Effect of pH, temperature, and potassium sorbate on amino acid uptake in Salmonella typhimurium 7136

The effect of sorbate on L-serine and L-histidine uptake in Salmonella typhimurium was studied at various pH levels, temperatures, and amino acid and sorbate concentrations. Low pH had an apparent synergistic effect on amino acid uptake inhibition caused by sorbate. The relationship between sorbate concentration and the amount of amino acid uptake inhibition was not linear. Compared with L-histidine, L-serine uptake was more sensitive to changes in pH, temperature, and sorbate concentration. Various degrees of amino acid uptake inhibition by sorbate may be related to differences between amino acid transport systems. The results of this study suggest that sorbate acts as a noncompetitive inhibitor of amino acid uptake in S. typhimurium.     

Neutral amino acid transport by membrane vesicles of Streptococcus cremoris is subject to regulation by internal pH.

The pH dependence of transport of the neutral amino acids L-serine and L-alanine by membrane vesicles of Streptococcus cremoris have been studied in detail. The rates of four modes of facilitated diffusion (e.g., influx, efflux, exchange, and counterflow) of L-serine and L-alanine increase with increasing H+ concentration. Rates of artificially imposed electrical potential across the membrane (delta psi)-driven transport of L-serine and L-alanine show an optimum at pH 6 to 6.5. Under similar conditions, delta psi- and pH gradient across the membrane (delta pH)-driven transport of L-leucine is observed within the pH range studied (pH 5.5 to 7.5). The effect of ionophores on the uptake of L-alanine and L-serine has been studied in membrane vesicles of S. cremoris fused with proteoliposomes containing beef heart mitochondrial cytochrome c oxidase as a proton motive force (delta p)-generating system (Driessen et al., Proc. Natl. Acad. Sci. USA 82:7555-7559, 1985). An increase in the initial rates of L-serine and L-alanine uptake is observed with decreasing pH, which is not consistent with the pH dependency of delta p. Nigericin, an ionophore that induced a nearly complete interconversion of delta pH into delta psi, stimulated both the rate and the final level of L-alanine and L-serine uptake. Valinomycin, an ionophore that induced a collapse of delta psi with a noncompensating increase in delta pH, inhibited L-alanine and L-serine uptake above pH 6.0 more efficiently than it decreased delta p. Experiments which discriminate between the effects of the internal pH and the driving force (delta pH) on solute transport indicate that at high internal pH the transport systems for L-alanine and L-serine are inactivated. A unique relation exists between the internal pH and the initial rate of uptake of L-serine and L-alanine with an apparent pK of 7.0. The rate of L-alanine and L-serine uptake decreases with increasing internal pH. The apparent complex relation between the delta p and transport of L-alanine and L-serine can be explained by a regulatory effect of the internal pH on the activity of the L-serine and L-alanine carriers.     

Inhibition of Growth and Uptake Processes in Bacteria by Some Chemical Food Preservatives

The effect on growth and uptake processes of some common chemical food preservatives [benzoate, sorbate, propionate and alkyl esters of p -hydroxybenzoic acid (parabens)] has been studied in strains of Escherichia coli, Bacillus subtilis and Pseudomonas aeruginosa. For parabens. the inhibitory action on growth and amino acid uptake in whole cells and bacterial membrane vesicles followed similar dose-response curves. Growth inhibition caused by parabens appears to be a consequence of transport inhibition. For benzoate, sorbate and propionate, uptake inhibition seems inadequate to explain growth inhibition.     

Proline uptake by monolayers of human intestinal absorptive (Caco-2) cells in vitro.

