The Sugar Model: Catalysis by Amines and Amino Acid Products

Ammonia and amines (including amino acids) were shown tocatalyze the formation of sugars from formaldehyde andglycolaldehyde, and the subsequent conversion of sugars tocarbonyl-containing products under the conditions studied (pH5.5 and 50°C). Sterically unhindered primary amineswere better catalysts than ammonia, secondary amines, andsterically hindered primary amines (i.e.-aminoisobutyric acid). Reactions catalyzed by primaryamines initially consumed formaldehyde and glycolaldehyde about15–20 times faster than an uncatalyzed control reaction. Theamine-catalyzed reactions yielded aldotriose (glyceraldehyde),ketotriose (dihydroxyacetone), aldotetroses (erythrose andthreose), ketotetrose (erythrulose), pyruvaldehyde, acetaldehyde,glyoxal, pyruvate, glyoxylate, and several unindentifiedcarbonyl products. The concentrations of the carbonyl products,except pyruvate and ketotetrose, initially increased and thendeclined during the reaction, indicating their ultimateconversion to other products (like larger sugars or pyruvate).The uncatalyzed control reaction yielded no pyruvate orglyoxylate, and only trace amounts of pyruvaldehyde, acetaldehyde and glyoxal. In the presence of 15 mM catalyticprimary amine, such as alanine, the rates of triose andpyruvaldehyde of synthesis were about 15-times and 1200-timesfaster, respectively, than the uncatalyzed reaction. Sinceprevious studies established that alanine is synthesized fromglycolaldehyde and formaldehyde via pyruvaldehyde as its directprecursor, the demonstration that the alanine catalyzes theconversion of glycolaldehyde and formaldehyde to pyruvaldehydeindicates that this synthetic pathway is capable ofautocatalysis. The relevance of this synthetic process, namedthe Sugar Model, to the origin of life is discussed.     

Novel formation of α-amino acid from α-oxo acids and ammonia in an aqueous medium

In the course of a study of possible mechanisms for chemical evolution in the primeval sea, we found the novel formation of -amino acids and N-acylamino acids from -oxo acids and ammonia in an aqueous medium. Glyoxylic acid reacted with ammonia to form N-oxalylglycine, which gave glycine in a 5–39% yield after hydrolysis with 6N HCl. Pyruvic acid and ammonia reacted to give N-acetylalanine, which formed alanine in a 3–7% overall yield upon hydrolysis. The pH optima in these reactions were between pH 3 and 4. These reactions were further extended to the formation of other amino acids. Glutamic acid, phenylalanine and alanine were formed from -ketoglutaric acid, phenylpyruvic acid and oxaloacetic acid, respectively, under similar conditions. N-Succinylglutamic acid was obtained as an intermediate in glutamic acid synthesis. Phenylacetylphenyl-alanineamide was also isolated as an intermediate in phenylalanine synthesis. Alanine, rather than aspartic acid, was produced from oxaloacetic acid. These reactions provide a novel route for the prebiotic synthesis of amino acids. A mechanism for the reactions will be proposed.     

Novel formation of alpha-amino acids from alpha-oxo acids and ammonia in an aqueous medium

In the course of a study of possible mechanisms for chemical evolution in the primeval sea, we found the novel formation of alpha-amino acids and N-acylamino acids from alpha-oxo acids and ammonia in an aqueous medium. Glyoxylic acid reacted with ammonia to form N-oxalylglycine, which gave glycine in a 5-39% yield after hydrolysis with 6N HC1. Pyruvic acid and ammonia reacted to give N-acetylalanine, which formed alanine in a 3-7% overall yield upon hydrolysis. The pH optima in these reactions were between pH 3 and 4. These reactions were further extended to the formation of other amino acids. Glutamic acid, phenylalanine and alanine were formed from alpha-ketoglutaric acid, phenylpyruvic acid and oxaloacetic acid, respectively, under similar conditions. N-Succinylglutamic acid was obtained as an intermediate in glutamic acid synthesis. Phenylacetylphenylalanineamide was also isolated as an intermediate in phenylalanine synthesis. Alanine, rather than aspartic acid, was produced from oxaloacetic acid. These reactions provide a novel route for the prebiotic synthesis of amino acids. A mechanism for the reactions will be proposed.     

