Amino Acid Fraction Research Articles

Kinetics of bacterial processes in natural aquatic systems based on biomass as determined by high-resolution flow cytometry.

The two primary kinetic constants for describing the concentration dependency of nutrient uptake by microorganisms are shown to be maximal rate of substrate uptake and, rather than the Michaelis constant for transport, specific affinity. Of the two, the specific affinity is more important for describing natural aquatic microbial processes because it can be used independently at small substrate concentrations. Flow cytometry was used to evaluate specific affinities in natural populations of aquatic bacteria because it gives a convenient measure of biomass, which is an essential measurement in the specific-affinity approach to microbial kinetics. Total biomass, biomass in various filter fractions, and the specific affinity of the bacteria in each fraction were determined in samples from a near-arctic lake. The partial growth rate of the pelagic bacteria from the 25 micrograms/liter of dissolved amino acids present (growth rate from the amino acid fraction alone) was determined to be 0.78 per day. By measuring activity in screened and whole-system populations, the biomass of the bacteria associated with particles was computed to be 427 micrograms/liter.

COMPOSITION EN PROTEINES ET EN ACIDES AMINES DES CELLULES DE CANNE A SUCRE D’UNE LIGNEE TOLERANTE A UN MILIEU RICHE EN LYSINE ET EN THREONINE

A cell line selection was carried out on sugarcane (Saccharum officinarum L.) callus, and the F. 156 (LT2) line having a high capability to produce lysine and threonine was isolated. This cell line contains more proteins, peptides and free amino acids than the control line. The amount and relative percentage of free and bound lysine and threonine were especially high in the LT2 line. In the free amino acid pool, the contents of the aspartic and glutamic acids of LT2 were also higher but that of alanine, valine, serine and proline tended to be lower than in the control. The biochemical modifications seem to be more important in the fractions of soluble proteins and free amino acids. This study showed the possibility of selection of overproducing amino acid cell lines in cereals and vegetable plants.Key words: Amino acids, lysine, threonine, cell line selection, sugarcane

タケノコのチロシン生成に関与するD-グルコース-U-<sup>14</sup>C及びシキミ酸-G-<sup>14</sup>Cのトレーサー実験

D-glucose-U-14C and shikimic acid-G-14C known as good precursors for biosynthesis of aromatic compounds were fed to the tissue slices of both the apical and the basal sections of bamboo shoots for 3, 6, 9 and 21 hours at 30°C. After incubations, alcohol soluble substances were extracted with 70% methanol and then separated into organic acid, sugar and amino acid fractions by ion-exchange chromatography. The homogentisic acid fraction was collected from organic acid fraction by HPLC. In the meantime, lignin aldehyde was separated from alcohol insoluble residue by the method of alkaline nitrobenzene oxidation. The radioactivity incorporated into each compound was measured using a Packard Tri-Carb Scintillation spectrometer after adding toluene scintillator.1. Glucose-U-14C was mainly incorporated into alcohol insoluble residue. The activity increased with the progress of incubation time and after 9 hours of incubation, it reached to 13.6% of the total radioactivity in the apical section and 52.5% in the basal section, but in which the activity of 14C incorporated into lignin aldehyde was very low. By determining the activity incorporated into each amino acid from glucose, it was found that the radioactivity was detected in almost all of amino acids, and also that the highest activity was found in alanine among amino acids in both sections. In the meantime, the radioactivity incorporated into tyrosine or phenylalanine was several percent in both sections.2. Shikimic acid-G-14C was steadily incorporated into the tissues during incubation and was rapidly converted to aromatic amino acids (tyrosine and phenylalanine). The incorporation of shikimic acid into lignin aldehyde increased during incubation and the activity was higher in the basal section than in the apical section. On determining the amount of homogentisic acid, it was found that the change from shikimic acid to homogentisic acid was very low in both sections.

Influence of L-methionine-deprived total parenteral nutrition on the tumor tissue and plasma amino acids fraction and the host metabolism: Experimental study with Sato lung carcinoma-bearing rats.

