Phenylalanine and Tyrosine Biosynthesis in Sporeforming Members of the Order Actinomycetales

Abstract The enzymes of the terminal steps of phenylalanine and tyrosine biosynthesis, chorismate mutase, prephenate dehydratase, arogenate dehydratase, prephenate dehydrogenase and aroge nate dehydrogenase were studied in 13 sporeforming members of the order Actinomycetales. In these organisms tyrosine is synthesized exclusively via arogenate, phenylalanine, however, via phenylpyruvate. The regulation pattern of the corresponding enzymes was determined: No feed back inhibition of arogenate dehydrogenase by L-phenylalanine and ʟ-tyrosine was observed. Chorismate mutase was found to be inhibited in all organisms by ʟ-tyrosine and in most organisms by ʟ-tryptophan. ʟ-Phenylalanine was shown to inhibit prephenate dehydratase in the majority of bacteria tested and ʟ-tyrosine activated this enzyme in most cases. The elution profiles for the phenylalanine and tyrosine biosynthetic enzymes were studied in three members of the order Actinomycetales by anion exchange chromatography on DEAE-cellulose.

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Purification and characterization of a Ca2+-dependent novel lectin from Nymphaea nouchali tuber with antiproliferative activities

Bioscience Reports ◽

10.1042/bsr20100126 ◽

2011 ◽

Vol 31 (6) ◽

pp. 465-475 ◽

Cited By ~ 20

Author(s):

Syed Rashel Kabir ◽

Md. Abu Zubair ◽

Md. Nurujjaman ◽

Md. Azizul Haque ◽

Imtiaj Hasan ◽

...

Keyword(s):

Anion Exchange ◽

Pathogenic Bacteria ◽

Sequence Similarity ◽

Deae Cellulose ◽

Ehrlich Ascites Carcinoma ◽

Anion Exchange Chromatography ◽

Exchange Chromatography ◽

Neutral Sugar ◽

The Stability

A lectin (termed NNTL) was purified from the extracts of Nymphaea nouchali tuber followed by anion-exchange chromatography on DEAE-cellulose, hydrophobic chromatography on HiTrap Phenyl HP and by repeated anion-exchange chromatography on HiTrap Q FF column. The molecular mass of the purified lectin was 27.0 ± 1.0 kDa, as estimated by SDS/PAGE both in the presence and in the absence of 2-mercaptoethanol. NNTL was an o-nitrophenyl β-D-galactopyranoside sugar-specific lectin that agglutinated rat, chicken and different groups of human blood cells and exhibited high agglutination activity over the pH range 5–9 and temperatures of 30–60°C. The N-terminal sequence of NNTL did not show sequence similarity with any other lectin and the amino acid analysis revealed that NNTL was rich in leucine, methionine and glycine residues. NNTL was a glycoprotein containing 8% neutral sugar and showed toxicity against brine shrimp nauplii with an LC50 value of 120 ± 29 μg/ml and exerted strong agglutination activity against four pathogenic bacteria (Bacillus subtilis, Sarcina lutea, Shigella shiga and Shigella sonnei). In addition, antiproliferative activity of this lectin against EAC (Ehrlich ascites carcinoma) cells showed 56% and 76% inhibition in vivo in mice at 1.5 and 3 mg·kg−1·day−1 respectively. NNTL was a divalent ion-dependent glycoprotein, which lost its activity markedly in the presence of denaturants. Furthermore, measurement of fluorescence spectra in the presence and absence of urea and CaCl2 indicated the requirement of Ca2+ for the stability of NNTL.

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Comparison of different anion-exchange chromatography resins for the purification of cyanobacterial microcystins

Water Science & Technology Water Supply ◽

10.2166/ws.2015.108 ◽

2015 ◽

Vol 16 (1) ◽

pp. 1-8 ◽

Cited By ~ 1

Author(s):

Theerasak Somdee ◽

Anchana Somdee

Keyword(s):

Liquid Chromatography ◽

Anion Exchange ◽

High Performance ◽

Total Yield ◽

Deae Cellulose ◽

Anion Exchange Chromatography ◽

Exchange Chromatography ◽

Ionization Mass ◽

Freeze Dried ◽

Wide Range

For the first time, different types of diethylaminoethyl (DEAE) anion-exchange resins, widely used in previous studies, were investigated to determine the most effective resin for the purification of microcystins (MCs). MCs were extracted from freeze-dried Microcystis aeruginosa cells that had been harvested from the Bueng Nong Khot reservoir, Khon Kaen, Thailand. The toxins were precipitated with ammonium sulfate and then fractionated using five different anion-exchange chromatography resins, followed by chromatography with a C18 cartridge. The toxins were further identified via liquid chromatography–electrospray ionization–mass spectrometry (LC-ESI-MS) analysis, and the yields and purity were determined by high-performance liquid chromatography (HPLC) with ultraviolet detection. DEAE Sephadex A-25 exhibited the best overall performance for MC purification regarding both yield and purity, followed by DEAE cellulose, DEAE Sephacel, DEAE Sepharose Fast Flow and Toyopearl DEAE. Four MC variants, MC-RR, MC-FR, [Dha7]MC-LR and MC-WR, were obtained, and [Dha7]MC-LR was the major variant, with a total yield of 53.08 mg and a purity of 95% using the Sephadex resin. This study indicates that protein precipitation and single-column chromatography using DEAE Sephadex A-25 constitute an effective method for the purification of a wide range of MC variants.

