Alanine Formation Research Articles

Formation of sugar-specific reactive intermediates from (13)C-labeled L-serines.

Analysis of the pyrolysis products of [1-(13)C], [2-(13)C], and [3-(13)C]-labeled L-serines has indicated the presence of three initial degradation pathways. Decarboxylation followed by deamination produces aminoethanol and acetaldehyde, respectively; a retro-aldol pathway generates formaldehyde and glycine. Dehydration of L-serine can lead to the formation of pyruvic acid, which eventually can be converted into the amino acid alanine. Formation of alanine and glycine was confirmed due to the detection of 2, 5-diketo-3,6-dimethylpiperazine and cycloglycylalanine. Most of the advanced decomposition products of L-serine can be rationalized on the basis of these initial degradation products. Label incorporation studies have elucidated the origin of carbonyl precursors of methyl- and 2,3-dimethylpyrazines formed in the thermal decomposition mixture of L-serine. Three mechanistic pathways were identified for the formation of carbonyl precursors of methyl- and 2, 3-dimethylpyrazines. The major pathway (70%) for the formation of the precursor of methylpyrazine involved aldol addition of formaldehyde to glycolaldehyde to form glyceraldehyde. On the other hand, the major pathway (60%) for the formation of the precursor of 2,3-dimethylpyrazine involved an aldol condensation of acetaldehyde with glycolaldehyde to form 2,3-butanedione.

Subcellular localization of enzyme activities in chemostat-grown murine myeloma cells

As part of the development of structured models for the metabolism of myeloma cells in suspension culture, a study was made of the subcellular localization of key enzymes of glucose and glutamine metabolism. Steady state chemostat cultures of the mouse myeloma SP2/0-Ag14 were used as a reproducible source of biomass. Homogenates of the cells, obtained via mechanical disruption, were separated into a mitochondrial and a cytosolic fraction via differential centrifugation. The following conclusions are drawn: (1) approximately one fifth of the hexokinase activity of cell-free homogenates is associated with the mitochondria; (2) a malate–aspartate shuttle may operate for oxidation of cytosolic NADH, as indicated by high levels of malate dehydrogenase and aspartate aminotransferase in both particulate and soluble fractions; (3) the pentose phosphate pathway and isocitrate dehydrogenase may contribute to the provision of cytosolic NADPH; (4) phosphoenolpyruvate carboxykinase and pyruvate kinase, which are present in high activities, are exclusively cytosolic and probably play a key role in glutamine metabolism; (5) oxidation of glutamine via these enzymes leads to the formation of pyruvate that enters the same pool as pyruvate generated by glycolysis. As a result, lactate and alanine formation can occur from both glucose and glutamine.

Silica, Alumina, and Clay-Catalyzed Alanine Peptide Bond Formation

The peptide bond formation of alanine (ala), ala + glycine (gly), ala + diglycine (gly2), and ala + gly cyclic anhydride (cyc-gly2) in drying/wetting cycles at 80°C was studied. Silica, alumina, and representative smectites—montmorillonite and hectorite—were used as catalysts, and the dependence of reaction yields on the available amount of water in the reaction systems was evaluated. Silica and alumina catalyze the formation of oligopeptide mainly in temperature fluctuation experiments, whereas higher amounts of water in the reaction system support clay-catalyzed reactions. Silica and alumina are much more efficient for amino acid dimerization than clays. Whereas only 0.1% of ala oligomerized on hectorite and no reaction proceeded on montmorillonite, about 0.9 and 3.8% alanine converted into its dimer and cyclic anhydride on silica and alumina, respectively. Clay minerals, on the other hand, seem to more efficiently catalyze peptide chain elongation than amino acid dimerization. The reaction yields of ala-gly-gly and gly-gly-ala from ala + gly2 and ala + cyc-gly2 reached about 0.3% on montmorillonite and 1.0% on hectorite. The possible mechanisms of these reactions and the relevance of the results for prebiotic chemistry are discussed.

