Biochemical characterization of soybean ovary growth from anthesis to abscission of aborting ovaries

Soybean (Glycine max [L.] Merr.) ovary growth was measured from anthesis to 6 days after anthesis (DAA) to establish a timetable of biochemical events that might be useful in identifying processes that initiate abscission. Two procedures were developed to provide samples with either high or low percent pod set for `IX93-100,' a semideterminate line having long racemes. Characteristics measured were fresh and dry weight, soluble and insoluble protein, soluble carbohydrate, starch, RNA, and DNA. Setting ovaries grew more rapidly than abscising ovaries. Since there was a daily increase in ovary weight in both groups, all measured characteristics showed daily increases when expressed on perovary basis. Statistically significant differences between groups were detected between 2 and 5 DAA for most characteristics. When chemical composition was expressed on concentration basis, starch level was significantly higher in setting ovaries at 5 and 6 DAA. Regression analysis showed that these deviations between setting and abscising samples started between anthesis and 1 DAA. We conclude that processes leading to eventual shedding of fertilized ovaries (called flower abortion in soybeans) commence soon after anthesis of the shed flower, and that setting and abscising ovaries do not differ in protein, soluble carbohydrate, starch, or nucleic acid content when abscission processes begin.     

Source/Sink relations of abscising and nonabscising soybean flowers

The partitioning of recently fixed ¹⁴C to setting and abscising flowers within the axillary raceme of `Clark' isoline E1t soybeans (Glycine max L. Merr) was examined as a function of time after anthesis of individual flowers. In such racemes, the first four flowers showed a 17% abscission while the next four flowers showed 47% abscission.

Source/sink relations of flowers I-IV (normally setting) were compared to those of flowers V-VIII (normally abscising) by pulse labeling source leaves with ¹⁴CO₂ and determining the radioactivity of individual flowers after a 4-hour chase period. The relative specific activity (RSA;% disintegrations per minute per% dry weight), sink strength (% disintegrations per minute), and its components, sink size (milligrams dry weight) and sink intensity (% disintegrations per milligram dry weight) were then calculated as a function of days after anthesis.

Sink intensity (i.e. the competitive ability to accumulate photoassimilate per unit mass) was very high prior to anthesis of both setting and abscising flowers. Sink intensity then became very low for the first 3 days following anthesis after which it recovered in normally setting flowers, but failed to recover in normally abscising flowers. It is concluded that soybean reproductive abscission is determined at or very near the day of anthesis.

     

Metabolism of amino acids in Ascaridia galli: decarboxylation reactions

Production of ¹⁴CO₂ from 12 carbon-labelled amino acids by Ascaridia galli was studied. Appreciable amounts of CO₂ were evolved from alanine, aspartate, glutamate, serine, leucine and valine by intestines, ovaries, cuticle and intact worms, in that order, but not from lysine, proline and tyrosine. Maximum CO₂ produced by whole worms was from serine, while with isolated organs it was from alanine. For cuticle, the decarboxylations of alanine, aspartate and glutamate were found to be associated with the mitochondrial fraction.     

Developmental Changes in the Free Amino Acid Pool of Rice(Oryza sativa L. subsp,japonica) Bmbryos

The level of/various amino acids in rice embryos rose sharply during embryo differentiation (7–9 days after anthesis). Then it increased steadily or tended to become stable at mid-maturation stage (13–18 days after anthesis), thereafter continuing to increase, except that the contents of aspartate and glutamate decreased significantly. The total free amino acid pool expanded rapidly during the differential stage. There after the pool capacity showed only a slightly increase until the end of embryogenesis. Both on embryo cell and dry weight bases, the capacity reached the maximum at the 9th day after anthesis, then decreased at the 13th day, and later remained stable. We deemed that the establishment of the free amino acid pool is one of the events which occur in the process of rice embryo differentiation. By the fulfillment of the differentiation (the 13th dray after anthesis), the pool capacity within the embryo cells remained stable on the whole. The free amino acid pool was dominated by serine, alanine, aspartate and glutamate during the differentiation stage. In the maturation stage, serine, alanine, arginine and lysine were the main components. These predominant; amino acids may play an important role in regulating the availability of the whole amino acid pool.     

