Amino Acid Pool Size Research Articles

Effect of UVB Radiation on Utilization of Inorganic Nitrogen by Antarctic Microalgae

Abstract— The impact of UVB (280‐315 nm) radiation (WG 305) on uptake of 15N‐ammonium and 15N‐nitrate of marine phytoplankton from station 219 (47°W, 61.5°S) and sea ice‐algae from station 265 (22.6°W, 73.29°S) was studied during the Polarstern Cruise (EPOS III, Leg 3) to the Weddell Sea, Antarctica 1989. Uptake rates of 15NH4+ were higher and more affected by UVB radiation than those of 15N03‐. Pool sizes of the main amino acids changed in response to the used inorganic nitrogen source and UV exposure. Pools of glutamine, serine and glycine decreased, whereas those of alanine, asparagine and glutamate increased after UVB irradiation. The 15N‐incorporation into the amino acids was reduced as a result of UVB exposure of phytoplankton and ice algae. Results are discussed with reference to an inhibitory effect on the enzymes of both carbon and nitrogen metabolism as well as to adaptation strategies.

Model of Milk Protein Synthesis. A Mechanistic Model of Milk Protein Synthesis in the Lactating Bovine Mammary Gland

A model of amino acid (AA) uptake and metabolism by the mammary gland (udder) of a high producing dairy cow in mid-lactation is presented. Two cell types, milk-secreting alveolar epithelial cells, and constitutive stromal cells are represented separately. Solution of the model at steady state, with appropriate assumptions, allows calculation of amino acid (AA) pool sizes in tissue bound AA and milk protein bound AA, and concentrations of intracellular free AA. Bidirectional flux rates of amino acids between each of these pools is also estimated. The flow of two amino acids, phenylalanine (Phe) and tyrosine (Tyr), is described using uptake kinetics of the L-system AA transporter. Model results suggest that AA uptake capacity of the alveolar cells, and the amount/activity of protein translation components limit the rate of milk protein synthesis in the high-producing dairy cow. A considerable amount of experimental work is necessary to provide data, if a comprehensive quantitative understanding of milk protein synthesis is to be achieved.

CHANGES IN PHOTOSYNTHETIC YIELD, AMINO ACIDS, AND ORGANIC ACIDS ARE INDUCED BY AMMONIUM ADDITION TO CELLS OF PHORMIDIUM LAMINOSUM (CYANOPHYCEAE)1

ABSTRACTThe effect of NH4+ addition to NO3−‐growing cells of the non‐N2‐fixing cyanobacterium Phormidium laminosum (Agardh) Gomont (strain OH‐1‐pCl1) on photo‐synthetic and respiratory electron transport as well as on the intracellular levels of amino acids and some organic acids was studied. Addition of ammonium to nitrate‐growing cells resulted in substantial increases in the pool size of most amino acids and a transient decrease in the pool size of organic acids. The high demand for organic acids was partially overcome by degradation of stored carbohydrates, more than by newly fixed carbon, as indicated by the large stimulation of the respiration rate upon ammonium addition. Following ammonium addition, the photosynthetic yield of the in vivo noncyclic electron transport decreased, and the sensitivity of photosystem II to photodamage increased. Results indicate that cells balance their photosynthetic and respiratory activities depending on nitrogen availability and point to an important involvement of respiration in providing energy for ammonium assimilation until adaptation of bioenergetic processes to the new nitrogen source is complete.

Changes in intracellular amino acids and organic acids induced by nitrogen starvation and nitrate or ammonium resupply in the cyanobacterium Phormidium laminosum.

In Phormidium laminosum cells, nitrogen starvation caused a decrease in the intracellular levels of all amino acids, except glutamate, and an increase in the total level of the analyzed organic acids. The addition of nitrate or ammonium to N-starved cells resulted in substantial increases in the pool size of most amino acids. Upon addition of ammonium the total level of organic acids diminished, whereas it increased upon addition of nitrate, after a transient decay during the first minutes. Nitrogen resupply stimulated amino acid synthesis, the effect being faster and higher when ammonium was assimilated. The data indicate that nitrate and ammonium assimilation induced an enhancement of carbon flow through the glycolytic and the tricarboxylic-acid pathways to amino acid biosynthesis, with a concurrent decrease in the carbohydrate reserves. The results suggest that the availability of carbon skeletons limited the rate of ammonium assimilation, whereas the availability of reducing equivalents limited the rate of nitrate assimilation.

