The inhibitory effect of choline and other quaternary ammonium compounds on thiamine transport in isolated rat hepatocytes

Quaternary ammonium compounds, such as choline and acetylcholine significantly inhibited thiamine uptake in isolated rat hepatocytes. Kinetic analysis using Lineweaver-Burk and Dixon plots of inhibition experiments revealed that choline and acetylcholine were purely competitive inhibitors for thiamine uptake with Ki values of 0.61 mM and 0.31 mM, respectively. Among quaternary ammonium compounds, hemicholinium-3 and curare were the strongest inhibitors, and kinetic studies showed that these compounds were also purely competitive inhibitors with Ki values of 12.5 microM and 4.3 microM, respectively. These results indicate that choline, acetylcholine and their structural analogs share a common binding site with thiamine in isolated rat hepatocytes. On the other hand, choline uptake by isolated rat hepatocytes occurred by a saturable mechanism with a Kt of 162 +/- 3.85 microM and Vmax of 80.1 +/- 1.30 pmol/10(5) cells per min as well as by a nonsaturable mechanism. Thiamine, pyrithiamine, oxythiamine, chloroethylthiamine and dimethialium inhibited choline uptake, while thiamine phosphates such as thiamine monophosphate and thiamine pyrophosphate insignificantly inhibited uptake. Although a Lineweaver-Burk plot of choline uptake in the presence of thiamine showed that thiamine also competitively inhibited choline uptake, a Dixon plot of the inhibition experiment was hyperbolic and indicated that the inhibition of choline uptake by thiamine was 'pseudo-competitive'. On the basis of these results, it is suggested that in isolated rat hepatocytes thiamine and choline do not share common transport sites.     

Active transport of dimethialium in isolated rat hepatocytes

The uptake of dimethialium, a thiamine analog having a methyl group in place of the hydroxyethyl group in the thiazole moiety, was studied in freshly isolated rat hepatocytes. In an Na+-medium, dimethialium at 10 microM was accumulated rapidly by the cells and an almost steady intra- to extracellular distribution ratio of 4.2 was attained in 5 min of incubation. The Kt and the Vmax for the saturable component were estimated to be 27 microM and 19 pmol/10(5) cells per min, respectively. In a K+ medium, the uptake of dimethialium was decreased to 58% of that of control. Ouabain and 2,4-dinitrophenol significantly lowered the rate of dimethialium uptake. Both phenylthiazinothiamine and oxythiamine were inhibitory on the uptake of dimethialium, which uptake was also inhibited by choline. These data indicate that dimethialium transport in liver cells proceeds via a carrier-mediated active process dependent on Na+ and biological energy. Furthermore, these results also suggest that thiamine transport in liver is dissociable from thiamine phosphorylation.     

Osmoregulation in Rhodobacter sphaeroides.

Betaine (N,N,N-trimethylglycine) functioned most effectively as an osmoprotectant in osmotically stressed Rhodobacter sphaeroides cells during aerobic growth in the dark and during anaerobic growth in the light. The presence of the amino acids L-glutamate, L-alanine, or L-proline in the growth medium did not result in a significant increase in the growth rate at increased osmotic strengths. The addition of choline to the medium stimulated growth at increased osmolarities but only under aerobic conditions. Under these conditions choline was converted via an oxygen-dependent pathway to betaine, which was not further metabolized. The initial rates of choline uptake by cells grown in media with low and high osmolarities were measured over a wide range of concentrations (1.9 microM to 2.0 mM). Only one kinetically distinguishable choline transport system could be detected. Kt values of 2.4 and 3.0 microM and maximal rates of choline uptake (Vmax) of 5.4 and 4.2 nmol of choline/min.mg of protein were found in cells grown in the minimal medium without or with 0.3 M NaCl, respectively. Choline transport was not inhibited by a 25-fold excess of L-proline or betaine. Only one kinetically distinguishable betaine transport system was found in cells grown in the low-osmolarity minimal medium as well as in a high-osmolarity medium containing 0.3 M NaCl. In cells grown and assayed in the absence of NaCl, betaine transport occurred with a Kt of 15.1 microM and a Vmax of 3.2 nmol/min . mg of protein, whereas in cells that were grown and assayed in the presence of 0.3 M NaCl, the corresponding values were 18.2 microM and 9.2 nmol of betaine/min . mg of protein. This system was also able to transport L-proline, but with a lower affinity than that for betaine. The addition of choline of betaine to the growth medium did not result in the induction of additional transport systems.     

