Threonine Transport Research Articles

A study on L-threonine and L-serine uptake in Escherichia coli K-12.

In the current study, we report the identification and characterization of the yifK gene product as a novel amino acid carrier in E. coli K-12 cells. Both phenotypic and biochemical analyses showed that YifK acts as a permease specific to L-threonine and, to a lesser extent, L-serine. An assay of the effect of uncouplers and composition of the reaction medium on the transport activity indicates that YifK utilizes a proton motive force to energize substrate uptake. To identify the remaining threonine carriers, we screened a genomic library prepared from the yifK-mutant strain and found that brnQ acts as a multicopy suppressor of the threonine transport defect caused by yifK disruption. Our results indicate that BrnQ is directly involved in threonine uptake as a low-affinity but high-flux transporter, which forms the main entry point when the threonine concentration in the external environment reaches a toxic level. By abolishing YifK and BrnQ activity, we unmasked and quantified the threonine transport activity of the LIV-I branched chain amino acid transport system and demonstrated that LIV-I contributes significantly to total threonine uptake. However, this contribution is likely smaller than that of YifK. We also observed the serine transport activity of LIV-I, which was much lower compared with that of the dedicated SdaC carrier, indicating that LIV-I plays a minor role in the serine uptake. Overall, these findings allow us to propose a comprehensive model of the threonine/serine uptake subsystem in E. coli cells.

Membrane transporter identification and modulation via adaptive laboratory evolution

Membrane transport proteins are potential targets for medical and biotechnological applications. However, more than 30% of reported membrane transporter families are either poorly characterized or lack adequate functional annotation. Here, adaptive laboratory evolution was leveraged to identify membrane transporters for a set of four amino acids as well as specific mutations that modulate the activities of these transporters. Specifically, Escherichia coli was adaptively evolved under increasing concentrations of L–histidine, L–phenylalanine, L–threonine, and L–methionine separately with multiple replicate evolutions. Evolved populations and isolated clones displayed growth rates comparable to the unstressed ancestral strain at elevated concentrations (four-to six-fold increases) of the targeted amino acids. Whole genome sequencing of the evolved strains revealed a diverse number of key mutations, including SNPs, small deletions, and copy number variants targeting the transporters leuE for histidine, yddG for phenylalanine, yedA for methionine, and brnQ and rhtC for threonine. Reverse engineering of the mutations in the ancestral strain established mutation causality of the specific mutations for the tolerant phenotypes. The functional roles of yedA and brnQ in the transport of methionine and threonine, respectively, are novel assignments and their functional roles were validated using a flow cytometry cellular accumulation assay. To demonstrate how the identified transporters can be leveraged for production, an L–phenylalanine overproduction strain was shown to be a superior producer when the identified yddG exporter was overexpressed. Overall, the results revealed the striking efficiency of laboratory evolution to identify transporters and specific mutational mechanisms to modulate their activities, thereby demonstrating promising applicability in transporter discovery efforts and strain engineering.

Fluorescent bacterial biosensor E. coli/pTdcR-TurboYFP sensitive to terahertz radiation.

A fluorescent biosensor E. coli/pTdcR-TurboYFP sensitive to terahertz (THz) radiation was developed via transformation of Escherichia coli (E. coli) cells with plasmid, in which the promotor of the tdcR gene controls the expression of yellow fluorescent protein TurboYFP. The biosensor was exposed to THz radiation in various vessels and nutrient media. The threshold and dynamics of fluorescence were found to depend on irradiation conditions. Heat shock or chemical stress yielded the absence of fluorescence induction. The biosensor is applicable to studying influence of THz radiation on the activity of tdcR promotor that is involved in the transport and metabolism of threonine and serine in E. coli.

Conformational Transitions in YddG Bacterial Transporter: A Mechanistic Picture

YddG is a DMT (Drug and Metabolite Transporter) protein found in the inner bacterial membrane and helps in maintaining cell homeostasis by expelling amino acids and exogenous toxic compounds. During the transport cycle it undergoes a large scale structural transitions from outward-(OF) to inward-facing (IF) state. We have used Steered Molecular Dynamics to drive the transitions and based on the non-equilibrium work we came up with the optimal reaction co-ordinate, which was then used to obtain the transition free energy landscape using well-tempered metadynamics. We were able to capture the occluded state which act like an intermediate between OF-IF transition. The transition relies on the movement of TM1, TM3, TM4 and TM9 in the outward facing conformation and TM4, TM6, TM8 and TM9 in the inward facing conformation. We have also explored the translocation of the substrate threonine in WT and mutant (H79A) YddG by performing 2D well-tempered metadynamics. The free energy landscape highlights the importance of hydrophilic nature of the lumen in threonine transport.

