Kinetics of D-glucose transport across the intestinal brush-border membrane of the cat

1. D-glucose transport across the intestinal brush-border membrane of the cat, a carnivorous animal, was investigated using isolated brush-border membrane vesicles (BBMV). Kinetic experiments were performed under zero-trans conditions (initial [Na+]in and [Gluc]in = O) with the transmembrane electrical potential difference clamped to zero. 2. D-glucose uptake by the BBMV was strongly stimulated by an inwardly directed Na+-gradient. Uptake under Na+-free conditions seemed to occur by simple diffusion. 3. The apparent kinetic constants (Vmax, Km) of Na+-dependent D-glucose transport were computed by forcing initial uptake rates at 0.002-10.0 mmol/l D-glucose to either a Michaelis-Menten type equation with a single or with two carrier-mediated components. 4. Best fit of the experimental data was obtained with the two-component model indicating the existence of two Na+-dependent carrier-mediated mechanisms. System 1 and system 2 differ with respect to the transport velocity as well as the substrate affinity constants with Vmax being 2.5-fold and Km being 5-fold higher for system 1 compared with system 2.     

Amino acid transport by the helicoidal colon of the new-born pig.

The proximal colon of the new-born pig maintains a stable short-circuit current which is partly dependent upon the presence of methionine. This interaction betweeen methionine and short-circuit current shows Michaelis-Menten knetics with a Km of 0.24 mM and a V of 27 muA.cm-2. The net flux of methionine to the serosal surface of proximal colons also shows a hyperbolic relation to the external concentration of methionine (Km 0.38 mM; V 10.4 nmol.cm-2. min-1). The proximal colon concentrates methionine within its epithelium giving a mucosal to medium ratio of 11.2 +/- 1.9 (90 min incubation in 1 mM methionine). The ability of the colon to transport methionine across and concentrate methionine within its mucosa is maintained for at least 24 h after birth. Colonic transport of amino acids could be physiologically important in the pig, where the immediate post-natal transfer of immune globulins has been shown to cause a temporary inhibition of normal intestinal function.     

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.     

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.     

Characterization of basal and methylxanthine-stimulated Ca2+ transport in abalone spermatozoa

Methylxanthines, such as 1-methyl-3-isobutylxanthine (MIX) and theophylline, stimulate abalone sperm 45Ca2+ uptake in a time- and concentration-dependent manner. MIX is the most potent compound tested, and the ability of these compounds to alter 45Ca2+ uptake resides with methyl or isobutyl substitution of the xanthine nucleus at multiple sites. Methylxanthine-stimulated 45Ca2+ uptake does not occur as a secondary consequence of cyclic nucleotide phosphodiesterase inhibition, and added cyclic nucleotides are also without effect. The dramatic elevation of intracellular cAMP concentrations and induction of the acrosome reaction of sperm incubated with methylxanthines in the presence of Ca2+ is mediated by a primary effect of methylxanthines on Ca2+ transport. Basal 45Ca2+ uptake occurs through a verapamil-insensitive site that obeys the properties of a simple diffusion-mediated process. MIX-stimulated 45Ca2+ uptake occurs through a carrier-mediated transport site that has low affinity for Ca2+ (Km = 19.9 mM) and is verapamil sensitive. 45Ca2+ uptake through both basal and MIX-stimulated sites is enhanced by low extracellular Na+ concentrations (less than or equal to 15 mM) and is not affected by either extracellular Mg2+ or K+ X 45Ca2+ uptake through both sites is pH sensitive, but this sensitivity is different for each site. These data suggest that methylxanthines can affect sperm function via primary effects on Ca2+ transport, which occur through a specific carrier-mediated site(s). It is possible that many of the previously described effects of methylxanthines on sperm function are mediated via such changes in Ca2+ conductance.     