Uptake of thymidine into isolated rat hepatocytes. Evidence for two transport systems

Thymidine transport was studied in isolated rat hepatocytes. In these cells no phosphorylation of the substrate by thymidine kinase occurred subsequent to transport. Results from studies of the concentration-dependent uptake of thymidine indicated two transport systems with about 80-fold differences in their kinetic constants. These systems were denoted as high affinity [Km = 5.3 micron, V = 0.47 pmol/(10(6) cells X s)] and low affinity systems [Km = 480 micron, V = 37.6 pmol/(10(6) cells X s)]. From intracellular to extracellular distribution ratios of [3H]thymidine it could be concluded that the uptake by the high affinity system was a concentrative process while the transport by the low affinity system was non-concentrative. The uptake of [3H]-thymidine by the high affinity system could only be inhibited by unlabeled thymidine. In contrast, all other nucleosides tested (uridine, 2'-deoxycytidine, and 2'-deoxyguanosine) were equally effective in inhibiting the low affinity system competitively. The results would suggest that in hepatocytes lacking phosphorylation by thymidine kinase, thymidine is taken up by a high and a low affinity system working in tandem. The high affinity system seems to be an active transport process with narrow substrate specificity. Thymidine uptake by the low affinity system is a facilitated diffusion process. This system is considered to be a common transport route for nucleosides of different structures.     

Inositol Metabolism During Neuroblastoma B50 Cell Differentiation: Effects of Differentiating Agents on Inositol Uptake

Inositol uptake was studied in the rat CNS neuroblastoma B50 cell line. Eadie-Hofstee analysis of the uptake pattern reveals two defined modes of inositol entry into the cell. The high-affinity uptake component requires the presence of extracellular sodium and is inhibited by phloridzin. Analysis of the uptake velocities of the high-affinity uptake component provided the following apparent kinetic parameters: Km = 13.7 microM and Vmax = 14.7 pmol/mg of protein/min (without correcting for residual diffusion) and Km = 12.9 microM and Vmax = 12.3 pmol/mg of protein/min (with correction). At physiological concentrations, the high-affinity transport process contributes approximately 70% to total uptake; the remainder is due to a low-affinity diffusion-like process. Uptake inhibition studies reveal that the uptake process is sensitive to ouabain, amiloride, and dichlorobenzamil inhibition but relatively insensitive to cytochalasin B or phloretin. When neuroblastoma B50 cells are induced to differentiate morphologically with high extracellular calcium or with dibutyryl cyclic AMP, a significant decrease in inositol uptake is observed. The dibutyryl cyclic AMP-mediated inhibition of uptake affects only the high-affinity uptake component and is noncompetitive in nature. The high extracellular calcium-mediated inhibition is less specific; it involves "disappearance" of the high-affinity process, some inhibition of the low-affinity process, and an increase of inositol efflux. The significance of these observations is discussed in the context of neuroblastoma B50 cell differentiation.     

