Intracellular Concentrations Of Alanine Research Articles

A novel limb in the osmoregulatory network of Methanosarcina mazei Gö1: Nε‐acetyl‐β‐lysine can be substituted by glutamate and alanine

N(epsilon)-acetyl-beta-lysine is a unique compatible solute found in methanogenic archaea grown at high salinities. Deletion of the genes that encode the lysine-2,3-aminomutase (ablA) and the beta-lysine acetyltransferase (ablB) abolished the production of N(epsilon)-acetyl-beta-lysine in Methanosarcina mazei Gö1. The mutant grew well at low and intermediate salinities. Interestingly, growth at high salt (800 mM NaCl) was only slowed down but not impaired demonstrating that in M. mazei Gö1 N(epsilon)-acetyl-beta-lysine is not essential for growth at high salinities. Nuclear magnetic resonance (NMR) analysis revealed an increased glutamate pool in the mutant. In addition to alpha-glutamate, a novel solute, alanine, was produced. The intracellular alanine concentration was as high as 0.36 +/- 0.05 micromol (mg protein)-1 representing up to 18% of the total solute pool at 800 mM NaCl. The cellular alanine concentration increased with the salinity of the medium and decreased in the presence of glycine betaine in the medium, indicating that alanine is used as compatible solute by M. mazei Gö1.

Glutamine kinetics and protein turnover in end-stage renal disease.

Alanine and glutamine constitute the two most important nitrogen carriers released from the muscle. We studied the intracellular amino acid transport kinetics and protein turnover in nine end-stage renal disease (ESRD) patients and eight controls by use of stable isotopes of phenylalanine, alanine, and glutamine. The amino acid transport kinetics and protein turnover were calculated with a three-pool model from the amino acid concentrations and enrichment in the artery, vein, and muscle compartments. Muscle protein breakdown was more than synthesis (nmol.min(-1).100 ml leg(-1)) during hemodialysis (HD) (169.8 +/- 20.0 vs. 125.9 +/- 21.8, P < 0.05) and in controls (126.9 +/- 6.9 vs. 98.4 +/- 7.5, P < 0.05), but synthesis and catabolism were comparable pre-HD (100.7 +/- 15.7 vs. 103.4 +/- 14.8). Whole body protein catabolism decreased by 15% during HD. The intracellular appearance of alanine (399.0 +/- 47.1 vs. 243.0 +/- 34.689) and glutamine (369.7 +/- 40.6 vs. 235.6 +/- 27.5) from muscle protein breakdown increased during dialysis (nmol.min(-1).100 ml leg(-1), P < 0.01). However, the de novo synthesis of alanine (3,468.9 +/- 572.2 vs. 3,140.5 +/- 467.7) and glutamine (1,751.4 +/- 82.6 vs. 1,782.2 +/- 86.4) did not change significantly intradialysis (nmol.min(-1).100 ml leg(-1)). Branched-chain amino acid catabolism (191.8 +/- 63.4 vs. -59.1 +/- 42.9) and nonprotein glutamate disposal (347.0 +/- 46.3 vs. 222.3 +/- 43.6) increased intradialysis compared with pre-HD (nmol.min(-1).100 ml leg(-1), P < 0.01). The mRNA levels of glutamine synthase (1.45 +/- 0.14 vs. 0.33 +/- 0.08, P < 0.001) and branched-chain keto acid dehydrogenase-E2 (3.86 +/- 0.48 vs. 2.14 +/- 0.27, P < 0.05) in the muscle increased during HD. Thus intracellular concentrations of alanine and glutamine are maintained during HD by augmented release of the amino acids from muscle protein catabolism. Although muscle protein breakdown increased intradialysis, the whole body protein catabolism decreased, suggesting central utilization of amino acids released from skeletal muscle.

