Affinity chromatography and inhibition of chorismate mutase-prephenate dehydrogenase by derivatives of phenylalanine and tyrosine

Several derivatives of phenylalanine and tyrosine were prepared and tested for inhibition of chorismate mutase-prephenate dehydrogenase (EC 1.3.1.12) from Escherichia coli K12 (strain JP 232). The best inhibitors were N-toluene-p-sulphonyl-L-phenylalanine, N-benzenesulphonyl-L-phenylalanine and N-benzloxycarbonyl-L-phenylalanine. Consequently two compounds, N-toluene-sulphonyl-L-p-aminophenylalanine and N-p-aminobenzenesulphonyl-L-phenylalanine, were synthesized for coupling to CNBr-activated Sepharose-4B. The N-toluene-p-sulphonyl-L-p-aminophenylalanine-Sepharose-4B conjugate was shown to bind the enzyme very strongly at pH 7.5. The enzyme was not eluted by various eluents, including 1 M-NaCl, but could be quantitatively recovered by washing with buffer of pH9. Elution was more effective in the presence of 10 mM-1-adamantaneacetic acid, a competitive inhibitor of the enzyme. This affinity-chromatography procedure results in a high degree of purification of the enzyme and can be used to prepare the enzyme in a one-step procedure from the bacterial crude extract. Such a procedure may therefore prove useful in studying this enzyme in a state that closely resembles that in vivo.

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General ligands in affinity chromatography. Cofactor–substrate elution of enzymes bound to the immobilized nucleotides adenosine 5′-monophosphate and nicotinamide–adenine dinucleotide

Biochemical Journal ◽

10.1042/bj1270625 ◽

1972 ◽

Vol 127 (4) ◽

pp. 625-631 ◽

Cited By ~ 169

Author(s):

K. Mosbach ◽

H. Guilford ◽

R. Ohlsson ◽

M. Scott

Keyword(s):

Lactate Dehydrogenase ◽

Affinity Chromatography ◽

Cyanogen Bromide ◽

Competitive Inhibitor ◽

Single Step ◽

Lactate Dehydrogenase Activity ◽

Step Procedure ◽

The Matrix ◽

Subsequent Elution ◽

Sepharose 4B

1. Two different gels have been prepared suitable for the separation of a number of enzymes, in particular NAD+-dependent dehydrogenases, by affinity chromatography. For both the matrix used was Sepharose 4B. For preparation (a), NAD+–Sepharose, 6-aminohexanoic acid has been coupled to the gel by the cyanogen bromide method and then NAD+ was attached by using dicyclohexylcarbodi-imide; for preparation (b), AMP–Sepharose, N6-(6-aminohexyl)-AMP has been coupled directly to cyanogen bromide-activated gel. 2. Affinity columns of both gels retain only the two enzymes when a mixture of bovine serum albumin, lactate dehydrogenase and glyceraldehyde 3-phosphate dehydrogenase is applied. Subsequent elution with the cofactor NAD+ yields glyceraldehyde 3-phosphate dehydrogenase whereas lactate dehydrogenase is eluted by applying the same molarity of the reduced cofactor. 3. The binding of both glyceraldehyde 3-phosphate dehydrogenase and lactate dehydrogenase to the gel tested, AMP–Sepharose, is strong enough to resist elution by gradients of KCl of up to at least 0.5m. A 0.0–0.15m gradient of the competitive inhibitor salicylate, however, elutes both enzymes efficiently and separately. 4. The elution efficiency of lactate dehydrogenase from AMP–Sepharose has been examined by using a series of eluents under comparable conditions of concentration etc. The approximate relative efficiencies are: 0 (lactate); 0 (lactate+semicarbazide); 0 (0.5mm-NAD+); 80 (lactate+NAD+); 95 (lactate+semicarbazide+NAD+); 100 (0.5mm-NADH). 5. All contaminating lactate dehydrogenase activity can be removed from commercially available crude pyruvate kinase in a single-step procedure by using AMP–Sepharose.

