Activity Of Phe Research Articles

A network pharmacology approach with experimental validation to discover protective mechanism of poly herbal extract on diabetes mellitus

ObjectivePolyherbal extracts (PHE) contain six traditional medicinal plants, and the efficacy of the medicinal plants used in the preparation of this PHE has been confirmed for the treatment of diseases like diabetes mellitus (DM). The aim of this study was to evaluate the efficacy and therapeutic mechanism of PHE through a network pharmacology approach to reveal the protective mechanism of Alpha-Tocospiro A (ATA) present in PHE on DM with experimental validation. MethodsIn this study, Lipinski's rule (Swiss ADME) and drug-likeness score (MolSoft's) web pages were used to confirm the drug-likeness of identified constituents in PHE. Swiss Target Prediction (STP) genes were found for ATA-related genes. The DisGeNet database was used to screen genes associated with DM. String created a network diagram of the interactions between the ATA and DM genes. Top-scoring genes from the string network through CytoNCA plugged into Cytoscape 3.8.2 were selected as hub genes. In addition, the ShinyGO database is used to predict GO and KEGG pathway enrichment analyses. ResultsA total of 675 and 105 therapeutic genes (STP) were associated with all bioactive compounds and ATA in the PHE screen, respectively. Additionally, a maximum of 2,803 DM-related genes (DisGeNet) were observed. Further, in the analysis, 331, 57 potential (intersecting) genes were identified in the correlation between the target genes of all compounds and ATA, respectively, of PHE and the target genes of DM. The identified hub gene “TNF” for both ATA and PHE was found to be precisely strengthened in 49 pathways, along with 14 signaling pathways out of more than 100 enriched KEGG pathways. This study predicted that ATA activates PI3K/Akt and MAPK pathways enriched with TNF by phosphorylating the insulin receptor (IR) β-subunit. The anti-diabetic activity of PHE was found to be good and primarily confirmed by in vitro α-glucosidase enzyme inhibition activity. ConclusionThe anti-diabetic activity of PHE was found to be effective and was confirmed by the enzyme inhibition activity in the primary study. This study predicted that ATA is a novel drug molecule in PHE that has a targeted mechanism of action and therapeutic effect on DM.

Perturbation of Hydrogen Bonding Network Plays an Important Role in Determining Antifreeze Activity

In order to develop new antifreeze agents, it is crucial to understand the antifreeze activity. The adsorption-inhibition mechanism has been widely accepted, and most previous studies focus on ice-binding affinity of additive molecules but rarely discuss their influence on the hydrogen bonding network of ice. Herein, we investigate the ice recrystallization inhibition (IRI) activity of two structurally similar molecules, histidine (His) and phenylalanine (Phe). It is verified by experiment that Phe has excellent IRI activity, while His does not. Molecular dynamics simulation is conducted to study the process of ice growth with amino acids and to explore the origin of the difference between His and Phe. We find that His is prone to be swallowed up by ice and thus no longer hinders its growth, whereas Phe can remain unswallowed on the ice surface for a long period, which is attributed to their different abilities to perturb the hydrogen bonding network. Based on a statistical analysis of oxygen–hydrogen bond orientations, we propose a method to evaluate the order of the hydrogen bonding network. With this method, the IRI activity of His and Phe can be well interpreted, and the influence of different groups on the hydrogen bonding network can be quantified. This study takes the structural integrity of ice into consideration and provides a possibility to investigate the influence of different additive molecules on hydrogen bonding network, which is expected to improve the understanding of antifreeze mechanism.

Fabrication of black NiO/Sr2FeTaO6 heterojunctions with rapid interface charge transfer for efficient photocatalytic hydrogen evolution.

