The use of a potential-sensitive cyanine dye for studying ion-dependent electrogenic renal transport of organic solutes. Uptake of l-malate and d-malate by luminal-membrane vesicles

The mechanisms of uptake of dicarboxylic acids by rabbit renal luminal-membrane vesicles were studied by the use of filtration and spectrophotometric techniques as described in an accompanying paper [Kragh-Hansen, Jørgensen & Sheikh (1982) Biochem. J. 208, 359–368]. Addition of l- or d-malate to dye-membrane-vesicle suspensions in the presence of Na⁺ gradients (extravesicular>intravesicular) resulted in spectral curves indicative of depolarization events. The renal uptake of dicarboxylic acids was dependent on the type of Na⁺-salt anion present and could be correlated with the ability of the anions to penetrate biological membranes (i.e. Cl⁻>SO₄²⁻>gluconate). Identical results were obtained by a filtration technique with Sartorius membrane filters. The results indicate that the dicarboxylic acids are taken up by the membrane vesicles in an electrically positive form (i.e. Na⁺/substrate coupling ratio 3:1) by an Na⁺-dependent transport system. This proposal was further supported by spectrophotometric experiments with various ionophores such as valinomycin, gramicidin and nigericin. The absorbance changes associated with simultaneous addition of l- and d-malate and spectrophotometric competition studies revealed that the two isomers are taken up by a common transport system. Spectral changes of the dye induced by addition of increasing concentrations of l- or d-malate indicated that the transport system favours the unphysiological d-form rather than the l-form of malate. Furthermore, it was observed that the affinity of both isomers for the transport system was dependent on the concentration of Na⁺ in the medium.     

In Vitro Analysis of the H-Hexose Symporter on the Plasma Membrane of Sugarbeets (Beta vulgaris L.)

The mechanism of hexose transport into plasma membrane vesicles isolated from mature sugarbeet leaves (Beta vulgaris L.) was investigated. The initial rate of glucose uptake into the vesicles was stimulated approximately fivefold by imposing a transmembrane pH gradient (ΔpH), alkaline inside, and approximately fourfold by a negative membrane potential (ΔΨ), generated as a K⁺-diffusion potential, negative inside. The -fold stimulation was directly related to the relative ΔpH or ΔΨ gradient imposed, which were determined by the uptake of acetate or tetraphenylphosphonium, respectively. ΔΨ- and ΔpH-dependent glucose uptake showed saturation kinetics with a K_m of 286 micromolar for glucose. Other hexose molecules (e.g. 2-deoxy-d-glucose, 3-O-methyl-d-glucose, and d-mannose) were also accumulated into plasma membrane vesicles in a ΔpH-dependent manner. Inhibition constants of a number of compounds for glucose uptake were determined. Effective inhibitors of glucose uptake included: 3-O-methyl-d-glucose, 5-thio-d-glucose, d-fructose, d-galactose, and d-mannose, but not 1-O-methyl-d-glucose, d- and l-xylose, l-glucose, d-ribose, and l-sorbose. Under all conditions of proton motive force magnitude and glucose and sucrose concentration tested, there was no effect of sucrose on glucose uptake. Thus, hexose transport on the sugarbeet leaf plasma membrane was by a H⁺-hexose symporter, and the carrier and possibly the energy source were not shared by the plasma membrane H⁺-sucrose symporter.     

Structural requirements for active intestinal transport. The nature of the carrier–sugar bonding at C-2 and the ring oxygen of the sugar