Transport of malic acid and other dicarboxylic acids in the yeast Hansenula anomala

DL-Malic acid-grown cells of the yeast Hansenula anomala formed a saturable transport system that mediated accumulative transport of L-malic acid with the following kinetic parameters at pH 5.0: Vmax, 0.20 nmol.s-1.mg (dry weight)-1; Km, 0.076 mM L-malate. Uptake of malic acid was accompanied by proton disappearance from the external medium with rates that followed Michaelis-Menten kinetics as a function of malic acid concentration. Fumaric acid, alpha-ketoglutaric acid, oxaloacetic acid, D-malic acid, and L-malic acid were competitive inhibitors of succinic acid transport, and all induced proton movements that followed Michaelis-Menten kinetics, suggesting that all of these dicarboxylates used the same transport system. Maleic acid, malonic acid, oxalic acid, and L-(+)-tartaric acid, as well as other Krebs cycle acids such as citric and isocitric acids, were not accepted by the malate transport system. Km measurements as a function of pH suggested that the anionic forms of the acids were transported by an accumulative dicarboxylate proton symporter. The accumulation ratio at pH 5.0 was about 40. The malate system was inducible and was subject to glucose repression. Undissociated succinic acid entered the cells slowly by simple diffusion. The permeability of the cells by undissociated acid increased with pH, with the diffusion constant increasing 100-fold between pH 3.0 and 6.0.     

Transport of malic acid and other dicarboxylic acids in the yeast Hansenula anomala.

DL-Malic acid-grown cells of the yeast Hansenula anomala formed a saturable transport system that mediated accumulative transport of L-malic acid with the following kinetic parameters at pH 5.0: Vmax, 0.20 nmol.s-1.mg (dry weight)-1; Km, 0.076 mM L-malate. Uptake of malic acid was accompanied by proton disappearance from the external medium with rates that followed Michaelis-Menten kinetics as a function of malic acid concentration. Fumaric acid, alpha-ketoglutaric acid, oxaloacetic acid, D-malic acid, and L-malic acid were competitive inhibitors of succinic acid transport, and all induced proton movements that followed Michaelis-Menten kinetics, suggesting that all of these dicarboxylates used the same transport system. Maleic acid, malonic acid, oxalic acid, and L-(+)-tartaric acid, as well as other Krebs cycle acids such as citric and isocitric acids, were not accepted by the malate transport system. Km measurements as a function of pH suggested that the anionic forms of the acids were transported by an accumulative dicarboxylate proton symporter. The accumulation ratio at pH 5.0 was about 40. The malate system was inducible and was subject to glucose repression. Undissociated succinic acid entered the cells slowly by simple diffusion. The permeability of the cells by undissociated acid increased with pH, with the diffusion constant increasing 100-fold between pH 3.0 and 6.0.     

Methionine transport in Salmonella typhimurium: evidence for at least one low-affinity transport system.

The systems which transport methionine in Salmonella typhimurium LT2 have been studied. Fourteen mutants, isolated by three different selection procedures, had similar growth characteristics and defects in the specific transport process showing a Km of 0.3 microM for L-methionine, and therefore lack the high-affinity, metP transport system. The sites of mutation in four of the mutants were shown by P1-mediated transduction to be linked (0.3 to 1.1%) with a proline marker located at unit 7 on the S. typhimurium chromosome. The high-affinity system was subject to both repression and transinhibition by methionine, and it may also be regulated by the metJ and metK genes. There appeared to be at least two additional transport systems with relatively low affinities for methionine in the metP763 mutant strain, with apparent Km values for methionine of 24 microM and approximately 1.8 mM. The latter system, with a very low affinity for methionine, was inhibited by leucine. In addition, methionine inhibited leucine transport, suggesting that one of the low-affinity methionine transport systems may actually be a leucine transport system.     

