Showing papers on "Phenylalanine published in 1970"

Alanine: Key Role in Gluconeogenesis

Abstract: Of 20 amino acids measured, alanine is the principal amino acid released by forearm muscle of man, in accord with its being the principal amino acid extracted by liver for gluconeogenesis. This occurs in both the postabsorptive state and after 4 to 6 weeks of starvation, when total amino acid release is markedly diminished.

Plasma Amino Acid Levels and Insulin Secretion in Obesity

Abstract: Of 20 plasma amino acids measured, valine, leucine, isoleucine, tyrosine and phenylalanine were increased, and glycine decreased, in obese subjects compared with age- and sex-matched controls. The concentration of each of the amino acids elevated in obesity correlated directly with serum insulin. In addition, these were the amino acids most sensitive to the action of insulin in lowering plasma amino acid levels, as evidenced by a diminution in concentration after glucose infusion. Despite a significantly greater increment in serum insulin in the obese group, the magnitude of the decline in amino acids after glucose administration was identical in the two groups. Hyperaminoacidemia appears to be a manifestation of the insulin ineffectiveness characteristic of obesity. Furthermore, hyperaminoacidemia may provide the feedback signal to the beta cell through which insulin resistance is accompanied by an appropriately augmented secretory rate of insulin.

Pancreozymin bioassay in man based on pancreatic enzyme secretion: potency of specific amino acids and other digestive products

Abstract: The ability of products of digestion to stimulate pancreozymin secretion in man was investigated using a bioassay procedure, based on duodenal perfusion, which quantified the total outputs of pancreatic enzymes evoked by intraduodenal stimuli under steady-state conditions. Patterns of response resulting from physiologic intraduodenal concentrations of test material were basal output (with isotonic saline), washout of enzymes (with dextrose, micellar fatty acid, and amino acids), and sustained output of enzymes (with amino acids and micellar fatty acid). The sustained secretion of pancreatic enzymes found during the 2nd hr of perfusion and subsequently was characteristic of pancreozymin-induced secretion. The enzyme output in response to a mixture of essential and nonessential amino acids was significantly higher than that evoked by micellar fatty acid and was comparable with that resulting from the maximally tolerated dose of pancreozymin given by vein. Perfusion with essential amino acids caused enzyme outputs comparable to those induced by perfusion with the original amino acid mixture, whereas perfusion with nonessential amino acids had no effect. When the essential amino acids were tested individually, only phenylalanine, methionine, and valine caused significant increases in pancreatic enzyme output; the effect of tryptophan was indeterminate. However, the pancreatic enzyme output was less in response to these three essential amino acids than to mixtures containing all of them.

[80] Tyrosine aminotransferase (rat liver)☆☆☆

Abstract: Publisher Summary This chapter describes the assay, purification, and properties of tyrosine aminotransferase (TAT). It catalyzes the first reaction in the pathway by which tyrosine is finally degraded to acetoacetate and fumarate. The assay for this enzyme is a modification of that described by Diamondstone and depends upon the alkali-catalyzed conversion of the reaction product–– p -hydroxyphenylpyruvate––to p -hydroxybenzaldehyde and oxalate. The basis of the assay is the absorbancy of the aromatic aldehyde in the reaction solution. One unit of enzyme activity is the quantity that catalyzes the formation of 1 micromole of p -hydroxyphenylpyruvate per minute at 37°. The assay due to the addition of KOH is applicable to turbid and crude extracts, which are clarified by the concentrated alkali. The enzyme is sensitive to sulfhydryl inactivation, and heavy metals may also cause loss of activity. α KG is the only keto acid that accepts the amino group of tyrosine. TAT also catalyzes the transamination of a number of aromatic amino acids, including phenylalanine and tryptophan, but at much lower rates than that of tyrosine.

Formation of aromatic amino acid pools in Escherichia coli K-12.