Several weakly transported sugars were tested for transport by the Na⁺-dependent sugar carrier with slices of everted hamster intestinal tissue. Sugars were assumed to be transported by this carrier if the accumulation was diminished in the absence of Na⁺ and in the presence of the competitive inhibitor 1,5-anhydro-d-glucitol. The extent of accumulation was correlated with the number of hydroxyl groups in the d-gluco configuration if the ring oxygen was placed in the normal d-glucose position. 5-Thio-d-glucose, with a sulphur atom in the ring, was transported at about the same rate as d-glucose and had a similar K_i for d-galactose transport, but myoinositol was poorly accumulated. It is suggested that there is no hydrogen bonding at the ring oxygen atom, but that the oxygen atom is found at this position as a result of steric constraints. No sugar without a hydroxyl group in the d-gluco position at C-2 of the sugar, including d-mannose, 2-deoxy-d-glucose, 2-chloro-2-deoxy-d-glucose and 2-deoxy-2-fluoro-d-glucose, was transported by the Na⁺-dependent carrier, but these sugars and l-fucose weakly and competitively inhibit the Na⁺-dependent accumulation of l-glucose into slices of everted hamster intestinal tissue. It is concluded that the bond between the carrier and C-2 of the sugar may be covalent, and a possible mechanism for active intestinal transport is proposed.     

Sodium-gradient-stimulated transport of l-alanine by plasma-membrane vesicles isolated from liver parenchymal cells of fed and starved rats. Crucial role of the adrenal glucocorticoids

The ability of liver efficiently to take up amino acids, particularly l-alanine, during starvation was studied in a cell-free system by isolating plasma-membrane vesicles in a transport-competent state from rat liver parenchymal cells. These membrane vesicles have the capacity to accumulate l-alanine against an apparent concentration gradient when exposed to an artificial and transient transmembrane Na⁺ gradient (extravesicular Na⁺ concentration greater than inside). The rate of accumulation of l-alanine is dependent on the plasma-membrane vesicle concentration, and the steady-state concentration attained is inversely related to the osmolarity of the medium. The Na⁺-mediated stimulation is not exhibited if the membrane vesicles are pre-equilibrated with NaCl, if K⁺ or Li⁺ are substituted for Na⁺, or if SO₄²⁻ replaces Cl⁻ as the counterion. The apparent active transport of l-alanine into the membrane vesicles appears to occur by an electrogenic mechanism: (1) the use of NaSCN significantly heightens the early concentrative phase of transport when compared with the effect of NaCl; (2) an enhanced active transport is also observed when a valinomycin-induced K⁺ efflux occurs concomitant with Na⁺ and l-alanine influx. Plasma-membrane vesicles isolated from liver parenchymal cells of a 24 h-starved rat exhibit an initial l-alanine transport rate that is 3–4 times that for membrane vesicles derived from a fed animal. The increased rate of l-alanine transport by plasma-membrane vesicles from starved animals can be obliterated by adrenalectomy and restored by administration of glucocorticoid. These results establish that stimulation of the gluconeogenic pathway by starvation involves a plasma-membrane-localized change affecting l-alanine transport which is regulated in part by the glucocorticoid hormones.     

Amino Acid Isomerization in the Production of l-Phenylalanine from d-Phenylalanine by Bacteria

To establish an advantageous method for the production of l-amino acids, microbial isomerization of d- and dl-amino acids to l-amino acids was studied. Screening experiments on a number of microorganisms showed that cell suspensions of Pseudomonas fluorescens and P. miyamizu were capable of isomerizing d- and dl-phenylalanines to l-phenylalanine. Various conditions suitable for isomerization by these organisms were investigated. Cells grown in a medium containing d-phenylalanine showed highest isomerization activity, and almost completely converted d- or dl-phenylalanine into l-phenylalanine within 24 to 48 hr of incubation. Enzymatic studies on this isomerizing system suggested that the isomerization of d- or dl-phenylalanine is not catalyzed by a single enzyme, “amino acid isomerase,” but the conversion proceeds by a two step system as follows: d-pheylalanine is oxidized to phenylpyruvic acid by d-amino acid oxidase, and the acid is converted to l-phenylalanine by transamination or reductive amination.     