Creation of an amino acid imbalance, particularly curtailment of L-methionine, at the tumor cell level is thought to have a favorable effect on the inhibition of tumor growth. In the present study, we examined the influence of a specially-formulated amino acid mixture, avoid of sulfur-containing amino acids (L-methionine and L-cysteine), on the growth and amino acid fraction of Sato lung carcinoma (SLC) and the host metabolism in SLC-bearing rats. The rats were treated by total parenteral nutrition containing the above amino acid mixture, plus other nutrients (methionine-deprived TPN) for 10 days. Tumor growth began to decrease 4 days after the start of this treatment and the size was significantly less at the end of the treatment than in rats receiving conventional TPN with general purpose Vuj-N type amino acid solution as a protein source. The tumor-to-carcass weight ratio also showed a similar trend. In biochemistry, the albumin level and albumin-to-globulin ratio were significantly lower than in the rats receiving conventional TPN but other parameters such as total protein, glucose, GOT and GPT were not affected by the treatment. In the amino acid fraction of the tumor tissue extraction, both L-methionine and L-tyrosine were decreased and L-serine was increased significantly compared with the control group.

Varietal differences in amino acid composition of wheat (Triticum aestivum L) grain

AbstractThe differences in the amino acid composition of the developing grain between the three winter wheat (Triticum aestivum L) varieties Splendeur, Hobbit and Maris Huntsman were analysed in a growth‐room experiment. At 15 days after anthesis, Maris Huntsman, the variety with the fastest leaf senescence, had a larger proportion of glutamate and smaller levels of threonine, glycine, alanine, valine, methionine and isoleucine in the total amino acid fraction; at later dates these differences were in general less pronounced, particularly compared with Splendeur. This suggests that, in the variety with the fastest leaf senescence, rapid gliadin synthesis in the grain occurs earlier. At any given percentage of nitrogen in the mature grain, Hobbit had a smaller proportion of glutamate and greater proportions of lysine, arginine and alanine; Splendeur, the only bread‐making variety, had smaller levels of aspartate and lysine; and Maris Huntsman had the greatest relative amount of phenylalanine. The possible differences among the varieties in the relative proportion of some protein fractions are discussed. Until 25 days after anthesis Maris Huntsman had smaller relative proportions of free alanine, valine, leucine, isoleocine and threonine, in particular when compared with Splendeur. In Maris Huntsman, the relative amount of aspartate increased from 15 days after anthesis onwards, whereas in Hobbit it only did so in mature grains.

Provenance, concentrations and nature of sedimentary organic nitrogen in the gulf of Maine

Factors controlling the provenance, concentrations, and nature of sedimentary organic matter (SOM), particularly the nitrogenous fraction, were examined for sites throughout the Gulf of Maine and two of its estuaries. Stable nitrogen isotope data corroborate previously reported Br: C ratios in indicating that macroalgae are important contributors to organic nitrogen pools in estuarine sediments. Significant contributions of terrigenous organic carbon in both the estuaries and the deep basins of the open Gulf are not accompanied by significant contributions of terrigenous organic nitrogen. Variations in concentrations of organic carbon, nitrogen, chlorophyll, and enzymatically hydrolyzable protein throughout the Gulf and its estuaries can be explained largely by variations in the delivery rate of organic matter to the benthos and the specific surface area of minerals in the sediments. Chlorophyll and protein concentrations represent relatively fresh organic matter and exhibit greater sensitivity to organic delivery variations than do total organic carbon or nitrogen. A strong, linear relationship between organic carbon and surface area is consistent with monolayer adsorption of SOM on mineral surfaces, which appears to inhibit degradation of the SOM. Amino acid compositions are remarkably constant throughout the Gulf, with large differences seen only with elevated tyrosine in estuaries and elevated basic amino acids and methionine in the open Gulf. Leucine and glutamic acid correlate with chlorophyll concentrations in open Gulf sites. Enzymatically hydrolyzable protein comprises small to major fractions of total hydrolyzable amino acids. Amino acid-nitrogen: total nitrogen correlates inversely with total nitrogen.