Factors Influencing the Free Amino Acid Content of Potato Solanum tuberosum L Tubers during Prolonged Storage

Tubers of the potato cultivars Pentland Dell and Record were stored for a period of up to 40 weeks at 5°C and 10°C over four consecutive storage seasons (1989–1990 to 1992–1993). Reconditioning treatments at 20°C were also performed at early, mid and late stages in storage. To assess the turnover of proteins during storage, tubers were analysed for free amino acid and soluble protein content. The net direction of nitrogen flow was dependent on the state of dormancy of the tuber and hence storage duration. An increase in free amino acid content commonly occurred during the latter part of storage, caused by an upturn of proteinase activity on the break of dormancy. This increased enzyme activity was probably due to de novosynthesis of proteinases, in particular of a 47 kDa aspartic proteinase which was purified to homogeneity and shown to have a marked substrate-specificity for endogenous tuber proteins such as patatin. The rate of protein turnover increased with storage temperature, although the net direction of nitrogen flow was temperature independent. However, when nitrogen flow exhibited a distinct direction, such as the protein breakdown in Pentland Dell during late storage, this was enhanced by higher temperatures. The accumulation of free amino acids in Pentland Dell at 10°C corresponded with a deterioration of processing quality that could not be accounted for by an upturn in reducing sugar content. Despite an excess of free amino acids with respect to reducing sugars, amino acids had a probable syn-ergistic influence on fry colour over the later stages of storage resulting in a darker colour per unit reducing sugar than in early storage. Reconditioning treatments were ineffective as a means of lowering the free amino acid pool size, these treatments operating solely through the decrease in reducing sugar content.

Factors Influencing the Free Amino AcidContent of Potato (Solanum tuberosum L) Tubers during Prolonged Storage

Tubers of the potato cultivars Pentland Dell and Record were stored for a period of up to 40 weeks at 5°C and 10°C over four consecutive storage seasons (1989-1990 to 1992-1993). Reconditioning treatments at 20°C were also performed at early, mid and late stages in storage. To assess the turnover of proteins during storage, tubers were analysed for free amino acid and soluble protein content. The net direction of nitrogen flow was dependent on the state of dormancy of the tuber and hence storage duration. An increase in free amino acid content commonly occurred during the latter part of storage, caused by an upturn of proteinase activity on the break of dormancy. This increased enzyme activity was probably due to de novo synthesis of proteinases, in particular of a 47 kDa aspartic proteinase which was purified to homogeneity and shown to have a marked substrate-specificity for endogenous tuber proteins such as patatin. The rate of protein turnover increased with storage temperature, although the net direction of nitrogen flow was temperature independent. However, when nitrogen flow exhibited a distinct direction, such as the protein breakdown in Pentland Dell during late storage, this was enhanced by higher temperatures. The accumulation of free amino acids in Pentland Dell at 10°C corresponded with a deterioration of processing quality that could not be accounted for by an upturn in reducing sugar content. Despite an excess of free amino acids with respect to reducing sugars, amino acids had a probable synergistic influence on fry colour over the later stages of storage resulting in a darker colour per unit reducing sugar than in early storage. Reconditioning treatments were ineffective as a means of lowering the free amino acid pool size, these treatments operating solely through the decrease in reducing sugar content.

