Effect of albumin binding and amino acid competition on tryptophan uptake into brain.

Abstract— In the present study we examine the influence of pH, palmitate, and neutral amino acids on the passage of tryptophan from blood into brain during a single capillary pass, and on the partitioning of tryptophan between free and albumin-bound forms. The results show that a considerable fraction of albumin-bound tryptophan is stripped from albumin sites during passage, that uptake is concentration-dependent, and that amino acid competition for carrier sites is quantitatively the most important factor in regulating tryptophan uptake into brain. The interaction between tryptophan concentration, tryptophan binding, and competing amino acids is of considerable influence on brain serotonin biosynthesis.     

Acid-stable and acid-unstable alpha-amylases of the mold fungi Aspergillus]

Acid-sable alpha-amylase of Asp. niger and acid-unstable, alpha-amylase of Asp. oryzae were studied. It was demonstrated, that beside being a more acid-stable properties, alpha-amylase Asp. niger has increased thermal stability as compared to alpha-amylase Asp. oryzae. The molecular weights of acid-stable alpha-amylase and acid-unstable alpha-amylase are 58 000 and 51 000, respectively. The amino acid composition, and the C- and N-terminal amino acids of both forms of alpha-amylases were determined. It was demonstrated, that the enzymes under study contain one sylfhydryl group per mole of enzyme, which in the Ca2+-bound form plays an important role in the maintenance of the catalytically active enzyme conformation.     

Influence of Protein Source on Growth-depressing Effect of Excess Amino Acids in Young Rats

The influence of protein quality on the growth-depressing effect of excessive amount of 12 individual essential and semiessential amino acids was examined. Growing rats were fed for 3 weeks diets containing either 10.5% egg albumin or 11.6% wheat gluten (equivalent to the protein content of a 10% casein diet) supplemented with 5% of each of the l-amino acids. In general, the pattern of growth depression produced by the addition of excess amino acids to the egg albumin or the wheat gluten diet was similar to that of the case of casein diet obtained previously under the same experimental conditions. However, the extent of these effects was dependent not only upon the kind of amino acid supplemented with but also upon the source of protein used, and the depressing effect of each of excess amino acids added to the wheat gluten diet was usually severer than those added to casein and egg albumin diets. No evidence was noted of any striking changes in the liver protein and nucleic acid concentrations by either diets, but total liver protein, RNA and DNA contents were decreased in some amino acid groups of the egg albumin diet and in all amino acid groups of the wheat gluten diet except the lysine addition. The free amino acid level in plasma generally showed extreme elevation for the amino acid supplemented in excess in the diet, and in most cases the extent of the elevation was correlated with the growth depression.     

Effects of Albumin, Amino Acids and Clofibrate on the Uptake of Tryptophan by the Rat Brain

Abstract: The influences of total tryptophan concentration, albumin binding and amino acid competition on the rate of tryptophan influx into rat brain were compared using a single-pass injection technique with tritiated water as a freely diffusible reference. Omission of 3% bovine albumin from a bolus containing tryptophan in Krebs–Ringer bicarbonate buffer injected into the carotid artery increased non-albumin bound (free) tryptophan concentration threefold but tryptophan uptake by only 35% and 30% into forebrain and hypothalamus, respectively. However, tryptophan uptake from injected rat plasma was more markedly elevated when free tryptophan concentration was raised. Thus, when free tryptophan was doubled, but total tryptophan unchanged, by in vitro addition of clofibrate to a plasma bolus, uptake was increased by 53% and 28% into forebrain and hypothalamus respectively. When clofibrate was injected in vivo so that plasma total tryptophan concentration was decreased by 45% but neither free tryptophan nor competing amino acid concentrations were altered, then uptake from a bolus of the rat's own plasma was unchanged. Addition of competing amino acids at physiological concentrations to tryptophan in Krebs-Ringer buffer significantly reduced tryptophan influx into both brain regions, but did not increase the effect of albumin binding. The results indicate that tryptophan uptake into rat forebrain is substantially influenced by albumin binding and competition from other amino acids, but that hypothalamic uptake is less influenced by these factors.     