The oxidation of d- and l-glycerate by rat liver

1. The interconversion of hydroxypyruvate and l-glycerate in the presence of NAD and rat-liver l-lactate dehydrogenase has been demonstrated. Michaelis constants for these substrates together with an equilibrium constant have been determined and compared with those for pyruvate and l-lactate. 2. The presence of d-glycerate dehydrogenase in rat liver has been confirmed and the enzyme has been purified 16–20-fold from the supernatant fraction of a homogenate, when it is free of l-lactate dehydrogenase, with a 23–29% recovery. The enzyme catalyses the interconversion of hydroxypyruvate and d-glycerate in the presence of either NAD or NADP with almost equal efficiency. d-Glycerate dehydrogenase also catalyses the reduction of glyoxylate, but is distinct from l-lactate dehydrogenase in that it fails to act on pyruvate, d-lactate or l-lactate. The enzyme is strongly dependent on free thiol groups, as shown by inhibition with p-chloromercuribenzoate, and in the presence of sodium chloride the reduction of hydroxypyruvate is activated. Michaelis constants for these substrates of d-glycerate dehydrogenase and an equilibrium constant for the NAD-catalysed reaction have been calculated. 3. An explanation for the lowered V_max. with d-glycerate as compared with dl-glycerate for the rabbit-kidney d-α-hydroxy acid dehydrogenase has been proposed.     

The metabolism of galactarate, d-glucarate and various pentoses by species of Pseudomonas

1. When NAD⁺ was present, cell extracts of Pseudomonas (A) grown with d-glucarate or galactarate converted 1mol. of either substrate into 1mol. each of 2-oxoglutarate and carbon dioxide; 70–80% of the gas originated from C-1 of the hexarate. 2. The enzyme system that liberated carbon dioxide from galactarate was inactive in air and was stabilized by galactarate or Fe²⁺ ions; the system that acted on d-glucarate was more stable and was stimulated by Mg²⁺ ions. 3. When NAD⁺ was not added, 2-oxoglutarate semialdehyde accumulated from either substrate. This compound was isolated as its bis-2,4-dinitrophenylhydrazone, and several properties of the derivative were compared with those of the chemically synthesized material. Methods were developed for the determination of 2-oxoglutarate semialdehyde. 4. Synthetic 2-oxoglutarate semialdehyde was converted into 2-oxoglutarate by an enzyme that required NAD⁺; the reaction rate with NADP⁺ was about one-sixth of that with NAD⁺. 5. For extracts of Pseudomonas (A) grown with d-glucarate or galactarate, or for those of Pseudomonas fragi grown with l-arabinose or d-xylose, specific activities of 2-oxoglutarate semialdehyde–NAD oxidoreductase were much higher than for extracts of the organisms grown with (+)-tartrate and d-glucose respectively. 6. Extracts of Pseudomonas fragi grown with l-arabinose or d-xylose converted l-arabonate or d-xylonate into 2-oxoglutarate when NAD⁺ was added to reaction mixtures and into 2-oxoglutarate semialdehyde when NAD⁺ was omitted.     

Uptake of monosaccharides by guinea-pig cerebral-cortex slices

By the use of 1mm-iodoacetate to inhibit glycolysis in guinea-pig cerebral tissue slices, the kinetics of the uptake of monosaccharides on transfer of tissue from 0° to 37° were studied. d-Ribose, d-galactose, d-mannose, l-sorbose, and d-fructose showed diffusion kinetics, whereas 2-deoxy-d-glucose, d-glucose, d-arabinose and d-xylose showed saturation kinetics.     

Preferential Utilization of d-Lactate by Shewanella oneidensis

Shewanella oneidensis couples oxidation of lactate to respiration of many substrates. Here we report that llpR (l-lactate-positive regulator, SO_3460) encodes a positive regulator of l-lactate utilization distinct from previously studied regulators. We also demonstrate d-lactate inhibition of l-lactate utilization in S. oneidensis, resulting in preferential utilization of the d isomer.     