65Zn2+ Transport by lobster hepatopancreatic lysosomal membrane vesicles

In crustaceans, the hepatopancreas is the major organ system responsible for heavy metal detoxification, and within this structure the lysosomes and the endoplasmic reticulum are two organelles that regulate cytoplasmic metal concentrations by selective sequestration processes. This study characterized the transport processes responsible for zinc uptake into hepatopancreatic lysosomal membrane vesicles (LMV) and the interactions between the transport of this metal and those of calcium, copper, and cadmium in the same preparation. Standard centrifugation methods were used to prepare purified hepatopancreatic LMV and a rapid filtration procedure, to quantify 65Zn2+ transfer across this organellar membrane. LMV were osmotically reactive and exhibited a time course of uptake that was linear for 15-30 sec and approached equilibrium by 300 sec. 65Zn2+ influx was a hyperbolic function of external zinc concentration and followed Michaelis-Menten kinetics for carrier transport (Km = 32.3 +/- 10.8 microM; Jmax = 20.7 +/- 2.6 pmol/mg protein x sec). This carrier transport was stimulated by the addition of 1 mM ATP (Km = 35.89 +/- 10.58 microM; Jmax = 31.94+/-3.72 pmol/mg protein/sec) and replaced by an apparent slow diffusional process by the simultaneous presence of 1 mM ATP+250 microM vanadate. Thapsigargin (10 microM) was also a significant inhibitor of zinc influx (Km = 72.87 +/- 42.75 microM; Jmax =22.86 +/- 4.03 pmol/mg protein/sec), but not as effective in this regard as was vanadate. Using Dixon analysis, cadmium and copper were shown to be competitive inhibitors of lysosomal membrane vesicle 65Zn2+ influx by the ATP-dependent transport process (cadmium Ki = 68.1 +/- 3.2 microM; copper Ki = 32.7 +/- 1.9 microM). In the absence of ATP, an outwardly directed H+ gradient stimulated 65Zn2+ uptake, while a proton gradient in the opposite direction inhibited metal influx. The present investigation showed that 65Zn2+ was transported by hepatopancreatic lysosomal vesicles by ATP-dependent, vanadate-, thapsigargin-, and divalent cation-inhibited, carrier processes that illustrated Michaelis-Menten influx kinetics and was stimulated by an outwardly directed proton gradient. These transport properties as a whole suggest that this transporter may be a lysosomal isoform of the ER Sarco-Endoplasmic Reticulum Calcium ATPase.     

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.     

Transport of L-methionine in human diploid fibroblast strain WI38

The transport of L-methionine in human diploid fibroblast strain WI38 was investigated. The uptake of L-methionine was measured in sparse cell cultures in a simple balanced salt solution buffered with either Tris.HCl of N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (HEPES). Similar results were obtained with these two buffers. Cultures were allowed to equilibrate with the buffered saline before transport was measured. The presence of glucose in the buffered saline results in a slight reduction in the initial rate of transport for the first 2 h of equilibration in buffered saline. L-Methionine is actively transported in WI38 by saturable, chemicallly specific mechanisms which are temperature, pH and, in part Na+ dependent, and are reactive with both L- and D-stereoisomers. Kinetic analysis of initial rates of transport at substrate concentrations from 0.0005 to 100 mM indicated the presence of two saturable transport systems. System 1 has an apparent KM of 21.7 micrometer and an apparent V of 3.57 nmol/mg per min. System 2 has an apparent KM of 547 micrometer and an apparent V of 22.6 nmol/mg per min. Kinetic analysis of initial rates of transport in Na+-free media or after treatment with ouabain suggested that system 1 is Na+ independent and that system 2 is Na+ dependent. Preloading of cells with unlabeled L-methionine greatly increases the initial rate of uptake. Efflux of transported methionine is temperature dependent, and is greatly increased in the presence of unlabeled L- or D-methionine or L-phenylalanine, but not in the presence of L-arginine. L-Methionine transport is strongly inhibited by other neutral amino acids, and is very weakly inhibited by dibasic amino acids, dicarboxylic amino acids, proline or glycine.     