Monolayers of the Caco-2 human intestinal cell line exhibit active and passive uptake systems for the imino acid L-proline. The active transport component is saturable and it is responsible for about two thirds of the observed flux over the nanomolar concentration range, at 37 degrees C and pH 7.4. In contrast to L-phenylalanine, specific L-proline uptake has a high degree of sodium dependency and the efficiency of the carrier system is significantly reduced when protein synthesis (cycloheximide), Na+/K(+)-ATPase (ouabain) or cellular metabolism (sodium azide) are inhibited. The expression of the L-proline carrier by Caco-2 cells was under some degree of nutritional control. Glucose deficiency, over the time scale of the experiment, had no effect. The temperature-dependence of the specific uptake process followed the Arrhenius model with an apparent activation energy of 93.5 kJ nmol-1. This pathway also displayed Michaelis-Menten concentration-dependence with a Ksdm of 5.28 mM and a maximal transport flux (Jsdmax) of 835 pmol min-1 (10(6) cells)-1. Although the passive component was unchanged, the pH of the donor phase exerted a profound effect on the active carrier component. Within the physiological pH range a local maximum efficiency was found at pH 7.4 but dramatic increases were noted as pH 5.0 was approached. In competition studies, with 100-fold excess of a second amino acid, strong inhibition of uptake was found with alpha-aminoisobutyric acid, L-alanine and L-serine whereas moderate inhibition was observed with glycine, D-proline and gamma-aminoisobutyric acid. Aromatic and branched amino acids showed weak (L-valine) or no interaction (L-phenylalanine, L-leucine) with the carrier system. These data indicate that the carrier system for the uptake of L-proline has many features in common with the A system for amino acid transport.     

Modelling the behaviour of Listeria monocytogenes in ground pork as a function of pH, water activity, nature and concentration of organic acid salts

AIMS: to study and model the effect of sodium acetate, sodium lactate, potassium sorbate and combination of acid salts on the behaviour of Listeria monocytogenes in ground pork. METHODS AND RESULTS: Water activity (a(w)), pH and concentration of acid salt of the meat were adjusted. The behaviour of inoculated L. monocytogenes was studied and modelled according to physicochemical parameters values. Whatever the acid salt concentration used, we observed an inhibition of the growth of L. monocytogenes at pH 5.6 and a(w) 0.95. At pH 6.2 and a(w) 0.97, addition of 402 mmol l(-1) of sodium lactate or 60 mmol l(-1) of potassium sorbate was required to observe a slower growth. CONCLUSIONS: The inhibitory effect of acid salts was a function of pH, a(w), as well as of the nature and concentration of acid salts added. When one acid salt was added, the Augustin's model (Augustin et al. 2005) yielded generally correct predictions of either the survival or growth of L. monocytogenes. SIGNIFICANCE AND IMPACT OF THE STUDY: The suggested model can be used for risk assessment concerning L. monocytogenes in pork products.     

Mechanism of energy coupling to entry and exit of neutral and branched chain amino acids in membrane vesicles of Streptococcus cremoris

The energetics of neutral and branched chain amino acid transport by membrane vesicles from Streptococcus cremoris have been studied with a novel model system in which beef heart mitochondrial cytochrome c oxidase functions as a proton-motive force (delta p) generating system. In the presence of reduced cytochrome c, a large delta p was generated with a maximum value at pH 6.0. Apparent H+/amino acid stoichiometries (napp) have been determined at external pH values between 5.5 and 8.0 from the steady state levels of accumulation and the delta p. For L-leucine napp (0.8) was nearly independent of the pH. For L-alanine and L-serine napp decreased from 0.9-1.0 at pH 5.5 to 0-0.2 at pH 8.0. The napp for the different amino acids decreased with increasing external amino acid concentration. At pH 6.0, first order rate constants for amino acid exit (kex) under steady state conditions for L-leucine, L-alanine, and L-serine were 1.1-1.3, 0.084, and 0.053 min-1, respectively. From the pH dependence of kex it is concluded that amino acid exit in steady state is the sum of two processes, pH-dependent carrier-mediated amino acid exit and pH-independent passive diffusion (external leak). The first order rate constant for passive diffusion increased with increasing hydrophobicity of the side chain of the amino acids. As a result of these processes the kinetic steady state attained is less than the amino acid accumulation ratio predicted by thermodynamic equilibrium. The napp determined from the steady state accumulation represents, therefore, a lower limit. It is concluded that the mechanistic stoichiometry (n) for L-leucine, L-alanine, and L-serine transport most likely equals 1.     

