Presence Of Phenylalanine Research Articles

Catalytical mechanism of the phenylalanyl-tRNA synthetase from yeast. Reactivity of ATP in the absence of phenylalanine.

Phenylalanyl-tRNA synthetase catalyses an AMP-ATP exchange under conditions where no aminoacylation of tRNA occurs. A plausible explanation for this reaction had not been given so far. The results of the present investigation provide evidence for the following interpretation. tRNAPhe induces a polarisation of the ATP in complex with the enzyme; this stimulates (a) the formation of phenylalanyl-adenylate in the presence of phenylalanine, (b) the hydrolysis of ATP in the absence of phenylalanine and AMP and (c) the transfer of diphosphoryl onto AMP in the presence of AMP, especially when phenylalanine is absent.

Regulation of L-phenylalanine ammonia-lyase by L-phenylalanine and nitrogen in Neurospora crassa.

Neurospora crassa possesses an inducible L-phenylalanine ammonia-lyase that is expressed only when cells are derepressed for nitrogen in the presence of L-phenylalanine. Enzyme synthesis requires both induction by L-phenylalanine and simultaneous nitrogen catabolite derepression. Carbon limitation in the presence of phenylalanine does not elicit induction of L-phenylalanine ammonia-lyase. Specific induction by L-phenylalanine is required, and other amino acids completely failed to induce any lyase activity. The nit-2 gene is a major regulatory locus which is believed to mediate nitrogen catabolite repression in Neurospora. Mutants of nit-2 fail to express any phenylalanine ammonia-lyase activity under conditions of derepression and induction which lead to good enzyme induction in the wild type and in nit-2 revertants. The loss of lyase activity in nit-2 mutants does not result from inducer exclusion, which suggests that the nit-2 gene product has a direct role in controlling the expression of this enzyme. Substantial amounts of the enzyme were detected in the growth medium as well as in cell extracts. Inhibitors of protein synthesis or RNA synthesis block the induction of L-phenylalanine ammonia-lyase, suggesting that expression of this enzyme is controlled at the level of transcription.

Studies on the Zn2+/Co2+ exchange with acylamino acid amidohydrolase from pig kidney.

The kinetics of inactivation of the Zn2+-metalloenzyme acyl-amino acid amido hydrolase by chelating ligands were studied. The rate of inactivation by 1,10-phenanthroline is enhanced by histidine and inhibited in the presence of phenyl-alanine. Removal of the metal ion increases the heat stability and decreases the pH stability of the enzyme. Reactivation of the inactive metal free enzyme is possible with Zn2+ and Co2+. Titration of the metal free protein with a free enzyme is possible with Zn2+ and Co2+. Titration of the metal free protein with a Co2+/nitrilotriacetate metal buffer revealed a dissociation constant of about 10(-7) M for the Co2+ enzyme (Zn2+ enzyme = 10(-10) M). The Co2+ substituted enzyme is less stable than the Zn2+ enzyme. Histidine and phenylalanine protect the Co2+ enzyme against inactivation by 1,10-phenanthroline.

Tyrosine transport by membrane vesicles isolated from rat brain

Tyrosine uptake by membrane vesicles derived from rat brain has been investigated. The uptake is dependent on an Na + gradient ([ Na +] outside > [ Na +] inside ). The uptake is transport into an osmotically active space and not a binding artifact as indicated by the effect of increasing the medium osmolarity. The process is stimulated by a membrane potential (negative inside) as demonstrated by the effect of the ionophores valinomycin and carbonyl cyanide m- chlorophenylhydrazone and anions with different permeabilities. Kinetic data show that tyrosine is accumulated by two systems with different affinities. Tyrosine uptake is inhibited by the presence of phenylalanine and tryptophan.

A novel enzymatic activity of phenylalanyl transfer ribonucleic acid synthetase from baker's yeast: zinc ion induced transfer ribonucleic acid independent hydrolysis of adenosine triphosphate.