Abstract: Phenylalanine, tyrosine, and tryptophan were taken up into cells of Escherichia coli K-12 by a general aromatic transport system. Apparent Michaelis constants for the three amino acids were 4.7 × 10−7, 5.7 × 10−7, and 4.0 × 10−7m, respectively. High concentrations (> 0.1 mm) of histidine, leucine, methionine, alanine, cysteine, and aspartic acid also had an affinity for this system. Mutants lacking the general aromatic transport system were resistant to p-fluorophenylalanine, β-2-thienylalanine, and 5-methyltryptophan. They mapped at a locus, aroP, between leu and pan on the chromosome, being 30% cotransducible with leu and 43% cotransducible with pan. Phenylalanine, tyrosine, and tryptophan were also transported by three specific transport systems. The apparent Michaelis constants of these systems were 2.0 × 10−6, 2.2 × 10−6, and 3.0 × 10−6m, respectively. An external energy source, such as glucose, was not required for activity of either general or specific aromatic transport systems. Azide and 2,4-dinitrophenol, however, inhibited all aromatic transport, indicating that energy production is necessary. Between 80 and 90% of the trichloroacetic acid-soluble pool formed from a particular exogenous aromatic amino acid was generated by the general aromatic transport system. This contribution was abolished when uptake was inhibited by competition by the other aromatic amino acids or by mutation in aroP. Incorporation of the former amino acid into protein was not affected by the reduction in its pool size, indicating that the general aromatic transport system is not essential for the supply of external aromatic amino acids to protein synthesis.

Intestinal absorption of two dipeptides in Hartnup disease

Abstract: The results of oral tolerance tests of two dipeptides and of their constitutent amino acids are compared in normal subjects and in a case of Hartnup disease. In the control subjects the rate of absorption of phenylalanine from phenylalanyl-phenylalanine and of tryptophan from glycyl-tryptophan was slower than after the equivalent amount of the free amino acids. Absorption of the two essential amino acids (tryptophan and phenylalanine) in the patient was almost zero after administration in the free form, but was much greater after the dipeptide. Results of experiments on absorption and hydrolysis of the two peptides in the rat small intestine are also reported. It is suggested that whereas normal subjects absorb essential amino acids by a dual mechanism of mucosal uptake of free amino acids and oligopeptides, nutrition in Hartnup disease is largely dependent on uptake of oligopeptides containing the amino acids affected by the intestinal transport defect of the disease.

Biogenesis of rosmarinic acid in Mentha

Abstract: The biogenesis of rosmarinic acid (alpha-O-caffeoyl-3,4-dihydroxyphenyl-lactic acid), the second most common ester of caffeic acid in the plant kingdom, was studied in Mentha arvense and Mentha piperita. Administration of (14)C-labelled compounds showed that, whereas the caffeoyl moiety was formed from phenylalanine via cinnamic acid and p-coumaric acid, the 3,4-dihydroxyphenyl-lactic acid moiety was formed from tyrosine and 3,4-dihydroxyphenylalanine. Time-course studies and the use of labelled rosmarinic acid showed that endogenous rosmarinic acid had a low turnover rate. The caffeoyl moiety did not appear to contribute to the formation of insoluble polymers, as has been suggested for chlorogenic acid in other plants.

Relationship of a Membrane-Bound D-(-)-Lactic Dehydrogenase to Amino Acid Transport in Isolated Bacterial Membrane Preparations

Abstract: The conversion of D-lactate to pyruvate in isolated membrane preparations of E. coli ML 308-225 markedly stimulates the transport of proline, glutamic acid, aspartic acid, aspargine, tryptophan, lysine, serine, alanine, and glycine. The uptake of histidine, phenylalanine, tyrosine, leucine, isoleucine, and valine by the membranes is also markedly stimulated by this conversion, although these amino acids are taken up much less effectively than those mentioned previously. The uptake of arginine, methionine, cystine, and cysteine is enhanced only about twofold in the presence of D-(-)-lactate, and these amino acids are not concentrated well by the membranes. With the exception of glutamate, asparate, asparagine, and methionine, which are converted to other metabolites to varying extents in the intramembranal pool, each of the other amino acids was recovered from the membranes as the unchanged amino acid. Succinate, L-(+)-lactate, D,L-α-hydroxybutyrate, and DPNH partially replace D-(-)-lactate but are less effective.

