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

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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Phenylalanine and Tyrosine Biosynthesis in Sporeforming Members of the Order Actinomycetales

Zeitschrift für Naturforschung C ◽

10.1515/znc-1987-0410 ◽

1987 ◽

Vol 42 (4) ◽

pp. 387-393 ◽

Cited By ~ 6

Author(s):

Hilda-K. Hund ◽

Brigitte Keller ◽

Franz Lingens

Keyword(s):

Anion Exchange ◽

Deae Cellulose ◽

Anion Exchange Chromatography ◽

Exchange Chromatography ◽

Chorismate Mutase ◽

Biosynthetic Enzymes ◽

Prephenate Dehydratase ◽

Prephenate Dehydrogenase ◽

Arogenate Dehydrogenase ◽

Tyrosine Biosynthesis

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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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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Purification and Characterization of Two Endo-β-1,4-glucanases from Mollusca, Ampullaria crossean

Acta Biochimica et Biophysica Sinica ◽

10.1111/j.1745-7270.2005.00099.x ◽

2005 ◽

Vol 37 (10) ◽

pp. 702-708 ◽

Cited By ~ 17

Author(s):

Yan-Hong Li ◽

Rui Guo ◽

Qiu-Yu Yin ◽

Ming Ding ◽

Si-Liang Zhang ◽

...

Keyword(s):

Anion Exchange ◽

Carboxymethyl Cellulose ◽

Polyacrylamide Gel Electrophoresis ◽

Sodium Dodecylsulfate ◽

Gel Filtration ◽

Residual Activity ◽

Hydrolytic Activity ◽

Anion Exchange Chromatography ◽

Exchange Chromatography ◽

The Stability

Abstract Two novel endo-β-1,4-glucanases, EG45 and EG27, were isolated from the gastric juice of mollusca, Ampullaria crossean, by anion exchange, hydrophobic interaction, gel filtration and a second round of anion exchange chromatography. The purified proteins EG45 and EG27 appeared as a single band on sodium dodecylsulfate polyacrylamide gel electrophoresis with a molecular mass of 45 kDa and 27 kDa, respectively. The optimum pH for CMC activity was 5.5 for EG45 and 4.4-4.8 for EG27. The optimum temperature range for EG27 was broad, between 50 °C and 60 °C; for EG45 it was 50 °C. The analysis on the stability of these two endo-β-1,4-glucanases showed that EG27 was acceptably stable at pH 3.0-11.0 even when the incubation time was prolonged to 24 h at 30 °C, whereas EG45 remained relatively stable at pH 5.0-8.0. About 85% of the activity of EG27 could be retained upon incubation at 60 °C for 24 h. However, less than 10% residual activity of EG45 was detected at 50 °C. Among different kinds of substrates, both enzymes showed a high preference for carboxymethyl cellulose. EG45, in particular, showed a carboxymethyl cellulose hydrolytic activity of 146.5 IU/mg protein. Both enzymes showed low activities to xylan (from oat spelt) and Sigmacell 101, and they were inactive to p-nitrophenyl-β-D-cellobioside, salicin and starch.

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The stability of oxaloacetate on anion-exchange chromatography

Biochemical Society Transactions ◽

10.1042/bst0100450 ◽

1982 ◽

Vol 10 (6) ◽

pp. 450-450

Author(s):

MAUREEN LEONARD ◽

ROBERT A. JOHN ◽

CHARLES F. A. BRYCE

Keyword(s):

Anion Exchange ◽

Anion Exchange Chromatography ◽

Exchange Chromatography ◽

The Stability

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Aberrant 3H labelling of ATP during in vivo labelling of Ehrlich mouse ascites tumour cells with [2-3H]inositol is significant in the study of isomers of InsP3 and InsP4

Biochemical Journal ◽

10.1042/bj3000859 ◽

1994 ◽

Vol 300 (3) ◽

pp. 859-863 ◽

Cited By ~ 3

Author(s):

S Christensen ◽

H Harbak ◽

L O Simonsen

Keyword(s):

Anion Exchange ◽

Ambient Pressure ◽

Anion Exchange Chromatography ◽

Exchange Chromatography ◽

Tumour Cells ◽

Ascites Tumour ◽

Small Column ◽

Mouse Ascites ◽

Ascites Tumour Cells

Neutralized perchloric acid extracts of intra-abdominally proliferating Ehrlich mouse ascites-tumour cells harvested after 24 h exposure to [2-3H]inositol were analysed by Mono Q HR5/5 anion-exchange h.p.l.c. using an ammonium formate/phosphoric acid gradient or by ambient-pressure small-column anion-exchange chromatography (Bio-Rad AG 1-X8, 200-400 mesh). The results showed that cellular ATP contained aberrant 3H label in excess of 3H in the isomers of InsP3 and InsP4. The putative ATP 3H radioactivity showed: (i) h.p.l.c. run time as the material causing the largest A254 peak traced, (ii) precise spiking with ATP and [14C]ATP and (iii) specific absorption to charcoal. Moreover, enzymic conversion of ATP into ADP changed putative ATP 3H into putative ADP 3H. In addition, aberrant 3H labelling of cellular ADP and GTP was detected, although at a lower level.