Metabolic Conversion of Amino Acids Loaded in the Vacuole of Chara australis Internodal Cells

Vacuoles of internodal cells of Chara australis (or Chara corallina) were loaded with a 10 millimolar amount of various amino acids by a perfusion method and incubated under continuous light. After 20 to 24 hours, the cell sap was collected, and free amino acids in it and the rest of the cell (cytoplasm) were analyzed. The only amino acid metabolized completely was alanine. About 40 to 80% of the aspartic acid, glutamine, serine, and glycine were metabolized, whereas less than 30% of the threonine, asparagine, isoasparagine, isoleucine, phenylalanine, γ-aminobutyric acid, lysine, and arginine were metabolized. The figure for glutamic acid fluctuated between 10 and 100%. The main metabolites of alanine were glutamine, glycine and ammonia, which accumulated in the vacuole. Alanine utilization was not affected by l-methionine-d,l-sulfoximine or azaserine, but was strongly inhibited by aminooxyacetate. The cell extract contained enough alanine aminotransferase activity to account for the rate of alanine metabolism.     

Amino Acid metabolism of pea leaves: diurnal changes and amino Acid synthesis from N-nitrate

In the young leaves of pea (Pisum sativum L.) plants, there was a diurnal variation in the levels of amino acids. In the light, total amino nitrogen increased for the first few hours, then stabilized; in the dark, there was a transient decrease followed by a gradual recovery. Asparagine, homoserine, alanine, and glutamine accounted for much of these changes. The incorporation of ¹⁵N into various components of the young leaves was followed after supply of ¹⁵N-nitrate. ¹⁵N appeared most rapidly in ammonia, due to reduction in the leaf, and this process took place predominantly in the light. A large proportion of the primary assimilation took place through the amide group of glutamine, which became labeled and turned over rapidly; labeling of glutamic acid and alanine was also rapid. Asparagine (amide group) soon became labeled and showed considerable turnover. Slower incorporation and turnover were found for aspartic acid, γ-aminobutyric acid, and homoserine. Synthesis and turnover of all of the amino acids continued at a low rate in the dark. γ-Aminobutyric acid was the only compound found to label more rapidly in the dark than in the light.     

Alanine synthesis from glyceraldehyde and ammonium ion in aqueous solution

Summary Alanine is formed under anaerobic conditions from glyceraldehyde and ammonium ion in aqueous solutions of sodium phosphate (pH 7.0) or imidazole-imidazolium chloride (pH 7.0) at ambient temperature. In 500 mM imidazole (pH 7.0), alanine synthesis from 10 mM glyceraldehyde and 15 mM ammonium ion is roughly 6 times more rapid in the presence of 10 mM 3-mercaptopropionate (0.62% yield at 60 days) than in its absence (0.10% yield at 60 days). Likewise, the formation of alanine in 500 mM sodium phosphate (pH 7.0) from 5 mM glyceraldehyde and 10 mM ammonium ion is more rapid in the presence of 10 mM N-acetylcysteine than in its absence. In this reaction with N-acetylcysteine, the ratio of the yield of alanine to the yield of lactate is fairly constant. The yield of alanine is about 4.5% that of lactate. Alanine synthesis in the presence of thiol probably proceeds via alanyl thioester, which is produced by rearrangement of the imine of the hemithioacetal of pyruvaldehyde, a product of glyceraldehyde dehydration. The significance of this reaction for molecular evolution is discussed.     