Characteristics of the methylammonium/ammonium transport systems of the nitrogen-fixing cyanobacterium Anabaena 7120 (ATCC 27893)

NH4(+)-transport in Anabaena 7120 was studied using the NH4+ analogue, 14CH3NH3+. At pH 7, two energy-dependent NH4(+)-transport systems were detected in both N2- and NO3(-)-grown cells, but none in NH4(+)-grown cells. Both transport systems showed a low and a high affinity mode of operation depending on the substrate concentration. One of the transport systems showed Km values of 8 microM (Vmax = 1 nmole min-1mg-1protein) and 80 microM (Vmax = 7 nmole min-1mg-1protein), and was insensitive to L-methionine-DL-sulphoximine, a glutamate analogue and irreversible inhibitor of glutamine synthetase. The other transport system showed Km values of 2.5 microM (Vmax = 0.1 nmole min-1mg-1protein) and 70 microM (Vmax = 0.7 nmole min-1mg-1protein), and was sensitive to L-methionine-DL-sulphoximine. Intracellular accumulation of free 14CH3NH3+ showed a biphasic pattern in response to variation in external 14CH3NH3+ concentrations. A maximum intracellular concentration of 2.5 mM and 7.5 mM was reached in the external 14CH3NH3+ concentration range of 1-50 microM and 1-500 microM, respectively. At pH 9, an energy-independent diffusion of 14CH3NH2 leading to a higher intracellular accumulation and assimilation rate, than that at pH 7, was observed.     

Transport and utilization of D-methionine and other methionine sources in Escherichia coli.

The transport and utilization of D-methionine was investigated in several strains of Escherichia coli K-12. Wild-type cells exhibit a single transport system with a Km of 1.16 muM. This activity exhibits a specificity similar to that of the uptake of L-methionine. The activity toward the D-isomer and the high-affinity uptake of L-methionine are lost in strains mutant in metD, along with the ability to utilize D-methionine as methionine source. Both activities respond identically to gene dosage of metD and are both restored in revertants or transductants. However, although L-methionine is a potent inhibitor of D-methionine uptake, D-methionine has little or no effect on the uptake of the L-isomer. No mutants altered in the uptake of only one of the two isomers were found in a screening. Regulation of both activities was similar in their response to the internal methionine pool, and some evidence was suggestive of partial repressive control of these activities. The evidence is most consistent with the role of the metD product as a common step for two methionine-specific uptake systems, but other gene products may represent the initial substrate binding sites. This system also appears to be involved in the uptake of N-acetyl methionine and methionine sulfoxide and methionine sulfoximine. The uptake of the keto analogue of methionine, alpha-keto-gamma-methiol butyrate, appears to be mediated by a separate system specific for alpha-keto straight-chain acids 5- to 6-carbon units in length.     

Biochemical characterization of a low-affinity arginine permease from the parasite Trypanosoma cruzi

Trypanosoma cruzi, the etiological agent of Chagas disease, uses arginine for several metabolic processes, including energy reserves management. In the present work, a novel low-affinity arginine transport system has been studied. Maximum velocity (97 pmol min(-1) per 10(7) cells), and an estimate for the apparent Km value (350 microM) of this arginine transporter, were 6-fold and 80-fold higher respectively, when compared with the previously described high-affinity arginine transport system. This transport activity seems to be H+ -mediated, presents a broad specificity by other amino acids such as methionine, and is regulated along the parasite growth curve and life cycle.     

Characterization of the ascorbic acid transport by 3T6 fibroblasts

Ascorbic acid transport by 3T6 mouse skin fibroblasts has been characterized using radiometric technique with L-[1-14C]ascorbic acid under the conditions in which oxidation of ascorbic acid was prevented by addition of 1 mM thiourea. The ascorbate transport is temperature-dependent with the energy of activation E and Q10 of 13.3 kcal/mol and 2.0, respectively. The transport requires energy and exhibits Michaelis-Menten kinetics with an apparent Km of 112 microM and Vmax of 158 pmol/min per mg protein, when the extracellular Na+ concentration is 150 mM. The ascorbate transport requires presence of extracellular Na+ and can be inhibited by ouabain treatment. At 40 and 200 microM ascorbate concentrations, respectively, 1.4 and 1.0 moles of Na+ bound the transporter molecule per each mole of ascorbate transported. Increased Na+ binding to the transporter at lower ascorbate concentration may signify multiple Na+-binding sites or ascorbate concentration dependent conformational changes in the transporter molecule. Increasing Na+ concentration decreases Km without affecting Vmax, suggesting that Na+ increases affinity of ascorbate for the transporter molecule without affecting translocation process. An increase in ascorbate concentration reduces the number of Na+ bound to the transporter from 1.4 to 1.0. The ascorbate transport is stimulated by Ca2+ and other divalent cations. The mechanism of stimulation by Ca2+ is not clear. Calcium increases both the Km and Vmax. The data presented support the hypothesis that the ascorbate transport by 3T6 fibroblasts is an energy and temperature-dependent active process driven by the Na+ electrochemical gradient. A potent inhibitor of ascorbate transport is also demonstrated in human serum.     