Aminotransfer from Alanine and Glutamate to Glycine and Serine during Photorespiration in Oat Leaves

¹⁵N-Labeled glutamate and alanine were used to examine the photorespiratory nitrogen metabolism in oat (Avena sativa L.) leaf slices. Glutamate and alanine supply amino groups for glycine formation during photorespiration. The nitrogen flux from alanine to glycine was estimated to be 3 times higher than that from glutamate. It is concluded from these results that alanine is a direct and important amino donor for photorespiratory glycine formation in oat leaves. The ¹⁵N labeling of serine was almost as high as that of glycine during the initial period of the labeling experiments. Thereafter, the ratio of ¹⁵N label in serine to ¹⁵N label in glycine declined substantially.     

Release of Endogenous Amino Acids from the Striatum from Developing and Adult Mice in Ischemia

In most other studies the release of amino acid neurotransmitters and modulators in vitro has been studied mostly using labeled preloaded compounds. For several reasons the estimated release may not reliably reflect the release of endogenous compounds. The magnitudes of the release cannot thus be quite correctly estimated using radioactive labels. The basal and K⁺-evoked release of the neuroactive endogenous amino acids γ-aminobutyrate (GABA), glycine, taurine, glutamate and aspartate was now studied in slices from the striatum from 7-day-old to 3-month-old mice under control (normoxic) and ischemic conditions. The release of alanine, threonine and serine was assessed as control. GABA and glutamate release was much greater in 3-month-old than in 7-day-old mice, whereas with taurine the situation was the opposite. Ischemia markedly enhanced the release of all these three amino acids. The release of aspartate and glycine was markedly enhanced as well whereas no effects were discernible in the release of glutamine, alanine, serine and threonine. K⁺ stimulation (50 mM) enhanced the release of GABA, glutamate, taurine, aspartate and glycine in most cases, except with taurine in 3-month-old mice under the ischemic conditions and with aspartate in 7-day-old mice under the control conditions. K⁺ stimulation did not affect the release of glutamine, alanine, serine or threonine. The results on endogenous amino acids are qualitatively similar to those obtained in our earlier experiments with labeled preloaded amino acids. In conclusion, in developing mice only inhibitory taurine is released in such amounts that may counteract the harmful effects of excitatory amino acids in ischemia.     

Decreases in Amino Acid and Acetylcholine Metabolism During Hypoxia

Abstract: Hypoxia impairs brain function by incompletely defined mechanisms. Mild hypoxia, which impairs memory and judgment, decreases acetylcholine (ACh) synthesis, but not the levels of ATP or the adenylate energy charge. However, the effects of mild hypoxia on the synthesis of the glucosederived amino acids [alanine, aspartate, γ-amino butyric acid (GABA), glutamate, glutamine, and serine] have not been characterized. Thus, we examined the incorporation of [U-¹⁴C]glucose into these amino acids and ACh during anemic hypoxia (injection of NaNO₂), hypoxic hypoxia (15 or 10% O₂), and hypoxic hypoxia plus hypercarbia (15 or 10% O₂ with 5% CO₂). In general, the synthesis of the amino acids and of ACh declined in parallel with each type of hypoxia we studied. For example, anemic hypoxia (75 mg/kg of NaNO₂) decreased the incorporation of [U-¹⁴C]glucose into the amino acids and into ACh similarly. [Percent inhibition: ACh (57.4), alanine (34.4), aspartate (49.2), GABA (61.9). glutamine (59.2), glutamate (51.0), and serine (36.7)]. A comparison of several levels (37.5, 75, 150, 225 mg/kg of NaNO₂) of anemic hypoxia showed a parallel decrease in the flux of glucose into ACh and into the amino acids whose synthesis depends on mitochondrial oxidation: GABA (r= 0.98), glutamate (r= 0.99), aspartate (r= 0.96), and glutamine (r= 0.97). The synthesis of the amino acids not dependent on mitochondrial oxidation did not correlate as well with changes in ACh metabolism: serine (r= 0.68) and alanine (r= 0.76). The decreases in glucose incorporation into ACh and into the amino acids with hypoxic hypoxia (15% or 10% O₂) or hypoxic hypoxia with 5% CO₂ were very similar to those with the two lowest levels of anemic hypoxia. Thus, any explanation of the brain's sensitivity to a decrease in oxygen availability must include the alterations in the metabolism of the amino acid neurotransmitters as well as ACh.     