Albumin synthesis in cultured hempatoma cells. Regulation by essential amino acids.

The effects of essential amino acids on albumin synthesis by a mouse hepatoma cell line have been investigated. The amino acids tested were tryptophan, phenylalanine, histidine, isoleucine and leucine. Cellular rates of synthesis (molecules albumin/cell per min) were determined from rates of [3H]leucine incorporation into immunoprecipitable albumin in the culture medium. The effects of amino acids on albumin synthesis fall into three distinct groups. The concentration of tryptophan producing half-maximal synthesis is 4 micronM. The corresponding concentration for leucine is 100 micronM. Histidine, phenylalanine and isoleucine were very similar, the half-maximal concentrations being approximately 15 micronM. The concentrations of amino acids producing half-maximal synthesis correlate directly with the amino acid composition of albumin. The levels of these essential amino acids necessary to saturate albumin synthesis have been compared with amino acid levels in normal plasma.     

The metabolism of L-tryptophan by isolated rat liver cells. Effect of albumin binding and amino acid competition on oxidatin of tryptophan by tryptophan 2,3-dioxygenase.

1. Novel methods, using L-[ring-2-14C]tryptophan, are described for the measurement of tryptophan 2,3-dioxygenase activity and tryptophan accumulation in isolated rat liver cells. 2. The effects of bovine serum albumin, non-esterified fatty acids and neutral amino acids on tryptophan oxidation by hepatocytes and on the partition of tryptophan between free and albumin-bound forms were investigated. 3. Oxidation of physiological concentrations (0.1 mM) of tryptophan was inhibited by approx. 50% in the presence of 2% (w/v) bovine serum albumin; no effects were found at tryptophan concentrations of 0.5 mM and above. 4. Increases in free tryptophan concentrations produced by displacement of 0.1 mM-tryptophan from albumin-binding sites by palmitate resulted in increased flux through tryptophan dioxygenase. 5. Addition of a mixture of neutral amino acids, at plasma concentrations, to hepatocyte incubations had no effect on the rate of tryptophan oxidation. 6. It is concluded that alterations in free tryptophan concentrations consequent to changes in albumin binding may be an important factor in regulating tryptophan uptake and catabolism by the liver. The results are briefly discussed with reference to possible consequences on brain tryptophan metabolism.     

The effect of yeast autolysates on the level and distribution of free amino acids in yeast cells of the Candida genus

The work was aimed at studying the effect of yeast autolysate on the content and subcellular distribution of free amino acids in yeast cells. The overall pool of free amino acids decreased 1.5-2 times when mineral nitrogen in the growth medium was substituted either completely or partly by yeast autolysate. As was shown using the technique of differential extraction, the vacuolar pool of the cell is mainly responsible for the decrease in the content of free amino acids.     

The effects of amino acids on albumin synthesis by the isolated perfused rat liver

Albumin synthesis was measured in the isolated perfused rat liver by using the livers of both well-fed and starved rats. Starvation markedly decreased albumin synthesis. The livers from starved rats were unable to increase synthesis rates after the addition to the perfusates of single amino acids or the addition of both glucagon and tryptophan. Arginine, asparagine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, threonine, tryptophan and valine, added together to ten times their normal peripheral blood concentrations, restored synthesis rates to normal. The plasma aminogram (i.e. the relative concentrations, of amino acids) was altered by depriving rats of protein for 48h. The use of blood from the deprived rats as perfusate, instead of normal blood, decreased albumin synthesis rates significantly by livers obtained from well-fed rats. The addition of single amino acids, including the non-metabolizable amino acid, alpha-aminoisobutyric acid, to the above mixture increased albumin synthesis rates to normal values. It is concluded that amino acids play an important role in the control of albumin synthesis and that more than one mechanism is probably involved.     