OXIDATION OF ERYTHROCYTE PROTEIN AND LIPID, AND HEMOLYSIS UN RABBIT RED BLOOD CELLS TREATED WITH BENZO[a]PYRENE OR ADRIAMYCIN

A number of free-radical-generating carcinogens catalyze the oxidative modification of macromolecules. Malondialdehyde (MDA), carbonyl content, alanine formation, and hemolysis were used as biomarkers of oxidative stress, and were determined in rabbit erythrocytes treated in vitro with benzolalpyrene or adriamycin. MDA and carbonyl content were significantly increased in a concentration-dependent manner by carcinogens. Alanine formation was also increased in a concentration-dependent manner in rabbit erythrocytes treated with carcinogens. Hemolysis occurred in erythrocytes treated with benzolalpyrene (540 µM) or adriamycin (300 µM) between 4 and 8 h of incubation, respectively. The hemolysis pattern correlated with increases in MDA, carbonyl content, and alanine formation. These data indicate that lipid peroxidation as measured by MDA may be the most sensitive indicator for oxidative stress in erythrocytes. Hemolysis could thus be applicable to free-radical-induced cellular damage as an alternative biomarker of oxidative stress.

Taurine reduction in anaerobic respiration of Bilophila wadsworthia RZATAU.

Organosulfonates are important natural and man-made compounds, but until recently (T. J. Lie, T. Pitta, E. R. Leadbetter, W. Godchaux III, and J. R. Leadbetter. Arch. Microbiol. 166:204-210, 1996), they were not believed to be dissimilated under anoxic conditions. We also chose to test whether alkane- and arenesulfonates could serve as electron sinks in respiratory metabolism. We generated 60 anoxic enrichment cultures in mineral salts medium which included several potential electron donors and a single organic sulfonate as an electron sink, and we used material from anaerobic digestors in communal sewage works as inocula. None of the four aromatic sulfonates, the three unsubstituted alkanesulfonates, or the N-sulfonate tested gave positive enrichment cultures requiring both the electron donor and electron sink for growth. Nine cultures utilizing the natural products taurine, cysteate, or isethionate were considered positive for growth, and all formed sulfide. Two clearly different pure cultures were examined. Putative Desulfovibrio sp. strain RZACYSA, with lactate as the electron donor, utilized sulfate, aminomethanesulfonate, taurine, isethionate, and cysteate, converting the latter to ammonia, acetate, and sulfide. Strain RZATAU was identified by 16S rDNA analysis as Bilophila wadsworthia. In the presence of, e.g., formate as the electron donor, it utilized, e.g., cysteate and isethionate and converted taurine quantitatively to cell material and products identified as ammonia, acetate, and sulfide. Sulfite and thiosulfate, but not sulfate, were utilized as electron sinks, as was nitrate, when lactate was provided as the electron donor and carbon source. A growth requirement for 1,4-naphthoquinone indicates a menaquinone electron carrier, and the presence of cytochrome c supports the presence of an electron transport chain. Pyruvate-dependent disappearance of taurine from cell extracts, as well as formation of alanine and release of ammonia and acetate, was detected. We suspected that sulfite is an intermediate, and we detected desulfoviridin (sulfite reductase). We thus believe that sulfonate reduction is one aspect of a respiratory system transferring electrons from, e.g., formate to sulfite reductase via an electron transport system which presumably generates a proton gradient across the cell membrane.

Effect of endurance training on ammonia and amino acid metabolism in humans

Few studies examine ammonia and amino acid metabolism in response to endurance training. Trained humans generally experience less increase in plasma ammonia during either prolonged or intense exercise. This is probably a reflection of reduced ammonia production and release from the active muscle; it could be a reflection of decreased AMP deaminase activity, decreased glutamate dehydrogenase activity, and/or increased alanine and glutamine formation. Little is known regarding the associated enzyme systems in humans, but in experiments with animal models, aerobic training decreases AMP deaminase and increases the enzymes of amino acid transamination and oxidation.

Enhanced metabolism of arginine and glutamine in enterocytes of cortisol-treated pigs.