Growth inhibition of putrefactive anaerobe 3679 caused by stringent-type response induced by protonophoric activity of sorbic acid

The inhibitory effects of potassium sorbate on the bioenergetics, phenylalanine uptake, protein synthesis, and certain aspects of cell regulation were examined in putrefactive anaerobe 3679. Undissociated sorbic acid appeared to act as a protonophore by lowering the intracellular pH and dissipating the proton motive force of the membrane. Sorbate inhibited the uptake of phenylalanine, decreased the rate of protein synthesis, and altered patterns of phosphorylated nucleotide accumulation, resulting in increased intracellular concentrations of GTP, ppGpp, and an unidentified compound (possibly pppGpp). The addition of a noninhibitory amount of tetracycline released the inhibition of growth by sorbate. Based on these results, we concluded that the inhibition of putrefactive anaerobe 3679 by sorbate resulted from a stringent-type regulatory response induced by the protonophoric activity of sorbic acid.     

Growth inhibition of putrefactive anaerobe 3679 caused by stringent-type response induced by protonophoric activity of sorbic acid.

The inhibitory effects of potassium sorbate on the bioenergetics, phenylalanine uptake, protein synthesis, and certain aspects of cell regulation were examined in putrefactive anaerobe 3679. Undissociated sorbic acid appeared to act as a protonophore by lowering the intracellular pH and dissipating the proton motive force of the membrane. Sorbate inhibited the uptake of phenylalanine, decreased the rate of protein synthesis, and altered patterns of phosphorylated nucleotide accumulation, resulting in increased intracellular concentrations of GTP, ppGpp, and an unidentified compound (possibly pppGpp). The addition of a noninhibitory amount of tetracycline released the inhibition of growth by sorbate. Based on these results, we concluded that the inhibition of putrefactive anaerobe 3679 by sorbate resulted from a stringent-type regulatory response induced by the protonophoric activity of sorbic acid.     

Effect of amino acids on choline uptake and phosphatidylcholine biosynthesis in the isolated hamster heart

Choline uptake by the hamster heart has been shown to be enhanced by exogenous glycine. In this study, the effect of neutral, basic, and acidic amino acids on choline uptake was assessed. Hamster hearts were perfused with labelled choline, and in the presence of L-alanine, L-serine, or L-phenylalanine (greater than or equal to 0.1 mM), choline uptake was enhanced 20-38%. L-Arginine, L-lysine, L-aspartate, and L-glutamate did not influence choline uptake. The rate of phosphatidylcholine biosynthesis was unaffected by all amino acids tested. Enhancement of choline uptake by neutral amino acids was not additive or dose dependent but required a concentration threshold. The enhancement of choline uptake by neutral amino acids was not influenced by preperfusion with the same amino acid. Exogenous choline had no effect on the uptake of amino acids. We postulate that choline and the neutral amino acids are not cotransported and modulation of choline uptake is facilitated by direct interaction of the neutral amino acids with the choline transport system.     

The dark respiration of Anacystis nidulans. Production of HCN from histidine and oxidation of basic amino acids.