Effect of chronic renal failure with metabolic acidosis on alanine metabolism in isolated liver cells

<h2>Abstract</h2> <i>Background & aims</i>: Decreased ureagenesis and gluconeogenesis from alanine have been reported during chronic renal failure in rat. This study addressed the respective roles of plasma-membrane transport and intracellular metabolism in these abnormalities of alanine pathways. <i>Methods</i>: In hepatocytes isolated from uremic and control rats, we investigated: (1) the influence of uremia on gluconeogenesis and ureagenesis during incubations with alanine; (2) the kinetics of alanine plasma-membrane transport; (3) the relationships between intracellular alanine concentrations and its metabolism. Plasma-membrane alanine transport was assessed after addition of alanine (2mM) by measuring its intracellular accumulation from 0 to 10min, in the presence of a transaminase inhibitor. Alanine metabolism was studied in perifused hepatocytes by measuring intracellular alanine concentration together with urea, glucose and lactate production in the presence of increasing concentrations of alanine (0–8mM). <i>Results</i>: Uremic rats showed decreased plasma bicarbonate. Uremia induced (<i>P</i><0.05) a decrease in both gluconeogenesis (36%) and ureagenesis (22%). Alanine plasma-membrane transport decreased by 20% during uremia. During perifusions, uremia induced a 30–40% decrease in urea, glucose, and lactate production without modifying intracellular alanine concentration. <i>Conclusions</i>: In uremic rats with acidosis, hepatocyte alanine utilization was impaired at both plasma-membrane transport and intracellular transamination steps.

Effect of chronic renal failure with metabolic acidosis on alanine metabolism in isolated liver cells

Background & aims: Decreased ureagenesis and gluconeogenesis from alanine have been reported during chronic renal failure in rat. This study addressed the respective roles of plasma-membrane transport and intracellular metabolism in these abnormalities of alanine pathways. Methods: In hepatocytes isolated from uremic and control rats, we investigated: (1) the influence of uremia on gluconeogenesis and ureagenesis during incubations with alanine; (2) the kinetics of alanine plasma-membrane transport; (3) the relationships between intracellular alanine concentrations and its metabolism. Plasma-membrane alanine transport was assessed after addition of alanine (2 mM) by measuring its intracellular accumulation from 0 to 10 min, in the presence of a transaminase inhibitor. Alanine metabolism was studied in perifused hepatocytes by measuring intracellular alanine concentration together with urea, glucose and lactate production in the presence of increasing concentrations of alanine (0–8 mM). Results: Uremic rats showed decreased plasma bicarbonate. Uremia induced ( P<0.05) a decrease in both gluconeogenesis (36%) and ureagenesis (22%). Alanine plasma-membrane transport decreased by 20% during uremia. During perifusions, uremia induced a 30–40% decrease in urea, glucose, and lactate production without modifying intracellular alanine concentration. Conclusions: In uremic rats with acidosis, hepatocyte alanine utilization was impaired at both plasma-membrane transport and intracellular transamination steps.

Alanine aminotransferase from gill tissue of the brackish-water bivalve Corbicula japonica (Prime): Subcellular localization and some enzymatic properties

Subcellular localization and some enzymatic properties of alanine aminotransferase (A1AT, EC 2.6.1.2) were investigated with regard to salinity-related changes in amino acid metabolism. Activities of AlAT were detected in both the cytosolic (cAlAT) and mitochondrial (mAlAT) fractions prepared from the gill tissue of the brackish-water bivalve Corbicula japonica. The cAlAT and mAlAT were partially purified and characterized with respect to their pH-dependency and K m values for the respective substrates in both directions of the A1AT reaction, pyruvate-forming and alanine-forming. These two enzymes differed from each other in the ratios of the pyruvate-forming vs. the alanine-forming activity and K m values for glutamate. The K m values for alanine were smaller than its intracellular concentration indicating that both the mAlAT and cAlAT may be physiologically functional in the pyruvate-forming direction. The A1AT is indicated to be involved in low salinity-induced decrement of intracellular alanine concentration.