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Two in one: bifunctional derivatives of trolox acting as antimalarial and antioxidant agents

Future Medicinal Chemistry ◽

10.4155/fmc-2020-0106 ◽

2020 ◽

Vol 12 (20) ◽

pp. 1845-1854

Author(s):

Florence Souard ◽

Edwige Nicolle ◽

Delphine Cressend ◽

Alexis Valentin ◽

Ahcène Boumendjel

Keyword(s):

Antioxidant Activity ◽

Ascorbic Acid ◽

In Vivo Studies ◽

Chemical Libraries ◽

Falciparum Strain ◽

Antioxidant Agents ◽

One Step ◽

Set Up ◽

Derivatives Of

Background: The aim of the present work was to set-up compounds that are able to act simultaneously as antimalarial and antioxidants. Trolox, a known antioxidant was chosen as a core structure to ensure the antioxidant activity and contribute to antiplasmodial effect. Results: Ten compounds were prepared in one step and evaluated on chloroquino-sensitive (3D7) and chloroquino-resistant (FcB1) strains of Plasmodium falciparum. The most active compound (3d) shows antiplasmodial activity in the range of chloroquine against chloroquino-sensitive and chloroquino-resistant P. falciparum strain. The antioxidant activity of (3d) was conducted through four tests and was found to be more potent than trolox itself and L-ascorbic acid. Conclusion: Compound (3d) can be considered as an excellent lead molecule for further in vivo studies. This study paves the way for building large chemical libraries to be investigated in the field of malaria.

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Complex Formation of Covalently Crosslinked Fibrin Oligomers with Agarose Coupled Fibrinogen and Fibrin

10.1055/s-0039-1682429 ◽

1977 ◽

Author(s):

R. von Hugo ◽

R. Hafter ◽

A. Stemberger ◽

H. Graeff

Keyword(s):

Molecular Weight ◽

Complex Formation ◽

Affinity Chromatography ◽

High Molecular Weight ◽

Calcium Chloride ◽

Gel Filtration ◽

Void Volume ◽

Soluble Fibrin ◽

Derivatives Of

Crosslinked high molecular weight derivatives of fibrin (fibrinoligomers) were observed during intravascular coagulation. It was the purpose of this study to investigate the complex formation of fibrin oligomers with fibrinogen and fibrinmonomer. Fibrinogen coupled to agarose (Fg-ag) as well as fi-brinmonomer coupled to agarose (Fm-ag) was used as substrate. Soluble oligomers of fibrin were produced by incubating fibrinogen with thrombin, calcium-chloride, cystein and F XIII. They were separated from fibrinogen by gel filtration. Γ-dimers were demonstrated in fractions from the void volume and the shoulder prior to the fibrinogen peak. These fractions were subjected to affinity chromatography. Crosslinked oligomers of fibrin were not adsorbed on Fg-ag, yet adsorption occured on Fm-ag. This indicates that fibrin oligomers have no affinity to fibrinogen, yet readily form complexes with fibrin. This could mean that in vivo they compete with fibrinogen for the fibrinmonomer part of soluble fibrin monomer complexes, and hence have a tendency to increase in size.

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Unmasking Snake Venom ofBothrops leucurus: Purification and Pharmacological and Structural Characterization of New PL Bleu TX-III

BioMed Research International ◽

10.1155/2013/941467 ◽

2013 ◽

Vol 2013 ◽

pp. 1-9 ◽

Cited By ~ 11

Author(s):

Fábio André Marangoni ◽

Luis Alberto Ponce-Soto ◽

Sergio Marangoni ◽

Elen Cristina Teizem Landucci

Keyword(s):

Snake Venom ◽

Intravenous Route ◽

Systemic Action ◽

Phospholipid Hydrolysis ◽

Multiple Alignments ◽

Sds Page ◽

One Step ◽

High Degree

Bleu TX-III was isolated fromBothrops leucurussnake venom on one-step analytical chromatography reverse phase HPLC, was homogeneous on SDS-PAGE, and was confirmed by Q-Tof Ultima API ESI/MS (TOF MS mode) mass spectrometry in 14243.8 Da. Multiple alignments of Bleu TX-III show high degree of homology with basic PLA2myotoxins from otherBothropsvenoms. Our studies on local and systemic myotoxicity “in vivo” reveal that Bleu TX-III is myotoxin with local but not systemic action due to the decrease in the plasmatic CK levels when Bleu TX-III is administrated by intravenous route in mice (dose 1 and 5 μg). And at a dose of 20 μg myotoxin behaves like a local and systemic action. Bleu TX-III induced moderate marked paw edema, evidencing the local increase in vascular permeability. The inflammatory events induced in the mice (I.M.) were investigated. The increase in the levels of IL-1, IL-6, and TNF-αwas observed in the plasma. It is concluded that Bleu TX-III induces inflammatory events in this model. The enzymatic phospholipid hydrolysis may be relevant to these phenomena.Bothrops leucurusvenom is still not extensively explored, and the knowledge of its toxins separately through the study of structure/function will contribute for a better understanding of its action mechanism.