The basic amino acids, L-arginine, L-lysine, LO-irnithine, and to a lesser extent L-histidine, strongly stimulate the O2 uptake of cell suspensions of the blue-green alga or cyanobacterium anacystis nidulans. In the case of L-histidine, the extra O2 consumption is associated with the formation in vivo of small amounts of HCN, particularly in an atmosphere of O2. The enzyme responsible for both the stimulated O2 uptake with the basic amino acids and the formation of HCN from histidine has been isolated and identified as an L-amino acid oxidase specific for the basic amino acids. The purification (15 000-fold) of this enzyme is described. The isolated enzyme is inhibited by o-phenanthroline, which has a similar inhibitory effect on the O2 uptake of cell suspensions with (and without) added amino acids. The basic amino acid oxidase, which is not inhibited by HCN, can be regarded as an 'alternate' oxidase in A. nidulans. An oxidase sensitive to HCN is apparently also operative. At high concentrations of lysine or arginine added HCN can almost double the initial rate of O2 consumption of cell suspensions. This can be attributed to the inhibition of catalase by HCN. At low concentrations of the amino acids, and with more prolonged incubation time, HCN becomes inhibitory. One interpretation could be that the HCN-sensitive terminal oxidase is also involved in the extra O2 uptake elicited by the basic amino acids, but other interpretations are possible. The extra O2 uptake elicited by histidine is almost completely inhibited by HCN, which is consistent with the finding that histidine is a relatively poor substrate for the basic amino acid oxidase.     

The gamma-glutamyltranspeptidase of Trypanosoma cruzi

Trypanosoma cruzi epimastigotes show gamma-glutamyltranspeptidase activity which has characteristics significantly different than the mammalian enzyme. The protozoan enzyme is localized in the cytosolic fraction, it has a Km of 1.6 mM and a Vmax of 17.4 nmol/min/mg protein with L-gamma-glutamyl-p-nitroanilide as gamma-glutamyl donor, and an optimun pH range from 7.5 to 8.0. The best amino acid acceptors were L-histidine, L-asparagine, L-aspartate, L-glutamate and L-proline, but L-glutamine was a very poor acceptor. The enzyme was very sensitive to inhibition by 6-diazo-5-oxo-L-norleucine (k2 = 4.0 X 10(5)/M per min) and O-diazo-acetyl-L-serine (k2 = 1.1 X 10(4)/M per min). Phenobarbital (k2 = 8.38/M per min) and L-serine borate (Ki = 34 mM) were poor inhibitors. The activity of the enzyme was not correlated with the logarithmic phase of growth of the parasites and steadily decreases with the age of the cultures.     

Stereoselective transport of histidine in rat lung microvascular endothelial cells

The transport characteristics of L- and D-histidine through the blood-lung barrier were studied in cultured rat lung microvascular endothelial cells (LMECs). L-Histidine uptake was a saturable process. The addition of metabolic inhibitors [2,4-dinitrophenol (DNP) and rotenone] reduced the uptake rate of L-histidine. Ouabain, an inhibitor of Na(+)-K(+)-ATPase, also reduced uptake of L-histidine. Moreover, the initial L-histidine uptake rate was reduced by the substitution of Na(+) with choline chloride and choline bicarbonate in the incubation buffer. The system N substrate, L-glutamic acid gamma-monohydroxamate, also inhibited uptake of L-histidine. However, system N-mediated transport was not pH sensitive. These results demonstrated that L-histidine is actively taken up by a system N transport mechanism into rat LMECs, with energy supplied by Na(+). Moreover, the Na(+)-independent system L substrate, 2-amino-2-norbornanecarboxylic acid (BCH), had an inhibitory effect on L-histidine uptake in Na(+) removal, indicating facilitated diffusion by a Na(+)-independent system L transport into the rat LMECs. These results provide evidence for there being at least two pathways for L-histidine uptake into rat LMECs, a Na(+)-dependent system N and Na(+)-independent system L process. On the other hand, the uptake of D-histidine into rat LMECs was not reduced by the addition of DNP, rotenone, or ouabain, or by Na(+) replacement. Although the uptake of D-histidine was reduced in the presence of BCH, the addition of L-glutamic acid gamma-monohydroxamate did not significantly decrease uptake of D-histidine. These results suggest that the uptake of D-histidine by rat LMECs has different characteristics compared with its isomer, L-histidine, indicating that system N transport did not involve D-histidine uptake.     