Influence of Amino Acid Supplementation to Egg Albumin Reacted with Oxidized Lipids on Liver Lipids of Rats

Fatty infiltration in the liver of rats fed a low egg albumin diet was apparently disappeared by substituting the protein source for the albumin reacted with oxidized lipids (the mixture of egg albumin-ethyl linoleate (2: 1, w/w) was kept at RH 80.4% and at 50°C for 7 days and defatted). The present study deals with the mechanism causing such a phenomenon.

The decrease in the content of liver triglyceride (TG) due to feeding the reacted albumin was essentially disturbed by supplementing amino acids for lost ones accompanied by lipid oxidation.

Feeding the amino acid mixture simulating the reacted albumin showed the same response to the reacted protein.

Supplementation of basic amino acids damaged to the amino acid mixture simulating the reacted albumin increased the content of liver TG, while that of sulfur amino acids inclined to decrease liver TG.

These results indicate that the disappearance of fatty liver in rats fed the reacted albumin is almost entirely, but not exclusively, ascribed to the change in the amino acid pattern, particulary to the loss of the basic amino acids. However, since the plasma lipid levels did not respond similarly, participation of other factor(s) could not be excluded.     

Induced Autolysis of Aspergillus oryzae (A. niger group): IV. Carbohydrates

The examination of substances formed during induced autolysis by Aspergillus niger was continued in this work, which dealt in particular with carbohydrates. The autolysate contained a large amount of d-glucose (14 to 20% dry wt) and traces of glycolic aldehyde, dihydroxyacetone, ribose, xylose, and fructose. It also contained glycopeptides (about 10% dry wt), which were split from the cell wall during autolysis and which differed from one another in their level of polymerization and their composition. They were constituted by glucose and mannose, glucose and galactose, or mannose, glucose, and galactose (mannose being the most abundant in this case), and amino acids (chiefly alanine, serine, glutamic acid, and aspartic acid). During autolysis, only a part of the cell wall was dissolved, since it retained its shape. Upon further chemical hydrolysis, it produced mostly glucose and glucosamine, and smaller amounts of mannose, galactose, and amino acids. Presumably, glucomannoproteins and glucogalactoproteins were present in the intact cell as a macromolecular complex, constituting, together with chitin, the major part of the cell wall of Aspergillus.     

Induction of tryptophan oxygenase by dexamethasone in isolated hepatocytes. Dependence on composition of medium and pH.

Hepatocytes were isolated from perfused rat livers. 4 x 10-6 cells/ml were incubated at at 37 degrees C in different media in the absence and presence of a steroid hormone, dexamethasone phosphate (2 x 10-5 M). 1. Hormonal enzyme induction occurred in cells suspended in a simple salt medium, devoid of amino acids and macromolecules. This induction was completely blocked by addition of either actinomycin D (2 mu-g/ml) or cycloheximide (50 mu-g/ml). 2. Incubation of cells in media containing defatted albumin did not enhance hormonal enzyme induction, although disintegration of cells during incubation was reduced. Addition of a crude albumin fraction reduced tryptophan oxygenase induction and dextran completely blocked enzyme induction by dexamethasone. 3. An increase of dexamethasone concentration in the presence of albumin to 9 x 10-5 M was unable to raise enzyme induction further, and a still higher concentration of hormone, 3 x 10-4 M, resulted in reduced enzyme induction. 4. The hormonal induction of tryptophan oxygenase was most pronounced when the pH of the medium was between 7.0 and 7.6, with an optium at 7.3. No induction was found when the pH of the medium was either 6.6 or 7.8. The basal tryptophan oxygenase activity was much less influenced by similar pH variations. It is concluded that hepatocytes in suspension are able to carry out hormone-stimulated enzyme synthesis and that factors influencing this process may be studied under controlled conditions in such systems.     