Nitrogen Assimilation in Mycorrhizas : Ammonium Assimilation in the N-Starved Ectomycorrhizal Fungus Cenococcum graniforme

Ammonium assimilation was followed in N-starved mycelia from the ectomycorrhizal Ascomycete Cenococcum graniforme. The evaluation of free amino acid pool levels after the addition of 5 millimolar NH₄⁺ indicated that the absorbed ammonium was assimilated rapidly. Post-feeding nitrogen content of amino acids was very different from the initial values. After 8 hours of NH₄⁺ feeding, glutamine accounted for the largest percentage of free amino acid nitrogen (43%). The addition of 5 millimolar methionine sulfoximine (MSX) to NH₄⁺-fed mycelia caused an inhibition of glutamine accumulation with a corresponding increase in glutamate and alanine levels.

Using ¹⁵N as a tracer, it was found that the greatest initial labeling was into glutamine and glutamate followed by aspartate, alanine, and ornithine. On inhibiting glutamine synthetase using MSX, ¹⁵N enrichment of glutamate, alanine, aspartate, and ornithine continued although labeling of glutamine was quite low. Moreover, the incorporation of ¹⁵N label in insoluble nitrogenous compounds was lower in the presence of MSX. From the composition of free amino acid pools, the ¹⁵N labeling pattern and effects of MSX, NH₄⁺ assimilation in C. graniforme mycelia appears to proceed via glutamate dehydrogenase pathway. This study also demonstrates that glutamine synthesis is an important reaction of ammonia utilization.

     

Chemotaxis toward amino acids in Escherichia coli

Escherichia coli cells are shown to be attracted to the l-amino acids alanine, asparagine, aspartate, cysteine, glutamate, glycine, methionine, serine, and threonine, but not to arginine, cystine, glutamine, histidine, isoleucine, leucine, lysine, phenylalanine, tryptophan, tyrosine, or valine. Bacteria grown in a proline-containing medium were, in addition, attracted to proline. Chemotaxis toward amino acids is shown to be mediated by at least two detection systems, the aspartate and serine chemoreceptors. The aspartate chemoreceptor was nonfunctional in the aspartate taxis mutant, which showed virtually no chemotaxis toward aspartate, glutamate, or methionine, and reduced taxis toward alanine, asparagine, cysteine, glycine, and serine. The serine chemoreceptor was nonfunctional in the serine taxis mutant, which was defective in taxis toward alanine, asparagine, cysteine, glycine, and serine, and which showed no chemotaxis toward threonine. Additional data concerning the specificities of the amino acid chemoreceptors with regard to amino acid analogues are also presented. Finally, two essentially nonoxidizable amino acid analogues, alpha-aminoisobutyrate and alpha-methylaspartate, are shown to be attractants for E. coli, demonstrating that extensive metabolism of attractants is not required for amino acid taxis.     

The effects of electrical stimulation and ionic alterations on the metabolism of amino acids and proteins in excised superior cervical ganglia of the rat