Phenylalanyl-tRNA synthetase from baker's yeast in the presence of phenylalanine or other amino acids misactivated by the enzyme, ATP, and low concentrations of Zn2+ is able to hydrolyze ATP to AMP and PPi very efficiently. After dialysis of the enzyme against ethylenediaminetetraacetic acid (EDTA), this amino acid dependent but tRNAPhe-independent hydrolysis is suppressed to negligible levels. The ATP hydrolysis can be restored by the addition of Zn2+ to the EDTA-dialyzed enzyme. During aminoacylation of tRNAPhe the Zn2+-induced ATP hydrolysis parallels the aminoacylation reaction, leading to nonstoichiometric production of AMP. Mechanistically, we conclude that Zn2+ can be bound to phenylalanyl-tRNA synthetase and can influence the stability of ATP if an activatable amino acid is present. The influence of Zn2+, if any, on the aminoacylation of tRNAPhe is not known. In practice, this side reaction is of the utmost importance in all cases in which the fate of ATP during aminoacylation is followed, especially if the stoichiometry of ATP consumption in relation to Phe-tRNAPhe formation has to be determined.

Purification of human parathyroid hormone: recent studies and further observations.

During the isolation of human parathyroid hormone there is an extensive loss of immuno-assayble hormone over the successive extraction steps, due in part to the presence of fragments that are soluble in 4% trichloroacetic acid. These fragments are derived from both the amino- and carboxyl-terminal regions of the hormone. The hormonal fractions precipitated with trichloroacetic acid were further purified by gel filtration and ion-exchange chromatography. At the final ion-exchange purification step, some preparations of the hormone eluted in multiple fractions. When the various components were characterized separately by immunoassay, amino acid composition, enzymic cleavage and partial sequence analysis, they were found to be closely comparable, although the most acidic fraction contained a blocked terminal amino group. Extraction of a number of batches of tissue permitted revision of the amino acid composition of human parathyroid hormone. Biosynthetic studies with labelled amino acids confirmed the absence of tyrosine and the presence of phenylalanine and threonine and localized these residues to definite regions of the molecule.

Isolation of amino acid activating subunit-pantetheine protein complexes: Their role in chain elongation in tyrocidine synthesis

Dissociation of the multienzymes of tyrocidine synthesis by prolonged incubation of crude extracts of Bacillus brevis (Dubos strain, ATCC 8185) has yielded, on Sephadex G-100 chromatography, two fractions of amino acid activating subunits, a larger one of 70,000 daltons and a smaller one of 90,000 daltons; the latter was a complex consisting of the 70,000 dalton subunit and the pantetheine-carrying protein of about 20,000 daltons. When it dissociated, the intermediate enzyme, which activates three amino acids, contained two-thirds of the subunits in the 70,000 dalton and one-third in the 90,000 dalton fraction; the heavy enzyme, which activates six amino acids, contained five-sixths of the subunits in the former fraction and one-sixth in the latter. Both fractions showed ATP-PP(i) exchange with all amino acids that are activated by the respective polyenzymes. With proline as an example, the 70,000 dalton subunit exhibited a single low-affinity binding site, which should correspond to the peripheral thiol acceptor site, whereas the 90,000 dalton subunit showed both a low-affinity binding site and an additional high-affinity site for proline; the high-affinity site is attributed to the pantetheine present on the pantetheine-carrying protein, and suggests that amino acids are translocated from the peripheral SH to the pantetheine-carrying moiety during chain elongation. This was confirmed by the observation that the 90,000 dalton complex, when incubated with the light enzyme in the presence of phenylalanine and proline, produced DPhe-Pro dipeptide that cyclized into DPhe-Pro diketopiperazine, but the 70,000 dalton activating subunit, when similarly incubated, did not. After subunit dissociation, however, no further elongation occurred after the transfer from phenylalanine to proline.

Studies on the Mixed Ligand Complexes Involving Ligands of Biological Importance (Ni(II)/Zn(II)/Cd(II)-1,10-phenanthroline-2,2′ -bipyridyl-phenylalanine)

pH-metric studies on the interaction of Ni(II), Zn(II) or Cd(II) with 1,10-phenanthroline (Phen) or 2,2′-bipyridyl (Bipy) in the presence of phenylalanine (Phe) indicate the formation of 1:1:1 mixed ligand chelates and their monohydroxo derivatives. The addition of Phe takes place after the combination of Phen or Bipy with the metal ion is complete. The formation constants of the resulting complexes have been determined at 30 ± 1 °C and ionic strength (µ) = 0.1 KNO3 and the ternary complexes involving Phen are found to be more stable than the corresponding complexes involving Bipy as primary ligand.