Effect of UV Irradiance on Utilization of Inorganic Nitrogen by the Antarctic Diatom Odontella weissflogii (Janisch) Grunow

Abstract: The impact of UV‐B irradiation on unialgal cultures of the temperate marine diatom Odontella sinensis and of the Antarctic Odontella weissflogii was tested under controlled laboratory conditions. Uptake rates of inorganic nitrogen by Odontella sinensis were more affected by UV‐B radiation than those of Odontella weissflogii. Utilization of 15N‐ammonium was reduced after 3 h of UV‐B exposure. Values of Km from several marine phytoplankton species were estimated from algae not exposed to UV‐B and from those after 3 h of UV‐B radiation. Calculation of Lineweaver‐Burk plot showed, under UV‐B stress, a noncompetitive inhibition for 15NH+4 uptake and a competitive inhibitory effect for 15NO−3 by Odontella weissflogii. The damage to 15NH+4 uptake by UV‐B was more pronounced under red than blue light; a contradictory result was obtained for 15NO−3 utilization by Odontella weissflogii. Reduction of the pigments by UV‐B under white light was dependent on the exposure time; a strong depression of the contents of chlorophyllide a, chlorophyll c, diatoxanthin and the fucoxanthins was found. Contents of the chlorophylls are markedly affected by UV‐B under red light whereas the chlorophyll a concentration is enhanced in blue and green light. UV‐B exposure in conjunction with blue and green light led to a reduction of the protein content and an increase in the amino acid contents. The pattern of pool sizes of free amino acids varied after UV‐B exposure. UV‐A irradiance had no effect. The possible targets of UV‐B irradiation on the uptake system of inorganic nitrogen are discussed in detail. Adaptation to the environmental conditions, e.g. via synthesis of UV stress proteins or mycosporine‐like amino acids, were also considered.

Mechanism of Action of C2H4 in Promoting the Germination of Cocklebur Seeds. III. A Further Enhancement of Priming Effect With Nitrogenous Compounds and C2H4 Responsiveness of Seeds

Efficiency of organic or inorganic osmotica for seed priming of cocklebur (Xanthium pennsylvanicum Wallr.) revealed that KNO3 was the most promising, and was more effective than mannitol or other salts at the same concentration (200 mM) and was independent of the C2H4 action. However, KNO3 applied as a priming reagent enhanced the effect of C2H4 or that of the water stress imposed with mannitol. Unlike the action of mannitol, both KNO3M and C2H4 augmented the pool size of amino acids in seed cells. However, below 50 mM KNO3 imposing no stress only slightly, though insignificantly, affected the germinability as well as the levels of total cyanogen. On the other hand, at a high concentration which imposed water stress on the seeds, 200 mM KNO3 remarkably elevated the contents of both cyanogenic glycosides and lipids in the excised cotyledons. When C2H4 was added with KNO3, the level of cyanogenic compounds significantly increased but when added without KNO3, the contrary effect was shown. Hence the enhancement of the mannitol-induced priming effect by nitrogenous reagents in cocklebur seeds could be implicated in the accumulation of cyanogenic compounds. Unlike cocklebur, both common chickweed and barnyard grass seeds are very responsive to 30 mM KNO3 on germination, and such species abundantly contain cyanogen. The amount of cyanogen was further augmented by contact with KNO3 at only 30 mM. The role of NO-3 -dependent cyanogenesis is highlighted in relation to germination response of seeds.

Mechanism of Action of C2H4 in Promoting the Germination of Cocklebur Seeds. II. The Role of C2H4 in the Enhancement of Priming Effects

In cocklebur (Xanthium pennsylvanicum Wallr.) seeds, the pre-exposure to water stress imposed by polyethylene glycol or mannitol (seed priming) increased osmotic pressure (OP) in cell saps and water extracts. Carbohydrates were the major components and soluble proteins also played a partial role as an osmoregulator in the primed seeds. C2H4, which was effective in stimulating the growth of both axial and cotyledonary tissues even under water-stressed conditions, changed the amino acid pool size regardless of water stress. This C2H4-induced amino acid accumulation also occurred under anoxic conditions. CO2 was capable of stimulating growth of axial tissues but it did not increase OP values or carbohydrate and amino acid contents. The effectiveness of seed primlng gradually declined with increasing duration of presoaking, but C2H4 prevented the reduction of the priming effect by effectively maintaining the amino acid levels. Thus, it is likely that C2H4 contributes to the enhancement of the priming effect by abundantly supplying amino acids.