Extra- and intracellular metabolite concentrations for murine hybridoma cells.

Intra- and extracellular metabolite concentrations were determined for hybridoma cells grown in tissue culture flasks in batch culture. Significant differences were found for intracellular lactate and amino acid concentrations depending on the culture medium. AB2-143.2 cells cultivated in Dulbecco's modified Eagle's (DME) medium supplemented with 50 mM lactate exhibited intracellular lactate and glutamine levels of 40 mM and 4 mM, respectively, whereas cells grown in standard DME medium had low intracellular glutamine and 20 mM lactate concentrations. For cells cultivated in medium supplemented with 6 mM pyruvate, intracellular lactate levels were estimated at approx. 40 mM, but these cells also showed an increased intracellular alanine concentration of 22 mM. The higher alanine pools probably result from increased transamination of pyruvate under these conditions.

Cell Volume Regulatory Responses of Isolated Perfused Rat Liver. The Effect of Amino Acids

1) Addition of glutamine, glycine, alanine, serine, phenylalanine, proline at a concentration of 3mM, each, or of an amino-acid mixture resembling the physiological amino-acid composition of portal venous blood, to influent perfusate of isolated perfused rat liver led to a 4-6% increase of liver mass without increase of the [3H]inulin space, and biphasic K+ movements across the plasma membrane. These K+ movements consisted of an initial net K+ uptake (0.4-0.9 mumol X g-1 liver) for about 2 min, being followed by a net K+ release (1.0-2.8 mumol X g-1 liver) during the next 10 min. Withdrawal of the amino acids from influent perfusate caused a slow net K+ reuptake by the liver and restored the initial liver mass. No effects on liver mass and K+ fluxes were observed following addition of glutamate or glucose at a concentration of 3mM, each. 2) Aminooxyacetate did not affect the alanine (3 mM) induced increase in liver mass. However, in presence of aminooxyacetate the alanine-induced net K+ release from the liver (i.e. K+ release from 2-10 min minus initial K+ uptake) increased from 0.1 to 2.2 mumol X g-1 liver, whereby simultaneously the alanine tissue level rose from 6.8 to 13.3 mumol X g-1 (corresponding to an increase of the intracellular alanine concentration from about 12 to 25 mM) in presence of aminooxyacetate. 3) When livers were perfused with different glutamine concentrations, a maximal increase in liver mass of 5-6% was observed at glutamine concentrations above 1.5-2mM. A halfmaximal increase in liver mass was observed at 0.6-1.0mM glutamine in influent, i.e. at the physiological portal glutamine concentration.(ABSTRACT TRUNCATED AT 250 WORDS)

Cytoskeletal control of alanine transport across the rat and turtle small intestine

1. 1. Procaine inhibited significantly ( P < 0.01) alanine accumulation in the rat intestinal strips in a concentration-dependent pattern, whereas it showed no effect on alanine uptake by the turtle intestinal cells. 2. 2. Colchicine and Vinca alkaloids at 5 × 10 −4 and 1.5 × 10 −6 M respectively caused a significant inhibition ( P < 0.01) of intracellular alanine concentration in the rat with no effect noticed in the turtle. 3. 3. Unidirectional influx of alanine across the brush border membrane of the rat was significantly ( P < 0.01) reduced in the presence of procaine, colchicine and vincristine in the preincubation medium. The same drugs did not show any effect on alanine influx into the turtle small intestine. 4. 4. Electron microscopy showed major structural alterations in the cytoskeletal organization of the turtle intestine in response to procaine, colchicine or vincristine treatment. 5. 5. It is proposed that microtubular system may participate in the overall transport mechanism of alanine across the small intestine.