The digestion and absorption of protein in man. 2. The form in which digested protein is absorbed.

Abstract: 1. Intestinal contents, collected from the human jejunum after a test meal (milk-protein, gelatin or low-protein) were fractionated by centrifugation and gel filtration on G-75Sephadex. The fractions were hydrolysed and the proportion of the total amino acid in each fraction was determined. The amino acids were measured with an EEL Amino Acid Analyser.2. The free amino acid concentrations were determined in samples of the contents of the small intestine collected from various levels after the three types of test meal.3. Intestinal contents collected from two levels of the jejunum after a milk-protein meal, were incubated in vitro at 37° for periods up to 80 min and the rates of release of the individual free amino acids were determined.4. There was a rapid breakdown of the proteins of the test meals to fragments of molecular weight under 5000. The further breakdown (during incubation in vitro) to free amino acids was sufficiently rapid to account for the absorption in the free form of arginine, lysine, tyrosine, valine, phenylalanine, methionine and leucine. It was not rapid enough to account for the absorption of glycine, threonine, serine, the imino acids or the dicarboxylic amino acids in the free form.5. The free amino acid concentrations in the intestinal lumen bore very little relationship to the concentrations in hydrolysates of the test meals or to those in hydrolysates of the intestinal contents. Many of the free amino acids in the intestinal samples were present in approximately equimolar concentrations.6. It is suggested that experiments in which amino acid mixtures, simulating a dietary protein, are fed to experimental animals to determine the rates of amino acid absorption do not present a true picture of the events in the small intestine following the ingestion of a protein meal.

DL-[2-14C]p-chlorophenylalanine as an inhibitor of tryptophan 5-hydroxylase.

Abstract: — The distribution in vivo of dl-[2-14C]p-chlorophenylalanine (p-CP) in regions and subcellular fractions of the rat brain was determined. The half-lives of p-CP and its metabolite p-chlorophenylpyruvic acid (p-CPPA) in plasma and brain were correlated with the development of inhibition of cerebral tryptophan 5-hydroxylase (EC 1.99.1.4). There was active transamination in vivo of p-CP and p-CPPA in the brain. Transport of indolealkylamino acids into brain was impaired by p-CP. Inhibition of tryptophan 5-hydroxylase could not be reversed by administration of large doses of l-tryptophan, l-tyrosine, or l-phenylalanine. After administration of [2-14C]p-CP in vivo, appreciable radioactivity was bound to cerebral proteins, including those with tryptophan 5-hydroxylase activity, as well as to phenylalanine 4-hydroxylase (EC 1.99.1.2) purified from liver. Amino acid analysis of the acid hydrolysate of purified, radioactive hepatic phenylalanine 4-hydroxylase showed over 80 per cent of the radioactivity to be present as p-CP. Neither the inhibition in vivo nor in vitro of tryptophan 5-hydroxylase could be reversed by dialysis; in controls, dialysis resulted in marked loss of enzyme activity. After incubation for 5 min with p-CP in vitro, enzymic activity was inhibited 60 per cent. In vitro, p-CPPA labelled protein much more extensively than p-CP, yet inhibited the enzyme less. Some of the label from p-CPPA was removable by dialysis.

Effect of phenylalanine on protein synthesis in the developing rat brain.