CORRELATION OF PLASMA AND BRAIN AMINO ACID AND PUTATIVE NEUROTRANSMITTER ALTERATIONS DURING ACUTE HEPATIC COMA IN THE RAT

During acute hepatic coma following two-stage hepatic devascularization in the rat, profound changes occurred in plasma and whole-brain amino acids and putative neurotransmitters. Brain ammonia, glutamine and GABA were increased, aspartate was decreased, while glutamate was unchanged. An increase in brain tryptophan was accompanied by a similar increase in plasma unbound tryptophan but decreased plasma total tryptophan. These changes occurred in the presence of high plasma levels of the other neutral amino acids, including the branched chain amino acids. Plasma insulin was unchanged while glucagon levels rose, resulting in a decreased insulin to glucagon ratio. These results suggest that while plasma unbound tryptophan may influence brain tryptophan levels, altered plasma concentrations of neutral amino acids which compete with tryptophan for transport into the brain do not contribute to the increase in brain tryptophan observed during acute hepatic coma.     

The Hydroxyl Radical Generated by an Iron(II)/EDTA/Ascorbate System Preferentially Attacks Tryptophan Residues of the Protein

Iron(II)/EDTA/ascorbate-mediated oxidative damage to specific amino acid residues (tryptophan) of serum albumin was studied. The active species generated by Fe(II)/EDTA/ascorbate preferred to react with tryptophan residues rather than histidine or other amino acids. The observation of preferential damage to tryptophan residues of the protein was fully suported by a model experiment using a tryptophan analogue. The reaction of Fe(II)/EDTA/ascorbate to the protein was significantly suppressed by mannitol and dimethysulfoxide, suggesting the participation of the hydroxyl radical generated via Fenton’s reaction. The result was supported by the hydroxyl radical assay using 2-deoxyribose.     

The influence of detergents and active components of detergent on bioproduction of organic matters and enzymatic activity of some species of fungi.

Detergent (Merix, "Merima " Krusevac) applied in concentration of 1% vol. showed specific influence on the bioproduction of some 15 different amino acids and on the enzyme activity of the species of fungi A. niger, A. alternata and T. roseum. Detergent has significantly stimulated the production of 15 analyzed amino acids of the fungi species A. niger. The same applied concentration of detergent has decreased or considerably decreased the production of some 14 of totally 15 analyzed amino acids of investigated fungi species A. alternata and T. roseum. The enzyme activity of the fungi A. niger was more intensive in relation to the species A. alternata and T. roseum during the experimental period or in some phases of the experimental period. The detergent component, ethoxyled oleyl-cetyl alcohol, in concentration of 0.01%, 0.1% and 1% showed an inhibitory effect, or significant inhibitory effect on the enzyme activity of the examined species of fungi (A. niger, A. alternata and T. roseum).     

The role of hemolymph proline as a nitrogen sink during blood meal digestion by the mosquito Aedes aegypti

Mosquitoes utilize the amino acids derived from blood meal protein to produce egg proteins. But the amino acids can also be used to produce egg lipid or can be oxidized for energy production. These latter two processes result in the release of nitrogen as toxic ammonia. Therefore, amino acids must be processed in such a way that amino acid nitrogen can be incorporated into non-toxic waste products. Proline is the predominant amino acid in the hemolymph of the adult female mosquito Aedes aegypti. After feeding on albumin meal, hemolymph proline levels increased five-fold over unfed levels, reached maximal levels in the first hours after feeding and remained high through oviposition. Hemolymph proline levels increased as the concentration of protein in the meal increased. When starved of sugar for 24 h prior to feeding on an albumin meal, hemolymph proline levels increased four-fold over the proline levels of non-starved mosquitoes. Proline levels after feeding on a protein deficient in essential amino acids, pike parvalbumin, increased to twice the levels of albumin fed mosquitoes. Based on these observations, we propose that mosquitoes utilize proline as a temporary nitrogen sink to store ammonia arising from deamination of blood meal amino acid.     

Synthesis of angiotensinogen by isolated rat liver cells and its regulation in comparison to serum albumin

Angiotensinogen (renin substrate) and albumin are synthesized by isolated hepatocytes almost linearly for 5 hr. The incorporation of radioactive leucine into total protein proceeded linearly for 3 hr. Without addition of amino acids to the incubation medium the synthesis of both proteins was still linear but fell off to 40% compared to the synthesis rate obtained by incubation with amino acids in serum concentrations. Higher amino acid concentrations could not further stimulate the synthesis. Addition or withdrawal of tryptophan had no effect on the synthesis rate of both proteins. After 5 hr incubation hydrocortisone had stimulated the incorporation of radioactive leucine into total protein by 13%, the albumin synthesis by 43%, and the angiotensinogen synthesis by 142%.     