Influence of Amino Acids on the Synthesis of an Extracellular Proteinase from Staphylococcus aureus

The influence of amino acids on the synthesis of staphylococcal serine-proteinase (proteinase I) by Staphylococcus aureus strain V8 was studied. The repression of proteinase synthesis during the exponential growth phase was not due to any single amino acid in the medium. Proline was essential for proteinase synthesis in post-exponential growth but not for bacterial growth. At low concentrations of glycine, serine, threonine and proline, proteinase synthesis was inhibited, whereas at high concentrations of any of these amino acids, proteinase synthesis was fully induced. During post-exponential growth in the absence of glycine, serine, threonine or proline, the intracellular concentrations of alanine, aspartic acid and glutamic acid increased two to threefold. In the presence of these amino acids, the pool of alanine and aspartic acid remained at a low level and the concentration of glutamic acid decreased. The synthesis of proteinase I is probably repressed at high intracellular concentrations of glutamic acid.

The effects of hyperphenylalaninaemia on the concentrations of aminoacyl-transfer ribonucleic acid in vivo. A mechanism for the inhibition of neural protein synthesis by phenylalanine.

An acute administration of phenylalanine to neonatal animals has been reported to result in large decreases in the intracellular concentrations of several essential amino acids in neural tissue, as well as an inhibition of neural protein synthesis. The present report evaluates the effects of the loss of amino acids on the concentrations of aminoacyl-tRNA in vivo, with the view that an alteration in the concentrations of specific aminoacyl-tRNA molecules could be the rate-limiting step in brain protein metabolism during hyperphenylalaninaemia. tRNA was isolated from saline- and phenylalanine-injected mice 30-45 min after injection, by using a procedure designed to maintain the concentrations of aminoacyl-tRNA present in vivo. Periodate oxidation of the non-acylated tRNA and aminoacylation with radioactively labelled amino acids was used to determine the proportion of tRNA that was present in vivo as aminoacyl-tRNA. Although decreases in the intracellular concentrations of alanine, lysine and leucine were observed after phenylalanine administration, the concentrations of alanyl-tRNA, lysyl-tRNA and leucyl-tRNA actually increased by 15%. Although tryptophan has been suggested to be rate-limiting during hyperphenylalaninaemia, the proportion of tryptophan tRNA that was acylated was maximal in both normal and hyperphenylalaninaemic animals. This unexpected increase in aminoacyl-tRNA concentration is discussed as perhaps a secondary effect resulting from the phenylalanine-induced inhibition of protein synthesis. In contrast, the proportion of methionine tRNA that was acylated in vivo after phenylalanine administration was demonstrated to be decreased by approx. 17%. When the isoaccepting species of methionine tRNA were separated by reverse-phase column chromatography, three species were separated, one of which was demonstrated to be the initiator species, tRNAfMet, by the selective aminoacylation and formylation with Escherichia coli enzymes. After the administration of phenylalanine, the acylation of each of the three methionine tRNA species was decreased, with the initiator species being lowered by 10%. This effect on aminoacylation of tRNAfMet may be the primary step by which phenylalanine affects neural protein synthesis, and this is consistent with previous reports that re-initiation may be inhibited during hyperphenylalaninaemia.

Placental amino acid uptake. II. Tissue preincubation, fluid distribution, and mechanisms of regulation.

