scholarly journals NAD(P)H-dependent aldose reductase from the xylose-assimilating yeast Candida tenuis: Isolation, characterization and biochemical properties of the enzyme

1997 ◽  
Vol 326 (3) ◽  
pp. 683-692 ◽  
Author(s):  
Wilfried NEUHAUSER ◽  
Dietmar HALTRICH ◽  
Klaus D. KULBE ◽  
Bernd NIDETZKY
Keyword(s):  
Aldose Reductase ◽  
Binding Constants ◽  
Catalytic Efficiency ◽  
Anion Exchange Chromatography ◽  
Competitive Inhibitor ◽  
Biochemical Properties ◽  
Product Inhibition ◽  
Exchange Chromatography ◽  
Binding Studies ◽  
Coenzyme Binding

During growth on D-xylose the yeast Candida tenuis produces one aldose reductase that is active with both NADPH and NADH as coenzyme. This enzyme has been isolated by dye ligand and anion-exchange chromatography in yields of 76%. Aldose reductase consists of a single 43 kDa polypeptide with an isoelectric point of 4.70. Initial velocity, product inhibition and binding studies are consistent with a compulsory-ordered, ternary-complex mechanism with coenzyme binding first and leaving last. The catalytic efficiency (kcat/Km) in D-xylose reduction at pH 7 is more than 60-fold higher than that in xylitol oxidation and reflects significant differences in the corresponding catalytic centre activities as well as apparent substrate-binding constants. The enzyme prefers NADP(H) approx. 2-fold to NAD(H), which is largely due to better apparent binding of the phosphorylated form of the coenzyme. NADP+ is a potent competitive inhibitor of the NADH-linked aldehyde reduction (Ki 1.5 μM), whereas NAD+ is not. Unlike mammalian aldose reductase, the enzyme from C. tenuisis not subject to oxidation-induced activation. Evidence of an essential lysine residue located in or near the coenzyme binding site has been obtained from chemical modification of aldose reductase with pyridoxal 5′-phosphate. The results are discussed in the context of a comparison of the enzymic properties of yeast and mammalian aldose reductase.

scholarly journals Synthesis of divalent ligands of β-thio- and β-N-galactopyranosides and related lactosides and their evaluation as substrates and inhibitors of Trypanosoma cruzi trans-sialidase

2014 ◽  
Vol 10 ◽  
pp. 3073-3086 ◽  
Author(s):  
María Emilia Cano ◽  
Rosalía Agusti ◽  
Alejandro J Cagnoni ◽  
María Florencia Tesoriero ◽  
José Kovensky ◽  
...  
Keyword(s):  
Sialic Acid ◽  
Trypanosoma Cruzi ◽  
High Performance ◽  
Transfer Reaction ◽  
Main Product ◽  
Amperometric Detection ◽  
Anion Exchange Chromatography ◽  
Competitive Inhibitor ◽  
Exchange Chromatography ◽  
Competitive Inhibitors

In this work we describe the synthesis of mono- and divalent β-N- and β-S-galactopyranosides and related lactosides built on sugar scaffolds and their evaluation as substrates and inhibitors of the Trypanosoma cruzi trans-sialidase (TcTS). This enzyme catalyzes the transfer of sialic acid from an oligosaccharidic donor in the host, to parasite βGalp terminal units and it has been demonstrated that it plays an important role in the infection. Herein, the enzyme was also tested as a tool for the chemoenzymatic synthesis of sialic acid containing glycoclusters. The transfer reaction of sialic acid was performed using a recombinant TcTS and 3’-sialyllactose as sialic acid donor, in the presence of the acceptor having βGalp non reducing ends. The products were analyzed by high performance anion exchange chromatography with pulse amperometric detection (HPAEC-PAD). The ability of the different S-linked and N-linked glycosides to inhibit the sialic acid transfer reaction from 3’-sialyllactose to the natural substrate N-acetyllactosamine, was also studied. Most of the substrates behaved as good acceptors and moderate competitive inhibitors. A di-N-lactoside showed to be the strongest competitive inhibitor among the compounds tested (70% inhibition at equimolar concentration). The usefulness of the enzymatic trans-sialylation for the preparation of sialylated ligands was assessed by performing a preparative sialylation of a divalent substrate, which afforded the monosialylated compound as main product, together with the disialylated glycocluster.

scholarly journals Expression in Escherichia Coli, Purification, and Functional Reconstitution of Human Steroid 5α-Reductases

Endocrinology ◽  
2020 ◽  
Vol 161 (8) ◽  
Author(s):  
Hwei-Ming Peng ◽  
Juan Valentín-Goyco ◽  
Sang-Choul Im ◽  
Bing Han ◽  
Jiayan Liu ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Functional Characterization ◽  
Catalytic Efficiency ◽  
Biochemical Properties ◽  
Exchange Chromatography ◽  
Structural Basis ◽  
Cancer Drug ◽  
Ph Optimum ◽  
Hek 293 ◽  
Triton X