Biochemistry and regulation of folate and methotrexate transport in Leishmania major

Promastigotes of the protozoan parasite Leishmania major exhibit high affinity uptake of folate (Kt = 0.7 microM) and methotrexate (MTX) (Kt = 1.8 microM) which is saturable and sensitive to metabolic poisons. Influx of folate and MTX is competitively inhibited by 5-formyltetrahydrofolate and p-aminobenzoic acid-glutamate, but not by 4-deoxy-4-amino-10-methylpteroate, biopterin, or pteroate. A single carrier is inferred for both folate and MTX transport, as the Ki of each inhibitor for both folate and MTX influx is the same, and the apparent affinities (Kt) of the substrates folate and MTX are identical to their respective Ki values for inhibition of MTX and folate uptake. Folate influx is specifically regulated according to cellular growth phase, as stationary phase cells exhibit 7% of the Vmax of log phase cells, while energy-dependent glucose uptake is only moderately reduced in stationary phase. Folate influx is also regulated by external folate levels, as cells grown in 5 microM folate exhibit 30% of the Vmax of cells grown in folate-depleted medium. Comparison of bacterial, mammalian, and Leishmania folate transport activities indicates considerable diversity in both biochemical and regulatory properties, and suggests the possibility that selective inhibition or manipulation of folate transport may be exploited in parasite chemotherapy.     

Mathematical analysis and experimental study of uridine transport and phosphorylation in 3T6 cells

A mathematical model has been analysed describing uridine uptake in mammalian cells as a tandem process that involves membrane transport and uridine phosphorylation within the cell. The measurement of kinetic parametres of uridine uptake in 3T6 cells showed that the transport system possesses a low affinity to uridine (Kt = 145 microM) and a high velocity (Vt = 10 microM/sec), whereas the phosphorylation system possesses a high affinity for uridine (Ke = 10 microM) and a low velocity (Ve = 0.17 microM/sec). A method of construction of "ideal" curves was proposed, describing the time dependence of uridine uptake which helps to verify values of kinetic parameters obtained. On the basis of the theoretical analysis and generalization of experimental data it was concluded that the optimum conditions of uridine transport parameters measuring at 25 degrees C involve the uridine concentration in the medium equal to 20-200 microM, and the time of cell incubation, 2-20 sec, while the optimum conditions of uridine phosphorilation parameters measuring being its concentration in the medium 5-20 microM and the cell incubation longer than 1 minute.     

The high and low affinity transport systems for dipeptides in kidney brush border membrane respond differently to alterations in pH gradient and membrane potential

The principal aim of the present study was to investigate the effects of variation in proton gradient and membrane potential on the transport of glycyl-L-glutamine (Gly-Gln) by renal brush border membrane vesicles. Under our conditions of transport assay, Gly-Gln was taken up by brush border membrane vesicles almost entirely as intact dipeptide. This uptake was mediated by two transporters shared by other dipeptides and characterized as the high affinity (Kt = 44.1 +/- 11.2 microM)/low capacity (Vmax = 0.41 +/- 0.03 nmol/mg protein/5 s) and low affinity (Kt = 2.62 +/- 0.50 mM)/high capacity (Vmax 4.04 +/- 0.80 nmol/mg protein/5 s) transporters. In the absence of a pH gradient, only the low affinity system was operational, but with a reduced transport capacity. Imposing a pH gradient of 1.6 pH units increased the Vmax of both transporters. Kinetic analysis of the rates of Gly-Gln uptake as a function of external pH revealed Hill coefficients of close or equal to 1, indicating that transporters contain only one binding site for the interaction with external H+. The effects of membrane potential on Gly-Gln uptake were investigated with valinomycin-induced K+ diffusion potentials. The velocity of the high affinity system but not of the low affinity system increased linearly with increasing inside-negative K+ diffusion potentials (p less than 0.01). The Kt of neither system was affected by alterations in either pH gradient or membrane potential. We conclude that (a) the high affinity transporter is far more sensitive to changes in proton gradient and membrane potential than the low affinity transporter and (b) in the presence of a pH gradient, transport of each dipeptide molecule requires cotransport of one hydrogen ion to serve as the driving force.     