Anaerobic Amino Acid Metabolism

Anoxic stress induces a strong change in sugar, protein, and amino acid metabolism in higher plants. Sugars are rapidly consumed through the anaerobic glycolysis to sustain energy production. Protein degradation under anoxia is a mechanism to release free amino acids contributing in this way to maintaining the osmotic potential of the tissue under stress. Among free amino acids, a particular role is played by glutamic acid, being a precursor of some characteristic compounds of the anaerobic metabolism (alanine, -aminobutyric acid, and putrescine). The glutamine synthetase/glutamate synthase cycle contributes to ammonia reassimilation and primary assimilation of nitrate, and resynthesizes constantly glutamate for the synthesis of other compounds. Some polypeptides involved in these pathways are expressed under anoxia. The importance of amino acid metabolism for the response to anaerobic stress is discussed.     

Prebiotic ribose synthesis: A critical analysis

The discovery of catalytic ability in RNA has given fresh impetus to speculations that RNA played a critical role in the origin of life. This question must rest on the plausibility of prebiotic oligonucleotide synthesis, rather than on the properties of the final product. Many cliams have been published to support the idea that the components of RNA were readily available on the prebiotic earth. In this article, the literature cited in support of the prebiotic availability of one subunit, D-ribose, is reviewed to determine whether it justifies the claim.Polymerization of formaldehyde (the formose reaction) has been the single reaction cited for prebiotic ribose synthesis. It has been conducted with different catalysts: numerous basic substances, neutral clays and heat, and various types of radiation. Ribose has been identified (yields are uncertain, but unlikely to be greater than 1%) in reactions run with concentrated (0.15 M or greater) formaldehyde. It has been claimed in reactions run at lower concentration, but characterization has been inadequate, and experimental details have not been provided.The complex sugar mixture produced in the formose reaction is rapidly destroyed under the reaction conditions. Nitrogenous substances (needed for prebiotic base synthesis) would interfere with the formose reaction by reacting with formaldehyde, the intermediates, and sugar products in undesirable ways.The evidence that is currently available does not support the availability of ribose on the prebiotic earth, except perhaps for brief periods of time, in low concentration as part of a complex mixture, and under conditions unsuitable for nucleoside synthesis.     

Aqueous Synthesis of Peptide Thioesters from Amino Acids and a Thiol Using 1,1′-Carbonyldiimidazole

A new method was developed for the synthesis of peptide thioesters from free amino acids and thiols in water. This one-pot simple method involves two steps: (1) activation in water of an amino acid presumably as its N-carboxyanhydride (NCA) using 1,1′-carbonyldiimidazole (CDI), and (2) subsequent condensation of the activated amino acid-NCA in the presence of a thiol. With this method citrulline peptide thioesters containing up to 10 amino acid residues were prepared in a single reaction. This aqueous synthetic method provides a simple way to prepare peptide thioesters for studies of peptide replication involving ligation of peptide thioesters on peptide templates. The relevance of peptide replication to the origin-of-life process is supported by previous studies showing that amino acid thioesters (peptide thioester precursors) can be synthesized under prebiotic conditions by reaction of small sugars with ammonia and a thiol.     

Sugar-Driven Prebiotic Synthesis of Ammonia from Nitrite

Reaction of 3–5 carbon sugars, glycolaldehyde, and α-ketoaldehydes with nitrite under mild anaerobic aqueous conditions yielded ammonia, an essential substrate for the synthesis of nitrogen-containing molecules during abiogenesis. Under the same conditions, ammonia synthesis was not driven by formaldehyde, glyoxylate, 2-deoxyribose, and glucose, a result indicating that the reduction process requires an organic reductant containing either an accessible α-hydroxycarbonyl group or an α-dicarbonyl group. Small amounts of aqueous Fe⁺³ catalyzed the sugar-driven synthesis of ammonia. The glyceraldehyde concentration dependence of ammonia synthesis, and control studies of ammonia’s reaction with glyceraldehyde, indicated that ammonia formation is accompanied by incorporation of part of the synthesized ammonia into sugar-derived organic products. The ability of sugars to drive the synthesis of ammonia is considered important to abiogenesis because it provides a way to generate photochemically unstable ammonia at sites of sugar-based origin-of-life processes from nitrite, a plausible prebiotic nitrogen species.     