Draft genome sequence of Microbacterium oleivorans strain Wellendorf implicates heterotrophic versatility and bioremediation potential

Microbacterium oleivorans is a predominant member of hydrocarbon-contaminated environments. We here report on the genomic analysis of M. oleivorans strain Wellendorf that was isolated from an indoor door handle. The partial genome of M. oleivorans strain Wellendorf consists of 2,916,870bp of DNA with 2831 protein-coding genes and 49 RNA genes. The organism appears to be a versatile mesophilic heterotroph potentially capable of hydrolysis a suite of carbohydrates and amino acids. Genomic analysis revealed metabolic versatility with genes involved in the metabolism and transport of glucose, fructose, rhamnose, galactose, xylose, arabinose, alanine, aspartate, asparagine, glutamate, serine, glycine, threonine and cysteine. This is the first detailed analysis of a Microbacterium oleivorans genome.

Role of estrogen receptor in the regulation of estrogen induced amino acid transport of System A in breast cancer and other receptor positive tumor cells

The transport of amino acids across the plasma membrane is a process of fundamental physiological importance. If this process is modified by estrogens and if estrogen receptors play a role in this regulation, then the alteration of metabolic events will be of significant importance in cancer cells which have high estrogen receptor content because estrogens modify cellular physiology through transactivational effects of their cognate receptors. In the present study, we investigated the role of 17 beta-estradiol on the regulation of different amino acid transport systems, in particular, Systems A, ASC and y+ in estrogen receptor positive (MCF-7, T47D, H-301) and estrogen receptor negative (MCF-10, SKBR-3, MDA-MB-231) cell lines. The cells were treated with 17 beta-estradiol (10 nM), ICI 182780 (1 microM), or 17 beta-estradiol plus ICI 182780. We discovered that 17 beta-estradiol specifically stimulates System A activity by 2- to 4-fold in estrogen receptor positive cell lines with a maximal stimulation 48 h after estrogen-treatment while no stimulation was observed in estrogen receptor negative cell lines. Estrogen-dependent activation of System A was inhibited by co-treatment with the antiestrogen ICI 182780. Northern blot analysis showed that System A mRNA levels are also increased following estrogen treatment, and this induction of mRNA transcript levels by estrogen can be inhibited by co-treatment with antiestrogen ICI 182780. The increase in System A transport activity following estrogen treatment was abbrogated when estrogen receptor positive cells were stably transfected with human antisense ER alpha cDNA. Kinetic analysis demonstrated that estrogen-induced stimulation results in a doubling of Vmax with no changes in Km. However, 17 beta-estradiol did not stimulate the activation of transport systems responsible for the transport of arginine (y+) or threonine (ASC). In summary, we have provided evidence that estrogen receptors play a role in the activation of System A by estrogen. This adaptation may have important physiological and nutritional significance on estrogen dependent growth of breast tumors.

Threonine metabolism in isolated rat hepatocytes

The removal of the 1-carbon of threonine can occur via threonine dehydrogenase or threonine aldolase, this carbon ending up in glycine to be liberated by the mitochondrial glycine cleavage system and producing CO(2). Alternatively, in the threonine dehydratase pathway, the 1-carbon ends up in alpha-ketobutyrate, which is oxidized in the mitochondria to CO(2). Rat hepatocytes, incubated in Krebs-Henseleit medium, were incubated with 0.5 mM L-[1-(14)C]threonine, and (14)CO(2) production was measured. Added glycine (0.3 mM) marginally suppressed threonine oxidation. Cysteamine (0.5 mM), a potent inhibitor of the glycine cleavage system, reduced threonine oxidation to 65% of controls. However, alpha-cyanocinnamate (0.5 mM), a competitive inhibitor of mitochondrial alpha-keto acid uptake, reduced threonine oxidation to 35% of controls. These data provided strong evidence that approximately 65% of threonine oxidation occurs through the glycine-independent threonine dehydratase pathway. Glucagon (10(-7) M) increased threonine oxidation and stimulated threonine uptake by these cells. In summary, the majority of threonine oxidation occurs through the threonine dehydratase pathway in rat hepatocytes, and threonine oxidation is increased by glucagon, which also increases threonine's transport.