Abstract: 1. Inhibition of the rate of incorporation of [35S]methionine into protein by phenylalanine was more effective in 18-day-old than in 8-day-old or adult rat brain. 2. Among the subcellular fractions incorporation of [35S]methionine into myelin proteins was most inhibited in 18-day-old rat brain. 3. Transport of [35S]methionine and [14C]leucine into the brain acid-soluble pool was significantly decreased in 18-day-old rats by phenylalanine (2mg/g body wt.). The decrease of the two amino acids in the acid-soluble pool equalled the inhibition of their rate of incorporation into the protein. 4. Under identical conditions, entry of [14C]glycine into the brain acid-soluble pool and incorporation into protein and uptake of [14C]acetate into lipid was not affected by phenylalanine. 5. It is proposed that decreased myelin synthesis seen in hyperphenylalaninaemia or phenylketonuria may be due to alteration of the free amino acid pool in the brain during the vulnerable period of brain development. Amyelination may be one of many causes of mental retardation seen in phenylketonuria.

The isolation and composition of two phosphoproteins from hen's egg

Abstract: 1. Phosvitin extracted from domestic hen's-egg yolk was resolved on Sephadex G-100 into two phosphoprotein components. 2. The major component has a molecular weight of about 3.4×104 and alanine as an N-terminal residue. Glucosamine is present, but tyrosine is virtually absent. 3. The minor component has a molecular weight of about 2.8×104 and lysine as an N-terminal residue. Missing residues are glucosamine, methionine and leucine. Lysine, histidine, threonine, glycine, phenylalanine and tyrosine contents differ significantly from those of the major component. 4. Sephadex G-100 also removes small amounts of an impurity with a much higher molecular weight.

Evaluation of amino acid and protein requirements in chronic uremia.

Abstract: Plasma amino acids were found to deviate extensively from normal in patients with end-stage uremia. Concentrations of all essential amino acids except phenylalanine and methionine were reduced. Progressive impairment of reabsorption of amino acids by the kidney is found as glomerular filtration rate (GFR) falls, renal clearances increasing fivefold to fortyfold at GFR less than 10 ml/min. Impairment of absorption of amino acids by the small intestine may explain the abnormal plasma curves after oral loading with tryptophan. These and other abnormalities may be the basis for development of abnormal protein metabolism in chronic, progressive renal failure in man.

Metabolism of amino acids in incubated slices of mouse brain.

Abstract: — 1 Slices of mouse brain were incubated with [U-14C]alanine, valine, leucine, phenylalanine, proline, histidine, lysine, arginine or aspartic acid, and the extent of metabolism was estimated by analyses utilizing paper chromatography of the tissue extracts and with an amino acid analyser. 2 The metabolism of Ala and Asp was high; of Leu and Pro, moderate; and of Lys, Arg and Phe, low; the metabolism of Val and His was not significant. The time-course of metabolism in most cases showed varying rates, indicating heterogeneous metabolic compartments for the amino acids. 3 Production of CO2 was high from Asp, moderate from Ala, and low from Leu; the other amino acids were not oxidized to CO2 to any significant extent. A large portion of the metabolized label was trapped in the form of Glu or Asp. 4 Metabolism increased with increasing concentration of amino acid to some extent and was largely inhibited by omission of glucose, by anaerobic conditions, or by cyanide. Although these conditions also inhibit uptake, the time-course and extent of inhibition uptake and metabolism were different. 5 With Asp, Ala and Phe, metabolism was lowest in slices from pons-medulla; the brain area exhibiting the highest metabolism differed for each amino acid. The metabolism of Asp was lower in brain samples from newborn than in those from adults; the metabolism of Leu was higher in slices from newborn brain. 6 The results indicate that the majority of the amino acids can be metabolized in brain tissue and that the metabolic rates are influenced by a number of factors, among them the level of amino acids and the level of available energy.

Partial purification and properties of L-phenylalanine ammonia-lyase from Streptomyces verticillatus.