Blood–Brain Transport of Thiamine Monophosphate in the Rat: A Kinetic Study In Vivo

To calculate the kinetic parameters of thiamine monophosphate transport across the rat blood-brain barrier in vivo, different doses of a [35S]thiamine monophosphate preparation with a specific activity of 14.8 mCi.mmol-1 were injected in the femoral vein and the radioactivity was measured in arterial femoral blood and in the cerebellum, cerebral cortex, pons, and medulla 20 s after the injection. This short experimental time was used to prevent thiamine monophosphate hydrolysis. Thiamine monophosphate was transported into the nervous tissue by a saturable mechanism. The maximal transport rate (Jmax) and the half-saturation concentration (Km) equaled 27-39 pmol.g-1.min-1 and 2.6-4.8 microM, respectively. When compared with that of thiamine, thiamine monophosphate transport seemed to be characterized by a lower affinity and a lower maximal influx rate. At physiological plasma concentrations, thiamine monophosphate transport rate ranged from 2.06 to 4.90 pmol.g-1.min-1, thus representing a significant component of thiamine supply to nervous tissue.     

A membrane transporter mediates access of uridine 5'-diphosphoglucuronic acid from the cytosol into the endoplasmic reticulum of rat hepatocytes: implications for glucuronidation reactions

Hepatic glucuronidation of a wide variety of substrates is catalyzed by the membrane-bound UDP-glucuronosyltransferases. Uridine 5'-diphosphoglucuronic acid (UDP-GlcUA) is the essential cosubstrate for all UDP-glucuronosyltransferase-mediated reactions. The mechanism by which this bulky, hydrophilic nucleotide-sugar is transported from the cytosol (where it is synthesized) to its binding site(s) on the enzyme is unknown. To determine whether a membrane carrier mediates the access of UDP-GlcUA into the endoplasmic reticulum, the transport of uridine 5'-diphospho-D-[U-14C]glucuronic acid into vesicles of rough and smooth endoplasmic reticulum isolated from rat liver was investigated at 38 degrees C using a rapid filtration technique. Uptake of UDP-GlcUA by both rough and smooth vesicles was extremely rapid (linear for only 10-20 s) and temperature-dependent (negligible at 4 degrees C). UDP-GlcUA uptake was saturable, and similar kinetic parameters were obtained for rough and smooth vesicles (Km 1.9 microM, Vmax 443 pmol/mg protein per min, and Km 1.3 microM, Vmax 503 pmol/mg protein per min, respectively). The uptake of UDP-GlcUA also exhibited a high degree of specificity, since many related compounds, including UMP, UDP and UDP-Glc, did not influence uptake. In addition, the non-penetrating inhibitors of anion transport, 4-acetamido-4'-isothiocyanostilbene-2,2'-disulfonic acid (SITS), 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (DIDS), and probenecid, markedly inhibited UDP-GlcUA uptake. Finally, osmotic modulation of the intravesicular volume did not affect total uptake of UDP-GlcUA by membrane vesicles at equilibrium, indicating that this nucleotide-sugar is transported into the membrane rather than the intravesicular space. Collectively, these data provide direct evidence for a specific, carrier-mediated uptake process, which transports UDP-GlcUA from the cytosol into the endoplasmic reticulum of hepatocytes. This UDP-GlcUA transporter may be involved in the regulation of hepatic glucuronidation reactions.     

Biotin uptake: influx, efflux and countertransport in Escherichia coli K12

Biotin uptake by Escherichia coli K12 has been reinvestigated. The vitamin uptake is an active process depending on energy and inhibited by uncouplers. The kinetic parameters (Km = 0.27 microM, Vmax = 6.8 pmol/min per mg dry cells) are close to those previously determined for a biotin-dependent strain E. coli C162 (Piffeteau, A., Zamboni, M. and Gaudry, M. (1982) Biochim. Biophys. Acta 688, 29-36). By use of biotin p-nitrophenyl ester, an affinity label of the biotin transport system, it was shown, under conditions of steady state, that the efflux of biotin is not energy dependent and is mainly mediated by a diffusion mechanism. Reexamination of the regulation of the biotin transport by biotin, revealed that only 50% of the biotin uptake system is under control by the vitamin.     