一些氨基酸对产甘油假丝酵母甘油生产的促进作用

以EMP途径与TCA循环中间代谢物的添加为对照,研究在尿素为氮源的产甘油假丝酵母发酵过程中添加氨基酸对甘油产量的影响。结果表明:对甘油产量有强促进作用的氨基酸有谷氨酸、谷氨酰胺、天冬氨酸、天冬酰胺、甘氨酸、赖氨酸、酪氨酸、脯氨酸、组氨酸和丝氨酸,其最适添加浓度在0.26～0.45g/L之间,丙酮酸、α_酮戊二酸、草酰乙酸、柠檬酸和琥珀酸的最适添加浓度在0.24～0.42g/L之间;赖氨酸最适于在0h添加,丙酮酸和草酰乙酸在第14h,谷氨酸、谷氨酰胺、组氨酸、脯氨酸、天冬氨酸、酪氨酸、甘氨酸、α_酮戊二酸和琥珀酸在第30h,天冬酰胺、丝氨酸和柠檬酸在第48h;在最适条件下添加这些促进剂,甘油产量均呈显著增加趋势,转化率和增加率分别达到60%和16%以上。氨基酸的作用机理为其脱氨形成的碳骨架经特定的分解代谢途径进入TCA循环,使其强化,导致碳代谢流在3_磷酸甘油醛节点处发生转移,使甘油合成途径的代谢流增加。     

A proton nuclear magnetic resonance investigation of histine-binding protein J of salmonella typhimurium: A model for transport of L-histidine across cytoplasmic membrane

Genetic evidence suggests that the high-affinity L-histidine transport in Salmonella typhimurium requires the participation of a periplasmic binding protein (histidine-binding protein J) and two other proteins (P and Q proteins). The histidine-binding protein J binds L-histidine as the first step in the high-affinity active transport of this amino acid across the cytoplasmic membrane. High-resolution proton nuclear magnetic resonance spectroscopy at 600 MHz is used to investigate the conformations of this protein in the absence and presence of substrate. Previous nuclear magnetic resonance results reported by this laboratory have shown that there are extensive spectral changes in this protein upon the addition of L-histidine. When resonances from individual amino acid residues of a protein can be resolved in the proton nuclear magnetic resonance spectrum, a great deal of detailed information about substrate-induced structural changes can be obtained. In order to gain a deeper insight into the nature of these structural changes, deuterated phenylalanine or tyrosine has been incorporated into the bacteria. Proton nuclear magnetic resonance spectra of selectively deuterated histidine-binding protein J were obtained and compared to the normal protein. Several of the proton resonances have been assigned to the various aromatic amino acid residues of this protein. A model for the high-affinity transport of L-histidine across the cytoplasmic membrane of S typhimurium is proposed. This model, which is a version of the pore model, assumes that both P and Q proteins are membrane-bound and that the interface between these two proteins forms the channel for the passage of substrate. The histidine-binding protein J serves as the “key” for the opening of the channel for the passage of L-histidine. In the absence of substrate, this channel or gate is closed owing to a lack of appropriate interactions among these three proteins. The channel can be opened upon receiving a specific signal from the “key”; namely, the substrate-induced conformational changes in the histidine-binding protein J molecule. This model is consistent with available experimental evidence for the high-affinity transport of L-histidine across the cytoplasmic membrane of S typhimurium.     