This study was designed to determine whether cortisol plays a role in arginine and glutamine metabolism in enterocytes and, more specifically, whether cortisol regulates metabolic changes in these cells during weaning. Twenty-eight 21-day-old suckling pigs were randomly assigned to one of four groups (7 animals in each) and received intramuscular injections of vehicle solution (sesame oil) (control group), hydrocortisone 21-acetate (HYD) (25 mg/kg body wt), RU-486 (10 mg/kg body wt) (a potent blocker of glucocorticoid receptors), or HYD plus RU-486. At 29 days of age, pigs were killed for preparation ofjejunal enterocytes. During the entire experimental period, pigs were nursed by sows. Activities of argininosuccinate synthase, argininosuccinate lyase (ASL), arginase, and pyrroline-5-carboxylate (P5C) synthase were measured. For metabolic studies, enterocytes were incubated for 30 min at 37 degrees C in 2 ml of Krebs-bicarbonate buffer (pH 7.4) containing 0, 0.5, or 2 mM [U-(14)C]arginine or [U-(14)C]glutamine. Compared with control, cortisol administration increased 1) the activities of ASL and arginase and the production of CO(2), ornithine, and proline from arginine, and 2) P5C synthase activity and the formation of glutamate, alanine, aspartate, ornithine, citrulline, proline, and CO(2) from glutamine in enterocytes. The stimulating effects of cortisol on the enzyme activities and the metabolism of arginine and glutamine were abolished by coadministration of RU-486. Our data suggest that cortisol plays an important role in regulating arginine and glutamine metabolism in enterocytes via a glucocorticoid receptor-mediated mechanism.

3-(1-Piperidinyl)alanine formation during the preparation ofC-terminal cysteine peptides with the Fmoc/t-Bu strategy

Several side reactions have been detected for cysteine-containing peptides. During the synthesis ofC-terminal cysteine peptides, a base-catalyzed elimination of the sulfhydryl-protected side-chain to afford the dehydroalanine derivative followed by a nucleophilic addition to the alkene was observed. MALDI-TOF analysis was a useful analytical technique to determine this phenomenon.

Metabolic engineering of animal cells.

Substrate-limited fed-batch cultures were used to study growth and overflow metabolism in hybridoma and insect cells. In hybridoma cells a glucose-limited fed-batch culture decreased lactate formation but increased glutamine consumption and ammonium formation. Glutamine limitation decreased ammonium and alanine formation but did not enhance glucose consumption. Instead lactate formation was reduced, indicating that glucose was used more efficiently. The formation of lactate, alanine, and ammonium was negligible in a dual substrate-limited fed-batch culture. The efficiency of the energy metabolism increased, as judged by the increase in the cellular yield coefficient for glucose of 100% and for glutamine of 150% and by the change in the metabolic ratios lac/glc, ala/gln, and NHx/gln, in the combined fed-batch culture. Insect cell metabolism was studied in Spodoptera frugiperda (Sf-9) cells. A stringent relation between glucose excess and alanine formation was found. In contrast, glucose limitation induced ammonium formation, while, at the same time, alanine formation was completely suppressed. Simultaneous glucose and glutamine limitation suppressed both alanine and ammonium formation. Alanine formation appears as wasteful as lactate formation because the growth rate of insect cells in substrate-limited cultures was the same as in batch cultures with substrate excess. In batch and fed-batch cultures of both cell lines, mu reaches it maximum early during growth and decreases thereafter so that no exponential growth occurs. The growth rate limiting factor for hybridoma cells was found to be a component of serum, because intermittent serum additions to batch cultures resulted in a high and constant growth rate. Insulin was identified as the main cause, inasmuch as intermittent insulin additions gave the same result as serum.

Sugar metabolism of hyperthermophiles

In recent years a number of hyperthermophiles with the ability to utilize sugars as source for carbon and energy have been isolated. Analysis of their central metabolism may reveal adaptations to the extreme environment, or give information about the evolution of the primary pathways involved. The best studied representative is Pyrococcus furiosus, which has become the model organism of the heterotrophic hyperthermophiles. This deeply branched archaeon utilizes a modified Embden-Meyerhof Pathway, which involves a set of unprecedented ADP-dependent kinases, and a unique glyceraldehyde-3-phosphate: ferredoxin oxidoreductase. Moreover, pyruvate is converted via acetyl-CoA to acetate, involving an ADP-forming acetyl-CoA synthetase, which is not encountered in Bacteria. Reductant generated by ferrodoxin-linked enzymes is released either by S0-reduction to H2S, by proton reduction to H2 or by the formation of alanine. Yield studies suggest that in addition to ATP synthesis by substrate level phosphorylation in the ultimate acetate-forming step, there are alternative energy conserving systems. The ADP-dependent Embden-Meyerhof pathway is probably shared by other members of the Thermococcales. In contrast, an ATP-dependent Embden-Meyerhof pathway is operating in the S0-respiring archaeon Thermoproteus tenax, although it involves a PPi-dependent phosphofructokinase. Finally, hyperthermophilic bacteria such as Thermotoga maritima utilize a classical Embden-Meyerhof pathway. Thus, the presence of the different versions of the Embden-Meyerhof pathway in these deeply rooted microbes indicates that the hypothesis that the Entner-Doudoroff pathway is more primitive is not correct.