Extract: Human placental villous tissue was incubated with the nonmetabolizable amino acid analog, α-aminoisobutyric acid (AIB). A previous investigation demonstrated that AIB uptake into intracellular fluid is markedly increased by preincubation of tissue in a balanced salt, glucose-containing medium. This increase in transport capacity with preincubation has now been found to be blocked completely by cycloheximide, dinitrophenol, or cyanide, which indicates that the process requires protein synthesis and aerobic metabolism. Inasmuch as increases in AIB uptake with preincubation were reported in other tissues to be regulated by amino acids in the medium, effects of amino acids were studied in the placenta. Preincubation of placental tissue with alanine did result in a transport activity which was lower than that of control preincubated tissue. However, two types of experiments indicated that this difference results from inhibition of transport activity by intracellular alanine accumulated during preincubation rather than from blockage of formation of new transport capacity during preincubation. (1) Much of the difference between control and alanine-preincubated tissue was eliminated by short additional preincubations in cycloheximide-containing, alanine-free medium; a treatment which should lower intracellular alanine concentration but not permit protein synthesis. (2) Preincubation with AIB permitted measurement of both intracellular concentration and transport activity. Addition of AIB to control preincubated tissue caused concentrating ability to fall as intracellular concentration increased. When AIB and control preincubated tissue were tested with the same intracellular AIB concentration, their transport activities were the same. The rate of increase in concentrating ability with preincubation was inversely related to the amount of tissue per volume of preincubation medium and was enhanced by changing the medium at intervals. Preincubation of new placental tissue in medium which had been used previously for preincubation resulted in a smaller increase in concentrating ability than did preincubation of the same tissue in fresh medium. These findings were not dependent on glucose concentration or pH. During preincubation, some as yet unidentified tissue substance is apparently released into the medium and inhibits the increase in transport activity. Speculation: Amino acids required by the fetus must be supplied by the placenta. In vitro incubation of placental tissue has identified and partially characterized two potential mechanisms for regulation of placental amino acid uptake. The first mechanism increases transport activity when tissue is preincubated in vitro before measurement of uptake. The second mechanism apparently decreases existing uptake activity when amino acids are present intracellularly in high concentrations. In vivo these mechanisms could be the basis for regulation of amino acid transfer from mother to fetus.

Alanine and sodium fluxes across mucosal border of rabbit ileum.

Unidirectional influxes of L-alanine and Na from the mucosal solution into the epithelium of in vitro rabbit ileum have been determined. In the presence of 140 mM Na, alanine influx is approximately 2.2 micromoles/hr cm(2), but is inhibited if the NaCl in the mucosal solution is replaced by choline Cl, Tris-Cl, mannitol, LiCl, or KCl. Although alanine influx is strongly dependent upon Na in the mucosal solution, it is uninfluenced by marked reduction of intracellular Na pools. In addition, alanine influx is unaffected by intracellular alanine concentration. Na influx is markedly inhibited by the presence of Li. Evidence is presented that Na transport across the mucosal border cannot be attributed to simple diffusion even though the net flux across this surface is in the direction of the electrochemical potential difference.

RELATION OF L-ALANINE METABOLISM TO THE ACTION OF TRIAMCINOLONE IN LIVER SLICES.

The hypothesis which proposes that glucocorticoids enhance the transport of glycogenic amino acids across the membrane of the liver cell was investigated using a rat liver slice system. In this system, slices respond to glucocorticoids with an increased rate of gluconeogenesis when glucocorticoids are added in vitro. It was concluded that the action of glucocorticoids, specifically triamcinolone (l,4-pregnadiene-9α-fluoro-llβ, 16α, 17α, 21-tetrol-3,20-dione), in this system is not manifested as an acceleration of transport of amino acids (specifically alanine) into the liver cell. The evidence for this conclusion was as follows: 1) L-Alanine disappearance from the incubation medium was not increased in the presence of triamcinolone, although glucose synthesis was stimulated; 2) in contrast, raising the concentration of Lalanine in the incubation medium led to a marked increase in alanine uptake but little or no increase in glucose synthesis; 3) there was no evidence of a sharp concentration gradient between extracellular and intracellular alanine; 4) no rise in intracellular alanine concentration was apparent when glucocorticoids were acting; and 5) by suitable manipulation of experimental conditions it was possible to demonstrate a stimulation of glucose synthesis when pyruvate was substituted for alanine. Thus, the action of triamcinolone, and presumably the action of other glucocorticoids, in this system is not evidenced as an effect upon the transport of alanine nor upon its conversion to pyruvate, but primarily at some reaction or reactions involved in the conversion of pyruvate to glucose. (Endocrinology75: 602, 1964)