myo-Inositol-1-Phosphatase from the Pollen of Lilium longiflorum Thunb

A Mg²⁺-dependent, alkaline phosphatase has been isolated from mature pollen of Lilium longiflorum Thunb., cv. Ace and partially purified. It hydrolyzes 1l- and 1d-myo-inositol 1-phosphate, myo-inositol 2-phosphate, and β-glycerophosphate at rates decreasing in the order named. The affinity of the enzyme for 1l- and 1d-myo-inositol 1-phosphate is approximately 10-fold greater than its affinity for myo-inositol 2-phosphate. Little or no activity is found with phytate, d-glucose 6-phosphate, d-glucose 1-phosphate, d-fructose 1-phosphate, d-fructose 6-phosphate, d-mannose 6-phosphate, or p-nitrophenyl phosphate. 3-Phosphosphoglycerate is a weak competitive inhibitor. myo-Inositol does not inhibit the reaction. Optimal activity is obtained at pH 8.5 and requires the presence of Mg²⁺. At 4 millimolar, Co²⁺, Fe²⁺ or Mn²⁺ are less effective. Substantial inhibition is obtained with 0.25 molar Li⁺. With β-glycerophosphate as substrate the K_m is 0.06 millimolar and the reaction remains linear at least 2 hours. In 0.1 molar Tris, β-glycerophosphate yields equivalent amounts of glycerol and inorganic phosphate, evidence that transphosphorylation does not occur.     

An l-glucose Catabolic Pathway in Paracoccus Species 43P

An l-glucose-utilizing bacterium, Paracoccus sp. 43P, was isolated from soil by enrichment cultivation in a minimal medium containing l-glucose as the sole carbon source. In cell-free extracts from this bacterium, NAD⁺-dependent l-glucose dehydrogenase was detected as having sole activity toward l-glucose. This enzyme, LgdA, was purified, and the lgdA gene was found to be located in a cluster of putative inositol catabolic genes. LgdA showed similar dehydrogenase activity toward scyllo- and myo-inositols. l-Gluconate dehydrogenase activity was also detected in cell-free extracts, which represents the reaction product of LgdA activity toward l-glucose. Enzyme purification and gene cloning revealed that the corresponding gene resides in a nine-gene cluster, the lgn cluster, which may participate in aldonate incorporation and assimilation. Kinetic and reaction product analysis of each gene product in the cluster indicated that they sequentially metabolize l-gluconate to glycolytic intermediates, d-glyceraldehyde-3-phosphate, and pyruvate through reactions of C-5 epimerization by dehydrogenase/reductase, dehydration, phosphorylation, and aldolase reaction, using a pathway similar to l-galactonate catabolism in Escherichia coli. Gene disruption studies indicated that the identified genes are responsible for l-glucose catabolism.     

Accumulation of d-Glucose from Pentoses by Metabolically Engineered Escherichia coli

Escherichia coli that is unable to metabolize d-glucose (with knockouts in ptsG, manZ, and glk) accumulates a small amount of d-glucose (yield of about 0.01 g/g) during growth on the pentoses d-xylose or l-arabinose as a sole carbon source. Additional knockouts in the zwf and pfkA genes, encoding, respectively, d-glucose-6-phosphate 1-dehydrogenase and 6-phosphofructokinase I (E. coli MEC143), increased accumulation to greater than 1 g/liter d-glucose and 100 mg/liter d-mannose from 5 g/liter d-xylose or l-arabinose. Knockouts of other genes associated with interconversions of d-glucose-phosphates demonstrate that d-glucose is formed primarily by the dephosphorylation of d-glucose-6-phosphate. Under controlled batch conditions with 20 g/liter d-xylose, MEC143 generated 4.4 g/liter d-glucose and 0.6 g/liter d-mannose. The results establish a direct link between pentoses and hexoses and provide a novel strategy to increase carbon backbone length from five to six carbons by directing flux through the pentose phosphate pathway.     

Moderate l-lactate administration suppresses adipose tissue macrophage M1 polarization to alleviate obesity-associated insulin resistance