Series of black NiO/Sr2FeTaO6 (NiO/SFT) composites were synthesized by the combined processes of hydrothermal method and calcination treatment. The formed NiO was deposited on the surface of Sr2FeTaO6 to form a closely interfacial contact, leading to the formation of NiO/Sr2FeTaO6 heterojunction. The resulted samples were fully characterized by XRD, TEM, XPS, and UV-Vis DRS to gain their microstructure, crystal phase, atomic states and optical absorption properties. Introducing narrow-bandgap semiconductor of black NiO in NiO/Sr2FeTaO6 heterojunctions exhibits two major advantages. On the one hand, coupling with black NiO can significantly increase the light harvesting capacity of Sr2FeTaO6. On the other hand, the formed NiO/Sr2FeTaO6 heterojunctions benefited the separation and transfer of photogenerated charge carriers, which was confirmed by photo-electrochemical measurement, PL and TR-PL spectra. The activity of as-prepared samples was evaluated by photocatalytic hydrogen (H2) evolution (PHE) under visible light irradiation. The resulted NiO/SFT composites showed the improved PHE efficiency than that of NiO and Sr2FeTaO6, owing to the synergistic effects of synergistic effects of heterojunction formation for the efficient charge carrier transfer/separation and increased light harvesting capacity. However, the excess amount of NiO loaded in NiO/SFT composites will restrain the light harvesting of Sr2FeTaO6 component and decrease, leading to the decreased PHE activity. Our work provided an insight on the construction of high-efficiency heterojunction photocatalysts for PHE reaction.

Interior and Surface Synergistic Modifications Modulate the SnNb2O6/Ni-Doped ZnIn2S4 S-Scheme Heterojunction for Efficient Photocatalytic H2 Evolution.

Interior and surface synergistic modifications can endow the photocatalytic reaction with tuned photogenerated carrier flow at the atomic level. Herein, a new class of 2D/2D SnNb2O6/Ni-doped ZnIn2S4 (SNO/Ni-ZIS) S-scheme heterojunctions is synthesized by a simple hydrothermal strategy, which was used to evaluate the synergy between interior and surface modifications. Theoretical calculations show that the S-scheme heterojunction boosts the desorption of H atoms for rapid H2 evolution. As a result, 25% SNO/Ni0.4-ZIS exhibits significantly improved PHE activity under visible light, roughly 4.49 and 2.00 times stronger than that of single ZIS and Ni0.4-ZIS, respectively. In addition, 25% SNO/Ni0.4-ZIS also shows superior structural stability. This work provides advanced insight for developing high-performance S-scheme systems from photocatalyst design to mechanistic insight.

Intramolecular π-conjugated channel expansion achieved by doping cross-linked dopants into carbon nitride frameworks for propelling photocatalytic hydrogen evolution and mechanism insight

PYM as a cross-linked bridge in CN frameworks via a multi-bonding mode expedites the transfer and separation of intramolecular photoproduced electron–hole pairs by π-conjugated channel expansion to improve the PHE activity.

Constructing Z-scheme heterojunction with a special electron transfer path and more active sites over MnS/D-PCN for photocatalytic H2 evolution

Given that low hydrogen active sites and serious photoexcited electron-hole recombination greatly limits the solar-to-chemical energy conversion efficiency, a synergistically integrated strategy of the defect engineering and the construction of Z-scheme heterojunction is introduced to the design of PCN photocatalyst, a strand-like polymer carbon nitride, in this work to resolve these problems and enhance the photocatalytic activity. The PHE activity was intimately related to MnS compositions. A maximum hydrogen evolution rate of 670.5 μmol/g/h could be achieved over 5% MnS/D-PCN composite, almost 5 times the rate of D-PCN and 18.4 times higher than that pristine PCN. This high PHE activity of MnS/D-PCN was attributed to the following factors: defects effects and Z-scheme heterojunction. We found that defects sites, on one hand, were the docking sites of MnS through a linkage to bridging the MnS and defected PCN, which offered an efficient spatial transfer path for electron-hole pairs at the interface, on the other hand, acted as the hydrogen evolving centers gathering the excited photoelectrons from D-PCN and MnS. Furthermore, Z-scheme pathway greatly enhanced the photoexcited charge separation due to their enormous driving force in internal electric field of p-n heterojunction.