Analysis of aspartic acid and asparagine metabolism in Xenopus laevis oocytes using a simple and sensitive HPLC method

The amino acids in methanol-soluble extracts of Xenopus oocytes were measured using a method involving precolumn derivatization with phenylisothiocyanate and reverse phase HPLC of the derivatized amino acids. This technique allows the estimation of asparagine and glutamine pools in oocytes, estimated as 70 and 283 pmoles per oocyte, respectively. The pool sizes of the other amino acids were similar to previously reported results obtained using conventional ion exchange chromatography and postcolumn derivatization with ninhydrin. The advantages of the method developed here include picomolar sensitivity and the enhanced resolution of asparagine and glutamine from other amino acids. The kinetics of aspartic acid and asparagine utilization were monitored following microinjection of oocytes with [3H]aspartic acid and [14C]asparagine. The aspartic acid pool turned over rapidly with a half-time of < 30 min. The asparagine pool was metabolized much more slowly and appeared to be utilized almost completely for protein synthesis. The absolute rate of protein synthesis in oocytes was calculated from the incorporation data and chemical pool measurements as approximately 25 ng/hr-oocyte. The methodology developed here may be useful in experimental situations involving limited amounts of biological material.

Energy metabolism, ion homeostasis, and cell damage in the brain

observed neither on metabolic deprivation nor on depolarization, but only after treatment with excitotoxins [7,9]. In related experiments (results not shown) using ‘T-m.r. to study flux through metabolic pathways, we observed quite different patterns between these various insults. NMDA caused rapidly decreased N-acetylaspartate and increased lactate, with slow decreases in pool sizes of amino acids [lo], in marked contrast to the changes caused by severe hypoxia [ l l ] or depolarization

Assimilation of 15N-ammonium by Lithodesmium variabile Takano during irradiation with UV-B (300–320 nm) and complemental monochromatic light

The temperate marine diatom Lithodesmium variabile (Takano) was grown at 18 °C, normal air (0.035 vol.% CO 2) and a light—dark rhythm of 12:12 h (16.8 W m −2) in an artificial sea-water medium (35‰ salinity). Algae were exposed to UV-B (Philips lamps TL 40 W/01; 1.6 W m −2) for up to 2 h in conjunction with monochromatic or white light (4.7 W m −2). The content of chlorophylls was more reduced by UV-B radiation in blue and green than in red and white light. As a result of UV-B irradiation the uptake of 15N-ammonia increased under blue and green and decreased in red and white light. The pattern of 15N incorporation into amino acids and of the pool sizes depended on the light quality used. UV-B radiation resulted in an increase in all the tested free amino acid pools under green and red light. Pools of glutamate and glutamine were enhanced by UV-B in blue and white light whereas aspartate and asparagine are reduced. 15N enrichment in the amino acids was different from the pool sizes of the same amino acids. A marked depression by UV-B irradiation of 15N labelling into the amino acids was found under blue light as expected of glutamate. Inhibition of 15N enrichment in asparagine in green and red light and of alanine in white light during UV-B exposure could be observed. The results were discussed with reference to possible damaging effects on the key enzymes and the supply with carbon skeletons.

Hind leg muscle amino acid balances in cold-exposed rats.

The changes in hind leg tissue (muscle and skin) amino acid pool size and arteriovenous balance were measured in rats subjected to 0-90 min of cold exposure (4 degrees C). Tissue free amino acid pools presented a different composition pattern from protein amino acids. Muscle rapidly reacted to cold exposure by releasing small amounts of some amino acids (alanine, aspartate), with only small changes in pool size during the first 30 min. Amino acid oxidation was very limited during the whole period of cold exposure, since at all times tested there was either nil ammonia efflux or net absorption of ammonia and glutamine; i.e. the muscle was in positive nitrogen balance throughout the period studied. Thus most of the amino acid nitrogen taken up from the blood and not found in the free amino acid pools must have been incorporated into protein, since it was not oxidized, as shown by the glutamine and ammonia balance. The data on amino acid incorporation into proteins indicate that hind leg protein turnover is rapidly and widely modulated from a low initial setting upon cold exposure to a higher protein synthesis rate immediately afterwards, suggesting that protein turnover may have an important role in short-term events in cold-exposed muscle, in addition to its influence in long-term adaptation.