Changes in the Levels of Free Amino Acids in Various Regions of <italic>Cuscuta</italic> during Growth

The angiospermous plant parasite Cuscuta derives reduced carbon and nitrogen compounds primarily from its host. Free amino acids along Cuscuta vines in three zones, viz., 0 to 5 cm, 5 to 15 cm, and 15 to 30 cm, which in a broad sense represent the region of cell division, cell elongation and differentiation and vascular tissue differentiation respectively, were quantitatively estimated. The free amino acid content was the highest in the 0 to 5 cm region and progressively decreased along the posterior regions of the vine. The haustorial region showed the lowest content of free amino acids. In general, the free amino acid content in samples collected at 7 p.m. was found to be higher than that in the samples collected at 7 a.m. Three basic amino acids, histidine, the uncommon amino acid γ-hydroxyarginine, and arginine constituted more than 50% of the total free amino acids in all the zones studied except the haustorial region. Aspartic acid and glutamic acid constituted the major portion in the acidic and neutral fraction of amino acids. Glutamine, asparagine, threonine, and serine were eluted together and occurred in substantial amounts. γ-Hydroxyarginine constituted the largest fraction in the cut end exudate of Cuscuta and presumably appeared to be the major form of transport amino acid. γ-Hydroxyarginine was also a major constituent of the basic amino acids in Cuscuta vines parasitizing host plants from widely separated families, suggesting that this amino acid is a biosynthetic product of the parasite rather than that of the hosts. Also, U-14C arginine was converted to γ-hydroxyarginine by cut Cuscuta vines, suggesting that γ-hydroxyarginine is synthesized de novo from arginine by Cuscuta.

A Mutant of Nicotiana sylvestris Lacking Serine:Glyoxylate Aminotransferase

The photorespiratory mutant of Nicotiana sylvestris, NS 349, lacking serine:glyoxylate aminotransferase (SGAT) grows in 1% CO(2) but not in normal air (NA McHale, EA Havir, I Zelitch 1988 Theor Appl Genet. In press). Alanine:hydroxypyruvate and asparagine:hydroxypyruvate aminotransferase activities were also lacking in the mutant, and plants heterozygous with respect to SGAT which grow in normal air had 50% of the activities present in homozygous plants. Therefore, all these activities are associated with the same enzyme. On feeding [2-(14)C]glycolate to leaf discs in the light, NS 349 showed reduced incorporation of radioactivity into the neutral and organic acid fractions and increased incorporation into the amino acid fraction, principally into serine. The effect of reducing SGAT by 50% in heterozygous plants produced little change in the metabolism of [2-(14)C]glycolate, showing there is a large excess of this enzyme in wild-type plants.

Studies on the uptake of (14C) amino acids derived from both dietary (14C) protein and dietary (14C) amino acids by rainbow trout, Salmo gairdneri Richardson

Previous studies have shown that rainbow trout fed on diets containing whole protein have superior growth rates compared to fish fed on diets of similar amino acid composition but containing a high proportion of free amino acids. The influence of several nutritional factors on the uptake of radioactivity from food pellets containing either [U‐I4C] protein or [U‐14C] amino acids into the systemic blood of trout has been investigated. The time taken for radioactivity in the free amino acid fraction of blood to reach a peak after a meal containing [U‐14C] protein had been given was much shorter, and the level of radioactivity in the blood higher, in trout with almost empty stomachs than in fish with almost full stomachs; uptake of radioactivity into blood amino acids was also more rapid and reached much higher concentrations when pellets containing [U‐14C] amino acids were fed than when [U‐14C] protein was fed. Incorporation of radioactivity into blood protein continued for a much longer period and reached higher levels when a pellet containing [U‐14C] protein was fed than when a pellet containing [U‐14C] amino acids was fed. Previous dietary history (low or high protein intake) did not appear to affect the rate of absorption of amino acids from either protein or free amino acid pellets. The uptake rates from pellets containing free amino acids could be slowed by mixing the dietary amino acids with albumin. The distribution, postabsorption, of radioactivity in the different fractions of blood and liver suggested that incorporation of carbon residues into glycogen and lipid from an amino acid diet was greater than from a protein diet. The converse was true of incorporation of radioactivity into tissue protein.