Amino Acid metabolism in pea leaves : utilization of nitrogen from amide and amino groups of [N]asparagine

The flow of nitrogen from the amino and amide groups of asparagine has been followed in young pea (Pisum sativum CV Little Marvel) leaves, supplied through the xylem with ¹⁵N-labeled asparagine. The results confirm that there are two main routes for asparagine metabolism: deamidation and transamination.

Nitrogen from the amide group is found predominantly in 2-hydroxy-succinamic acid (derived from transamination of asparagine) and in the amide group of glutamine. The amide nitrogen is also found in glutamate and dispersed through a range of amino acids. Transfer to glutamineamide results from assimilation of ammonia produced by deamidation of both asparagine and its transamination products: this assimilation is blocked by methionine sulfoximine. The release of amide nitrogen as ammonia is greatly reduced by aminooxyacetate, suggesting that, for much of the metabolized asparagine, transamination precedes deamidation.

The amino group of asparagine is widely distributed in amino acids, especially aspartate, glutamate, alanine, and homoserine. For homoserine, a comparison of N and C labeling, and use of a transaminase inhibitor, suggests that it is not produced from the main pool of aspartate, and transamination may play a role in the accumulation of homoserine in peas.

     

Lipoic Acid Synthesis and Attachment in Yeast Mitochondria

Lipoic acid is a sulfur-containing cofactor required for the function of several multienzyme complexes involved in the oxidative decarboxylation of α-keto acids and glycine. Mechanistic details of lipoic acid metabolism are unclear in eukaryotes, despite two well defined pathways for synthesis and covalent attachment of lipoic acid in prokaryotes. We report here the involvement of four genes in the synthesis and attachment of lipoic acid in Saccharomyces cerevisiae. LIP2 and LIP5 are required for lipoylation of all three mitochondrial target proteins: Lat1 and Kgd2, the respective E2 subunits of pyruvate dehydrogenase and α-ketoglutarate dehydrogenase, and Gcv3, the H protein of the glycine cleavage enzyme. LIP3, which encodes a lipoate-protein ligase homolog, is necessary for lipoylation of Lat1 and Kgd2, and the enzymatic activity of Lip3 is essential for this function. Finally, GCV3, encoding the H protein target of lipoylation, is itself absolutely required for lipoylation of Lat1 and Kgd2. We show that lipoylated Gcv3, and not glycine cleavage activity per se, is responsible for this function. Demonstration that a target of lipoylation is required for lipoylation is a novel result. Through analysis of the role of these genes in protein lipoylation, we conclude that only one pathway for de novo synthesis and attachment of lipoic acid exists in yeast. We propose a model for protein lipoylation in which Lip2, Lip3, Lip5, and Gcv3 function in a complex, which may be regulated by the availability of acetyl-CoA, and which in turn may regulate mitochondrial gene expression.Several oxidative decarboxylation reactions are carried out in prokaryotes and eukaryotes by multienzyme complexes. The function of these complexes requires the action of a sulfur-containing cofactor, lipoic acid (6,8-thioctic acid) (1, 2). Lipoic acid is covalently attached via an amide linkage to a specific lysine residue on the surface of the conserved lipoyl domain of the E2 subunits of pyruvate dehydrogenase (PDH),³ α-ketoglutarate dehydrogenase (α-KDH), the branched chain α-keto acid dehydrogenase complexes, and the H protein of the glycine cleavage (GC) enzyme (3). The lipoyl moiety serves as a swinging arm that shuttles reaction intermediates between active sites within the complexes (1). Despite the well characterized function of lipoic acid as a prosthetic group, the mechanisms of its synthesis and attachment to proteins are the subject of ongoing investigations (4–7).These reactions are best understood in Escherichia coli, which has two well defined pathways for lipoic acid synthesis and attachment: a de novo pathway and a salvage pathway (8). Octanoic acid, synthesized on the acyl carrier protein (ACP) (9), is the substrate for the de novo pathway. Lipoyl synthase (LipA) catalyzes the addition of two sulfur atoms to form lipoic acid from octanoic acid either before or after transfer to the target protein (10) by lipoyl(octanoyl)-ACP:protein transferase (LipB) (11, 12). The preferred order of these two reactions is attachment of octanoic acid by LipB, followed by addition of sulfur by LipA (13). By contrast, in the salvage pathway, lipoate-protein ligase (LplA) attaches free lipoic acid to proteins in a two-step reaction. Lipoic acid, which can be scavenged from the medium, is first activated to lipoyl-AMP and then the lipoyl group is transferred to the proteins (14).Lipoic acid synthesis and attachment to target proteins are less well understood in eukaryotes. Homologs of the E. coli enzymes have been found in fungi, plants, protists, and mammals, but many mechanistic details are unclear (15–17). In Saccharomyces cerevisiae, the mitochondrial type II fatty acid biosynthetic pathway (FAS II) synthesizes octanoyl-ACP, which is the substrate for de novo lipoic acid synthesis (18). Lip2 and Lip5, the respective yeast homologs of E. coli LipB and LipA, were shown to be required for respiratory growth on glycerol medium, PDH activity (19), and lipoic acid synthesis (20), indicating functional roles in de novo lipoic acid synthesis and attachment. However, there has been no previous report of an LplA-like lipoate-protein ligase homolog in yeast. Furthermore, lip2 and lip5 mutant strains cannot grow on medium containing lipoic acid (19, 20), suggesting that yeast either cannot use exogenously supplied lipoic acid or there is no yeast equivalent of the E. coli LplA-driven salvage pathway.Here we report the involvement of two additional enzymes in protein lipoylation in yeast mitochondria. The first, Lip3, is a lipoate-protein ligase homolog and is required with Lip2 and Lip5 for lipoylation of the E2 subunits of PDH (Lat1) and α-KDH (Kgd2). The second enzyme, Gcv3, the H protein of the GC enzyme, is absolutely required for lipoylation of all proteins in yeast.     