Iodoacetate inhibits the biosynthesis of alanine in glial cells and its utilization in photoreceptors of the honeybee drone (Apis mellifera) retina

The aim of this work was to study the effects of iodoacetate on the metabolism of the honeybee drone retina. In the superfused retina, iodoacetate only at high concentration (3 mmol·1-1) causes a 77% decrease in the O2 consumption induced by a flash of light. Chromatographic analysis showed that 3 mmol·1-1 iodoacetate strongly inhibited glycolysis in the retinal glial cells and consequently suppressed the biosynthesis of alanine, which is the fuel transferred from the glia to the photoreceptors. However, the synthesis of 14C-alanine from [1-14C]-pyruvate was not affected by iodoacetate. It was therefore surprising to find that superfusion of the retina with 10 mmol·1-1 pyruvate had no protective effect on the decrease in O2 consumption, and that the 14CO2 production from [1-14C]-pyruvate was inhibited 60% by iodoacetate. Also, no protection from the effect of iodoacetate was obtained by adding 10 and 20 mmol·1-1 alanine in the superfusate, even though the transport of 14C-alanine in the photoreceptor cells was not significantly affected by 3 mmol·1-1 iodoacetate. However, exposure to iodoacetate strongly inhibited the production of 14C-glutamate from 14C-alanine. In contrast, the transformation of 14C-proline to 14C-glutamate was not affected by iodoacetate. Indeed, in the presence of iodoacetate, photostimulation caused a decrease in the total concentration of proline and glutamate. It appears therefore that 3 mmol·1-1 iodoacetate inhibits not only glycolysis and, consecutively, the formation of alanine, but also its use in the photoreceptors. Possibly a large intracellular store of proline, whose mitochondrial use was not affected, contributed in slowing down the inhibition of O2-consumption by iodoacetate.

Properties of Glutamate Dehydrogenase and Its Involvement in Alanine Production in a Hyperthermophilic Archaeon, Thermococcus profundus1

Thermococcus profundus, a hyperthermophilic archaeon, did not exhibit detectable glutamine synthetase activity, although the organism possessed an extraordinarily high level of glutamate dehydrogenase (GDH), the content of which reached over 10% of total soluble proteins. This GDH was purified to homogeneity. The enzyme had a molecular weight of 263,000 and was composed of six homogeneous subunits of molecular weight 43,000. The enzyme was extremely thermostable with a half life of 1 h at 90 degrees C. Circular dichroism (CD) spectra of the enzyme revealed gradual unfolding of alpha-helices upon exposure to increasing temperature. The enzyme reaction was strongly biased toward glutamate formation. T. profundus excreted L-alanine into the medium, and the concentration reached mM. High activity of alanine aminotransferase (AAT) was present in the cells, while no alanine dehydrogenase activity was detected. The alanine formation may be initiated by ammonia uptake by GDH followed by aminotransfer from glutamate to pyruvate by AAT.

Prostaglandin F2 alpha- and 12-O-tetradecanoylphorbol-13-acetate-induced alterations in the pathways of renal ammoniagenesis.