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A 63 kDa phosphoprotein undergoing rapid dephosphorylation during exocytosis in Paramecium cells shares biochemical characteristics with phosphoglucomutase

Biochemical Journal ◽

10.1042/bj3090557 ◽

1995 ◽

Vol 309 (2) ◽

pp. 557-567 ◽

Cited By ~ 10

Author(s):

T Treptau ◽

R Kissmehl ◽

J D Wissmann ◽

H Plattner

Keyword(s):

Sarcoplasmic Reticulum ◽

Molecular Mass ◽

Protein Kinases ◽

Anion Exchange ◽

Anion Exchange Chromatography ◽

Enzymic Activity ◽

Exchange Chromatography ◽

Paramecium Tetraurelia ◽

Release Channel

We have enriched phosphoglucomutase (PGM; EC 5.4.2.2) approximately 20-fold from Paramecium tetraurelia cells by combined fractional precipitation with (NH4)2SO4, gel filtration and anion-exchange chromatography yielding two PGM peaks. Several parameters affecting PGM enzymic activity, molecular mass and pI were determined. Phosphorylation studies were done with isolated endogenous protein kinases. Like the 63 kDa phosphoprotein PP63, which is dephosphorylated within 80 ms during synchronous trichocyst exocytosis [Höhne-Zell, Knoll, Riedel-Gras, Hofer and Plattner (1992) Biochem. J. 286, 843-849], PGM has a molecular mass of 63 kDa and forms of identical pI. Since mammalian PGM activity depends on the presence of glucose 1,6-bisphosphate (Glc-1,6-P2) (which is lost during anion-exchange chromatography), we analysed this aspect with Paramecium PGM. In this case PGM activity was shown not to be lost, due to p-nitrophenyl phosphate-detectable phosphatase(s) (which we have separated from PGM), but also due to loss of Glc-1,6-P2. Like PGM from various vertebrate species, PGM activity from Paramecium can be fully re-established by addition of Glc-1,6-P2 at 10 nM, and it is also stimulated by bivalent cations and insensitive to chelating or thiol reagents. The PGM which we have isolated can be phosphorylated by endogenous cyclic-GMP-dependent protein kinase or by endogenous casein kinase. This results in three phosphorylated bands of identical molecular mass and pI values, as we have shown to occur with PP63 after phosphorylation in vivo (forms with pI 6.05, 5.95, 5.85). In ELISA, antibodies raised against PGM from rabbit skeletal muscle were reactive not only with original PGM but also with PGM fractions from Paramecium. Therefore, PGM and PP63 seem to be identical with regard to widely different parameters, i.e. co-elution by chromatography, molecular mass, phosphorylation by the two protein kinases tested, pI values of isoforms, and immuno-binding. Recent claims that PP63 (‘parafusin’) would not be identical with PGM specifically in Paramecium are critically evaluated. Since some glycolytic enzymes are discussed as being associated with the Ca(2+)-release channel in muscle sarcoplasmic reticulum, and since sub-plasmalemmal Ca2+ stores in Paramecium closely resemble sarcoplasmic reticulum, a possible function of PP63/PGM in exocytosis regulation is discussed, particularly since dephosphorylation strictly parallels exocytosis.

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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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Decorating sdAbs with Chelators: Effect of Conjugation on Biodistribution and Functionality

Pharmaceuticals ◽

10.3390/ph14050407 ◽

2021 ◽

Vol 14 (5) ◽

pp. 407

Author(s):

Henri Baudhuin ◽

Janik Puttemans ◽

Heleen Hanssens ◽

Philippe Vanwolleghem ◽

Sophie Hernot ◽

...

Keyword(s):

Anion Exchange ◽

Anion Exchange Chromatography ◽

Exchange Chromatography ◽

In Vivo Studies ◽

Liver Uptake ◽

Organ Uptake ◽

Pharmacokinetic Properties ◽

Single Domain Antibodies ◽

The Impact

Single domain antibodies (sdAbs) have proven to be valuable probes for molecular imaging. In order to produce such probes, one strategy is the functionalization of the reactive amine side chain of lysines with a chelator, resulting in a mixture of compounds with a different degree of conjugation. In this study, we implemented anion exchange chromatography (AEX) to separate the different compounds or fractions that were further characterized and evaluated to study the impact of the conjugation degree on pharmacokinetic properties and functionality. Anti-HER2 and anti-MMR sdAbs were functionalized with NOTA or DTPA chelator. Anion exchange chromatography was performed using 0.02 mol/L Tris pH 7.5 as the first solvent and 0.25 M or 0.4 M NaCl (in case of NOTA chelator or DTPA chelator, respectively) as the second solvent applied as a gradient. The fractions were characterized via mass spectrometry (MS), surface plasmon resonance (SPR), and isoelectric focusing gel electrophoresis (IEF), while in vivo studies were performed after radiolabeling with either 68Ga (NOTA) or 111In (DTPA) to assess the impact of the conjugation degree on pharmacokinetics. AEX could successfully be applied to separate fractions of (chelator)n-anti-HER2 and (chelator)n-anti-MMR sdAb constructs. MS confirmed the identity of different peaks obtained in the separation process. SPR measurement suggests a small loss of affinity for (chelator)3-anti-sdAb, while IEF revealed a correlated decrease in isoelectric point (pI) with the number of conjugated chelators. Interestingly, both the reduction in affinity and in pI was stronger with the DTPA chelator than with NOTA for both sdAbs. In vivo data showed no significant differences in organ uptake for any construct, except for (DTPA)n-anti-MMR, which showed a significantly higher liver uptake for (DTPA)1-anti-MMR compared to (DTPA)2-anti-MMR and (DTPA)3-anti-MMR. For all constructs in general, high kidney uptake was observed, due to the typical renal clearance of sdAb-based tracers. The kidney uptake showed significant differences between fractions of a same construct and indicates that a higher conjugation degree improves kidney clearance. AEX allows the separation of sdAbs with a different degree of conjugation and provides the opportunity to further characterize individual fractions. The conjugation of a chelator to sdAbs can alter some properties of the tracers, such as pI; however, the impact on the general biodistribution profile and tumor targeting was minimal.

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