Contribution of erythrocytes and plasma in threonine and lysine transfer across the portal drained viscera and the liver in pigs. Effect of threonine and lysine dietary supply.

Contributions of erythrocytes and plasma to threonine and lysine transport across the PDV and the liver were determined in growing pigs successively fed a threonine deficient diet and a control well-balanced diet (experiment 1) or a lysine deficient or a well-balanced diet (experiment 2). The animals were surgically prepared for insertion of chronic catheters in the mesenteric vein (MV), the portal vein (PV), a hepatic vein (HV) and the carotid artery (CA). Plasma and whole blood AA concentrations in PV, HV and CA and PV and HV blood flows were determined during 6 hours of para-aminohippuric acid constant infusion. During this period the pigs were continuously fed (1 meal per hour). The contribution of plasma to lysine and threonine transport was higher in pigs fed the well balanced diets. More than 50% of threonine and lysine appearing in the PV and in the HV are transported by the plasma. Our results suggest that erythrocytes are probably little involved in lysine and threonine transfer across the liver and digestive tract of pig continuously fed.

Placental transport of threonine and its utilization in the normal and growth-restricted fetus.

Placental transport and fetoplacental utilization of threonine (Thr) were compared at 130 +/- 1 days gestational age between seven control ewes (C) and six ewes in which intrauterine growth restriction (IUGR) had been induced by exposure to high ambient temperature from 33 +/- 1 to 112 +/- 2 days of gestation. The fluxes were measured using simultaneous intravenous infusions of L-[1-13C]Thr into the mother and L-[U-14C]Thr into the fetus. The IUGR group had less fetal weight (1.27 +/- 0.14 vs. 3.10 +/- 0.10 kg, P < 0.01) and placental weight (120 +/- 17 vs. 295 +/- 14 g, P < 0.01) than the C group. The direct flux of maternal Thr into the fetal systemic circulation was less in the IUGR fetuses, both relative to fetal weight (1.40 +/- 0.19 vs. 2.19 +/- 0.18 mumol.min-1.kg fetus-1, P = 0.0107) and placental weight (1.5 +/- 0.2 vs. 2.3 +/- 0.2 mumol.min-1.100 g placenta-1, P = 0.0187). In both groups, there was excretion of CO2 produced from fetal Thr. The rate of CO2 production from fetal plasma Thr carbon by fetus plus placenta was reduced in the IUGR group (1.50 +/- 0.23 vs. 2.86 +/- 0.32 mumol.min-1.kg fetus-1, P = 0.0065). We conclude that the flux of maternal Thr into the IUGR fetus is markedly reduced because of a reduction in placental mass and because of a weight-specific reduction in Thr placental transport. The reduced flux is routed into fetal Thr accretion via a decrease in fetal Thr oxidation.

Tissue localization of threonine oxidation in pigs.

Two experiments were designed to determine the tissue distribution of threonine oxidation through the threonine dehydrogenase (EC 1.1.1.103) pathway in pigs. The first experiment was conducted on eleven Piétrain x Large White piglets. The piglets were slaughtered at 5, 12 or 20 kg after 1 h of infusion with L-[U-14C]threonine (55 kBq/kg) mixed with unlabelled threonine (100 mg/kg). In the second experiment, four Piétrain x Large White and four large White piglets (10 kg body weight) were infused with L-[1-13C]threonine (50 mg/kg) mixed with 50 mg/kg unlabelled threonine for 1 h, then killed for tissue sampling. In the two experiments, threonine dehydrogenase specific activity and threonine and glycine specific radioactivities and enrichments were measured in several tissues and in plasma. The higher level of labelling of threonine in the pancreas than in the liver suggested either a lower protein degradation rate or a faster rate of threonine transport in the liver than in the pancreas. Threonine dehydrogenase activity was found only in the liver and the pancreas. Whereas liver and pancreas threonine dehydrogenase specific activities were similar, glycine specific radioactivity and enrichment were 12- to 14-fold higher in the pancreas than in the liver. This is probably the consequence of a higher production rate of glycine from sources other than threonine (protein degradation, de novo synthesis from serine) in the liver than in the pancreas. Our results showed that Large White pigs could oxidize more threonine than Piétrain x Large White pigs. This could be related to the difference in growth performance and dietary N efficiency for protein deposition between these two genotypes.

In vivo threonine oxidation in growing pigs fed on diets with graded levels of threonine.