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.     

Effects of halothane on transport of 5-hydroxytryptamine by platelet membranes.

Na+-dependent uptake of 5-HT (5-hydroxytryptamine) into plasma membrane vesicles derived from bovine blood platelets and ATP-dependent 5-HT uptake into storage vesicles in platelet lysates were measured. Na+-dependent uptake was temperature-dependent, inhibited by imipramine and exhibited Michaelis-Menten kinetics (apparent Km, 0.12 +/- 0.02 microM; Vmax. 559 +/- 54 pmol/min per mg of protein. Halothane had no effect on Na+-dependent transport of 5-HT in plasma-membrane vesicles. ATP-dependent 5-HT transport into storage granules also exhibited Michaelis-Menten kinetics (apparent Km 0.34 +/- 0.03 microM; Vmax. 34.3 +/- 1.7 pmol/min per mg of protein) and was inhibited by noradrenaline (norepinephrine), but not by imipramine. Exposure of the granules to halothane resulted in a progressive decrease in Vmax. The results demonstrate a possible site for disruption of platelet function by anaesthetics.     

Transport of carnosine by mouse intestinal brush-border membrane vesicles

The characteristics of carnosine (beta-alanyl-L-histidine) transport have been studied using purified brush-border membrane vesicles from mouse small intestine. Uptake curves did not exhibit any overshoot phenomena, and were similar under Na+, K+ or choline+ gradient conditions (extravesicular greater than intravesicular). However, uptake of histidine showed an overshoot phenomenon in the presence of a Na+-gradient. There was no detectable hydrolysis of carnosine during 15 min of incubation with membrane vesicles under conditions used for transport experiments. Analysis of intravesicular contents further showed the complete absence of the constituent free amino acids of carnosine, and indicates that intact carnosine is transported. Studies on the effect of concentration on peptide uptake revealed that transport occurred by a saturable process conforming to Michaelis-Menten kinetics with a Km of 9.6 +/- 1.4 mM and a Vmax of 2.9 +/- 0.2 nmol/mg protein per 0.4 min. Uptake of carnosine was inhibited by both di- and tripeptides with a maximum inhibition of 68% by glycyl-L-leucyltyrosine. These results clearly demonstrate that carnosine is transported intact by a carrier-mediated, Na+-independent process.     

Choline transport in Saccharomyces cerevisiae.

Choline transport of Saccharomyces cerevisiae was measured by the filtration method with the use of glass microfiber paper. The uptake was time and temperature dependent. The kinetics of choline transport showed Michaelis behavior; an appearent Km for choline was 0.56 microM. N-Methylethanolamine, N,N-dimethylethanolamine, and beta-methylcholine were competitive inhibitors of choline transport, with Ki values of 40.1, 3.1, and 6.9 microM, respectively. Ethanolamine, phosphorylcholine, and various amino acids examined had no effect. Choline transport required metabolic energy; removal of glucose resulted in a great loss of transport activity, and the remaining activity was abolished by 2,4-dinitrophenol, carbonyl cyanide p-trifluoromethoxyphenyl hydrazone, arsenate, and cyanide. External Na+ was not required, and the transport was not effected by ionophores, valinomycin, and gramicidin D. These results indicate that S. cerevisiae possess an active choline transport system mediated by a specific carrier. This view is further supported by the isolation and characterization of a choline transport mutant. The choline transport activity in this mutant was very low, whereas the transport of L-leucine, L-methionine, D-glucose, and myo-inositol was normal. Together with the choline transport mutant, mutants defective in choline kinase were also isolated.