Alanine metabolism in skeletal muscle in tissue culture.

Alanine production by skeletal muscle in tissue culture was studied using an established myogenic line (L6) of rat skeletal muscle cells. Correlation analyses were performed on rates of metabolism of alanine, glucose, lactate and pyruvate over incubation periods up to 96 h. Alanine production did not correlate significantly with glucose utilization (r = 0.24, P less than 0.20). Alanine production, however, did correlate with lactate production (r = 0.72, P less than 0.0005) as well as medium (r = 0.50, P less than 0.025) and intracellular (r = 0.85, P less than 0.0005) pyruvate concentrations. The intercepts of the latter two correlation analyses indicated that when medium or cell pyruvate fell below 0.28 mM or 1 nmol/mg protein, respectively, net alanine consumption occurred. Alanine synthesis also correlated (r = 0.71, P less than 0.0005) with the percent change in the cell mass action ratio for the sum of the alanine and aspartate aminotransferase reactions, i.e., [alanine] [malate]/[aspartate] [lactate]. These results suggest that alanine production is not necessarily linked to the rate of glucose utilization but rater to pyruvate overflow above a critical intracellular level; under conditions of pyruvate overflow, alanine synthesis is driven by the tendency to establish equilibrium between metabolites of the linked amino acid transaminases in skeletal muscle.     