Interactions between electrokinetics and rhizoremediation on the remediation of crude oil-contaminated soil

An electrokinetics (EK)-enhanced phytoremediation system with ryegrass was constructed to remediate crude oil-polluted soil. The four treatments employed in this study included (1) without EK or ryegrass (CK-NR), (2) EK only (EK-NR), (3) ryegrass only (CK-R), and (4) EK and ryegrass (EK-R). After 30d of ryegrass growth, EK at 1.0 V·cm−1 with polarity reversal (PR-EK) was supplied for another 30 d. The electric current was recorded during remediation. The pH, electrical conductivity, total petroleum hydrocarbon content (TPH), 16S rDNA, functional genes of AlkB, Nah, and Phe, DGGE, and dehydrogenase activity in soil were measured. The physical-chemical indexes of the plant included the length, dry mass, and chlorophyll contents of the ryegrass. Results showed that EK-R removed 18.53 ± 0.53% of TPH, which was higher than that of other treatments (13.34–14.31%). Meanwhile, the values of 16S rDNA, AlkB, Nah, Phe, and dehydrogenase activity in the bulk soil of EK-R all increased. Further clustering analysis with numbers of genes and DGGE demonstrated that EK-R was similar to the ryegrass rhizosphere soils in both EK-R and CK-R, while the EK treatment of EK-NR was similar to that of CK-NR without EK and ryegrass. These results indicate that the PR-EK treatment used in this experiment successfully enlarged the existing scale of the rhizosphere microorganisms, improved microbial activity and enhanced degradation of TPH.

ANTIOXIDANT, ANTI-LIPID PEROXIDATIVE AND ANTIMICROBIAL PROPERTIES OF THE EPIPHYTIC FERN, PYRROSIA HETEROPHYLLA (L.) M. G. PRICE

Objective: The present study was aimed at establishing the antioxidant, free radical scavenging, anti-lipid peroxidative and antimicrobial properties of the plant Pyrrosia heterophylla (L.) M. G. PRICE.Methods: Standard protocols were used to estimate the antioxidant potential of the hexane, ethyl acetate and methanolic extracts of the plant. Radical scavenging ability of the extracts was assayed for 2, 2'-azino-bis (3-ethylbenzothiazoline-6-sulphonicacid) (ABTS), 1,1-diphenyl-2-picryl-hydrazil (DPPH) and hydroxyl radicals. Total antioxidant activity assay was done following the phospho- molybdenum method. The reductive potential was measured by ferric reducing antioxidant power (FRAP) assay. Lipid peroxidation assay was done in vitro. Total phenolic content was measured by the Folin-Ciocalteu method. Antimicrobial activity was identified by well diffusion method, and minimum inhibitory concentration (MIC) was determined by serial dilution method.Results: Results revealed that the ethyl acetate extract (PHE) exhibited the highest antioxidant capacity followed by the methanolic extract (PHM) whereas the hexane extract (PHH) had the lowest activity. The percentage radical scavenging by PHE was found to be 86.63±0.85, 89.48±2.08 and 70.89±1.46 for DPPH, ABTS and hydroxyl radicals respectively, at a concentration of 800μg/ml. The total antioxidant activity of PHE, PHM and PHH was found to be 538.33±3.51, 283.33±7.57and 13.76±3.95 ascorbic acid equivalents/g of extract respectively. Phenolic content of PHE was the highest (207.22±1.95 gallic acid equivalents (GAE)/g of extract), followed by PHM and PHH (197.92±2.00 and 37.50±2.18 GAE/g respectively). Total reducing power was also found to be the highest in PHE followed by PHM and PHH as per the FRAP assay. All the extracts were found to possess inhibitory activity against the tested microorganisms. MIC50 value of all the extracts was below 40 µg/ml.Conclusion: The results of this study confirmed the antioxidant, antimicrobial and anti-lipid peroxidation potentials of the plant P. heterophylla (L.) M. G. PRICE.