The mechanisms whereby prostaglandin F2 alpha (PGF2 alpha) and the phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA) inhibit ammoniagenesis and the reason why they behave differently at pH 7.4, were examined with (15N)glutamine to assess the metabolic pathways and 2'-7'-bis(2-carboxyethyl)-5-(and-6)-carboxylfluorescein, acetoxymethylester (BCECF-AM) to evaluate Na+/H+ antiporter activity. LLC-PK1 cultures were incubated for 1 h in a Krebs-Hensleit bicarbonate buffer of pH 7.4 and pH 6.8 supplemented either with 5-15N- or 2-15N-labeled glutamine, followed by the assessment of (15N)ammonia and (15N)amino acid formation. Exposure of cells to either PGF2 alpha or TPA completely inhibited the low pH-induced increases in (15N)ammonia formation from incubations with 5-15N, reflecting reduced flux through the mitochondrial phosphate-dependent glutaminase, and from (2-15N)glutamine, reflecting reduced flux through the mitochondrial glutamate dehydrogenase pathway. They also qualitatively reversed the acute acidosis-induced changes in (15N)alanine formation and (15N)glutamate accumulation in the media. By contrast only TPA, but not PGF2 alpha, modified glutamine metabolism at pH 7.4. Na+/H+ antiporter activity was assessed under both acidified and basal (pH 7.4) conditions by measuring changes in intracellular pH in cells loaded with BCECF. TPA and PGF2 alpha both stimulated Na+/H+ antiporter activity comparably under acidified conditions. When cells were studied at pH 7.4, TPA but not PGF2 alpha stimulated the Na+/H+ antiporter and increased steady-state intracellular pH.(ABSTRACT TRUNCATED AT 250 WORDS)

Glutamine metabolism in chick enterocytes: absence of pyrroline-5-carboxylase synthase and citrulline synthesis.

This study was designed to determine whether pyrroline-5-carboxylate (P-5-C) synthase is deficient in chick enterocytes therefore resulting in the lack of synthesis of ornithine and citrulline from glutamine. Post-weaning pig enterocytes, which are known to contain P-5-C synthase and to synthesize both ornithine and citrulline from glutamine, were used as positive controls. Enterocytes were incubated at 37 degrees C for 0-30 min in the presence of 2 mM [U-14C]glutamine or 2 mM ornithine plus 2 mM NH4Cl. In chick enterocytes, glutamine was metabolized to NH3, CO2, glutamate, alanine and aspartate, but not to ornithine, citrulline, arginine or proline. Likewise, there was no formation of citrulline, arginine, alanine or aspartate from ornithine in chick enterocytes. Furthermore, the rate of conversion of ornithine into proline in chick enterocytes was only about 4% of that in cells from pigs. To elucidate the reason for the inability of chick enterocytes to synthesize ornithine and citrulline from glutamine, the activities of the enzymes involved were measured. No activity of P-5-C synthase or ornithine carbamoyltransferase was found in chick enterocytes, in contrast with cells from post-weaning pigs. It was also demonstrated that the activity of ornithine aminotransferase in chick enterocytes was only 3% of that in cells from pigs. Thus the present findings elucidate the biochemical reason for the lack of endogenous synthesis of ornithine and citrulline in chicks. Our results also explain previous observations that ornithine cannot replace arginine or proline in the diet of chicks. We suggest that the absence of P-5-C synthase and ornithine carbamoyltransferase in enterocytes is the metabolic basis for the nutritional requirement of arginine in the chick.

Glutamine and glucose metabolism in enterocytes of the neonatal pig.

Glutamine and glucose metabolism was studied in 0- to 21-day-old pig enterocytes. Cells were incubated at 37 degrees C for 30 min in Krebs-Henseleit bicarbonate buffer (pH 7.4) in the presence of 2 mM [U-14C]glutamine with or without 5 mM glucose, or 5 mM [U-14C]glucose with or without 2 mM glutamine. Glutamine was metabolized to ammonia, glutamate, alanine, aspartate, CO2, citrulline, ornithine, and proline, whereas glucose was converted to lactate, pyruvate, and CO2 in pig enterocytes. CO2 production from glutamine accounted for 32-36% and 3-4% of utilized glutamine carbons in 0- to 7-day-old and 14- to 21-day-old pigs, respectively. The rates of O2 consumption and metabolism of glutamine and glucose decreased in enterocytes from 2- to 14-day-old pigs compared with 0-day-old pigs. By day 14 after birth, the oxidation of glutamine and glucose as well as citrulline production had decreased by 90-95%. Arginine synthesis from glutamine occurred in cells from 0- to 7-day-old pigs but not 14- to 21-day-old ones. Glucose (5 mM) had no effect on glutamine utilization and oxidation or the production of glutamate and arginine but stimulated the formation of alanine, citrulline, and proline at the expense of aspartate. In contrast, glutamine (2 mM) inhibited glycolysis and glucose oxidation in cells from 0- to 7-day-old pigs and had no effects in 14- to 21-day-old pigs. As a result, glutamine contributed approximately 2-fold greater amounts of ATP to 0- to 7-day-old pig enterocytes than glucose.(ABSTRACT TRUNCATED AT 250 WORDS)