Non-equivalence of the Sites of Yeast Phenylalanyl-tRNA Synthetase during Catalysis

Yeast phenylalanyl-tRNA synthetase, an enzyme with an alpha2beta2 structure, has two active sites for phenylalanine, tRNAphe, phenylalanyladenylate and phenylalanyl-tRNAphe. Determination of phenylalanine binding properties to the free enzyme by equilibrium dialysis shows that only one mole of amino acid binds per mole of enzyme, i.e. absolute negative cooperativity. Binding of the amino acid in the presence of tRNA or of ATP and PPi unmasks the second phenylalanine binding site. The difference between the affinities at the tight and loose binding sites under such conditions is about 10--15. Titration of phenylalanyladenylate sites by the burst of ATP consumption shows the formation of a (enzyme-phenylalanyladenylate)2 complex in the presence of pyrophosphatase; however, the two sites differ widely in their affinity as shown by dialysis experiments. Measurements of hydrolysis rates of enzyme-bound phenylalanyladenylate suggests that when only the high-affinity adenylate site is occupied, the other protomer can still bind phenylalanine and ATP (in the presence of phenylalanine). Two moles of Phe-tRNAphe bind to the enzyme with a very high affinity (Kd less than 48 nM). The presence of millimolar concentrations of ATP, phenylalanine and pyrophosphate triggers negative cooperativity and under these conditions only one mole of Phe-tRNAphe is bound per mole of enzyme with a Kd value of 0.15 muM. The present results give support to interprotomer catalytic cooperativity in the mechanism of action of yeast phenylalanyl-tRNA synthetase.

26] Detection of peptides by fluorescence methods

The recent introduction of fluorescamine, a novel reagent designed after the fluorogenic reaction of ninhydrin with primary amines in the presence of phenylalanine, has now provided a strong alternative to ninhydrin for detection of peptides. Fluorescamine reacts with primary amino groups of peptides almost instantaneously at room temperature in aqueous solution at pH 7.5-9, to form a fuorescent compound. The reagent is subsequently hydrolyzed to yield a watersoluble, nonfluorescent product. Fluorescamine itself is not fluorescent. Ammonia in solution yields little or no fluorescence with fluorescamine. These features and the great sensitivity attainable in the fluorescence measurement have made fiuorescamine increasingly popular in amino acid and peptide analysis. Another development in recent years is the rediscovery of o-phthalaldehyde as a general reagent for amino acids and peptides when used in the presence of 2-mercaptoethanol. This reagent has recently been applied to the microanalysis of amino acids and peptides.

Kinetic analysis of phenylalanine-induced inhibition in the saturable influx of tyrosine, tryptophan, leucine and histidine into brain cortex slices from adult and 7-day-old rats.

Abstract—An attempt was made to isolate the saturable uptake from the unidirectional influx of amino acids into tissue slices and to estimate the transport constants and maximal velocities of saturable transport. The method was applied to studies on the inhibition of phenylalanine in the saturable influx of tyrosine, tryptophan, histidine and leucine into brain cortex slices from adult and 7‐day‐old rats. In both age groups phenylalanine inhibited the influx of the other amino acids, and vice versa. The apparent transport constants of the other amino acids increased in the presence of phenylalanine more noticeably in the slices from 7‐day‐old rats than in those from adult rats, whereas the concomitant influx of phenylalanine was inhibited less in the slices from 7‐day‐old rats. In immature animals in vivo competition between amino acids may play a more marked role in the supply of amino acids from plasma to brain, as the transport systems in brain slices from 7‐day‐old rats become saturated with extracellular amino acids more readily than do the transport systems in brain slices from adult rats.

Protein synthesis requirement for the cytokinin effect upon tobacco cell division

In tobacco cell suspensions, protein synthesis and mitotic activity were inhibited by amino acid analogues: p-fluorophenylalanine (pFPA) or 5-methyltryptophan (5MT). After inhibition by pFPA, when the mitotic activity recovered in the presence of phenylalanine and casein hydrolysate, the time table of the mitotic phases was permanently altered. The inhibiting effects of 5MT were effectively reversed by tryptophan addition to the medium. Therefore 5MT was selected for reversible protein synthesis inhibition in partially synchronized cell suspensions. When cytokinin was added in a culture where protein synthesis was inhibited by 5MT, no mitosis was observed after the cells were transferred to a hormone-free medium and protein synthesis restored by tryptophan. Cytokinin must again be added in order to restore mitosis. Thus, the hormone effectiveness of cytokinins required that protein synthesis remained undisturbed. The effect of the protein synthesis inhibition by 5MT upon the metabolism of N 6 -benzyladenine was investigated: the intracellular concentration of this cytokinin was not altered, whereas the metabolic pool of its derivatives was quantitatively reduced.