Abstract— The effects of supramaximal electrical stimulation on the metabolism of amino acids and proteins in incubated superior cervical ganglia of the rat were studied by the use of a gas-liquid chromatographic (GLC) assay procedure. Stimulation at 5 Hz for 2 h caused an apparent increase in tissue levels of free amino acids, with alanine, serine, glycine, valine, threonine, isoleucine and aspartate (+ asparagine) most noticeably affected. The amino acid composition (partial) of the TCA-insoluble proteins of resting and stimulated ganglia was approximately the same after 60 min of incubation, but there was less TCA-insoluble protein in the stimulated ganglia. The addition of amino acids (at plasma concentrations) to the standard media had no apparent affect on the amino acid composition of this protein fraction. Stimulation for 0 , 5 h initially increased the efflux of alanine, valine, proline and ornithine into the incubation media but prolonged stimulation (for 4–0 h) decreased the efflux of alanine, serine, glycine and isoleucine and increased the efflux of lysine into the incubation media. The leakage of amino acids from the ganglia appeared to be a sodium-dependent process. The incorporation of ¹⁴C from [U-¹⁴C]glucose into glutamate (+ glutamine) and aspartate (+ asparagine) was greater in stimulated than in resting ganglia. However, the conversion of glutamate carbons from [U-¹⁴C]l -glutamate into aspartate was not affected by stimulation. Incorporation of ¹⁴C from [U-¹⁴C]glucose into glycine and serine was apparently not affected by stimulation during the 60 min of incubation. However, serine was the only amino acid which exhibited a higher specific radioactivity in stimulated ganglia than in resting ganglia incubated for 4 h in standard media. Lithium ions had the apparent specific effect of increasing the labelling with ¹⁴C from [U-¹⁴C]glucose into ornithine, and increasing the efflux and overall metabolism of serine in the ganglia. Incorporation of ¹⁴C from [U-¹⁴C]glucose into proteins was lower in the stimulated than in the resting ganglia if compensation was made for the higher radioactivity available in the total free amino acid pool of the stimulated ganglia. The rate of ¹⁴C incorporation from [U-¹⁴C]glutamate into the TCA-insoluble proteins of resting ganglia was greater when no other amino acids at concentrations approximating plasma levels were added to the bathing media; this rate was lower in stimulated than in resting ganglia.     

Labeling of Carbon Pools in Bradyrhizobium japonicum and Rhizobium leguminosarum bv viciae Bacteroids following Incubation of Intact Nodules with CO(2)

The aim of the work reported here was to ascertain that the patterns of labeling seen in isolated bacteroids also occurred in bacteroids in intact nodules and to observe early metabolic events following exposure of intact nodules to ¹⁴CO₂. Intact nodules of soybean (Glycine max L. Merr. cv Ripley) inoculated with Bradyrhizobium japonicum USDA 110 and pea (Pisum sativum L. cv Progress 9) inoculated with Rhizobium leguminosarum bv viciae isolate 128C53 were detached and immediately fed ¹⁴CO₂ for 1 to 6 min. Bacteroids were purified from these nodules in 5 to 7 min after the feeding period. In the cytosol from both soybean and pea nodules, malate had the highest radioactivity, followed by citrate and aspartate. In peas, asparagine labeling equaled that of aspartate. In B. japonicum bacteroids, malate was the most rapidly labeled compound, and the rate of glutamate labeling was 67% of the rate of malate labeling. Aspartate and alanine were the next most rapidly labeled compounds. R. leguminosarum bacteroids had very low amounts of ¹⁴C and, after a 1-min feeding, malate contained 90% of the radioactivity in the organic acid fraction. Only a trace of activity was found in aspartate, whereas the rate of glutamate and alanine labeling approached that of malate after 6 min of feeding. Under the conditions studied, malate was the major form of labeled carbon supplied to both types of bacteroids. These results with intact nodules confirm our earlier results with isolated bacteroids, which showed that a significant proportion of provided labeled substrate, such as malate, is diverted to glutamate. This supports the conclusion that microaerobic conditions in nodules influence carbon metabolism in bacteroids.     

Changes in Amino Acid Content and the Metabolism of γ-Aminobutyrate in Cucurbita moschata Seedlings