Mechanism of copper transport from plasma to hepatocytes

The effects of plasma components on the kinetics of copper transport by rat hepatocytes were examined in an attempt to determine how copper is mobilized from plasma for uptake by the liver. Specific protein-facilitated transport was indicated by saturation kinetics, competition by related substrates, and similar kinetic parameters for uptake and efflux. For copper uptake, Km = 11 +/- 0.6 microM and Vmax = 2.7 +/- 0.6 nmol Cu/(min X mg protein). Zinc is a competitive inhibitor of copper uptake, and copper competes for zinc uptake. Copper efflux from preloaded cells is biphasic. The kinetic parameters for the initial rapid phase are similar to the parameters for uptake. Copper transport by hepatocytes is strictly passive. A variety of metabolic inhibitors have no effect on uptake and initial rates are solely dependent on extracellular-intracellular concentration gradients. Albumin markedly inhibits copper uptake by a substrate removal mechanism, and histidine facilitates albumin-inhibited copper uptake. The active species that delivers copper to hepatocytes under conditions of excess albumin and excess histidine is the His2Cu complex. Experiments with [3H]His2 64Cu showed that the transported species is free ionic copper. The kinetic parameters of copper transport by hepatocytes isolated from the brindled mouse model of Menkes' disease are normal. However, these cells show a decreased capacity to accumulate copper on prolonged incubation. An intracellular metabolic defect seems to be involved.     

Leucine transport in membrane vesicles from Chironomus riparius larvae displays a mélange of crown-group features

Leucine uptake into membrane vesicles from larvae of the midge Chironomus riparius was studied. The membrane preparation was highly enriched in typical brush border membrane enzymes and depleted of other membrane contaminants. In the absence of cations, there was a stereospecific uptake of l-leucine, which exhibited saturation kinetics. Parameters were determined both at neutral (Km 33 +/- 5 microM and Vmax 22.6 +/- 6.8 pmol/7s/mg protein) and alkaline (Km 46 +/- 5 microM and Vmax 15.5 +/- 2.5 pmol/7s/mg protein) pH values. At alkaline pH, external sodium increased the affinity for leucine (Km 17 +/- 1 microM) and the maximal uptake rate (Vmax 74.0 +/- 12.5 pmol/7s/mg protein). Stimulation of leucine uptake by external alkaline pH agreed with lumen pH measurements in vivo. Competition experiments indicated that at alkaline pH, the transport system readily accepts most L-amino acids, including branched, unbranched, and alpha-methylated amino acids, histidine and lysine, but has a low affinity for phenylalanine, beta-amino acids, and N-methylated amino acids. At neutral pH, the transport has a decreased affinity for lysine, glycine, and alpha-methylleucine. Taken together, these data are consistent with the presence in midges of two distinct leucine transport systems, which combine characters of the lepidopteran amino acid transport system and of the sodium-dependent system from lower neopterans.     

Further characterization of adenosine transport in renal brush-border membranes

Adenosine transport has been further characterized in rat renal brush-border membranes (BBM). The uptake shows two components, one sodium-independent and one sodium-dependent. Both components reflect, at least partly, translocation via a carrier mechanism, since the presence of adenosine inside the vesicles stimulates adenosine uptake in the presence as well as in the absence of sodium outside the vesicles. The sodium-dependent component is saturable (Km adenosine = 2.9 microM, Vmax = 142 pmol/min per mg protein) and is abolished at low temperatures. The sodium-independent uptake has apparently two components: one saturable (Km = 4-10 microM, Vmax = 174 pmol/min per mg protein) and one non-saturable (Vmax = 3.4 pmol/min per mg protein, Km greater than 2000 microM). Inosine, guanosine, 2-chloroadenosine and 2'-deoxyadenosine inhibit the sodium-dependent and -independent transport, as shown by trans-stimulation experiments, probably because of translocation via the respective transporter. Uridine and dipyridamole inhibited only the sodium-dependent uptake. Other analogs of adenosine showed no inhibition. The kinetic parameters of the inhibitors of the sodium-dependent component were further investigated. Inosine was the most potent inhibitor with a Ki (1.9 microM) less than the Km of adenosine. This suggests a physiological role for the BBM ecto-adenosine deaminase (enzyme which extracellularly converts adenosine to inosine), balancing the amount of nucleoside taken up as adenosine or inosine by the renal proximal tubule cell.     