Modification of phenylalanyl-tRNA synthetase from Escherichia coli by histidine-specific reagents. Effects on structure and function.

Phenylalanyl‐tRNA synthetase from Escherichia coli was subjected to chemical modification with diethylpyrocarbonate at pH 6.0 and to photochemical oxidation in the presence of rose Bengal, both methods giving rise to loss of enzymic activity. The highly sensitive reaction of the enzyme with either reagent is pseudo‐first order. The specificity of the modification methods employed has been investigated.All substrates were studied for their ability to protect the enzyme against inactivation. Presence of phenylalanine, ATP and Mg2+ provides the most pronounced effect. Under this condition the tRNA‐aminoacylation activity may be completely abolished, whereas the pyrophosphate–ATP exchange activity is only partly affected. However, such enzyme is still able to form a specific complex with tRNAPhe. The results presented therefore suggest that histidine residues of phenylalanyl‐tRNA synthetase accessible to modification by diethylpyrocarbonate or to photooxidation do not seem to be involved in the binding of tRNA but might possibly take part in the esterification reaction.Quantitative analysis showed that a total of 50 histidine residues are accessible to diethylpyrocarbonate in the unprotected enzyme under non‐denaturing conditions. In the presence of phenylalanine, ATP and Mg2+ the loss of aminoacylation activity is correlated with the modification of 2–4 residues.During extensive modification of the enzyme a dissociation process takes place. It was found that the observed loss of quaternary structure is not correlated with the decrease of the aminoacylation activity of the enzyme upon modification.

Yeast phenylalanyl-tRNA synthetase. Molecular weight and interaction with tRNA Phe and phenylalanine.

The stoichiometries of phenylalanyl‐tRNA‐synthetase · tRNAPhe complexes were found close to 2, as determined by the following methods: zone centrifugation in sucrose gradient, Sephadex filtration, sedimentation boundary technique and fluorescence quenching of Y base of tRNAPhe. The two former techniques allow the isolation of a complex composed of active enzyme (which is only a fraction of total enzyme used) and tRNA; the two last methods take into account the input protein. All the observed stoichiometries are consistent with the existence of two binding sites for tRNAPhe. Under the experimental conditions used (pH 7.0 and 12°C) a dissociation constant of 3 × 10−8 M was calculated from the fluoresence quenching of tRNAPhe by the enzyme.tRNAPhe protects the enzyme against heat denaturation. A protection constant of 9 × 10−7 M was found at 55°C, pH 7.0. The presence of phenylalanine and ATP did modify the strength of interaction of the enzyme‐tRNA system. They induce, however, a synergistic effect of the protection afforded by tRNAPhe.The binding of phenylalanine was examined by equilibrium dialysis in presence of tRNAPhe. The stoichiometry obtained indicates two binding sites for phenylalanine. The two sites however do not possess the same affinity: the corresponding Kd values are 6 and 30μM. The presence of more than one active site is also manifested by the curvature of the double‐reciprocal plots of v versus [S] for phenylalanine, when measuring the initial rates of phenylalanyl‐tRNA formation. With regard to the site having the highest affinity, a 20‐fold higher Kd value was found, in the absence of tRNAPhe, when binding of the amino acid was followed by the phase partition technique.