Amino Acid Racemization in Pseudomonas putida KT2440

d-Amino acids have been shown to play an increasingly diverse role in bacterial physiology, yet much remains to be learned about their synthesis and catabolism. Here we used the model soil- and rhizosphere-dwelling organism Pseudomonas putida KT2440 to elaborate on the genomics and enzymology of d-amino acid metabolism. P. putida KT2440 catabolized the d-stereoisomers of lysine, phenylalanine, arginine, alanine, and hydroxyproline as the sole carbon and nitrogen sources. With the exception of phenylalanine, each of these amino acids was racemized by P. putida KT2440 enzymes. Three amino acid racemases were identified from a genomic screen, and the enzymes were further characterized in vitro. The putative biosynthetic alanine racemase Alr showed broad substrate specificity, exhibiting measurable racemase activity with 9 of the 19 chiral amino acids. Among these amino acids, activity was the highest with lysine, and the k_cat/K_m values with l- and d-lysine were 3 orders of magnitude greater than the k_cat/K_m values with l- and d-alanine. Conversely, the putative catabolic alanine racemase DadX showed narrow substrate specificity, clearly preferring only the alanine stereoisomers as the substrates. However, DadX did show 6- and 9-fold higher k_cat/K_m values than Alr with l- and d-alanine, respectively. The annotated proline racemase ProR of P. putida KT2440 showed negligible activity with either stereoisomer of the 19 chiral amino acids but exhibited strong epimerization activity with hydroxyproline as the substrate. Comparative genomic analysis revealed differences among pseudomonads with respect to alanine racemase genes that may point to different roles for these genes among closely related species.     

Behavior of Free Aromatic Amino Acid Pools in Rosmarinic Acid-Producing Cell Cultures of Anchusa officinalis L

The pool sizes of free l-phenylalanine and l-tyrosine, the precursors of rosmarinic acid in Anchusa officinalis L. cell suspension cultures, fluctuated during the culture cycle. The major increase in pool sizes was preceded by a peak of prephenate aminotransferase activity, while the subsequent decrease coincided with the presence of high activities of phenylalanine ammonia-lyase and tyrosine aminotransferase, the two entrypoint enzymes of the rosmarinic acid biosynthesis pathway. Timecourse feeding studies with linear growth stage cells revealed that the tyrosine pool turned over rapidly, consistent with direct participation in rosmarinic acid synthesis. Since externally applied l-tyrosine was rapidly incorporated into rosmarinic acid with little evidence of radioactively labeled intermediates, it is suggested that there exists a close coupling between the l-tyrosine pool and the rosmarinic acid biosynthetic pathway, which may involve the channelling of intermediates both into and within the pathway.     

Effect of Penicillin on Amino Acid Fermentation

The effect of penicillin G(k) was first investigated on l-homoserine production by Micrococcus glutamicus 534-Co 147 (a threonine requiring mutant). The addition of 4 u/ml of penicillin, 7 to 9 hours after inoculation, brought about the conversion of l-homoserine to l-glutamic acid production. Similar phenomena were observed in l-lysine and l-valine fermentations. In these cases, a homoserine requiring and a leucine requiring mutant of M. glutamicus were used respectively. A marked conversion from lysine and valine to glutamate accumulation occured by penicillin addition. However, in l-isoleucine fermentation with Brevibacterium ammoniagenes ATCC 6871, no glutamate accumulation took place and isoleucine yields were remarkably decreased.     

Alanine metabolism in skeletal muscle in tissue culture

Alanine production by skeletal muscle in tissue culture was studied using an established myogenic line (L₆) of rat skeletal muscle cells. Correlation analyses were performed on rates of metabolism of alanine, glucose, lactate and pyruvate over incubation periods up to 96 h. Alanine production did not correlate significantly with glucose utilization (r = 0.24, P < 0.20). Alanine production, however, did correlate with lactate production (r = 0.72, P < 0.0005) as well as medium (r = 0.50, P < 0.025) and intracellular (r = 0.85, P < 0.0005) pyruvate concentrations. The intercepts of the latter two correlation analyses indicated that when medium or cell pyruvate fell below 0.28 mM or 1 nmol/mg protein, respectively, net alanine consumption occurred. Alanine synthesis also correlated (r = 0.71, P < 0.0005) with the percent change in the cell mass action ratio for the sum of the alanine and aspartate aminotransferase reactions, i.e., [alanine] [malate]/[aspartate] [lactate]. These results suggest that alanine production is not necessarily linked to the rate of glucose utilization but rather to pyruvate overflow above a critical intracellular level; under conditions of pyruvate overflow, alanine synthesis is driven by the tendency to establish equilibrium between metabolites of the linked amino acid transaminases in skeletal muscle.     