Abstract: Phenylalanine ammonia-lyase (EC 4.3.1.5) was purified 40-fold from a cell homogenate of Streptomyces verticillatus. In many respects the enzyme was similar to phenylalanine ammonia-lyases isolated from plants and fungi. It was most active at pH 9.0 and the Km for L-phenylalanine was 1.6 × 10−4 M. It showed no requirement for metal ions but was inhibited by heavy metals, some sulfhydryl reagents, and carbonyl reagents. The Stokes' radius was estimated by gel filtration to be 5.45 nm. Sucrose gradient centrifugation gave an s20,w of 10.0, leading to calculated values of 226 000 and 1.61 for the molecular weight and frictional ratio, respectively, if a partial specific volume of 0.725 ml/g is assumed. The enzyme deaminated o-, m-, and p-fluoro-, p-chloro-, and p-methyl-phenylalanine but was without action on L-tyrosine. It was inhibited by trans-cinnamic acid and certain phenylalanine derivatives, as well as by some less closely related aromatic compounds, but not by trans-cinnamamide.

Synergistic Effect of Certain Amino Acid Pairs upon Insulin Secretion in Man

Abstract: Individual amino acids were administered intravenously in amounts of 15 gm. and again together with 15 gm. of a second amino acid as 30-gm. mixtures. Synergism in the stimulation of the release of insulin was demonstrated when the sum of the increases in plasma insulin which resulted from the separate infusions of two amino acids was exceeded by that obtained when they were administered as a mixture. A synergistic effect was exerted by arginine and leucine, arginine and phenylalanine, but not by arginine and lysine, arginine and histidine, or by leucine and histidine. Synergism was greater with arginine and leucine than with arginine and phenylalanine. It appears to result from the effects of these amino acid pairs upon the pancreatic beta cell.

Effect of excess phenylalanine and of other amino acids on brain development in the infant rat.

Abstract: Extract: The effects of excess phenylalanine and of a number of other amino acids on brain weight, total brain lipid, and formation of the myelin lipid cerebroside sulfate (sulfatide) have been studied in the newborn rat. Brain weight, amount of brain lipids, and the formation in vivo of sulfatide were reduced as a result of injections of phenylalanine or of certain other amino acids. Total brain cholisterol, cerebroside, and sulfatide were all significantly reduced in phenylalanine-injected animals, although sulfatide was the only lipid that was reduced per gram wet weight of brain. The in vitro activity of the brain enzyme, galactolipid sulfotransferase, which catalyzes the formation of sulfatide from cerebroside and adenosine 3′-phosphate 5′-phosphosulfate (PAPS), was significantly reduced only when exogenous PAPS was not added to the assay medium. This finding suggests that in the brains of animals injected with phenylalanine there was impairement in the formation of PAPS from ATP and sulfate. In both cerebrums and cerebellums of animals injected with phenylalanine for the first 18 days of life, the amount of brain DNA and protein was reduced. Speculation: Increased ambient tissue levels of a number of amino acids, including phenylalanine, may interfere with normal development of the infant rat brain.

[70a] Preparation of prephenic acid

Abstract: Publisher Summary This chapter describes the preparation of prephenic acid. In Escherichia coli and in Salmonella, chorismic acid is converted to phenylpyruvate and to 4-hydroxyphenylpyruvate, respectively, by bifunctional enzymes that catalyze as a first step, the rearrangement of chorismate to prephenate. This method uses a tyrosine auxotroph of S. typhimurium, which has chorismate mutase but no prephenate dehydrogenase activity to accumulate prephenate under resting conditions. The cells are suspended in a medium that is devoid of tyrosine but contains tryptophan and phenylalanine to inhibit anthranilate synthase and prephenate dehydratase, respectively. Under resting conditions, the tyrosine auxotrophs of Salmonella produce less acid than phenylalanine auxotrophs. Certain phenylalanine and tyrosine auxotrophs are partially blocked. They are able to carry out only the first reaction in the sequence and may, therefore, excrete prephenate. The advantages of the method considered include a shorter incubation time, smaller volume of medium to process, and accumulation under nonsterile conditions.