The assimilation of aspartic and glutamic acids by the root parasites Alectra orobanchoides Benth. and Alectra vogelii Benth. (scrophulariaceae)

In this study seedlings of the root parasites A. orobanchoides and A. vogelii were grown independently of their respective hosts on an MS medium supplemented with aspartic and glutamic acids to which 18.5 kBq of the respective U- 14C-labelled amino acids were added. In order to investigate the metabolic fate of the added amino acids, extracts of seedlings were fractionated with ion exchange resins. The highest radioactivity was found to be in malic acid and to a lesser extent in succinic and fumaric acids 7 days after application, but it was also present in the sugar as well as the amino acid fractions in small quantities. It is concluded that the growth promotory effect previously observed might in part be due to the supplementation of the respiratory pool by carbon skeletons obtained from these amino acids.

The effect of whole milk, milk protein and some constituent amino acids on the in vitro availability of iron from cereal meals

Studies were carried out on the in vitro availability of iron from habitually consumed cereal based meals of preschool children, with and without the addition of whole milk, milk protein, soluble fraction of milk or certain constituent amino acids of milk namely lysine, cysteine or lysine + cysteine. Addition of 200 ml milk to the cereal meal brought about a significant increase (p<0.001) in the in vitro iron availability although there was no significant increase in total iron content. The protein fraction of milk accounted for most of the increase brought about by whole milk in available iron. On the other hand soluble fraction of milk as well as the amino acids lysine, cysteine or lysine + cysteine failed to show any effect on iron availability from the cereal meals studied.

Biogeochemical cycling in an organic-rich coastal marine basin: 10. The role of amino acids in sedimentary carbon and nitrogen cycling

Hydrolyzable amino acids were measured in cores and surface sediment samples collected over a 14 month period from the rapidly accumulating, anoxic sediments of Cape Lookout Bight, NC. The concentration of total hydrolyzable amino acids (THAAs) shows an exponential decrease with depth, in a manner similar to total organic carbon and total nitrogen. Carbon and nitrogen in THAAs averages 10–15% of the total organic carbon and 30–40% of the total nitrogen in these sediments (both on a mole/mole basis). In surface sediments the concentration of THAAs do not show strong seasonal variations, with the exception of a small apparent decrease during the winter months. Aspartic acid, glutamic acid, glycine and alanine are the most abundant individual amino acids in Cape Lookout Bight sediments. The distribution of individual amino acids in these sediments is very similar to that observed in the two major sources of organic matter, vascular salt marsh plants and marine plankton. The mole fractions of most amino acids show no depth variation in Cape Lookout Bight sediments. However the mole fractions of alanine and glutamic acid decrease with depth and the mole fraction of glycine increases with depth. These changes are attributed to the selective microbial utilization of alanine and glutamic acid, and the selective preservation of glycine in these sediments relative to other amino acids. Kinetic modelling of these data indicates that the deposition of amino acids to the surface of these sediments is 5.8 ± 1.0 mol· m −2· yr −1. Approximately 46 ± 16% of this input is remineralized in the upper 40 cm. The recycling of amino acids accounts for 82 ± 43% of the total nitrogen regeneration and 27 ± 11% of the regeneration of total organic carbon in these sediments. The mean residence time of metabolizable amino acids is approx. 9 months, a value which is comparable to the mean residence times of both metabolizable organic carbon and nitrogen in these sediments.

