was lower in BDL animals when compared with sham-operated rats (P less than 0.05). Total trunk blood amino acid levels were not significantly different in the two groups; however, there was a decreased serum level of branched-chain amino acids in the BDL group, P less than 0.05. No differences in plasma glucose or serum insulin were found in the two groups; lactate levels were lower in the BDL group, and plasma triglyceride levels were three times higher in the BDL animals. These data suggest that obstructive jaundice in the rat is associated with organ-specific metabolic abnormalities consistent with impaired peripheral amino acid uptake.

Amino Acids↗
PubMed2002

Involvement of active transport systems in the mobilization of cadmium by dithiocarbamates in vivo.

Previously, we reported that the action of cadmium (Cd) complexing dithiocarbamates, such as N-benzyl-D-glucamine dithiocarbamate (BGD) and N-p-hydroxymethylbenzyl-D-glucamine dithiocarbamate (HBGD), in removing Cd from the kidney involves a probenecid-sensitive organic anion transport system. However, other mechanisms responsible for Cd mobilizing effects of BGD and HBGD are still unclear. Therefore, in the present study we examined the effects of phloretin (an inhibitor of plasma membrane glucose carrier), phloridzin (an inhibitor of Na(+)-dependent active hexose transport) and alpha-aminoisobutyric acid (AIB, an inhibitor of amino acid transport) on the excretion and distribution of the chelating agents and Cd in mice. Phloretin pretreatment markedly decreased the biliary and urinary excretions of BGD and HBGD. Phloridzin pretreatment also decreased the biliary and urinary excretions of HBGD, but had no effect on the BGD. AIB pretreatment had no effect on the excretions of either BGD or HBGD. Phloretin pretreatment increased the hepatic and renal contents of BGD and HBGD. Contrary to this, phloridzin pretreatment decreased the hepatic content of BGD and hepatic and renal contents of HBGD, while AIB pretreatment decreased the renal contents of BGD and HBGD. The mobilizing effects of BGD and HBGD on the hepatic and renal Cd was also investigated using Cd-exposed mice. Phloretin or phloridzin pretreatment decreased the mobilizing effect of BGD and HBGD on the hepatic Cd, but had no effect on the renal Cd. These results suggest that BGD and HBGD are taken up into the liver and kidney by phloridzin- and phloretin-sensitive transport system, respectively; that Cd-BGD and Cd-HBGD complexes formed in the hepatic cells are secreted to the bile by phloridzin- and phloretin-sensitive transport systems; and that free BGD and HBGD secreted from these organ to the bile and urine might have occurred, at least in part, by different mechanisms.

Amino Acid Transport Systems↗
PubMed2002

Radiolabeled amino acids for tumor imaging with PET: radiosynthesis and biological evaluation of 2-amino-3-[18F]fluoro-2-methylpropanoic acid and 3-[18F]fluoro-2-methyl-2-(methylamino)propanoic acid.

Novel radiopharmaceuticals, including amino acids, that target neoplasms through their altered metabolic states have shown promising results in preclinical and clinical studies. Two fluorinated analogues of alpha-aminoisobutyric acid, 2-amino-3-fluoro-2-methylpropanoic acid (FAMP) and 3-fluoro-2-methyl-2-(methylamino)propanoic acid (N-MeFAMP), have been radiolabeled with fluorine-18, characterized in amino acid uptake assays, and evaluated in vivo in normal rats and a rodent tumor model. The key steps in the syntheses of both radiotracers involved the preparation of cyclic sulfamidate precursors. Radiosyntheses of both [18F]FAMP and [18F]N-MeFAMP via no-carrier-added nucleophilic substitution provided high yields (>78% decay-corrected) in high radiochemical purity (>99%). Amino acid transport assays using 9L gliosarcoma cells demonstrated that both compounds are substrates for the A type amino acid transport system, with [18F]N-MeFAMP showing higher specificity than [18F]FAMP for A type transport. Tissue distribution studies in normal Fischer rats and Fischer rats implanted intracranially with 9L gliosarcoma tumor cells were also performed. At 60 min postinjection, the tumor vs normal brain ratio of radioactivity was 36:1 in animals receiving [18F]FAMP and 104:1 in animals receiving [18F]N-MeFAMP. On the basis of these studies, both [18F]FAMP and [18F]N-MeFAMP are promising imaging agents for the detection of intracranial neoplasms via positron emission tomography.

Amino Acid Transport System A↗
PubMed1979

Amino acid transport in isolated hepatocytes after partial hepatectomy in the rat.

The effect of partial (70%) hepatectomy on amino acid transport was investigated by measuring the uptake of the nonmetabolizable amino acid analogue alpha-amino-[1(-14C]isobutyric acid in hepatocytes freshly isolated from the liver remnant. In hepatocytes from partially hepatectomized rats, both the influx and the efflux of alpha-aminoisobutyric acid were increased. Quantitative analysis of the relationship between the initial rate of uptake and the substrate concentration indicated that a high-affinity component was responsible for the increased rate of amino acid entry in hepatocytes from partially hepatectomized rats. This component could be detected 90 min after operation, but its Vmax increased with time. The high-affinity component was strictly dependent on sodium and had the properties of a pure A system; its mode of energization partly involved cationic transmembrane gradients. The L system of amino acid transport was not affected after partial hepatectomy. The rapid emergence of a high-affinity amino acid transport system in the liver remnant following partial hepatectomy may play an important role in the regulation of liver regeneration.