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Hydrophobic-interaction chromatography and anion-exchange chromatography in the presence of acetonitrile. A two-step purification method for human prolactin

Biochemical Journal ◽

10.1042/bj1990619 ◽

1981 ◽

Vol 199 (3) ◽

pp. 619-627 ◽

Cited By ~ 13

Author(s):

Steven C. Hodgkinson ◽

Philip J. Lowry

Keyword(s):

Anion Exchange ◽

Hydrophobic Interaction ◽

Polyacrylamide Gel Electrophoresis ◽

Sodium Dodecyl ◽

Deae Cellulose ◽

Hydrophobic Interaction Chromatography ◽

Anion Exchange Chromatography ◽

Exchange Chromatography ◽

Preparative Scale ◽

Human Prolactin

Described is a two-chromatographic-step preparative-scale technique for the purification of human prolactin from a frozen pituitary homogenate. The method utilizes hydrophobic interaction chromatography on the mildly hydrophobic adsorbent phenyl-Sepharose CL-4B and anion-exchange chromatography on DEAE-cellulose in the presence of acetonitrile. Human prolactin was solubilized at pH10.0 after a prior extraction of pituitaries at pH4.0, the acid pH being ineffective at solubilizing human prolactin but capable of solubilizing large amounts of interfering protein. An 11-fold increase in the potency of the solubilized human prolactin was achieved in this manner. Prolactin could be adsorbed to phenyl-Sepharose at low ionic strengths (I<0.01); few other proteins were adsorbed under these conditions. This is a demonstration of the hydrophobic nature of human prolactin. The amount of phenyl-Sepharose was limited to the minimum (35mg of protein/g of phenyl-Sepharose) necessary to adsorb human prolactin, further reducing the uptake of other pituitary protein. Desorption was achieved by using an acetonitrile gradient (0–30%, v/v), resulting in a purification of human prolactin of 85-fold and recovery of 78%. Acetonitrile (20%, v/v) was also included in all buffers for DEAE-cellulose chromatography, increasing the resolution and recovery of human prolactin, apparently by minimizing non-ionic interactions with the matrix. Prolactin (10mg) was recovered from 63g if pituitaries, an overall recovery of 58%. It was homogeneous by gel filtration and sodium dodecyl sulphate/polyacrylamide-gel electrophoresis, contained less than 0.1% somatotropin (growth hormone), on iodination demonstrated more than 95% binding to excess anti-(human prolactin) serum and could be displaced from anti-(human prolactin) serum in a manner indistinguishable from the serum of a patient with a human prolactin-secreting adenoma.

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Tyrosine Biosynthesis in Sorghum bicolor: Isolation and Regulatory Properties of Arogenate Dehydrogenase

Zeitschrift für Naturforschung C ◽

10.1515/znc-1986-1-212 ◽

1986 ◽

Vol 41 (1-2) ◽

pp. 69-78 ◽

Cited By ~ 37

Author(s):

James A. Connelly ◽

Eric E. Conn

Keyword(s):

Amino Acid ◽

Deae Cellulose ◽

Shikimate Pathway ◽

Oxidative Decarboxylation ◽

Strong Inhibition ◽

High Recovery ◽

Prephenate Dehydrogenase ◽

Arogenate Dehydrogenase ◽

Tyrosine Biosynthesis ◽

Regulatory Properties

Abstract The conversion of prephenic acid to tyrosine can occur by two different routes: (a) oxidative decarboxylation (prephenate dehydrogenase) followed by transamination (aromatic aminotrans ferase); (b) transamination of prephenate forming the non-aromatic amino acid arogenic acid (prephenate am inotransferase) followed by oxidative decarboxylation (arogenate dehydrogenase). High activity of arogenate dehydrogenase was found in extracts of etiolated sorghum seedlings, while no evidence of prephenate dehydrogenase was observed. Arogenate dehydrogenase from sorghum eluted, with high recovery of activity (93%), as a single peak on DEAE-cellulose chromatography. The enzyme was strongly inhibited by tyrosine but was unaffected by phenylalanine, prephenate, or tryptophan. Kinetic analysis showed that tyrosine inhibition was competitive with arogenate and that the Ki for tyrosine (61 μm) was much smaller than the Km for arogenate (350 μm). The properties of arogenate dehydrogenase indicate that this enzyme is important in the regulation of tyrosine biosynthesis in sorghum. Strong inhibition of the enzyme by tyrosine may indicate that arogenate is a branch point in the shikimate pathway in plants and therefore arogenate may be a precursor to phenylalanine and the numerous phenylpropanoid secondary metabolites derived from phenylalanine.

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