Cycling of selenite and selenate in marine phytoplankton

The marine phytoplankton Dunaliella tertiolecta, Cachonina niei, Thalassiosira nordenskioldii, Phaeodactylum tricornutum, and Chaetoceros sp. were incubated with a range of molar concentrations of sodium-selenite (Na2-SeIVO3) and sodium-selenate (Na2-SeVIO4) to examine further their role in metabolic cycling of selenium in ocean waters. At low selenium concentrations, approaching those found naturally in seawater (10-10 to 10-9 M), all species distinguished between selenite and selenate, and actively concentrated selenite from the incubating medium while only marginally accumulating selenate. At much higher concentrations (10-8 to 10-6 M), selenate was also taken up. At the highest concentration tested, i.e., 10-5 M with C. niei, after an immediate rapid uptake in the first 24 h, the intracellular selenite and selenate levels dropped to about 35 to 50% of the initial peak values. These observations suggest an uptake mechanism in these algae which, at normal ambient concentrations of selenium (10-9 M), preferentially selects selenite and excludes selenate. At much higher concentrations (10-8 M), the mechanism becomes overloaded and both selenium species enter the cells. Intracellularly, selenite became associated primarily with protein and amino acid fractions, in approximately equal proportions, while only ca. 4% of total intracellular selenium was found in the lipid fraction. Trace amounts of selenate that entered the cells, mainly during the first minutes of exposure, also entered the protein and amino acid components, but over time were increasingly associated with the protein fraction only. At the end of a 10-d incubation of algal cells in selenite-spiked medium, less than 25% of total Se in the medium could in fact be identified analytically as selenite. This suggests the presence of a non-selenite metabolite, possibly released back into the medium from the algae.

Optimisation of the reaction conditions for the direct determination of the dissolved combined amino acid levels in sea water

An investigation into the conditions necessary to determine accurately the amino acid composition of the dissolved combined amino acid (DCAA) fraction of sea water was undertaken. Maximum recoveries were obtained by hydrolysing sea water samples in 6 M HCl in tubes evacuated to 0.05mm Hg pressure and heated at 110°C for 16h. Full details of the method are reported, together with environmental data indicating the range of total DCAA found in the coastal waters off Plymouth, U.K. The need for further studies of this fraction in the context of phytoplankton nutrition is commented upon.

Photosynthetic carbon metabolism in French bean leaves under saline conditions

The influence of sodium chloride and sodium sulphate salinities on photosynthetic processes in French bean leaves was studied. Chlorophyll content was reduced in plants subjected to chloride salinity; an opposite trend was observed in sulphate-salinized plants. The rate of photosynthetic carbon assimilation was also markedly affected by chloride salinity. An analysis of long-term (1-hr) photosynthetic products revealed that incorporation of radioactivity in sucrose was reduced with both salinities. This reduction was accompanied by an increase in carbon assimilation in amino acid and sugar phosphate fractions. These observations are discussed in relation to salt sensitivity of the cultivar.

Dynamics of amino acid and protein metabolism of laying hens after administration of 15N-labeled wheat protein. 7. Incorporation into the amino acids and peptides of the gastrointestinal tract and the pancreas

In a 15N labelling experiment 12 colostomized laying hens received 15N labelled wheat with 14.37 atom-% 15N excess (15N') over 4 days. 3 hens each were butchered after 12 h, 36 h, 60 h and 108 h after the last 15N' application. The gastro-intestinal tract was divided into 3 parts (oesophagus with crop and gizzard as well as glandular stomach, small intestine, large intestine). These parts and the pancreas were hydrolysed with 6 N HCl and the individual basic as well as the sum of acid and neutral amino acids were determined in the hydrolysed fractions. In addition, the amino acids and peptides were determined in the TCA soluble N fraction. The atom-% 15N' was determined in the individual amino acid and peptide fractions. The labelling of the basic amino acids in the individual tract segments was lower than in the acid and neutral amino acids. In comparison to the peptides, a higher atom-% 15N' could be determined in the free amino acids.

Approaching the structures of mammalian propylamine transferases and their genes.