Ammonia assimilation and glutamate incorporation in coenzyme F420 derivatives ofMethanosarcina barkeri

Methanosarcina barkeri was able to grow on L-alanine and L-glutamate as sole nitrogen sources. Cell yields were 0.5 g/l and 0.7 g/l (wet wt), respectively. The mechanism of ammonia assimilation inMethanosarcina barkeri strain MS was studied by analysis of enzyme activities. Activity levels of nitrogen-assimilating enzymes in extracts of cells grown on different nitrogen sources (ammonia, 0.05–100 mM; L-alanine, 10 mM; L-glutamate, 10 mM) were compared. Activities of glutamate dehydrogenase, glutamate synthase, glutamine synthetase, glutamate oxaloacetate transaminase and glutamate pyruvate transaminase could be measured in cells grown on these three nitrogen sources. Alanine dehydrogenase was not detected under the growth conditions used. None of the measured enzyme activities varied significantly in response to the NH₄ ⁺ concentration. The length of the poly--glutamyl side chain of F₄₂₀ derivatives turned out to be independent of the concentration of ammonia in the culture medium.Abbreviations ADH alanine dehydrogenase - FO 7,8-didemethyl-8-hydroxy-5-deazariboflavin - GDH glutamate dehydrogenase - GOGAT glutamate synthase - GOT glutamate oxaloacetate transaminase - GPT glutamate pyruvate transaminase - GS glutamine synthetase - H₄MPT tetrahydromethanopterin     

A possible role of alanine for ammonia transfer between astrocytes and glutamatergic neurons

The metabolism of [U-(13)C]lactate (1 mM) in the presence of unlabeled glucose (2.5 mM) was investigated in glutamatergic cerebellar granule cells, cerebellar astrocytes, and corresponding co-cultures. It was evident that lactate is primarily a neuronal substrate and that lactate produced glycolytically from glucose in astrocytes serves as a substrate in neurons. Alanine was highly enriched with (13)C in the neurons, whereas this was not the case in the astrocytes. Moreover, the cellular content and the amount of alanine released into the medium were higher in neurons than astrocytes. On incubation of the different cell types in medium containing alanine (1 mM), the astrocytes exhibited the highest level of accumulation. Altogether, these results indicate a preferential synthesis and release of alanine in glutamatergic neurons and uptake in cerebellar astrocytes. A new functional role of alanine may be suggested as a carrier of nitrogen from glutamatergic neurons to astrocytes, a transport that may operate to provide ammonia for glutamine synthesis in astrocytes and dispose of ammonia generated by the glutaminase reaction in glutamatergic neurons. Hence, a model of a glutamate-glutamine/lactate-alanine shuttle is presented. To elucidate if this hypothesis is compatible with the pattern of alanine metabolism observed in the astrocytes and neurons from cerebellum, the cells were incubated in a medium containing [(15)N]alanine (1 mM) and [5-(15)N]glutamine (0.5 mM), respectively. Additionally, neurons were incubated with [U-(13)C]glutamine to estimate the magnitude of glutamine conversion to glutamate. Alanine was labeled from [5-(15)N]glutamine to 3.3% and [U-(13)C]glutamate generated from [U-(13)C]glutamine was labeled to 16%. In spite of the modest labeling in alanine, it is clear that nitrogen from ammonia is transferred to alanine via transamination with glutamate formed by reductive amination of alpha-ketoglutarate. With regard to the astrocytic part of the shuttle, glutamine was labeled to 22% in one nitrogen atom whereas 3.2% was labeled in two when astrocytes were incubated in [(15)N]alanine. Moreover, in co-cultures, [U-(13)C]alanine labeled glutamate and glutamine equally, whereas [U-(13)C]lactate preferentially labeled glutamate. Altogether, these results support the role proposed above of alanine as a possible ammonia nitrogen carrier between glutamatergic neurons and surrounding astrocytes and they show that lactate is preferentially metabolized in neurons and alanine in astrocytes.