PGF2α activation of Na/H antiporter and ammoniagenesis in parent/variant LLC-PK1 cells

A novel variant of the LLC-PK1 cell line was used to examine directly the mechanism whereby PGF2 alpha and TPA inhibit renal ammoniagenesis. The variant cells, which exhibit a growth pattern and morphology similar to the parent cell line, were isolated by a self selection process utilizing long-term cultures of parent cells maintained under conditions of continuous gentle rocking of the media fluid. Incubation of both parent and variant LLC-PK1 cells for one hour in a glutamine supplemented Krebs-Hensleit media of low pH (pH 6.8) increased ammonia and alanine production in comparison to the basal rates at pH 7.4. The phorbol ester TPA and also PGF2 alpha inhibited the low pH-induced increases in ammonia and alanine formation in parent cells; however, neither TPA nor PGF2 alpha inhibited ammonia or alanine metabolism in variant cells. TPA and PGF2 alpha activated PKC similarly in the parent and variant cells as demonstrated by a significant increase in membrane bound enzyme activity. BCECF labeling of cells indicated that the parent and variant cells possess an amiloride sensitive Na+/H+ antiporter of comparable activity. Exposure of parent cells to PGF2 alpha or TPA resulted in the activation of Na+/H+ antiporter activity. By contrast, neither compound stimulated antiporter activity in variant cells. These studies strongly suggest that PKC mediated activation of the Na+/H+ antiporter accounts for the inhibition of ammonia production produced by both PGF2 alpha and TPA. In addition, this novel variant of LLC-PK1 cells should provide a valuable tool to investigate various normal and pathophysiological functions involving mediation by PKC and/or Na+/H+ antiporter activity.

Catabolic control of hybridoma cells by glucose and glutamine limited fed batch cultures

Substrate limited fed batch cultures were used to study growth and overflow metabolism in hybridoma cells. A glucose limited fed batch, a glutamine limited fed batch, and a combined glucose and glutamine limited red batch culture were compared with batch cultures. In all cultures mu reaches its maximum early during growth and decreases thereafter so that no exponential growth and decreases thereafter so that no exponential growth rate limiting, although the glutamine concentration (>0.085mM) was lower than reported K(s) vales and glucose was below 0.9mM; but some other nutrients (s) was the cause as verified by simulations. Slightly more cells and antibodies were produced in the combined fed batch compared with the batch culture. The specific rates for consumption of glucose and glutamine were dramatically influenced in fed batch cultures resulting in major metabolic changes. Glucose limitation decreased lactate formation, but increased glutamine consumption and ammonium formation. Glutamine limitation decreased ammonium and alanine formation of lactate, alanine, and ammonium was negligible in the dual-substrate limited fed batch culture. The efficiency of the energy metabolism increased, as judged by the increase in the cellular yield coefficient for glucose by 100% and for glutamine by 150% and by the change in the metabolic ratios lac/glc, ala/ln, and NH(x)/ln, in the combined fed culture. The data indicate that a larger proportion of consumed glutamine enters the TCA cycle through the glutamate dehydrogenase pathway, which releases more energy from glutamine than the transamination pathway. We suggest that the main reasons for these changes are decreased uptake rates of glucose and glutamine, which in turn lead to a reduction of the pyruvate pool and a restriction of the flux through glutaminase and lactate dehydrogenase. There appears to be potential for further cell growth in the dual-substrate-limited fed batch culture as judged by a comparison of mu in the different cultures. (c) 1994 John Wiley & Sons, Inc.