The effect of Maltose release on growth and Nitrogen metabolism of symbioticChlorella

Indicators of nitrogen limitation were compared in cultured, ex-symbiotic, maltose-releasing Chlorella and in Chlorella freshly isolated from symbiosis with green hydra. Culture of Chlorella at low pH (which stimulates maltose release) caused a reduction in amino acid pool size but no change in the nitrogen to carbon ratio (N: C). In nitrogen-starved cells, both amino acid pool size and N: C decreased. Chlorophyll and protein content, and glutamine to glutamate ratios, indicated that freshly isolated symbiotic Chlorella were more similar to symbionts cultured at low pH than those cultured without nitrogen, while N: C values were intermediate

Boron nutrition and the composition of glucosinolates and soluble nitrogen compounds in two broccoli (Brassica oleracea var. italica) cultivars

The effects of boron (B) nutrition on the composition of glucosinolate and soluble nitrogen (amino acids, NO3−, NH4+) compounds, as well as the distribution of B, were investigated in two cultivars of broccoli (Brassica oleracea var. italica ’Baccus’ and ’Commander’). The plants were grown to commercial maturity in vermiculite under glasshouse conditions and supplied continuously with B levels ranging from 0 to 2.5 mg L−1 of nutrient solution. Visible symptoms and inflorescence characteristics confirmed that Commander was more tolerant to B deficiency than Baccus. Eight glucosinolates were identified by high performance liquid chromatography and mass spectrometric analysis including 4-hydroxy-3-indolylmethyl-glucosinolate and 4-methoxy-3-indolylmethyl-glucosinolate which have not previously been reported for broccoli. The predominant glucosinolates were 3-indolylmethyl-glucosinolate, 1-methyoxy-3-indolylmethyl-glucosinolate and 4-methylsulfinylbutyl-glucosinolate. Baccus, but not Commander, also had considerable 2-hydroxy-3-butenyl-glucosinolate. In response to induced B deficiency [tissue B concentrations from 9–18 μg (g DW)−1], the total glucosinolates increased by about threefold in the young leaves, but not in the florets. In both the young leaves and florets, there were increases (54–175%) in the proportion of indolyl-glucosinolates. In both cultivars, the pool sizes of the glucosinolate precursor amino acids (phenylalanine, tryptophan and methionine), and of NO3− and NH4+ were not markedly affected.Key words: Boron nutrition, Brassica, broccoli, cultivars, glucosinolates, nitrogen

Patterns of Assimilate Production and Translocation in Muskmelon (Cucumis melo L.)

Continuous monitoring of steady-state carbon dioxide exchange rates in mature muskmelon (Cucumis melo L.) leaves showed diurnal patterns of photosynthesis and respiration that were translated into distinct patterns of accumulation and phloem export of soluble sugars and amino acids. Leaf soluble sugar patterns in general followed the pattern of photosynthetic activity observed in the leaf, whereas starch accumulated steadily throughout the light period. Sugar and starch levels declined through the dark phase. Phloem exudate analysis revealed that diurnal levels of the major transport sugars (stachyose and sucrose) in the phloem did not appear to correlate directly with the photosynthetic activity of the leaf but instead were inversely correlated with leaf starch accumulation and degradation. The amino acid pool in leaf tissues remained constant throughout the diurnal period; however, the relative contribution of individual amino acids to the total pool varied with the diurnal photosynthetic and respiratory activity of the leaf. In contrast, the phloem sap amino acid pool size was substantially larger in the light than in the dark, a result primarily due to enhanced export of glutamine, glutamate, and citrulline during the light period. The results indicate that the sugar and amino acid composition of cucurbit phloem sap is not constant but varies throughout the diurnal cycle in response to the metabolic activities of the source leaf.

Dissolved free amino acid pool composition and size in a hypertrophic, warm lake

Dissolved free amino acid pool composition and size in a hypertrophic, warm lake

Is there a relationship between branched amino acid pool size and cell cycle inhibition in roots treated with imidazolinone herbicides?