Ungerminated pumpkin (Cucurbita moschata Poir.) cotyledons contained 30 % of their dry weight as lipid and 26 % as protein, of which 93 % was globulin. There was a rapid degradation of these reserves 4 to 8 days after planting when the cotyledons had their maximum metabolic activity. About half of the mole percent of amino acids found in the globulin reserve was in arginine, glutamate, aspartate, and their amides. The cotyledons had a large soluble pool of arginine, glutamine, glutamate, and leucine. Most amino acids increased steadily in amount in the cotyledons during germination, except glutamine, ornithine, alanine, serine, glycine, and γ-aminobutyrate and these appeared in large amounts in the translocation stream to the axis tissue. Little arginine or proline was translocated. By 10 days, when translocation had decreased, amino acids accumulated. Ornithine, γ-aminobutyrate, and aspartate were rapidly utilized in the hypocotyl, while glutamine, glycine, and alanine accumulated there. Cysteine and methionine levels were low in the reserve, trans-location stream and soluble fractions. γ-Aminobutyrate-U^?14C injected into cotyledons or incubated with hypocotyls was utilized in a similar fashion. The label appeared in citric acid cycle acids and in the amino acids closely related to this cycle, but the bulk of the label appeared in CO₂. The labeling pattern suggests that γ-aminobutyrate was utilized via succinate, and thus entered the citric acid cycle. A close relationship between arginine, ornithine, glutamate, and γ-aminobutyrate exists in the cotyledon with all but arginine being translocated rapidly to the axis tissue where these amino acids are rapidly metabolized.     

Spermidine and related-metabolic inhibitors modulate sugar and amino acid levels in Vitis vinifera L.: possible relationships with initial fruitlet abscission

The relationships between free polyamines (PAs), sugar and amino acid status were investigated in cuttings from two cultivars of Vitis vinifera L., Pinot noir (PN), a low abscising cultivar and Merlot (MRT), a high abscising one. In both cultivars free PAs decreased in inflorescences, but more drastically in MRT plants. Upon anthesis, this was associated with a decreased sugar content, especially sucrose, and an increase in total free amino acids. Thereafter, sucrose and amino acids showed opposite trends. In addition, darkening the PN plants at full flowering resulted in a dramatic decrease of PAs and sugars in inflorescences, but an increase in amino acid content, followed by high abscission. The concept that initial fruitlet abscission might be regulated by free PAs through changes in primary metabolites was hypothesized. Hence, the application of exogenous spermidine (Spd), but not putrescine (Put), prior to flowering markedly inhibits abscission. The Spd treatment also increased soluble sugar content but reduced amino acids in both leaves and inflorescences, while Put had no significant effect. By contrast, cyclohexylamine and beta-hydroxyethylhydrazine, as potent inhibitors of Spd synthase and PA-oxidases, respectively, exerted inverse effects on sugar, amino acid and abscission levels. Sucrose and free proline seemed to be highly sensitive to these treatments. This study suggests that Spd could regulate fruitlet abscission in grapevine by modulating, in a reverse way, the levels of sugars and amino acids in inflorescences.     

Amino acid degradation by the mesophilic sulfate-reducing bacterium Desulfobacterium vacuolatum

Desulfobacterium vacuolatum strain IbRM was able to grow using casamino acids as a source of carbon, energy and nitrogen. Growth was accompanied by utilization of several amino acids and sulfide production. Proline and glutamate were used preferentially and to the greatest extent. Glycine, serine and alanine were used more slowly and only after proline and glutamate were used. Isoleucine, valine, leucine and aspartate decrease was slowest and occurred in a linear fashion throughout the growth phase. Amino acids used from casamino acids, excluding aspartate, were also used as single carbon, energy and nitrogen sources. As a single amino acid, aspartate could only be used as a nitrogen source. Aspartate was not used as an electron acceptor. No growth occurred on any amino acid in the absence of sulfate. As single substrates, isoleucine, proline and glutamate were oxidized without formation of acetate and with molar yields of 13.1, 9.4 and 7.7 g mol^–1, respectively. Received: 24 June 1997 / Accepted: 10 September 1997     

Studies on Amino Acid Metabolism in the Brain Using 15N-Labeled Precursors

The transfer of label from ¹⁵N-alanine and ¹⁵N-glutamate into amino acids in incubated brain slices has been followed using gas chromatography/mass spectrometry (GC/MS). ¹⁵N from alanine appeared in both amino and amide groups of glutamine more rapidly than into aspartate, glutamate and GABA, which were all labeled at similar rates. Maximum labelling of approx. 50% enrichment of these three metabolites was achieved in 3 hr. The ¹⁵N present in doubly-labeled glutamine exceeded that in the singly-labelled after 30 min. ¹⁵N from glutamate was rapidly transferred to aspartate and to alanine, with slower incorporation into glutamine and GABA. As was seen with labeling from alanine, doubly-labeled glutamine was higher than the singly-labeled species, also reaching some 50% enrichment in 3 hr. Depolarisation with 40 mM extracellular K⁺ caused a considerable reversal of the ratio of doubly- to singly-labeled glutamine species from both alanine and glutamate. The results are discussed in terms of the effects of depolarization on the glutamate/glutamine cycle.     