Participation of protein kinases in cytotoxic and proapoptotic effects of ethylene glycol ethers and their metabolites in SH-SY5Y cells

Ethylene glycol ethers (EGEs) are compounds widely used in many branches of industry. Their toxicological profile in the peripheral tissues is relatively well described, but little is known about their action on the central nervous system (CNS). In this study, we evaluated the effect of 2-ethoxyethanol (EE), 2-butoxyethanol (BE), 2-phenoxyethanol (PHE) and their metabolites on necrotic (estimated by cell viability and lactate dehydrogenase release) and apoptotic (caspase-3 activity and mitochondrial membrane potential) processes and reactive oxygen species' (ROS) production in human neuroblastoma (SH-SY5Y) cells. We have shown that, similar to the peripheral tissues, EGE metabolites in most of the performed assays revealed greater potential to damage than the parent compounds in the CNS cells. Subsequently, we investigated the participation of some selected protein kinases in the degenerative activity of PHE and its main metabolite, phenoxyacetic acid (PHA). It has been found that a GSK3β inhibitor weakened the damaging effects of PHE and PHA in each of the performed assays. Furthermore, the kinases, p38-MAPK, JNK-MAPK and PKC, had a significant role in the cytotoxic and proapoptotic effects of PHA. These results indicate that the neurotoxic effect of EGEs may stem from their impact on many intracellular signal transduction pathways.

Molecular characterization of a eukaryotic-like phenol hydroxylase from Corynebacterium glutamicum.

This study focuses on the genetic and biochemical characterization of phenol hydroxylase (Phe, NCgl2588) from Corynebacterium glutamicum that shares 31% identity in amino acids with phenol hydroxylase from yeast Trichosporon cutaneum but less similarity with that from bacteria. The phe deletion mutant significantly reduced its ability to grow with phenol as the sole carbon and energy source. Expression of the phe gene was strongly induced with phenol and also subject to the control of carbon catabolite repression (CCR). The molecular weight of purified Phe protein determined by gel filtration chromatography was 70 kDa, indicating that Phe exists as a monomer in the purification condition. However, Phe protein pre-incubated with phenol showed a molecular weight of 140 kDa, suggesting that Phe is likely active as a dimer. In addition to phenol, the Phe protein could utilize various other phenolic compounds as substrates. Site-directed mutagenesis revealed that D75, P261, R262, R269, C349 and C476 are key amino acid residues closely related to the enzyme activity of Phe.

The effect of carbonic anhydrase IX on focal contacts during cell spreading and migration

Carbonic anhydrase IX is a hypoxia-induced transmembrane enzyme linked with solid tumors. It catalyzes the reversible hydration of CO2 providing bicarbonate ions for intracellular neutralization and protons for extracellular acidosis, thereby supporting tumor cell survival and invasiveness. CA IX is the only human CA isoform containing the proteoglycan (PG) domain in its extracellular part. The PG domain appears to enhance the catalytic activity of CA IX and mediate its binding to the extracellular matrix. Moreover, manipulation of the CA IX level by siRNA or overexpression modulates cell adhesion pathway so that in the presence of CA IX, cells display an increased rate of adhesion and spreading. Here we show that deletion of the PG domain as well as treatment with the PG-binding monoclonal antibody M75 can impair this CA IX effect. Accordingly, CA IX-expressing cells show more prominent and elongated maturing paxillin-stained focal contacts (FC) than CA IX-negative controls, proving the role of CA IX in cell spreading. However, during active cell movement, CA IX is relocalized to lamellipodia and improves migration via its catalytic domain. Thus, we examined the influence of CA IX on FC turnover in these structures. While the lamellipodial regions lacking CA IX display dash-like adhesions, the CA IX-enriched neighboring regions exhibit dynamic dot-like FCs. These results suggest that CA IX can promote initial adhesion through its PG domain, but at the same time it facilitates formation of nascent adhesions at the leading edge of moving cells. Thereby it may allow for transmission of large forces and enhanced migration rate, presumably through catalytic activity and impact of pHe on FC dynamics. Thus, we provide the first evidence that CA IX protein localizes directly in focal adhesion (FA) structures and propose its functional relationship with the proteins involved in the regulation of FC turnover and maturation.