Amino Acids↗
PubMed1988

Inhibition of amino acid uptake and incorporation into proteins in Friend erythroleukemia cells by the anthracycline antitumor antibiotic aclacinomycin A.

Treatment of Friend erythroleukemia cells with aclacinomycin A caused a concomitant inhibition of the uptake of 14C-alpha-aminoisobutyric acid (AIB) and of the incorporation of 3H-alanine into proteins. The decrease in amino acid uptake and incorporation into proteins was dose-dependent and reached a maximum of 60% within 3 hours at the concentration of aclacinomycin A, 200 ng/ml. A comparison of the effect on protein incorporation of 3H-alanine and cell proliferation by various anthracycline antitumor antibiotics in a concentration range of 50-200 ng/ml revealed that two other N-alkylated anthracyclines, pyrromycin and marcellomycin, are also potent inhibitors of the incorporation of amino acids into proteins. Inhibition of amino acid incorporation into proteins correlated well with the reduction of cell number at a later time. In contrast, adriamycin and daunomycin inhibited the incorporation of 3H-alanine into proteins only weakly, although these substances were highly active at inhibiting cell proliferation. Studies with an inhibitor of RNA synthesis, actinomycin D, suggest that the concomitant inhibition of amino acid uptake and incorporation into proteins observed with aclacinomycin A is not due to a reduced RNA synthesis. In addition, aclacinomycin A, up to a concentration of 10 micrograms/ml, did not inhibit protein synthesis in a cell-free translational system from rabbit reticulocytes. These results indicate that the reduction of amino acid incorporation into proteins after treatment of Friend erythroleukemia cells with aclacinomycin A may be due to a reduced uptake of amino acids. Inhibition of the transport of 14C-AIB may be indicative for an interaction of aclacinomycin A with the plasma membrane.

Aclarubicin↗
PubMed1976

Sodium-stimulated amino acid uptake into isolated membrane vesicles from Balb/c 3T3 cells transformed by simian virus 40.

Mediated uptake of amino acids by membrane vesicles isolated from Balb/c 3T3 cells transformed by simian virus 40 has been demonstrated. Initial rates of transport of radioactively labeled L-leucine and alpha-aminoisobutyric acid were enhanced by the addition of NaCl (100 mM) to the reaction mixture at the start of the uptake process. This enhancement included a prominent "overshoot" during initial uptake. Slight stimulation of alpha-aminoisobutyric acid uptake was seen with K+, but none with Li+. The mediated nature of the uptake event for L-leucine was shown by saturation kinetics and by inhibition with L-valine. The transport assay measured predominantly intravesicular amino acid uptake rather than binding, as shown by the variation of uptake in response to changes in extravesicular osmolarity. Electron microscopy confirmed the presence of closed vesicles. Thus, amino acid transport has been characterized in an in vitro membrane vesicles system which should prove useful for studies of growth control.

Amino Acids↗
PubMed1983

Consecutive beta-turn conformation of a synthetic model peptide corresponding to the active site of thioredoxin.

A synthetic model peptide, (formula; see text) which mimics the active-site disulfide loop of thioredoxin has been prepared. 270 MHz 1H-NMR studies establish that Cys-4 and methylamide NH groups are solvent-shielded, using hydrogen-deuterium exchange, solvent and temperature dependence of chemical shifts and nitroxide radical-induced broadening as diagnostic criteria. Infrared measurements provide supporting evidence for intramolecularly hydrogen-bonded conformations. The related peptide in which Gly-2 is replaced by alpha-aminoisobutyric acid has been shown to adopt a similar backbone conformation based on NMR and CD data. Based on the known stereochemical preferences of alpha-aminoisobutyric acid residues, a consecutive beta-turn conformation involving two intramolecular 4 leads to 1 hydrogen bonds is proposed for both disulfides. Vicinal coupling constants and CD data are discussed with reference to the side-chain conformation of the cysteine residues. Large structural differences have been established between the thioredoxin active-site model disulfide and its acyclic precursor.

Bacterial Proteins↗
PubMed1982

Binding and action of insulin and glucagon in monolayer cultures and fresh suspensions of rat hepatocytes.