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Affinity chromatography of pig heart fumarase

Biochemical Journal ◽

10.1042/bj1770115 ◽

1979 ◽

Vol 177 (1) ◽

pp. 115-119 ◽

Cited By ~ 3

Author(s):

S Chaudhuri ◽

E W Thomas

Keyword(s):

Affinity Chromatography ◽

Aromatic Ring ◽

Fumaric Acid ◽

Competitive Inhibitor ◽

Sepharose 4B

2-(5′-Phenylpentyl)fumaric acid was shown to be a competitive inhibitor (Ki 0.5 mM) of pig heart fumarase. After nitration of the aromatic ring, reduction to the amine and diazotization, the acid was attached via azo linkages to a Sepharose 4B-tyramine matrix. The resulting adsorbent was used for the affinity chromatography of crude fumarase, purifications of approx. 20-fold being obtained by specific elution with 0.01 M-citrate.

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Easy assembly of ligands for glycosidase affinity chromatography

Biochemical Journal ◽

10.1042/bj2700539 ◽

1990 ◽

Vol 270 (2) ◽

pp. 539-540 ◽

Cited By ~ 6

Author(s):

R C Bernotas ◽

B Ganem

Keyword(s):

Amino Acids ◽

Affinity Chromatography ◽

Enzyme Purification ◽

Reductive Amination ◽

Water Soluble ◽

High Yield ◽

Glycosidase Inhibitors ◽

Neutral Ph ◽

Sepharose 4B ◽

Derivatives Of

An improved, high-yield synthesis of the corresponding N-carboxypentyl derivatives of three iminoalditol glycosidase inhibitors has been developed for affinity chromatography enzyme purification. Reductive amination of 1-deoxynojirimycin (or its D-manno or D-galacto analogues) with methyl 5-formylvalerate and NaBH3CN at neutral pH afforted an aminoester which upon hydrolysis with aqueous 5% HCl gave the desired aminoacid in 97% overall yield. These amino acids could then be covalently attached using water-soluble carbodi-imide to 6-aminohexyl Sepharose 4B.

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Isolation of the membrane-bound 26 000-Mr penicillin-binding protein of Streptomyces strain K15 in the form of a penicillin-sensitive d-alanyl-d-alanine-cleaving transpeptidase

Biochemical Journal ◽

10.1042/bj2070109 ◽

1982 ◽

Vol 207 (1) ◽

pp. 109-115 ◽

Cited By ~ 14

Author(s):

M Nguyen-Distèche ◽

M Leyh-Bouille ◽

J M Ghuysen

Keyword(s):

Affinity Chromatography ◽

Binding Protein ◽

Peptidase Activity ◽

Penicillin Binding Protein ◽

Streptomyces Strain ◽

Step Procedure ◽

Membrane Bound ◽

Sepharose 4B

The membrane-bound, 26 000-Mr penicillin-binding protein of Streptomyces K15 has been isolated in the form of an effective, penicillin-sensitive D-alanyl-D-alanine-cleaving peptidase exhibiting high transpeptidase activity (greater than 95%) and very low carboxy-peptidase activity (less than 5%). The penicillin-binding protein/transpeptidase can be extracted directly from the mycelium with N-cetyl-NNN-trimethylammonium bromide (Cetavlon) and subsequently obtained at 90% purity and with an 8000-fold specific enrichment (when compared with the activity of the isolated membranes) by a two-step procedure involving Sephadex filtration and affinity chromatography on ampicillin-linked CH Sepharose 4B in the presence of detergent. At saturating concentrations of the co-substrates diacetyl-L-Lys-D-Ala-D-Ala and Gly-Gly, the catalytic-centre activity is about 0.3 s-1.