Characteristics of taurine transport in rat hepatocytes in primary culture

Characteristics of taurine transport in rat hepatocytes maintained in primary culture for 24 h (cultured hepatocytes) have been investigated. The uptake of [3H] taurine by cultured hepatocytes at 2 degrees C was unsaturable, whereas that at 37 degrees C consisted of unsaturable and saturable processes. The saturable transport system was sodium-dependent and consisted of two processes with low and with high affinities. The latter process (Km, 76.9 microM; Vmax, 0.256 nmole/mg protein/min; activation energy (EA), 37.8 kcal mol-1) was competitively inhibited by 2,4-dinitrophenol and ouabain, as well as by taurine analogues such as hypotaurine and guanidinoethyl sulphonate. The Vmax and EA values found in cultured hepatocytes at 37 degrees C were 6.0 and 6.8 times higher than those found in freshly isolated hepatocytes. These results indicate that taurine transport in hepatocytes in primary culture consisted of unsaturable, and saturable, sodium and energy-dependent carrier-mediated transport processes, respectively. The facilitation of the latter transport system by primary culture of hepatocytes is also suggested.     

L-carnitine uptake in differentiating human cultured muscle

We studied carnitine uptake in human skeletal muscle growing in culture for up to 30 days, and correlated it to the degree of muscle differentiation revealed by myotube formation and muscle-specific creatine-kinase isozyme accumulation. In our study carnitine uptake was a saturable specific process with two distinct components: a high affinity uptake at carnitine concentration between 0.5 and 10 microM and a low affinity uptake at carnitine concentration between 25 and 200 microM. High affinity uptake (Km 4.17-5.50 microM, Vmax 11.78-19.6 pmol/h per mg protein) did not change during muscle maturation in culture. Low affinity uptake showed significant changes in Km and Vmax in the various stages of muscle differentiation. Our studies suggest the existence of a muscle-specific system, operating at physiological carnitine concentration, which gradually develops during muscle maturation in culture. We hypothesize that a defect of the low affinity muscle-specific uptake might be the cause of the primary muscle carnitine deficiency syndrome.     

Active transport of amino acids in Thiobacillus thioparus is a low-affinity process.

A method for the isolation of amino acid auxotrophs of Thiobacillus thioparus is described. Characterization of a leucine auxotroph indicated that leucine biosynthesis in T. thioparus was not different from that of heterotrophic bacteria. T. thioparus cells accumulated amino acids via an active mechanism. Kt values of amino acid transport were between 15 and 330 microM, and Vmax values were 200 to 350 pmol min-1 mg of protein-1. Amino acid transport was carried out by a limited number of systems, each responsible for the uptake of several amino acids. Amino acid auxotrophs of T. thioparus exhibited transport and growth properties similar to those of transport-deficient mutants of heterotrophs which lost the high-affinity, but retained the low-affinity, amino acid transport systems.     