Insulin and glucagon binding, and the subsequent stimulation of amino-acid transport, were investigated in adult-rat hepatocytes. Cells were used either in suspension shortly after isolation, or as monolayers after 20 h of culture in a serum-free medium. At 37 degrees C, hepatocytes in monolayer cultures bound 2.5 times as much insulin and glucagon as did freshly isolated cells, owing to an increase in the total number of binding sites per cell. For both hormones, these differences could be accounted for mainly by a greater number of low-affinity binding sites in primary cultured hepatocytes compared with freshly isolated cells. Exposure of hepatocytes to insulin or glucagon for 2-3 h at 37 degrees C in a medium free from amino acids increased the capacity (primary cultures) or induced the emergence (fresh suspensions) of a similar high-affinity component (Km approximately mM) of alpha-aminoisobutyric-acid (AIB) transport. Primary cultured hepatocytes were more sensitive to insulin (half-maximal effect occurred with insulin at approximately 0.3 nM) than freshly isolated cells (half-maximal effect approximately 0.7 nM) for the stimulation of AIB transport, whereas the dose-response curves were virtually indistinguishable for the glucagon stimulation of AIB transport in both preparations of cells (half-maximal effect occurred with glucagon at approximately 1.5 nM). These results indicate that, despite differences in the apparent insulin- and glucagon-binding capacities (which involved mainly a low affinity site), both freshly isolated and primary cultured (20-h monolayers) hepatocytes behave similarly in response to insulin and glucagon with regard to the stimulation of amino acid transport.

Amino Acids↗
PubMed1977

Derepression of amino acid transport by amino acid starvation in rat hepatoma cells.

Amino acid starvation causes an adaptive increase in the initial rate of transport of selected neutral amino acids in an established line of rat hepatoma cells in tissue culture. After a lag of 30 min, the initial rate of transport of alpha-aminoisobutyric acid (AIB) increases to a maximum after 4 to 6 h starvation of 2 to 3 times that seen in control cells. The increased rate of transport is accompanied by an increase in the Vmax and a modest decrease in the Km for this transport system, and is reversed by readdition of amino acids. The enhancement is specific for amino acids transported by the A or alanine-preferring system (AIB, glycine, proline); uptake of amino acids transported by the L or leucine-preferring system (threonine, phenylalanine, tyrosine, leucine) or the Ly+ system for dibasci amino acids (lysine) is decreased under these conditions. Amino acids which compete with AIB for transport also prevent the starvation-induced increase in AIB transport; amino acids which do not compete fail to prevent the enhancement. Paradoxically threonine, phenylalanine, tryptophan, and tyrosine, which do not compete with AIB for transport, block the enhancement of transport upon amino acid starvation. The starvation-induced enhancement of amino acid transport does not appear to be the result of a release from transinhibition. After 30 min of amino acid starvation, AIB transport is either unchanged or slightly decreased even though amino acid pools are already depleted. Furthermore, loading cells with high concentrations of a single amino acid following a period of amino acid starvation fails to prevent the enhancement of AIB transport, whereas incubation of the cells with the single amino acid for the entire duration of amino acid starvation prevents the enhancement; intracellular amino acid pools are similar under both conditions. The enhancement of amino acid transport requires concomitant RNA and protein synthesis, consistent with the view that the adaptive increase reflects an increased amount of a rate-limiting protein involved in the transport process. Dexamethasone, which dramatically inhibits AIB transport in cells incubated in amino acid-containing medium, both blocks the starvation-induced increase in AIB transport, and causes a time-dependent decrease in transport velocity in cells whose transport has previously been enhanced by starvation.

Amino Acids↗
PubMed1983

The use of HgCl2 to evaluate the cosubstrate: amino acid transport stoichiometry in Ehrlich ascites tumor cells.

Recent investigations have indicated that cellular rheogenic properties may interfere with the correct estimation of Na+ and amino transport stoichiometry. We have reevaluated the stoichiometry of Na+ and alpha-aminoisobutyric acid (alpha-AIB) cotransport in Ehrlich ascites tumor cells depleted of Na+ and ATP by incubation in Na+-free HEPES-buffered medium (pH 7.2) containing 160 mM K+ and 2.5 microM valinomycin. Transfer of the cells to a medium with 10 mM 22Na+, 10 mM 3H-AIB, and 150 mM K+ resulted in an enhancement of Na+ flux above basal levels, which represents 0.6 of the AIB uptake. Under these conditions the membrane potential, -7.0 +/- 0.1 mV (SEM), does not change with the addition of AIB, -7.3 +/- 0.6 mV (SEM). HgCl2 (10 microM) added to the medium inhibited AIB flux and AIB-stimulated Na+ flux by 45-50% but did not change the coupling ratio. HgCl2 (10 microM) does not inhibit the basal Na+ flux nor does it affect cellular Na+ or K+ content. In physiological medium cotransport is electrogenic. The membrane potential of Ehrlich cells in physiological medium is -22.3 +/- 0.8 mV (SEM) and depolarizes to -16.7 +/- 0.7 mV (SEM) upon addition of AIB. Under these conditions the coupling ratio was highly variable but the ratio of codepression is 0.90 +/- 0.02 (SEM) in the presence of HgCl2 (10 microM). These results are consistent with a model (Smith and Robinson, 1981) in which the stoichiometry is one cosubstrate molecule per molecule of alpha-AIB. We suggest that H+ provides the alternative cosubstrate in this low Na+ environment and that in high Na+ medium the Na+:AIB stoichiometry approaches 1:1.

Amino Acids↗