As a crucial metabolic intermediate, l-lactate is involved in redox balance, energy balance, and acid–base balance in organisms. Moderate exercise training transiently elevates plasma l-lactate levels and ameliorates obesity-associated type 2 diabetes. However, whether moderate l-lactate administration improves obesity-associated insulin resistance remains unclear. In this study, we defined 800 mg/kg/day as the dose of moderate l-lactate administration. In mice fed with a high-fat diet (HFD), moderate l-lactate administration for 12 weeks was shown to alleviate weight gain, fat accumulation, and insulin resistance. Along with the phenotype alterations, white adipose tissue thermogenesis was also found to be elevated in HFD-fed mice. Meanwhile, moderate l-lactate administration suppressed the infiltration and proinflammatory M1 polarization of adipose tissue macrophages (ATMs) in HFD-fed mice. Furthermore, l-lactate treatment suppressed the lipopolysaccharide-induced M1 polarization of bone marrow–derived macrophages (BMDMs). l-lactate can bind to the surface receptor GPR132, which typically drives the downstream cAMP–PKA signaling. As a nutrient sensor, AMP-activated protein kinase (AMPK) critically controls macrophage inflammatory signaling and phenotype. Thus, utilizing inhibitors of the kinases PKA and AMPK as well as siRNA against GPR132, we demonstrated that GPR132–PKA–AMPKα1 signaling mediated the suppression caused by l-lactate treatment on BMDM M1 polarization. Finally, l-lactate addition remarkably resisted the impairment of lipopolysaccharide-treated BMDM conditional media on adipocyte insulin sensitivity. In summary, moderate l-lactate administration suppresses ATM proinflammatory M1 polarization through activation of the GPR132–PKA–AMPKα1 signaling pathway to improve insulin resistance in HFD-fed mice, suggesting a new therapeutic and interventional approach to obesity-associated type 2 diabetes.     

A Novel Type of Peptidoglycan-binding Domain Highly Specific for Amidated d-Asp Cross-bridge,Identified in Lactobacillus casei Bacteriophage Endolysins

Peptidoglycan hydrolases (PGHs) are responsible for bacterial cell lysis. Most PGHs have a modular structure comprising a catalytic domain and a cell wall-binding domain (CWBD). PGHs of bacteriophage origin, called endolysins, are involved in bacterial lysis at the end of the infection cycle. We have characterized two endolysins, Lc-Lys and Lc-Lys-2, identified in prophages present in the genome of Lactobacillus casei BL23. These two enzymes have different catalytic domains but similar putative C-terminal CWBDs. By analyzing purified peptidoglycan (PG) degradation products, we showed that Lc-Lys is an N-acetylmuramoyl-l-alanine amidase, whereas Lc-Lys-2 is a γ-d-glutamyl-l-lysyl endopeptidase. Remarkably, both lysins were able to lyse only Gram-positive bacterial strains that possess PG with d-Ala⁴→d-Asx-l-Lys³ in their cross-bridge, such as Lactococcus casei, Lactococcus lactis, and Enterococcus faecium. By testing a panel of L. lactis cell wall mutants, we observed that Lc-Lys and Lc-Lys-2 were not able to lyse mutants with a modified PG cross-bridge, constituting d-Ala⁴→l-Ala-(l-Ala/l-Ser)-l-Lys³; moreover, they do not lyse the L. lactis mutant containing only the nonamidated d-Asp cross-bridge, i.e. d-Ala⁴→d-Asp-l-Lys³. In contrast, Lc-Lys could lyse the ampicillin-resistant E. faecium mutant with 3→3 l-Lys³-d-Asn-l-Lys³ bridges replacing the wild-type 4→3 d-Ala⁴-d-Asn-l-Lys³ bridges. We showed that the C-terminal CWBD of Lc-Lys binds PG containing mainly d-Asn but not PG with only the nonamidated d-Asp-containing cross-bridge, indicating that the CWBD confers to Lc-Lys its narrow specificity. In conclusion, the CWBD characterized in this study is a novel type of PG-binding domain targeting specifically the d-Asn interpeptide bridge of PG.     

Properties of an Aminotransferase of Pea (Pisum sativum L.)