Propylamine transferases, spermidine synthase (EC 2.5.1.16) and spermine synthase (EC 2.5.1.22) catalyze the two final, irreversible steps in the conversion of arginine and methionine to spermidine and spermine, respectively. Both of the amino acid precursors are nutritionally essential for animals and are present in animal tissues at low, well-conserved concentrations. Arginine plays a central role in protein catabolism by being an essential intermediate in the conversion of toxic ammonia to excretable nontoxic urea. Methionine, on the other hand, is vitally important for protein synthesis by being the amino acid needed for peptide chain initiation and for the transmethylation reactions involved in the control of metabolism at several steps of transcriptional, translational and post-translational level. The fraction of the precursor amino acids consumed in polyamine synthesis varies a great deal depending on the cell type, the stage of differentiation and the proliferation rate. As to methionine consumption, the share of polyamine synthesis is less than 1% in rat liver (Eloranta and Kajander, 1984) but may exceed 20% in some cultured cells (Iizasa and Carson, 1985). Although detailed action mechanisms of polyamines in animal physiology are poorly known, their essentiality for cellular functions have been widely demonstrated (for references see Tabor and Tabor, 1984; Pegg, 1986).

Unusual stable isotope ratios in amino acid and carboxylic acid extracts from the Murchison meteorite.

Much effort has been directed to analyses of organic compounds in carbonaceous chondrites because of their implications for organic chemical evolution and the origin of life. We have determined the isotopic composition of hydrogen, nitrogen and carbon in amino acid and monocarboxylic acid extracts from the Murchison meteorite. The unusually high D/H and 15N/14N ratios in the amino acid fraction (delta D = 1,370% after correction for isotope exchange; delta 15N = 90) are uniquely characteristic of known interstellar organic materials. The delta D value of the monocarboxylic acid fraction is lower (377%), but still consistent with an interstellar origin. These results confirm the extraterrestrial origin of both classes of compound, and provide the first evidence suggesting a direct relationship between the massive organo-synthesis occurring in interstellar clouds and the presence of pre-biotic compounds in primitive planetary bodies. The isotope data also bear on the historical problem of distinguishing indigenous material from terrestrial contaminants.

THE EFFECTS OF TERATOGENIC ANTISERA ON PROTEIN SYNTHESIS AND DEGRADATION BY THE CULTURED RAT CONCEPTUS

Rat embryo culture was initiated on the 10th day of gestation to study embryonic site protein synthesis in normal and pathologic states. Three culture media were employedsrat serum, serum plus teratogenic sheep anti-rat visceral yolk sac (VYS) serum, or rat serum plus normal sheep serum. Two studies were performed: a) 3H-leucine was added to produce incubation periods of 20 min. to 24 hr.; and after an initial period of culture in the presence of 3H-leucine to obtain a constant precursor specific radioactivity, culture was continued in the absence of 3H-leucine for a total of 1-30 hr. Embryos, VYS and culture media were analysed for total radioactivity and 3H-leucine specific radioactivity in the protein-bound and free amino acid fractions. The results suggest that: a) anti-VYS serum inhibited protein synthesis by the embryo and VYS but had no detectable effect on protein degradation; b) anti-VYS serum increased intracellular labeled leucine concentration. The teratogenic mechanism of anti-VYS serum appears to involve both the suppression yolk sac amino acid nutritive support of the embryo and the inhibition of the utilization of amino acids by the embryo for protein synthesis. These techniques provide us with the ability to determine the amino acid requirements which will support normal organogenesis and to quantitate the amino acid deprivation which will result in embryopathology. (Supported by NIH)

Preparative separation of an enzymatic hydrolysate of 14C-labelled proteins

The preparative separation of an enzymatic hydrolysate by a step gradient of a lithium—sodium citrate buffer was studied. Using a two-column arrangement, 10 GBq of 14C-labelled enzymatic hydrolysate or 250 mg of inactive hydrolysate were separated within 36 h. After the chromatographic separation, the fractions of amino acids were isolated from the citrate eluents on weak cation exchangers with an ethylene glycol—poly(methyl methacrylate) matrix. The use of volatile organic acids as an eluent was possible due to the weak interactions with the cation matrix.