Critical Residue That Promotes Protein Dimerization: A Story of Partially Exposed Phe25 in 14-3-3σ

Many proteins exist and function as oligomers. While hydrophobic interactions have been recognized as the major driving force for oligomerization, detailed molecular mechanisms for the assembly are unknown. Here, we used 14-3-3σ as a model protein and investigated the role of hydrophobic residues at the dimeric interface using MD simulations and coimmunoprecipitations. We found that a half-exposed and half-buried residue in the interface, Phe(25), plays a more important role in promoting homodimerization than the hydrophobic core residues by organizing both favorable hydrophobic and hydrophilic interactions. Phe(25) is critical in packing and stabilizing hydrophobic core residues. We conclude that the structural stability of hydrophobic cores is critical for a stable homodimer complex and this stable property can be bestowed by residues outside of hydrophobic core. The important organizing activity of Phe(25) for homodimerization of 14-3-3σ originates from its unique physical location, rigidity, size, and hydrophobicity. Thus, hydrophobic residues that are not deeply buried at the oligomeric interface may play important but different roles from the buried core residues and they may promote oligomerization by organizing co-operativity of core and other residues for favorable hydrophobic and electrostatic interactions.

PH dependence of melanoma cell migration: protons extruded by NHE1 dominate protons of the bulk solution

Migration and morphology of human melanoma cells (MV3) depend on extracellular pH (pHe) and the activity of the Na+/H+ exchanger NHE1. To distinguish effects of NHE1 activity per se from effects of pHe we compared an NHE1-deficient mutant with rescued and wild-type cells. Time lapse video microscopy was used to investigate migratory and morphological effects caused by pHe and NHE1 activity, and a membrane-bound fluorescein conjugate was employed for ratiometric pH measurements at the outer leaflet of the cell membrane. As long as NHE1 remained inactive due to deficiency or inhibition by cariporide (HOE642) neither migration nor morphology was affected by changes in pHe. Under these conditions pH at the outer leaflet of the plasma membrane was uniform all over the cell surface. The typical pH dependence of MV3 cell migration and morphology could be reconstituted by restoring NHE1 activity. At the same time the proton gradient at the outer leaflet of the plasma membrane with the higher proton concentration at the leading edge and the lower one at the cell rear was re-established as well. Hence, NHE1 activity generates a proton gradient at the cell surface accompanied by the cells' ability to respond to changes in pHe (bulk pH). We conclude that NHE1 activity contributes to the generation of a well-defined cell surface pH by creating a proton gradient at the outer leaflet of the plasma membrane that is needed for (i) the development of a variety of morphologies including a distinct polarity and (ii) migration. A missing proton gradient at the cell surface cannot be compensated for by varying pHe.

Migration of human melanoma cells depends on extracellular pH and Na+/H+ exchange

Their glycolytic metabolism imposes an increased acid load upon tumour cells. The surplus protons are extruded by the Na+/H+ exchanger (NHE) which causes an extracellular acidification. It is not yet known by what mechanism extracellular pH (pHe) and NHE activity affect tumour cell migration and thus metastasis. We studied the impact of pHe and NHE activity on the motility of human melanoma (MV3) cells. Cells were seeded on/in collagen I matrices. Migration was monitored employing time lapse video microscopy and then quantified as the movement of the cell centre. Intracellular pH (pHi) was measured fluorometrically. Cell-matrix interactions were tested in cell adhesion assays and by the displacement of microbeads inside a collagen matrix. Migration depended on the integrin alpha2beta1. Cells reached their maximum motility at pHe approximately 7.0. They hardly migrated at pHe 6.6 or 7.5, when NHE was inhibited, or when NHE activity was stimulated by loading cells with propionic acid. These procedures also caused characteristic changes in cell morphology and pHi. The changes in pHi, however, did not account for the changes in morphology and migratory behaviour. Migration and morphology more likely correlate with the strength of cell-matrix interactions. Adhesion was the strongest at pHe 6.6. It weakened at basic pHe, upon NHE inhibition, or upon blockage of the integrin alpha2beta1. We propose that pHe and NHE activity affect migration of human melanoma cells by modulating cell-matrix interactions. Migration is hindered when the interaction is too strong (acidic pHe) or too weak (alkaline pHe or NHE inhibition).

Alanine scanning mutagenesis of a type 1 insulin-like growth factor receptor ligand binding site.