Amino acid fermentation and hydrogen transfer in mixed cultures

Abstract The degradation of the following amino acids was investigated in mixed cultures obtained from a waste water purification plant: aspartate, glutamate, serine, alanine, valine and leucine. Inhibition of sulfate-reducing bacteria in these mixed cultures by molybdate was found to inhibit amino acid degradation. The degradation of serine, alanine, valine and leucine was accelerated considerably by active sulfate reduction. The fermentation of aspartate and glutamate was not stimulated by the presence of sulfate-reducing bacteria. The existence of species which are able to ferment valine and leucine by coupling their oxidation to the reduction of exogenous acetate to butyrate was demonstrated.     

Amino Acid Metabolism of Lemna minor L. : I. Responses to Methionine Sulfoximine

When Lemna minor L. is supplied with the potent inhibitor of glutamine synthetase, methionine sulfoximine, rapid changes in free amino acid levels occur. Glutamine, glutamate, asparagine, aspartate, alanine, and serine levels decline concomitantly with ammonia accumulation. However, not all free amino acid pools deplete in response to this inhibitor. Several free amino acids including proline, valine, leucine, isoleucine, threonine, lysine, phenylalanine, tyrosine, histidine, and methionine exhibit severalfold accumulations within 24 hours of methionine sulfoximine treatment. To investigate whether these latter amino acid accumulations result from de novo synthesis via a methionine sulfoximine insensitive pathway of ammonia assimilation (e.g. glutamate dehydrogenase) or from protein turnover, fronds of Lemna minor were prelabeled with [¹⁵N]H₄⁺ prior to supplying the inhibitor. Analyses of the ¹⁵N abundance of free amino acids suggest that protein turnover is the major source of these methionine sulfoximine induced amino acid accumulations. Thus, the pools of valine, leucine, isoleucine, proline, and threonine accumulated in response to the inhibitor in the presence of [¹⁵N]H₄⁺, are ¹⁴N enriched and are not apparently derived from ¹⁵N-labeled precursors. To account for the selective accumulation of amino acids, such as valine, leucine, isoleucine, proline, and threonine, it is necessary to envisage that these free amino acids are relatively poorly catabolized in vivo. The amino acids which deplete in response to methionine sulfoximine (i.e. glutamate, glutamine, alanine, aspartate, asparagine, and serine) are all presumably rapidly catabolized to ammonia, either in the photorespiratory pathway or by alternative routes.     

Glutamate Uptake and Metabolism in C-6 Glioma Cells: Alterations by Potassium Ion and Dibutyryl cAMP

Abstract: Uptake and metabolism of glutamate was studied in the C-6 glioma cell line grown in the absence or presence of dibutyryl cyclic AMP (dbcAMP). Glutamate and aspartate uptake were competitive in cells grown under both conditions. Increased [K⁺] in the medium caused a significant decrease in the uptake of both amino acids. A small part of this decrease (<25%) was due to an enhanced efflux of tissue amino acid. The effects of increased [K⁺] were observed whether or not the [Na⁺] in the medium was concomitantly decreased. In cells grown in the presence of 1 mM dbcAMP for 48 h, glutamate uptake and metabolism were altered. Tissue levels of glutamate, aspartate, glutamine, GABA, and alanine were generally less in treated than in naive cells. When incubated with 50 μM [U-¹⁴C]glutamate, there was significantly less incorporation of radioactivity into treated cells with time, resulting in greatly lowered specific radioactivities of glutamate, aspartate, and GABA. However, the rate of labeling of glutamine was greatly increased; this was consistent with the previously observed doubling in glutamine synthetase activity in dbcAMP-treated C-6 cells. Tissue glutamate decarboxylase activity was halved in treated cells, accounting for the large decrease in GABA labeling. The metabolic data suggested a decreased uptake of exogenous glutamate; in studies on initial rates of uptake, the V_max of high-affinity glutamate uptake was decreased by 40%. This decrease was of the same order of magnitude as that observed in the metabolic experiments. Thus, in this glial model, both rapid, acute changes and slower, long-term changes in neuroactive amino acid metabolism were observed. Each of these conditions mimics a stimulus of neuronal origin, and the resulting changes could modulate extrasynaptic activity of neuroactive amino acids.     