Effect of l-phenylalanine on 1-methyladenine production and spontaneous oocyte maturation in starfish

Production of 1-methyladenine (1-MA) in isolated ovarian fragment under the influence of the gonad-stimulating substance (GSS) was found to be inhibited by treatment with phenylalanine at concentrations higher than 10 mM in the starfish, Asterina pectinifera. The so-called ‘spontaneous’ maturation of the oocytes of Asterias amurensis isolated in sea water was also inhibited in the presence of phenylalanine. 1-MA was produced in the isolated follicle cells of this species in sea water in the absence of GSS. Such production of 1-MA was greatly reduced by lack of calcium ions, corresponding to the failure of spontaneous maturation in calcium-free sea water. Production of 1-MA in the isolated follicle cells of Asterias amurensis in sea water was suppressed by phenylalanine. The so-called ‘spontaneous’ oocyte maturation observed in the genus Asterias appears to be induced by 1-MA produced in the follicle cells in the absence of GSS. Therefore, it is concluded that oocyte maturation in starfish is invariably induced by 1-MA produced in the follicle cells. Failure of oocyte maturation in the presence of phenylalanine or in calcium deficiency does not seem to be due to the failure of release of pooled 1-MA from the follicle cells but to the inhibition of its synthesis in these cells.

Host-cell reactivation of phiX174 RF-DNA damaged by -ray-induced phenylalanine and water-radicals.

SummaryAqueous solutions of the double-stranded form of ΦX174 DNA have been irradiated with γ-rays in the presence of phenylalanine. Evidence is presented showing that under these circumstances DNA inactivation is largely due to secondary amino acid radicals. Such reactions of organic radicals might play a role in living cells.If the irradiation is carried out under N2O, the apparent sensitivity of the DNA is markedly increased by testing its virus-producing capacity on spheroplasts prepared from Uvr− strains of Escherichia coli. Such a difference in survival on Uvr− and Uvr+ spheroplasts is not found in the presence of oxygen, nor does it occur after irradiation in the absence of the amino acid.Testing of irradiated DNA on spheroplasts of a Pol− strain results in a somewhat increased apparent radiosensitivity in the presence, but also in the absence of phenylalanine. This effect is not suppressed by irradiation under oxygen.

Activation and transfer of oxygen. V. Spontaneous oxidation of 1,3,10‐trimethyl‐5,10‐dihydroalloxazine in acidic media in connection with its hydroxylating ability

AbstractOn autoxidation several dihydroalloxazines show a strong hydroxylating ability without addition of metal compounds. The assumption in the literature that N1 ‐alkylated dihydroalloxazines (I) cannot activate oxygen was proved to be wrong. The material balance was studied both in the absence and in the presence of phenylalanine as a substrate. Both autoxidation and hydroxylation to tyrosines were in agreement with calculations based on a theory which has already been put forward.Hydroxylation of organic compounds may be inhibited by some inorganic anions. An explanation is given for the inhibition occurring in hydrochloric acid.

Incorporation of glycine in pyrimidine and thiazole moieties of thiamine in Escherichia coli

[U− 14C]Glycine was incorporated into hydroxymethylpyrimidine and thiamine by a thiamine regulatory mutant of Escherichia coli with 5.1 and 2.5 × dilutions, respectively. The incorporation of 14C radioactivity into the pyrimidine moiety of thiamine was effectively decreased by the addition of hydroxymethylpyrimidine to the medium in the presence of adenine, whereas the incorporation into the thiazole moiety was decreased by thiazole in the presence of phenylalanine.

The Phenylalanine-binding Protein of Comamonas sp. (ATCC 11299a)

Abstract The phenylalanine-binding protein from Comamonas sp. (11299a) has been purified to homogeneity by ammonium sulfate treatment, carboxymethyl Sephadex chromatography in the presence of phenylalanine, and Sephadex G-100 filtration. Some properties of the protein have been determined including its amino acid composition, its isoelectric point, and its molecular weight. The binding is very tight with a Km of about 1 x 10-7 m but the phenylalanine bound is readily exchangeable. Other aromatic amino acids can bind to the protein. Urea and strong acid or base inhibit the binding, but these effects are reversible. Reagents which are known to react with the e-amino groups of lysine prevent the binding.

The Further Purification of Motilin, a Gastric Motor Activity Stimulating Polypeptide from the Mucosa of the Small Intestine of Hogs

A polypeptide has been isolated from the duodenal mucosa of hogs and has been named motilin. Motilin stimulates motor activity in both antral and fundic gland area pouches of the stomach of dogs. It will stimulate pepsin output, with no change in H+ secretion, from fundic gland area pouches. Motilin differs from the other characterized gastrointestinal polypeptides both chemically and in its physiological actions. In particular, the presence of phenylalanine as N-terminal residue, the absence of histidine, and presence of large amounts of glutamic acid and glutamine distinguish it from cholecystokinin–pancreozymin and secretin.