A transaminase (aminotransferase, EC 2.6.1) fraction was partially purified from shoot tips of pea (Pisum sativum L. cv. Alaska) seedlings. With α-ketoglutarate as co-substrate, the enzyme transaminated the following aromatic amino acids: d,l-tryptophan, d,l-tyrosine, and d,l-phenylalanine, as well as the following aliphatic amino acids: d,l-alanine, d,l-methionine, and d,l-leucine. Of other α-keto acids tested, pyruvate and oxalacetate were more active than α-ketoglutarate with d,l-tryptophan. Stoichiometric yields of indolepyruvate and glutamate were obtained with d,l-tryptophan and α-ketoglutarate as co-substrates. The specific activity was three times higher with d-tryptophan than with l-tryptophan.     

Active Sugar Transport by the Small Intestine : The effects of sugars,amino acids,hexosamines, sulfhydryl-reacting compounds,and cations on the preferential binding of D-glucose to Tris-disrupted brush borders

Tris-disrupted and intact brush border membrane preparations from mucosa of hamster jejunum were capable of preferentially binding actively transported D-glucose in a similar manner. Density gradient centrifugation of the Tris-disrupted brush borders indicated that D-glucose was bound to a fraction containing the cores or inner material of the microvilli. The properties of this binding were examined with the Tris-disrupted brush border preparation. Actively transported sugars competitively inhibited preferential D-glucose binding, whereas no effect was observed with nonactively transported sugars. Neither actively nor nonactively transported amino acids affected D-glucose binding. D-Glucosamine, which is not actively transported, was inhibitory to preferential D-glucose binding as well as to the active transport of D-glucose by everted sacs of hamster jejunum. No inhibitory effect was observed with the same concentration of D-galactosamine. Preferential D-glucose binding was also inhibited by sulfhydryl-reacting compounds, Ca²⁺, and Li⁺ ions. On the other hand, Mg²⁺ was shown to be stimulatory and Na⁺, NH₄ ⁺, and K⁺ had no effect on this phenomenon. The results of these experiments suggest that preferential D-glucose binding to brush borders is related to the initial step in active sugar transport by the small intestine.     

The biosynthesis of polysaccharides. Incorporation of d-[1-14C]glucose and d-[6-14C]glucose into plum-leaf polysaccharides

1. The utilization of specifically labelled d-glucose in the biosynthesis of plum-leaf polysaccharides has been studied. After these precursors had been metabolized in plum leaves, the polysaccharides were isolated from the leaves, and their monosaccharide constituents isolated and purified. 2. Both the specific activities and the distribution of ¹⁴C along the carbon chains of the monosaccharides were determined. Significant ¹⁴C activity was found in units of d-galactose, d-glucose, d-xylose and l-arabinose, but their specific activities varied widely. The labelling patterns suggest that in the leaves the other monosaccharides all arise directly from d-glucose without any skeletal change in the carbon chain, other than the loss of a terminal carbon atom in the synthesis of pentoses. 3. The results indicated that within the leaf there are various precursor pools for polysaccharide synthesis and that these pools are not in equilibrium with one another.     

K+-dependent 3H-d-glucose transport by hepatopancreatic brush border membrane vesicles of a marine shrimp

The effects of sodium, potassium, sugar inhibitors, and membrane potential on ³H-d-glucose uptake by hepatopancreatic epithelial brush border membrane vesicles (BBMV) of the Atlantic marine shrimp, Litopenaeus setiferus, were investigated. Brush border membrane vesicles were prepared using a MgCl₂/EGTA precipitation method and uptake experiments were conducted using a high speed filtration technique. ³H-d-Glucose uptake was stimulated by both sodium and potassium and these transport rates were almost doubled in the presence of an inside-negative-induced membrane potential. Kinetics of ³H-d-glucose influx were hyperbolic functions of both external Na⁺ or K⁺, and an induced membrane potential increased influx J _max and lowered K_m in both salts. ³H-d-Glucose influx versus [glucose] in both Na⁺ or K⁺ media also displayed Michaelis–Menten properties that were only slightly affected by induced membrane potential. Phloridzin was a poor inhibitor of 0.5 mM ³H-d-glucose influx, requiring at least 5 mM in NaCl and 10 mM in KCl to significantly reduce hexose transport. Several sugars (d-galactose, α-methyl-d-gluco-pyranoside, unlabeled d-glucose, d-fructose, and d-mannose) were used at 75 mM as potential inhibitors of 0.1 mM ³H-d-glucose influx. Only unlabeled d-glucose, d-fructose, and d-mannose significantly (p < 0.05) reduced labeled glucose transport. An additional experiment using increasing concentrations of d-mannose (0, 10, 25, 75, and 100 mM) showed this hexose to be an effective inhibitor of 0.1 mM ³H-d-glucose uptake at concentrations of 75 mM and higher. As a whole these results suggest that ³H-d-glucose transport by hepatopancreatic BBMV occurs by a carrier system that is able to use both Na⁺ and K⁺ as drivers, is enhanced by membrane potential, is relatively refractory to phloridzin, and is only inhibited by itself, d-fructose, and d-mannose. These properties are similar to those exhibited by the mammalian SLC5A9/SGLT4 transporter, suggesting that an invertebrate analogue of this protein may occur in shrimp.     