Evidence for the operation of a novel Embden-Meyerhof pathway that involves ADP-dependent kinases during sugar fermentation by Pyrococcus furiosus

The main pathway for the fermentation of maltose or cellobiose by the hyperthermophile Pyrococcus furiosus was investigated by in vivo NMR and by enzyme measurements. Addition of [1-13C]glucose to cell suspensions resulted in the formation of C2-labeled acetate and C3-labeled alanine. No label was recovered in CO2 or HCO3-. In the presence of [3-13C]glucose, the label ended up in the C1 atom of alanine and in HCO3- and CO2. These labeling patterns indicate that glucose is converted along an Embden-Meyerhof pathway, and they disagree with the previously proposed nonphosphorylated Entner-Doudoroff pathway (pyroglycolysis). The NMR data were supported by enzyme measurements. Hexokinase (8.7 units/mg), phosphoglucose isomerase (6.8 units/mg), phosphofructokinase (0.81 unit/mg), and aldolase (0.26 unit/mg) were present in cell-free extracts (specific activities at 90 degrees C). Remarkably, the two kinases required ADP as the phosphoryl group donor instead of ATP. No activity was found with pyrophosphate. These are the first descriptions of ADP-dependent (AMP-forming) kinases to date. Since P. furiosus is a phylogenetically ancient organism, these enzymes may represent an ancestral kind of metabolism.

Cerebral Alanine Transport and Alanine Aminotransferase Reaction: Alanine as a Source of Neuronal Glutamate

Alanine transport and the role of alanine amino-transferase in the synthesis and consumption of glutamate were investigated in the preparation of rat brain synaptosomes. Alanine was accumulated rapidly via both the high- and low-affinity uptake systems. The high-affinity transport was dependent on the sodium concentration gradient and membrane electrical potential, which suggests a cotransport with Na+. Rapid accumulation of the Na(+)-alanine complex by synaptosomes stimulated activity of the Na+/K+ pump and increased energy utilization; this, in turn, activated the ATP-producing pathways, glycolysis and oxidative phosphorylation. Accumulation of Na+ also caused a small depolarization of the plasma membrane, a rise in [Ca2+]i, and a release of glutamate. Intra-synaptosomal metabolism of alanine via alanine amino-transferase, as estimated from measurements of N fluxes from labeled precursors, was much slower than the rate of alanine uptake, even in the presence of added oxoacids. The velocity of [15N]alanine formation from [15N]glutamine was seven to eight times higher than the rate of [15N]-glutamate generation from [15N]alanine. It is concluded that (a) overloading of nerve endings with alanine could be deleterious to neuronal function because it increases release of glutamate; (b) the activity of synaptosomal alanine aminotransferase is much slower than that of glutaminase and hence unlikely to play a major role in maintaining [glutamate] during neuronal activity; and (c) alanine amino-transferase might serve as a source of glutamate during recovery from ischemia/hypoxia when the alanine concentration rises and that of glutamate falls.

The intensity of acidosis differentially alters the pathways of ammoniagenesis in LLC-PK1 cells

Utilizing [5-15N] and [2-15N]-labeled glutamine and gas chromatography mass spectrometry methodology, we examined the pathways of ammoniagenesis under basal and acute acidotic conditions of pH 7.0 and pH 6.8, respectively. LLC-PK1 cultures were incubated for one hour with gentle rocking in a bicarbonate buffer of pH 7.4, pH 7.0, or pH 6.8 supplemented either with [5-15N] or [2-15N] glutamine at 37 degrees C in a 5% CO2/95% air incubator atmosphere. Incubation of cultures with [5-15N] glutamine at pH 7.4 resulted in a substantial amount of 15N ammonia formation which was not significantly altered by incubations at pH 7.0. By contrast, exposure to pH 6.8 significantly increased 15N ammonia formation in comparison with its production at pH 7.0 or 7.4. However, 15N ammonia production from [2-15N] glutamine was significantly stimulated at pH 7.0 and was further increased at pH 6.8. Incubation of the cells with [2-15N] glutamine resulted in a substantially lower amounts of 15N ammonia formation than produced with [5-15N] glutamine. Alanine formation from [2-15N] glutamine increased significantly at pH 7.0; but in contrast to 15N ammonia formation, pH 6.8 had no additional stimulatory effect on 15N alanine formation. Cells incubated with [2-15N] glutamine resulted in a significant decrement in 15N glutamate production at both pH 7.0 and 6.8 when compared with pH 7.4. 15N aspartate formation was unaltered by the changes in media pH.(ABSTRACT TRUNCATED AT 250 WORDS)