The influence of detergent, the active component of detergent and sodiumtripolyphosphate on the biochemical process of some fungi.

Detergent "Merix" (Merima-Krusevac) and its components, sodiumtripolyphosphate and ethoxyled oleyl-cetyl alcohol in defined concentrations have influence on the enzymatic activity, bioproduction of amino acids and proteins and total biomass of species Aspergillus niger (A. niger) and Fusarium oxysporum (F. oxysporum). Detergent in concentration of 1% vol., in some cases, significantly stimulated production of the majority of amino acids. Detergent and its components (ethoxyled oleyl-cetyl alcohol and sodiumtripolyphosphate) in concentration of 1% vol. showed different influences on the production of proteins by the species of fungi A. niger and F. oxysporum. The enzymatic activity of fungi A. niger and F. oxysporum changed from total inhibition up to partial or total stimulation under influence of investigated pollutants. Detergent, sodiumtripolyphosphate and ethoxyled oleyl-cetyl alcohol, all in concentration of 1% vol. have reduced the production of the biomass of fungi A. niger and F. oxysporum. Investigated fungi have shown different response to the used pollutants.     

Structural studies of metalloporphyrins. Part XIa: Complexes of water-soluble zinc(II) porphyrins with amino acids: influence of ligand-ligand interactions on the stability of the complexes

The stability constants of a series of complexes of the cationic water-soluble porphyrin ZnTMPyP with various amino acids have been determined by 1H NMR spectroscopy at pH 10.5. The following stability order has been observed: Tyr greater than Phe, Glu greater than Asp greater than Ile greater than Val greater than Gly. These results can be best rationalized by invoking complex stabilization due to ligand-ligand (e.g., stacking or electrostatic) interactions. Evidence for stacking interactions between the porphyrin ring and the aromatic ring of phenylalanine, tyrosine, and tryptophan was further provided by study of the complexation of these amino acids with the free-base porphyrin TMPyPH2. In this case, complexation constants increased in the order: Phe less than Tyr less than Trp. Attempts to form complexes of the amino acids with the anionic porphyrin ZnTCPP proved unsuccessful, indicating that electrostatic interactions play a major role in the stability of the zinc porphyrin-amino acids complexes.     

The effect of AmtR on growth and amino acids production in Corynebacterium glutamicum

AmtR, the master regulator of nitrogen control in Corynebacterium glutamicum, plays important roles in nitrogen metabolism. To investigate the influence of AmtR on amino acids production in C. glutamicum ATCC 13032, the amtR deletion strain C. glutamicum Q1 was constructed and cultured in modified CGXII minimal medium for 60 h. The ammonium consumption rates as well as amino acids production of both strains cultured in modified CGXII minimal medium were determined. The amtR deletion in C. glutamicum caused an obvious growth defect in the exponential growth phase, but both strains had the same biomass in the stationary phases. Maybe the less alpha-oxoglutarate was used for the tricarboxylic acid cycle to influence the growth of strains. During 12 h, the rate of ammonium consumption and the concentration of Glu, Pro, Arg and Ser were higher but Asp, Gly, Ile, Leu, Lys were lower in the mutation strain. During 48 h, the Q1 had higher levels of Asp, Lys, Pro, Ala and Val,and lower levels of Glu, Arg, Leu and Ile, compared to the wild. The more Glu was synthesized by the activated GS/GOGAT pathway in Q1, and then the accumulation of relative amino acids (Pro, Arg and Ser) were up-regulated within 12 h growth. After 48 h growth, the amtR deletion obviously influenced accumulation of Ala, Asp and Pro. The amtR deletion could influence the growth and amino acids production, which could be useful to the production of amino acids.