Excised pea root tips were cultured in White's medium for 24 h and then treated for 12 h with one of the imidazolinone herbicides at 0.2, 2, 20, or 200 μM. Pursuit and Assert were almost ineffective in inhibiting the mitotic index (MI), except at the highest concentrations. Arsenal (ARS) and Scepter both showed good inhibition with 20 μM by 8 h. Adding all three branched amino acids (BAA) (VAL, ILE, and LEU) at 0.1 mM blocked herbicide action. Treatment with the BAAs singly had no protective effect. Experiments were performed to determine the BAA pool size and MI after an 8-h treatment with ARS at 2 and 200 μM and Chlorsulfuron (CS), a sulfonylurea herbicide, at 28 nM. Both CS and ARS at 200 μM inhibited the MI to almost 0 by 8 h. ARS at 2 μM inhibited the MI by about 40%. The BAA pool size in all three treatments was reduced by approximately 50%, whether the MI was totally blocked or not. The 1-mm root tips had a greater amount of VAL than did the mature portions of the roots, whereas ILE and LEU were slightly less in the root tip. Other soluble amino acids did not show consistent differences between herbicide-treated roots and controls. The implications of the pool size reduction, in instances where the MI was not totally inhibited, is discussed in light of new data from other laboratories on the mode of action of the imidazolinone herbicides.

Hepatic Sulfur Amino Acid Metabolism in Rats with Chronic Renal Failure

We recently demonstrated elevated plasma amino acid concentrations and abnormal responses to amino acid supplementation (e.g., elevated methionine and phenylalanine) in children with chronic renal failure (CRF). We also recently developed an improved model of CRF in which animals manifest abnormal tissue amino acid levels, marked anorexia and growth failure. The objective of the current study was to determine the etiology of elevations of sulfur amino acids in animals with chronic renal failure. Chronic renal failure, defined as creatinine clearance less than 30% of control values, was induced in male rats in a two-stage surgical procedure. Four groups were studied over 2, 4 and 6 wk: control (non-operated) control (sham-operated), pair-fed (sham-operated and pair-fed with uremics) and CRF. Animals with CRF were anorexic and growth-retarded. Although plasma sulfur amino acid levels tended to be lower in the uremic animals than in controls, hepatic tissue concentrations were higher. Methionine adenosyltransferase was higher, but cystathione synthase and cystathionase activities were not significantly different in rats with CRF compared to pair-fed controls. We conclude that uremia, not malnutrition, affected sulfur amino acid metabolism and that with CRF, a normal adaptive response to elevated methionine levels was occurring, sufficient to normalize sulfur amino acid pool size. Alternative causes of elevated sulfur amino acids must be sought.

Influence of ammonium and extracellular pH on the amino and organic acid contents of suspension culture cells of Acer pseudoplatanus

The effects of supplied ammonium and nitrate on the amino and organic acid contents and enzyme activities of cell suspension cultures of Acer pseudoplatanus L. were examined. Regardless of nitrogen source the pH of the culture medium strongly affected the malate and citrate contents of the cells; these organic acid pools declined at pH 5, but increased at pH 7 and 8. Over a period of two days, ammonium had little effect on the responses of the organic acid pool sizes to the pH of the medium. In contrast, ammonium had a strong influence on amino acid pool sizes, and this effect was dependent on the pH of the medium. At pH 5 there was no increase in cell ammonium or amino acid contents, but at higher pH values cellular ammonium content rose, accompanied by accumulation of glutamine, glutamate and asparagine. Over several days, supplied ammonium led to an increase in activity of glutamate dehydrogenase irrespective of any changes in internal ammonium and amino acid contents. If the pH of the medium was allowed to fall below pH 4 in the presence of ammonium, phosphoenolpyruvate (PEP) carboxylase activity declined to a very low value over several days; at higher pH, the activity of this enzyme, and that of NAD malic enzyme and NAD malate dehydrogenase, remained substantial irrespective of whether the nitrogen source was NH+4 or NO‐3.