Compartmentation of glutamate metabolism in the developing brain: experiments with labelled glucose, acetate, phenylalanine, tyrosine and proline

Abstract: —(1) Phenylalanine, proline and presumably tyrosine are precursors of the small glutamate pool in brain. This follows from the finding that with these precursors the specific radioactivity of glutamine is higher than the specific radioactivity of glutamate. (2) Glucose is not as efficient a precursor of glutamate and related amino acids in the brain of 10-day-old mice as it is in the adult brain. (3) Acetate, phenylalanine, tyrosine and proline are incorporated to about the same extent in glutamate, aspartate and glutamine in the brains of 10-day-old and adult mice. (4) The results suggest that the brain of the immature animal uses substrates other than glucose, relative to glucose better than the brain of adult animals.

The difference in sensitivity to cardiac steriods of (Na+ + K+)-stimulated ATPase and amino acid transport in the intestinal mucosa of the rat and other species

Abstract: 1. The effect of various cardioactive steroids on the activity of a microsomal (Na+ + K+)-activated ATPase from rat intestinal mucosa has been studied and compared with their effects on L-phenylalanine and D-galactose transport by rings of rat intestine in vitro. A similar comparison between the sensitivities to ouabain of microsomal (Na+ + K+)-ATPase and of phenylalanine transport in the intestines of the mouse, guinea-pig and toad has been made. 2. The rat intestinal enzyme is 50% inhibited by a concentration of 1 × 10−4 M ouabain, 1 × 10−5 M scillaren A and 4 × 10−6 M scilliroside. At concentrations which almost completely inhibit the (Na+ + K+)-ATPase activity, these steroids have no effect on the transport of phenylalanine or galactose by the rat intestine. Only at concentrations of 1 × 10−3 M are scillaren A and scilliroside able to reduce phenylalanine accumulation significantly, the same concentration of ouabain being effective only in the absence of external potassium ions. Digitoxin, 1 × 10−4 M, a comparatively apolar glycoside, had no action on phenylalanine transport in the rat intestine. 3. The effect of ouabain on the (Na+ + K+)-ATPase and phenylalanine transport system in the mouse intestine is completely analogous to its effect on these parameters in the rat. 4. A half-maximal inhibition of guinea-pig intestinal (Na+ + K+)-ATPase by ouabain occurs at an inhibitor concentration of 2 × 10−6 M, but phenylalanine transport by this tissue is only half-maximally reduced at a concentration of 3 × 10−5 M. Similarly, in the rabbit intestine, there appears to be a difference of an order of magnitude between the sensitivities of the two parameters. 5. In the toad, 50% inhibition of the enzymic activity is observed at a concentration of 3 × 10−5 M ouabain, whereas a concentration of 8 × 10−4 M is required to reduce phenylalanine accumulation by one half. 6. These findings are consistent with the suggestion that an (Na+ + K+)-stimulated ATPase is not the only enzyme in the epithelial cells of the intestinal mucosa that is responsible for sodium extrusion (the mechanism for sodium extrusion being intimately coupled with the mechanism for active amino acid transport); therefore, a second, as yet unidentified, enzyme system must be postulated to account for bulk sodium flow through the intestine.

Clinical and biochemical observations of patients with atypical phenylketonuria

Abstract: Patients with hyperphenylalaninemia appear to represent a heterogeneous group. The enzyme phenylalanine hydroxylase may be implicated, either by indirect measurement of activity (phenylalanine loading studies) or through direct enzyme assays in the patients studied. When phenylalanine tolerance tests were used, the measurement of phenylalanine levels alone did not distinguish the subject with classical phenylketonuria from the atypical subject. Rise in plasma tyrosine levels as well as the excretion of breakdown products of phenylalanine and tyrosine better distinguished the two types of subjects. A significant rise in plasma tyrosine level was demonstrable in the atypical subject in sharp contrast to the classical homozygous subject with phenylketonuria.