Abstract The potent androgen 5α-dihydrotestosterone irreversibly derives from testosterone via the activity of steroid 5α-reductases (5αRs). The major 5αR isoforms in most species, 5αR1 and 5αR2, have not been purified to homogeneity. We report here the heterologous expression of polyhistidine-tagged, codon-optimized human 5αR1 and 5αR2 cDNAs in Escherichia coli. A combination of the nonionic detergents Triton X-100 and Nonidet P-40 enabled solubilization of these extremely hydrophobic integral membrane proteins and facilitated purification with affinity and cation-exchange chromatography methods. For functional reconstitution, we incorporated the purified isoenzymes into Triton X-100-saturated dioleoylphosphatidylcholine liposomes and removed excess detergent with polystyrene beads. Kinetic studies indicated that the 2 isozymes differ in biochemical properties, with 5αR2 having a lower apparent Km for testosterone, androstenedione, progesterone, and 17-hydroxyprogesterone than 5αR1; however, 5αR1 had a greater capacity for steroid conversion, as reflected by a higher Vmax than 5αR2. Both enzymes preferred progesterone as substrate over other steroids, and the catalytic efficiency of purified reconstituted 5αR2 exhibited a sharp pH optimum at pH 5. Intriguingly, we found that the prostate-cancer drug-metabolite 3-keto-∆ 4-abiraterone is metabolized by 5αR1 but not 5αR2, which may serve as a structural basis for isoform selectivity and inhibitor design. The functional characterization results with the purified reconstituted isoenzymes paralleled trends obtained with HEK-293 cell lines stably expressing native 5αR1 and 5αR2. Access to purified human 5αR1 and 5αR2 will advance studies of these important enzymes and might help to clarify their contributions to steroid anabolism and catabolism.

Production and characterization of recombinant chicken insulin-like growth factor-II from Escherichia coli

1995 ◽  
Vol 14 (1) ◽  
pp. 79-90 ◽  
Author(s):  
Z Upton ◽  
G L Francis ◽  
K Kita ◽  
J C Wallace ◽  
F J Ballard
Keyword(s):  
Escherichia Coli ◽  
Fusion Protein ◽  
Anion Exchange Chromatography ◽  
Exchange Chromatography ◽  
Genetically Engineered ◽  
Binding Studies ◽  
Igf Binding Proteins ◽  
Igf Receptor

ABSTRACT Recombinant chicken (c)IGF-II has been produced in Escherichia coli after first modifying a plasmid that coded for a human (h)IGF-II fusion protein. The cIGF-II fusion protein, deposited in bacterial inclusion bodies, was dissolved under reducing conditions, desalted, subjected to anion-exchange chromatography and refolded. Recombinant cIGF-II was then released from the fusion protein using a genetically engineered serine protease and purified to homogeneity by reverse-phase HPLC. In vitro analysis of recombinant cIGF-II revealed differences between cIGF-II and its human counterpart. Recombinant cIGF-II was less potent than hIGF-II in stimulating protein synthesis in rat myoblasts. This appeared to be due to a decreased affinity for the type-1 IGF receptor. The human and chicken peptides were similar, however, in studies assessing binding to the type-2 IGF receptor and to IGF-binding proteins. Moreover, recombinant cIGF-II and hIGF-II were equipotent in both biological and receptor binding studies in chick embryo fibroblasts, suggesting that there may be a difference between mammalian and avian type-1 IGF receptors.

Purification, Biochemical Characterization and Decolorization Efficiency of Laccases from Peach and Cherry Cultures of Pleutorus eryngii: A Comparative Study

2020 ◽  
Vol 27 (7) ◽  
pp. 623-634 ◽  
Author(s):  
Merve Akpinar ◽  
Raziye Ozturk Urek
Keyword(s):  
Biochemical Characterization ◽  
Gel Filtration ◽  
Industrial Effluents ◽  
Anion Exchange Chromatography ◽  
Biochemical Properties ◽  
Exchange Chromatography ◽  
Kinetic Characteristics ◽  
Chemical Agents ◽  
Decolorization Efficiency