Analysis of energetic amino acid metabolism in Acyrthosiphon pisum: A multidimensional approach to amino acid metabolism in aphids

Aphids are highly specialized insects that feed on the phloem-sap of plants, the amino acid composition of which is very unbalanced. Amino acid metabolism is thus crucial in aphids, and we describe a novel investigation method based on the use of ¹⁴C-labeled amino acids added in an artificial diet. A metabolism cage for aphids was constructed, allowing for the collection and analysis of the radioactivity incorporated into the aphid body, expired as CO₂, and rejected in the honeydew and exuviae. This method was applied to the study of the metabolism of eight energetic amino acids (aspartate, glutamate, glutamine, glycine, serine, alanine, proline, and threonine) in the pea aphid, Acyrthosiphon pisum. All these amino acids except threonine were subject to substantial catabolism as measured by high ¹⁴CO₂ production. The highest turnover was displayed by aspartate, with 60% of its carbons expired as CO₂. For the first time in an aphid, we directly demonstrated the synthesis of three essential amino acids (threonine, isoleucine, and lysine) from carbons of common amino acids. The synthesis of these three compounds was only observed from amino acids that were previously converted into glutamate. This conversion was important for aspartate, and lower for alanine and proline. To explain the quantitative results of interconversion between amino acids, we propose a compartmentation model with the intervention of bacterial endosymbiotes for the synthesis of essential amino acids and with glutamate as the only amino acid supplied by the insect to the symbiotes. Moreover, proline exhibited partial conversion into arginine, and it is suggested that proline is probably indirectly involved in excretory nitrogen metabolism. © 1995 Wiley-Liss, Inc.     

Glucose and amino acid metabolism in neonatal rat skeletal muscle in tissue culture

The characteristics of glucose and amino acid metabolism over a 98-hour incubation period were studied in a primary culture of neonatal rat skeletal muscle cells. The cells formed large myotubes in culture, were spontaneously highly contractile, and had cell phosphocreatine levels exceeding ATP concentrations. Medium glucose fell from 7.2±0.2 to 1.5±0.1 mM between 0 and 98 hours; intracellular glucose was readily detectable, indicating glycolysis was limited by phosphorylation, not glucose transport. Alanine levels in the medium increased from 0.06±0.01 to 0.82±0.04 mM between 0 and 48 hours and decreased to 0.72±0.04 mM by 98 hours. The period of net alanine production correlated with the rise in the cell mass action ratio of the alanine aminotransferase reaction. Cell aspartate, glutamate, and calculated oxalacetate levels were inversely related to the cell NADH/NAD⁺ ratio, as represented by the intracellular lactate/pyruvate ratio (r=0.78–0.88). The branched chain amino acids (leucine, isoleucine, valine) were actively utilized, e.g., medium leucine fell from 0.70±0.01 to 0.30±0.06 mM between 0 and 98 hours. In addition, arginine and serine consumption was observed in conjunction with ornithine, proline, and glycine production. Conclusions: (1) A major driving force for the high rates of alanine production by skeletal muscle cells in tissue culture is the active utilization of branched chain amino acids. (2) Intracellular aspartate and glutamate pools are linked, probably via the malate-aspartate shuttle, to the cell NADH/NAD⁺ redox state. (3) Muscle cells in tissue culture metabolize significant amounts of arginine and serine in association with the production of ornithine and proline, and these pathways may possibly be related to creatine production.