Threonine oxidation to glycine was investigated in vivo in twelve growing pigs (27.4 kg live weight) fed on one of the following three diets with graded levels of threonine supply: a low-threonine diet (LT), a control well-balanced diet (C) or a high-threonine diet (HT), during 10 h constant infusion of L-[1-13C]threonine and [2-3H]glycine in the cranial vena cava and [1-14C]glycine in the portal vein. 13C-threonine and glycine enrichments and [3H]glycine and [14C]glycine specific radioactivities (SR) were determined at plateau in peripheral venous plasma, liver and pancreas. Glycine production rates calculated from plasma [2-3H]glycine or [1-14C]glycine SR gave similar values suggesting that [1-14C]glycine SR could be used in order to estimate whole-body glycine flux. The high pancreas [1-13C]glycine enrichment provided evidence that the pancreas may be, with the liver, a major site of threonine oxidation to glycine. Moreover, the present findings suggest that threonine transport into the liver could be the limiting step of threonine oxidation in this tissue when dietary threonine supply is low. Total threonine oxidation to glycine, calculated from plasma values of enrichment and specific radioactivity, was low and constant when the estimated absorbed threonine was lower than 4 g/d and increased for higher amounts of absorbed threonine.

Threonine diffusion and threonine transport in Corynebacterium glutamicum and their role in threonine production

Transmembrane threonine fluxes (i.e., uptake, diffusion, and carrier-mediated excretion) all contribut-ing to threonine production by a recombinant strain of Corynebacterium glutamicum, were analyzed and quantitated. A threonine-uptake carrier that transports threonine in symport with sodium ions was identified. Under production conditions (i.e., when internal threonine is high), this uptake system catalyzed predominantly threonine/threonine exchange. Threonine export via the uptake system was excluded. Threonine efflux from the cells was shown to comprise both carrier-mediated excretion and passive diffusion. The latter process was analyzed after inhibition of all carrier-mediated fluxes. Threonine diffusion was found to proceed with a first-order rate constant of 0.003 min–1 or 0.004 μl min–1 (mg dry wt.)–1, which corresponds to a permeability of 8 × 10–10 cm s–1. According to this permeability, less than 10% of the efflux observed under optimal conditions takes place via diffusion, and more than 90% must result from the activity of the excretion carrier. In addition, the excretion carrier was identified by (1) inhibition of its activity by amino acid modifying reagents and (2) its dependence on metabolic energy in the form of the membrane potential. Activity of the excretion system depended on the membrane potential, but not on the presence of sodium ions. Threonine export in antiport against protons is proposed.

TdcA, a transcriptional activator of the tdcABC operon of Escherichia coli, is a member of the LysR family of proteins

The tdcB and tdcC genes of the tdcABC operon of Escherichia coli encode threonine dehydratase and a threonine-serine permease, respectively. These proteins are involved in transport and metabolism of threonine and serine during anaerobic growth. In this study, we functionally characterized tdcA, which encodes a 35 kDa polypeptide consisting of 312 amino acid residues. Non-polar and partially polar mutations introduced into tdcA drastically reduced the expression of the genes down-stream from tdcA. Complementation studies using single-copy chromosomal integrants of a tdcB-lacZ fusion harboring an in-frame deletion of tdcA with chromosomal or plasmid-borne tdcA+ in trans showed complete restoration of tdc operon expression in vivo. The amino acid sequence at the amino-terminal end of TdcA revealed a significant homology to the helix-turn-helix motifs of typical DNA binding proteins. Sequence alignment of TdcA with LysR also showed considerable sequence similarity throughout their entire lengths. Our results suggest that TdcA is related to the LysR family of proteins by common ancestry and, based on its functional role in tdc expression, belongs to the LysR family of transcriptional activators.