Transport of 2-oxoisocaproate in isolated hepatocytes and liver mitochondria

The transport of 2-oxoisocaproate into isolated hepatocytes and liver mitochondria of rat was studied using [U-14C]2-oxoisocaproate and the silicone oil filtration procedure. 2-Oxoisocaproate uptake by hepatocytes was composed of: rapid adsorption, unmediated diffusion and carrier-mediated transport. The carrier-mediated transport was strongly inhibited by 4,4'-diisothiocyano-2,2'-stilbenedisulphonic acid and p-chloromercuribenzoate, was less sensitive to alpha-cyano-4-hydroxycinnamate and insensitive to p-chloromercuriphenylsulphonate. Other 2-oxo acids: pyruvate, 2-oxoisovalerate and 2-oxo-3-methylvalerate, were also inhibitory. The kinetic parameters of the carrier-mediated transport were Km 30.6 mM and Vmax 23.4 nmol/min per mg wet wt, at 37 degrees C. It is concluded that at its low, physiological, concentration, 2-oxoisocaproate penetrates the hepatocyte membrane mainly by unmediated diffusion. The uptake of 2-oxoisocaproate by isolated liver mitochondria was partly inhibited by alpha-cyano-4-hydroxycinnamate, the inhibitor of mitochondrial monocarboxylate carrier. The remaining uptake was linearly dependent on 2-oxoisocaproate concentration and represented unmediated diffusion. The carrier-mediated transport exhibited the following kinetic parameters: Km 0.47 mM, Vmax 1.0 nmol/min per mg protein at 6 degrees C; and Km 0.075 mM and Vmax about 8 nmol/min per mg protein at 37 degrees C.     

Inward fluxes of adenosine in erythrocytes and cultured cells measured by a quenched-flow method.

Dilazep, a vasodilator previously recognized as an inhibitor of adenosine permeation, very rapidly blocked the uptake of adenosine by cultured L5178Y cells, and accordingly was used as a quencher in a simple quenched-flow system for measuring cellular uptake of nucleosides during very short intervals. Time courses of cellular uptake of adenosine, assayed during intervals between 0.05 and 0.5s with the quenched-flow system, were linear and defined initial rates of adenosine uptake. The latter are rates of inward transport of adenosine. Kinetic constants for that process in cultured S49 cells determined with the quenched-flow procedure were similar to those determined with an assay dependent on manual timing. In studies of adenosine uptake kinetics in human erythrocytes at 22 degrees C and 37 degrees C in which the quenched-flow procedure was used, time courses of adenosine uptake were linear at both temperatures and defined initial uptake rates; kinetic constants (means +/- S.E.M.) at 22 degrees C (n = 8) were Km 25 +/- 14 microM and Vmax. 15 +/- 5 pmol/s per microliter of cell water and at 37 degrees C (n = 3) were Km 98 +/- 17 microM and Vmax. 80 +/- 9 pmol/s per microliter of cell water.     

Sodium ion-dependent amino acid transport in membrane vesicles of Bacillus stearothermophilus.

Amino acid transport in membrane vesicles of Bacillus stearothermophilus was studied. A relatively high concentration of sodium ions is needed for uptake of L-alanine (Kt = 1.0 mM) and L-leucine (Kt = 0.4 mM). In contrast, the Na(+)-H(+)-L-glutamate transport system has a high affinity for sodium ions (Kt less than 5.5 microM). Lithium ions, but no other cations tested, can replace sodium ions in neutral amino acid transport. The stimulatory effect of monensin on the steady-state accumulation level of these amino acids and the absence of transport in the presence of nonactin indicate that these amino acids are translocated by a Na+ symport mechanism. This is confirmed by the observation that an artificial delta psi and delta mu Na+/F but not a delta pH can act as a driving force for uptake. The transport system for L-alanine is rather specific. L-Serine, but not L-glycine or other amino acids tested, was found to be a competitive inhibitor of L-alanine uptake. On the other hand, the transport carrier for L-leucine also translocates the amino acids L-isoleucine and L-valine. The initial rates of L-glutamate and L-alanine uptake are strongly dependent on the medium pH. The uptake rates of both amino acids are highest at low external pH (5.5 to 6.0) and decline with increasing pH. The pH allosterically affects the L-glutamate and L-alanine transport systems. The maximal rate of L-glutamate uptake (Vmax) is independent of the external pH between pH 5.5 and 8.5, whereas the affinity constant (Kt) increases with increasing pH. A specific transport system for the basic amino acids L-lysine and L-arginine in the membrane vesicles has also been observed. Transport of these amino acids occurs most likely by a uniport mechanism.