Amino acid transport in a polyaromatic amino acid auxotroph of Saccharomyces cerevisiae

The initiation of growth of a polyaromatic auxotrophic mutant of Saccharomyces cerevisiae was inhibited by several amino acids, whereas growth of the parent prototroph was unaffected. A comparative investigation of amino acid transport in the two strains employing (14)C-labeled amino acids revealed that the transport of amino acids in S. cerevisiae was mediated by a general transport system responsible for the uptake of all neutral as well as basic amino acids. Both auxotrophic and prototrophic strains exhibited stereospecificity for l-amino acids and a K(m) ranging from 1.5 x 10(-5) to 5.0 x 10(-5) M. Optimal transport activity occurred at pH 5.7. Cycloheximide had no effect on amino acid uptake, indicating that protein synthesis was not a direct requirement for amino acid transport. Regulation of amino acid transport was subject to the concentration of amino acids in the free amino acid pool. Amino acid inhibition of the uptake of the aromatic amino acids by the aromatic auxotroph did not correlate directly with the effect of amino acids on the initiation of growth of the auxotroph but provides a partial explanation of this effect.     

Effects of amino acids on the isolated chicken retina, and on its response to glutamate stimulation

Abstract— The effect of a number of amino acids on the transparency and on the release of of [¹⁴C]glutamate from isolated chicken retinae charged with this compound was investigated. Also the effect of various amino acids on the response of the retina to stimulation with unlabelled glutamate, which causes an increase in transparency and a release of the label, was examined. In parallel experiments the effect of these same amino acids on the transparency and spreading depression (SD) was investigated in preparations consisting of the posterior part of the eye. A number of amino acids such as L-leucine, L-phenylalanine, L-tryptophan, L-lysine, L-histidine, L-arginine and others had little or no effect on these preparations. DL-valine and DL-homoserine caused an increase in transparency but no release of the label and did not affect the response to glutamate. Another group of amino acids comprising DL-a-alanine, L-serine, L-threonine, L-proline and glycine also caused an increase in the transparency of the retina without a release of labelled glutamate, but prevented the increase in transparency resulting from glutamate stimulation without affecting the release of the label. A final group of amino acids which included L-glutamic acid diethyl ester, DL-a-methyl glutamate, L-glutamine, L-asparagine, DL-homocysteate and L-cysteine caused a change in transparency of the retina accompanied by a release of the label; they prevented the change in transparency as well as the release of the label during stimulation by glutamate. Some amino acids, L-serine, L-threonine, DL-a-methyl glutamate, L-asparagine, DL-homocysteate and L-cysteine, caused wrinkling and folding of the retinae which furthermore became opaque. Of the amino acids investigated, proline gave promise of being a practical antagonist to the action of glutamate on the retina.     

Mechanism of L-serine oxidation in Entamoeba histolytica.

1. The enzymatic mechanism of oxygen uptake elicited by L-serine in axenically cultivated trophozoites of Entamoeba histolytica was investigated. 2. Of 22 amino acids examined, only L-serine stimulated oxygen consumption by intact and disrupted amoebae. 3. Pyruvate, a product of serine metabolism, also stimulated oxygen consumption in the amoebae. 4. Characterization of the oxygen uptake elicited by both L-serine and pyruvate, and analysis of the products of L-serine metabolism indicate that the amino acid is first converted to pyruvate. 5. L-Serine dehydratase, which catalyzes the deamination of serine to pyruvate, was detected primarily in the soluble fraction of the amoebae. D-Serine potently inhibited the enzyme, as well as oxygen uptake in the presence of L-serine but not in the presence of pyruvate. 6. The pyruvate formed is oxidized, at least in part, by a novel pyruvate oxidase involving the uptake of molecular oxygen.     