The high resolution crystal structure of an N-terminal fragment of the IGF-I receptor, has been reported. While this fragment is itself devoid of ligand binding activity, mutational analysis has indicated that its N terminus (L1, amino acids 1-150) and the C terminus of its cysteine-rich domain (amino acids 190-300) contain ligand binding determinants. Mutational analysis also suggests that amino acids 692-702 from the C terminus of the alpha subunit are critical for ligand binding. A fusion protein, formed from these fragments, binds IGF-I with an affinity similar to that of the whole extracellular domain, suggesting that these are the minimal structural elements of the IGF-I binding site. To further characterize the binding site, we have performed structure directed and alanine-scanning mutagenesis of L1, the cysteine-rich domain and amino acids 692-702. Alanine mutants of residues in these regions were transiently expressed as secreted recombinant receptors and their affinity was determined. In L1 alanine mutants of Asp(8), Asn(11), Tyr(28), His(30), Leu(33), Leu(56), Phe(58), Arg(59), and Trp(79) produced a 2- to 10-fold decrease in affinity and alanine mutation of Phe(90) resulted in a 23-fold decrease in affinity. In the cysteine-rich domain, mutation of Arg(240), Phe(241), Glu(242), and Phe(251) produced a 2- to 10-fold decrease in affinity. In the region between amino acids 692 and 702, alanine mutation of Phe(701) produced a receptor devoid of binding activity and alanine mutations of Phe(693), Glu(693), Asn(694), Leu(696), His(697), Asn(698), and Ile(700) exhibited decreases in affinity ranging from 10- to 30-fold. With the exception of Trp(79), the disruptive mutants in L1 form a discrete epitope on the surface of the receptor. Those in the cysteine-rich domain essential for intact affinity also form a discrete epitope together with Trp(79).

Phe1phi(CH2-NH)Gly2]nociceptin-(1-13)-NH2 acts as a partial agonist at ORL1 receptor endogenously expressed in mouse N1E-115 neuroblastoma cells.

The nociceptin derivative [Phe1phi(CH2-NH)Gly2]-nociceptin-(1-13)-NH2 (Phe(phi)noc) has been reported to act either as a simple antagonist or as a full agonist at the opioid receptor-like (ORL1) receptor. In the present study, we identified the expression of the ORL1 receptor in murine N1E-115 neuroblastoma cells and used this neuronal system to investigate the pharmacological activity of Phe(phi)noc. Like nociceptin, Phe(phi)noc stimulated the binding of [35S]GTPgammaS (EC50 = 120 nM) and inhibited forskolin-stimulated [3H]cAMP formation (EC50 = 3.3 nM). However, Phe(phi)noc elicited maximal effects lower than those induced by nociceptin, and when combined with nociceptin potently antagonized the responses to the natural agonist (Ki = 0.9 nM). These data indicate that Phe(phi)noc acts as a partial agonist at the ORL1 receptor endogenously expressed in N1E-115 cells.

Influence of copolymer composition on protein adsorption and structural rearrangements at the polymer surface