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Specific interaction of human Tamm-Horsfall gylcoprotein with leucoagglutinin, a lectin from Phaseolus vulgaris (red kidney bean)

Biochemical Journal ◽

10.1042/bj1830381 ◽

1979 ◽

Vol 183 (2) ◽

pp. 381-388 ◽

Cited By ~ 36

Author(s):

F Serafini-Cessi ◽

C Franceschi ◽

S Sperti

Keyword(s):

Phaseolus Vulgaris ◽

Affinity Chromatography ◽

Specific Interaction ◽

Lymphocyte Transformation ◽

Kidney Bean ◽

Step Procedure ◽

One Step ◽

Red Kidney Bean ◽

Antibody Reaction ◽

Antigen Antibody

Human Tamm-Horsfall glycoprotein inhibits lymphocyte transformation induced by leucoagglutinin and haemagglutinin from Phaseolus vulgaris (red kidney bean). The glycoprotein interacts with the two lectins, giving insoluble precipitates. The interaction with leucoagglutinin is highly specific, and the shape of the precipitin curve is that of an antigen-antibody reaction; precipitation is specifically inhibited by N-acetyl-D-galactosamine. Results are discussed, and it is suggested that inhibition of lymphocyte transformation is due to competition between human Tamm-Horsfall glycoprotein and carbohydrate receptors on lymphocytes for the two lectins. The interaction between human Tamm-Horsfall glycoprotein and Phaseolus vulgaris lectins has been used to develop a one-step procedure for the separation of the two lectins by affinity chromatography on (human Tamm-Horsfall-glycoprotein)-Sepharose.

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Expansion of Submucosal Bladder Wall TissueIn VitroandIn Vivo

BioMed Research International ◽

10.1155/2016/5415012 ◽

2016 ◽

Vol 2016 ◽

pp. 1-9

Author(s):

Gisela Reinfeldt Engberg ◽

Clara Ibel Chamorro ◽

Agneta Nordenskjöld ◽

Magdalena Fossum

Keyword(s):

Smooth Muscle ◽

Bladder Wall ◽

Subcutaneous Fat ◽

Culture Conditions ◽

Detrusor Muscle ◽

Step Procedure ◽

Standard Culture ◽

One Step

In order to develop autologous tissue engineering of the whole wall in the urinary excretory system, we studied the regenerative capacity of the muscular bladder wall. Smooth muscle cell expansion on minced detrusor musclein vitroandin vivowith or without urothelial tissue was studied. Porcine minced detrusor muscle and urothelium were culturedin vitrounder standard culture conditions for evaluation of the explant technique and in collagen for tissue sectioning and histology. Autografts of minced detrusor muscle with or without minced urothelium were expanded on 3D cylinder moulds by grafting into the subcutaneous fat of the pig abdominal wall. Moulds without autografts were used as controls. Tissue harvesting, mincing, and transplantation were performed as a one-step procedure. Cells from minced detrusor muscle specimens migrated and expandedin vitroon culture plastic and in collagen.In vivostudies with minced detrusor autografts demonstrated expansion and regeneration in all specimens. Minced urothelium autografts showed multilayered transitional urothelium when transplanted alone but not in cotransplantation with detrusor muscle; thus, minced bladder mucosa was not favored by cografting with minced detrusor. No regeneration of smooth muscle or epithelium was seen in controls.

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Galactose transfer to endogenous acceptors within Golgi fractions of rat liver.

The Journal of Cell Biology ◽

10.1083/jcb.92.1.139 ◽

1982 ◽

Vol 92 (1) ◽

pp. 139-146 ◽

Cited By ~ 63

Author(s):

J J Bergeron ◽

R A Rachubinski ◽

R A Sikstrom ◽

B I Posner ◽

J Paiement

Keyword(s):

Rat Liver ◽

Transferase Activity ◽

Cis Elements ◽

Galactosyl Transferase ◽

Highly Active ◽

Cell Biol ◽

Step Procedure ◽

One Step ◽

Low Activity

The distribution of galactosyl transferase was studied using trans and cis Golgi fractions isolated by a modification of the Ehrenreich et al. procedure (1973. J. Cell Biol. 59:45-72) as well as an intact Golgi fraction isolated by a new one-step procedure. Two methods of assay were used. The first method analyzed the ability of Golgi fractions to transfer galactose (from uridine diphosphogalactose [UDP-gal] substrate) to the defined exogenous acceptor ovomucoid. The second method assessed the transfer of galactose from UDP-gal substrate to endogenous acceptors (endogenous glycosylation). The trans Golgi fraction (Golgi light) was highly active by the first method but revealed only low activity by the second method. Golgi fractions enriched in central and cis elements (the Golgi intermediate, heavy and especially the intact Golgi fraction) were highly active in both methods of assay. The endogenous glycosylation approach was validated by gel fluorography of the endogenous acceptors. For all Golgi fractions, transfer of galactose was revealed to secretory glycopeptides. It is concluded that galactosyl transferase activity in vivo occurs primarily in central and cis Golgi elements but not trans Golgi vesicles.

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