Glutamate, glutamine, aspartate, asparagine, glucose and ketone-body metabolism in chick intestinal brush-border cells

1. Suspensions of isolated chick jejunal columnar absorptive (brush-border) cells respired on endogenous substrates at a rate 40% higher than that shown by rat brush-border cells. 2. Added d-glucose (5 or 10mm), l-glutamine (2.5mm) and l-glutamate (2.5mm) were the only individual substrates which stimulated respiration by chick cells; l-aspartate (2.5 or 6.7mm), glutamate (6.7mm), glutamine (6.7mm), l-alanine (1 or 10mm), pyruvate (1 or 2mm), l-lactate (5 or 10mm), butyrate (10mm) and oleate (1mm) did not stimulate chick cell respiration; l-asparagine (6.7mm) inhibited slightly; glucose (5mm) stimulated more than did 10mm-glucose. 3. Acetoacetate (10mm) and d-3-hydroxybutyrate (10mm) were rapidly consumed but, in contrast to rat brush-border cells, did not stimulate respiration. 4. Glucose (10mm) was consumed more slowly than 5mm-glucose; the dominant product of glucose metabolism during vigorous respiration was lactate; the proportion of glucose converted to lactate was greater with 10mm- than with 5mm-glucose. 5. Glutamate and aspartate consumption rates decreased, and alanine and glutamine consumption rates increased when their initial concentrations were raised from 2.5 to 6.7 or 10mm. 6. The metabolic fate of glucose was little affected by concomitant metabolism of any one of aspartate, glutamate or glutamine except for an increased production of alanine; the glucose-stimulated respiration rate was unaffected by concomitant metabolism of these individual amino acids. 7. Chick cells produced very little alanine from aspartate and, in contrast to rat cells, likewise produced very little alanine from glutamate or glutamine; in chick cells alanine appeared to be predominantly a product of transmination of pyruvate derived from glucose metabolism. 8. In chick cells, glutamate and glutamine were formed from aspartate (2.5 or 6.7mm); aspartate and glutamine were formed from glutamate (2.5mm) but only aspartate from 6.7mm-glutamate; glutamate was the dominant product formed from glutamine (6.7mm) but aspartate only was formed from 2.5mm-glutamine. 9. Chick brush-border cells can thus both catabolize and synthesize glutamine; glutamine synthesis is always diminished by concomitant metabolism of glucose, presumably by allosteric inhibition of glutamine synthetase by alanine. 10. Proline was formed from glutamine (2.5mm) but not from glutamine (2.5mm)+glucose (5mm) and not from 2.5mm-glutamate; ornithine was formed from glutamine (2.5mm)+glucose (5.0mm) but not from glutamine alone; serine was formed from glutamine (2.5mm)+glucose (5mm) and from these two substrates plus aspartate (2.5mm). 11. Total intracellular adenine nucleotides (22μmol/g dry wt.) remained unchanged during incubation of chick cells with glucose. 12. Intracellular glutathione (0.7–0.8mm) was depleted by 40% during incubation of respiring chick cells without added substrates for 75min at 37°C; partial restoration of the lost glutathione was achieved by incubating cells with l-glutamate+l-cysteine+glycine.