Characterization of threonine transport into a kidney epithelial cell line (BSC-1). Evidence for the presence of Na(+)-independent system asc [corrected

The transport routes for threonine in a primate kidney epithelial cell line (BSC-1) grown as monolayer in continuous cell culture were studied. We discovered at least four different transport systems for threonine uptake. The Na(+)-dependent route shows biphasic kinetics with a low and high affinity parameter. The apparent kinetic constants for Km1 and Km2 were 0.3 and 36 mM with apparent Vmax values of 6.3 and 90 nmol/mg protein/min, respectively. The high affinity, low Km component resembles system ASC activity, with respect to substrate selectivity. The Na(+)-independent route also exhibits biphasic kinetics. A high affinity component (apparent Km of 1.0 mM, and apparent Vmax of 7.2 nmol/mg protein/min) is sensitive to inhibition by leucine and the aminoendolevo-rotatory isomer of 2-aminobicyclo[2,2,1]heptane-2-carboxylic acid, suggesting participation by system L. The low affinity component (apparent Km of 10.2 mM, and apparent Vmax of 71 nmol/mg protein/min) was specifically inhibited by threonine, serine, and alanine and could be assigned to system asc. The discrimination between system L and asc is based upon differences in pH sensitivity, trans stimulation, and Ki values. In addition, the effects of harmaline, a suspected sodium transport site inhibitor, have been studied. Harmaline noncompetitively inhibited Na(+)-dependent threonine uptake but had no effect on Na(+)-independent transport of threonine. This report is the first to present evidence for the presence of system asc in renal epithelial cells. The physiological and biochemical significance of our findings are discussed.

?-threonine transport in the obligate methylotroph Methylobacillus flagellatum KT

?-threonine transport in the obligate methylotroph Methylobacillus flagellatum KT

Some characteristics of threonine transport across the blood-brain barrier of the rat.

Threonine entry into brain is altered by diet-induced changes in concentrations of plasma amino acids, especially the small neutrals. To study this finding further, we compared effects of various amino acids (large and small neutrals, analogues, and transport models) on transport of threonine and phenylalanine across the blood-brain barrier. Threonine transport was saturable and was usually depressed more by natural large than small neutrals. Norvaline and 2-amino-n-butyrate (AABA) were stronger competitors than norleucine. 2-Aminobicyclo[2.2.1]heptane-2-carboxylate (BCH), a model in other preparations for the large neutral (L) system, and cysteine, a proposed model for the ASC system only in certain preparations, reduced threonine transport; 2-(methylamino)isobutyrate (MeAIB; a model for the A system for small neutrals) did not. Phenylalanine transport was most depressed by cold phenylalanine and other large neutrals; threonine and other small neutrals had little effect. Norleucine, but not AABA, was a strong competitor; BCH was more competitive than cysteine or MeAIB. Absence of sodium did not affect phenylalanine transport, but decreased threonine uptake by 25% (p less than 0.001). Our results with natural, analogue, and model amino acids, and especially with sodium, suggest that threonine, but not phenylalanine, may enter the brain partly by the sodium-dependent ASC system.

Characterization of l-threonine and l-glutamine transport in murine P388 leukemia cells in vitro. Presence of an N-like amino acid transport system

The transport of l-threonine and l-glutamine into murine P388 leukemia cells has been characterized. Threonine appears to be a specific substrate for a Na +-dependent amino acid transport system similar to system ASC of the HTC hepatoma cell. Threonine transport is uninhibited by 2-aminobicyclo[2,2,1]heptane-2-carboxylic acid and α-(methylamino)isobutyric acid, shows a pattern of transport similar to that seen in HTC hepatoma cells over the pH range of 5.5–7.5, and is inhibited by l-serine and l-cysteine. Approximately two-thirds of glutamine transport into P388 cells also appears to enter P388 cells via this ASC-analogous system. However, based upon (a) inhibition studies with threonine (where the K 1 of threonine inhibition of glutamine transport was 7-fold the K m of threonine transport), (b) inhibition analysis of glutamine transport with various amino acids and amino acid analogues, and (c) different patterns of transport between threonine and glutamine over the pH range of 5.5–7.5, approximately one-third of glutamine transport can be attributed to a second Na +-dependent amino acid transport system. This system appears to be similar to the system N of rat hepatocytes. Glutamine and threonine do not appear to enter P388 cells via systems A or L to any significant degree. P388 cells do not appear to exhibit ‘adaptive regulation’ of amino acid transport. Differences in ‘adaptive regulation’ could therefore not be utilized for comparing threonine and glutamine transport.

Discrimination of Na+-independent transport systems L, T, and asc in erythrocytes. Na+ independence of the latter a consequence of cell maturation?