Background:: Laccases (Lacs) are used potentially in industrial and biotechnological applications such as decolorization of dyes, degradation of industrial effluents, delignification, etc. thanks to their large varieties of substrate specificities and excellent catalytic efficiencies. The efficient utilizations of Lacs in these applications mostly depend on the identifying their biochemical properties. Objective: The goal of this research is to investigate the purification, biochemical characterization and decolorization efficiencies of Lacs. Methods: Pleurotus eryngii was incubated on peach (PC) and cherry (CC) wastes under optimized solid state fermentation conditions. Then, the enzymes extracts were purified by ammonium sulfate precipitation, anion exchange chromatography, gel filtration, respectively. Lacs fractions were subjected to electrophoretic analyses as well as their structural and kinetic characteristics. Also, the effects of selected chemical agents on purified Lacs activities and determination of decolorization efficiencies were studied. Results: As the results of purification processes of Lacs from both cultures, 3.94-fold purification was obtained for PC, while it was 5.34 for CC. The electrophoretic results of purified Lacs illustrated the single bands of protein (30±1 kDa) in accordance with the results after gel filtration. The Km values of Lacs from PC and CC were respectively detected as 1.1381 and 0.329 mM for ABTS. The selected agents partially/completely inhibited Lac activities. The highest decolorization efficiencies of purified Lacs from PC and CC were separately obtained as 53 and 11.8%. Conclusion: The results clearly indicated that the performances of Lacs from both cultures in decolorization application are different from each other depending their activities, biochemical and kinetic characteristics.

Functional reconstitution of an eicosanoid-modulated Cl− channel from bovine tracheal smooth muscle

2002 ◽  
Vol 282 (3) ◽  
pp. C567-C577 ◽  
Author(s):  
Dany Salvail ◽  
Martin Cloutier ◽  
Eric Rousseau
Keyword(s):  
Inhibitory Concentration ◽  
Biochemical Characterization ◽  
Anion Exchange Chromatography ◽  
Biochemical Properties ◽  
Exchange Chromatography ◽  
Dependent Manner ◽  
Open Probability ◽  
Concentration Dependent Manner ◽  
Lipid Environment ◽  
Channel Control

We describe the biochemical properties of an eicosanoid-modulated Cl−channel and assess the mechanisms by which the epoxyeicosatrienoic acids (EETs) alter both its unitary conductance and its open probability ( P o). After a purification protocol involving wheat-germ agglutinin affinity and anion-exchange chromatography, the proteins were sequentially inserted into liposomes, which were then fused into PLBs. Functional and biochemical characterization tests confirm that the Cl− channel is a 55-kDa glycosylated monomer with voltage- and Ca2+concentration-independent activity. 5,6- and 8,9-EET decreased the conductance of the native channel (control conductance: 70 ± 5 pS in asymmetrical 50 mM trans/250 mM cis CsCl) in a concentration-dependent manner, with respective 50% inhibitory concentration values of 0.31 and 0.42 μM. These regioisomers similarly decreased the conductance of the purified channel (control conductance value: 75 ± 5 pS in asymmetrical 50 mM trans/250 mM cis CsCl), which had been stripped of its native proteic and lipidic environment. On the other hand, 5,6- and 8,9-EETs decreased the P o of the native channel with respective 50% inhibitory concentration values of 0.27 and 0.30 μM but failed to alter the P o of the purified protein. Thus we suggest that the effects of these EETs on channel conductance likely result from direct interactions of EET− anions with the channel pore, whereas the alteration of P o requires a lipid environment of specific composition that is lost on solubilization and purification of the protein.

Extracellular polysaccharides from suspension cultures of Nicotiana plumbaginifolia

2020 ◽  
Author(s):  
Ian Sims ◽  
A Bacic
Keyword(s):  
Uronic Acid ◽  
Cell Suspension Cultures ◽  
Anion Exchange Chromatography ◽  
Nicotiana Plumbaginifolia ◽  
Suspension Cultures ◽  
Exchange Chromatography ◽  
Arabinogalactan Protein ◽  
Extracellular Polysaccharides ◽  
Selective Precipitation ◽  
The Individual

The soluble polymers secreted by cell-suspension cultures of Nicotiana plumbaginifolia contained 78% carbohydrate, 6% protein and 4% inorganic material. The extracellular polysaccharides were separated into three fractions by anion-exchange chromatography using a gradient of imidazole-HCl at pH 7 and the individual polysaccharides in each fraction were then isolated by selective precipitation and enzymic treatment. Monosaccharide and linkage compositions were determined for each polysaccharide after reduction of uronic acid residues and the degree of esterification of the various uronic acid residues in each polysaccharide was determined concurrently with the linkage types. Six components were identified: an arabinoxyloglucan (comprising 34% of the total polysaccharide) and a galactoglucomannan (15%) in the unbound neutral fraction, a type II arabinogalactan (an arabinogalactan-protein, 11%) and an acidic xylan (3%) in the first bound fraction, and an arabinoglucuronomannan (11%) and a galacturonan (26%) in the second bound fraction. © 1995.

Correlation between protein desorption behavior and its adsorption enthalpy change in polymer grafted anion exchange chromatography

2021 ◽  
pp. 111853
Author(s):  
Joao Carlos Simoes-Cardoso ◽  
Nanako Hoshino ◽  
Yusuke Yoshimura ◽  
Chyi-Shin Chen ◽  
Cristina Dias-Cabral ◽  
...  
Keyword(s):  
Anion Exchange ◽  
Anion Exchange Chromatography ◽  
Exchange Chromatography ◽  
Enthalpy Change ◽  
Adsorption Enthalpy
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