The influence of copolymer composition on protein adsorption and subsequent structural rearrangements of the adsorbed protein has been studied by (1) investigating the ability of adsorbed proteins to be displaced by proteins in solution and (2) by determining the ability of an immobilized antibody to recognize an antigen—enzyme conjugate. Surfactant-free polystyrene copolymeric latexes were used to study the effect of specific comonomers [acrylic acid (AA), methacrylic acid (MAA), 2-hydroxyethyl acrylate (HEA), and acrylamide (A)] on protein adsorption. Although the classical surface techniques of microelectrophoresis and surfactant titration could not distinguish between the different polystyrene copolymers, the extent and nature of protein adsorption were influenced substantially by the copolymer composition. Adsorption was essentially irreversible with respect to dilution but adsorbed proteins could be displaced by proteins in solution. The order of effectiveness was fibrinogen > immunoglobulin > albumin. the ability of adsorbed proteins to be displaced from surfaces followed the order PS/PHEA > PS/PAA > PS/PMAA > PS (easiest to most difficult displacement). The retention of activity of an immobilized monoclonal antibody (Phe 1.9) at the surface followed the order PS/PHEA > PS/PAA = PS/PMAA > PS (greatest to least active). Protein adsorbed to polystyrene under low protein concentrations was less readily displaced than protein adsorbed at saturating protein concentrations. Similarly, the activity of the monoclonal antibody Phe 1.9 was dependent on the final adsorbed protein concentration at the surface of polystyrene homopolymer. In contrast, the activity of Phe 1.9 immobilized on copolymer containing HEA, AA, or MAA varied little if any with the final protein concentration at the surface. The retention of activity of covalently immobilized Phe 1.9 on poly(styrene—co-chloromethylstyrene) copolymers followed the order PS/PCS/PA > PS/PCS/PHEA(3) > PC/PCS/PHEA(1) > PS/PCS/PAA > PS/PCS/PMAA = PS/PCS (greatest to least activity). Thus, the inclusion of “modifier monomers” that are hydrophilic or are capable of hydrogen bonding with proteins appears to moderate structural rearrangements of an immobilized protein reflected by both the ease of displacement from the surface and the retention of biological activity.

Comparative Utilization of the Isomers of Phenylalanine and Phenyllactic Acid by Chicks and Rats

A series of experiments was conducted to assess quantitatively the growth-promoting capacity of the isomers of phenylalanine (Phe) and phenyllactic acid (PLA). The slope-ratio procedure was used to determine percent efficacy relative to l-Phe. Addition of the isomers of Phe to a Phe-deficient chemically-defined diet produced linear growth and feed efficiency responses. Only the l-isomer and racemic mixture of PLA elicited a gain or gain:feed response. Efficacy estimates for male chicks were: dl-Phe, 90.1%; d-Phe, 74.6%; l-PLA, 70.1%; dl-PLA, 36.9%; d-PLA, 5.9%. The comparative utilization of d-Phe, phenypyruvic acid (PPA) and l-PLA by chicks was also evaluated when these compounds were fed to male chicks as the sole source of dietary Phe. PPA activity, 84.8%, was slightly greater than that of either d-Phe, 75.3%, or l-PLA, 67.4%. Efficacies of d-Phe and l-PLA were similar whether fed as supplements to a Phe-deficient diet or when fed as the sole source of dietary Phe activity. Isomers of Phe and PLA were also fed as the sole dietary source of Phe to growing rats. dl-Phe (80.4%) and d-Phe (68.1%) were well utilized by the rat. Efficacies of l-PLA and dl-PLA were 46.1 and 28.0%, respectively. d-PLA had no growth-promoting activity for the rat.L-, DL-, and D-phenylalanine phenylpyruvic acid L-, DL-, and D--phenyllactic acid analog efficacy

Studies on 3-deoxy-d-arabinoheptulosonate-7-phosphate synthetase(phe) from escherichia coli k12. 3. Structural studies.

1. Investigations with structural analogues of phenylalanine indicated an absolute requirement for the aromatic ring and both the alpha-carboxyl and alpha-amino groups of phenylalanine for inhibition of 3-deoxy-D-arabinoheptulosonate-7-phosphate synthetase(phe) activity. Replacement of the alpha-H atom with a methyl group does not decrease the inhibition greatly. Varying degrees of inhibition were observed with o, m and p mono-substituted fluoro, chloro and hydroxy phenylalanines. D-Phenylalanine and several metabolites of the aromatic biosynthetic pathways do not inhibit enzymic activity. 2. Circular dichroism studies indicated that the native enzyme possesses approximately 26% alpha-helix. Both circular dichroic and ultraviolet difference spectra indicated that the addition of phenylalanine to the synthetase induces a conformational change involving a small alteration of the secondary structure and large alterations in th interactions of some of the aromatic residues of the enzyme. In particular, a tryptophan residue moves from an extremly hydrophobic environment to one less hydrophobic. 3. Kd for the binding of phenylalanine to the enzyme was determined spectrophotometrically to be 75 muM. 4. Chemical modification studies suggested that a sulphydryl group and possibly a lysine residue may be implicated in the catalytic activity of the enzyme.