Protective effect of casein toward Salmonella typhimurium in acid-milk

Lactic acid is the inhibitory agent in yoghurt responsible for the inhibition of Salmonella typhimurium. Casein, however, may exert a protective effect toward the survival of the salmonella in acid-milk products. Salmonella typhimurium was found to die-off 21.2% more rapidly in 18-h yoghurt-whey than in 18-h yoghurt at 37 degrees C with a pH of 3.85 and 1.42% lactic acid. When casein was added to yoghurt-whey, the die-off rate of the salmonellas was reduced to that found in yoghurt. The rate remained unchanged when 4.8% sodium caseinate was added to the whey. When 0 to 14% casein was added to the acid-whey the die-off rate changed from 9.7 to 24.0 min/log reduction of cells, respectively. There was a direct correlation between the increase in casein concentration and length of survival of the salmonellas. At a pH of 3.85, 4.2 or 4.5, the die-off rate was 6.5, 13.0 or 40 min/log reduction of cells in milk containing 1.42% lactic acid, and was 4.0, 10.0 or 33.3 min/log reduction, respectively, in whey with 1.42% lactic acid. Thus, the protective effect of casein toward Salm. typhimurium increased as the pH increased. This indicated that casein exerts a protective effect on Salm. typhimurium in acid dairy products and the degree of protection depends on the casein concentration, the form of the casein molecule and the pH.     

Protective effect of casein toward Salmonella typhimurium in acid-milk

Lactic acid is the inhibitory agent in yoghurt responsible for the inhibition of Salmonella typhimurium. Casein, however, may exert a protective effect toward the survival of the salmonella in acid-milk products. Salmonella typhimurium was found to die-off 21.2% more rapidly in 18-h yoghurt-whey than in 18-h yoghurt at 37 C with a pH of 3.85 and 1.42% lactic acid. When casein was added to yoghurt-whey, the die-off rate of the salmonellas was reduced to that found in yoghurt. The rate remained unchanged when 4.8% sodium caseinate was added to the whey. When 0 to 14% casein was added to the acid-whey the die-off rate changed from 9.7 to 24.0 min/log reduction of cells, respectively. There was a direct correlation between the increase in casein concentration and length of survival of the salmonellas. At a pH of 3.85, 4.2 or 4.5, the die-off rate was 6.5, 13.0 or 40 min/log reduction of cells in milk containing 1.42% lactic acid, and was 4.0, 10.0 or 33.3 min/log reduction, respectively, in whey with 1.42% lactic acid. Thus, the protective effect of casein toward Salm. typhimurium incieased as the pH increased. This indicated that casein exerts a protective effect on Salm. typhimurium in acid dairy products and the degree of protection depends on the casein concentration, the form of the casein molecule and the pH.     

Effects of amino acids and ethanolamine on choline uptake and phosphatidylcholine biosynthesis in baby hamster kidney-21 cells.

The effects of amino acids and ethanolamine on choline uptake and phosphatidylcholine biosynthesis in baby hamster kidney (BHK-21) cells were investigated. The cells were incubated with labelled choline in the presence of an amino acid or ethanolamine. The uptake of labelled choline was noncompetitively inhibited by amino acids. Glycine, L-alanine, L-serine, L-leucine, L-aspartate, and L-arginine were effective inhibitors and a maximum of 22% inhibition of choline uptake was obtained with 5 mM glycine. Analyses of the labelings in the choline-containing metabolites revealed that the conversion of choline to CDP-choline and subsequently phosphatidylcholine was not affected by the presence of amino acids. The uptake of choline was also inhibited by ethanolamine in a concentration-dependent manner. Kinetic studies on the uptake of choline indicated that the inhibition by ethanolamine was competitive in nature. Although ethanolamine is a potent inhibitor of choline kinase, analyses of the labelings in the choline-containing metabolites indicated that the conversion of choline to phosphocholine was not affected in the cells incubated with ethanolamine. Ethanolamine did not change the pool sizes of phosphocholine and CDP-choline. Based on the specific radioactivity of CDP-choline and the labeling of phosphatidylcholine, the rates of phosphatidylcholine biosynthesis were not significantly different between the control and the ethanolamine-treated cells. In view of the concentrations of amino acids (millimolar) and ethanolamine (micromolar) in most cell culture media, it appeared that only amino acids were important metabolites for the regulation of choline uptake in BHK-21 cells. We conclude that both amino acids and ethanolamine have no direct effect on the biosynthesis of phosphatidylcholine.