On the basis of inhibition analysis two bicyclic amino acid analogs appear to enter human red blood cells by much the same Na+-independent mediation, whereas striking differences are apparent in the routes for tryptophan and leucine, confirming a role for System T, but also suggesting the participation of a third system of low affinity somewhat selective for weakly basic amino acids. System T of the human cell is specifically inhibited by 4-azidophenylalanine, and is highly sensitive, relative to System L, to N-ethylmaleimide inhibition. Uptake by System T approaches its steady state much more slowly than does System L, and its participation in trans-stimulation is questionable, whereas that of System L is as usual strong. A different added transport system became apparent in the slow approach of the Na+-independent mediation of uptake of 3- and 4-carbon dipolar amino acids by the nucleated pigeon red cell to its steady state. In that cell System T makes at most a minor contribution. The patterns of trans-stimulation of fluxes among selected pairs of amino acids in the pigeon cell correspond to a usual participation in transmembrane exchange by System L, and also by the new transport system. An important but not the sole source of the heterogeneity in the pigeon cell is the participation of the system conspicuously involved in the transport of alanine, serine, and threonine, among other amino acids. This route of transport of these amino acids is made conspicuous by their small transport by other Na+-independent agencies, notably System L. Reactivity with this system is enhanced by a side change hydroxyl or sulfhydryl group. Uptake by this route as tested by threonine showed little inhibition by cysteinesulfinate under conditions inhibitory to System asc; also a sensitivity to lowering of pH unlike that seen with System asc. The new Na+-dependent transport system appears to be a species variant of quite similar Na+-independent systems discovered by Young et al. (Young, J. D., Ellory, J. C., and Tucker, E. M. (1975) Nature (Lond.) 254, 156-157; Fincham, D. A., Mason, D. K., and Young, J. D. (1982) Biochem. Soc. Trans. 11, 776-777) in sheep and horse erythrocytes on the basis of their absence in phenotypes. These authors have emphasized several similarities in these two cases to Na+-dependent System asc, and they propose that Na+ dependence has specifically been lost on maturation of the red cells without major changes in amino acid selectivity.(ABSTRACT TRUNCATED AT 400 WORDS)

Induction of Threonine Imbalance by Dispensable Amino Acids: Relation to Competition for Amino Acid Transport into Brain

The ability of low protein diets containing small neutral, dispensable amino acids to induce threonine imbalance has been examined. Diets containing amino acids which compete for threonine transport in vitro (serine, alanine, alpha-amino-n-butyrate) caused depressions of growth and food intake which could be corrected to varying degrees by adding threonine to the diet. Large neutral, indispensable amino acids, moderately inhibitory of threonine transport, also induced the imbalance. Some amino acids that had little or no effect on threonine transport in vitro (acidic amino acids and proline) did not cause growth and food intake depressions. Other non-inhibitory amino acids (arginine and lysine) caused growth depressions which were not satisfactorily corrected by additional threonine alone, but were prevented by supplements of all the indispensable amino acids including threonine. Ornithine which was also not inhibitory of threonine transport was an exception. It induced a moderate growth depression which was corrected by additional threonine. Similar studies showed that histidine or tryptophan imbalance could be induced by feeding diets containing only those large neutral amino acids which compete for histidine or tryptophan transport in vitro. These experiments show that, based on the results of transport competition experiments, it is generally possible to devise amino acid supplements which can induce a dietary imbalance of a given amino acid.

Glycoprotein secretion in vitro by human airway: Normal and chronic bronchitis

Precursor uptake, synthesis, and discharge of glycoproteins by mucous and serous cells of submucosal glands and surface epithelial secretory cells (SES cells) of normal and chronic bronchitic human airways were studied in explants of bronchial mucosa maintained in organ culture. Quantitative assessment of precursor incorporation and secretory rate was carried out by autoradiographic analysis of explants pulse-labeled with tritiated sugars or amino acids. Studies of precursor uptake showed that (i) [ 3H]threonine transport into mucous and serous cells was largely ouabain-sensitive, (ii) [ 3H]glucose transport was only partially ouabain-sensitive and was more sensitive in mucous than in serous cells, and (iii) the uptake of neither precursor was markedly ouabain-sensitive in SES cells. Studies of glycoprotein synthesis within mucous, serous, and SES cells showed that, in all three, precursor incorporation into intracellular macromolecules was inhibited to a similar degree by cycloheximide and salicylate. The effects of post-pulse addition of ouabain, cyclo-heximide, and salicylate indicated that precursor uptake, glycoprotein synthesis, and discharge by mucous and serous cells, are not coupled events and that each process is essentially rate-independent. In explants from patients with chronic bronchitis, both threonine uptake and glycoprotein synthesis appeared to occur at a higher rate than in explants from normal subjects.