scholarly journals Characterization of a metalloprotease from ovine chromaffin granules which cleaves a proenkephalin fragment (BAM12P) at a single arginine residue

1994 ◽  
Vol 301 (2) ◽  
pp. 607-614 ◽  
Author(s):  
N Tezapsidis ◽  
D C Parish
Keyword(s):  
Ion Exchange ◽  
Metal Chelate ◽  
Ion Exchange Chromatography ◽  
Exchange Chromatography ◽  
Arginine Residue ◽  
Affinity Column ◽  
Intermediate Step ◽  
Chromaffin Granules ◽  
Reverse Phase ◽  
Ph Optimum

A metalloprotease has been identified in ovine chromaffin granules which cleaves the proenkephalin fragment BAM12P to produce adrenorphin-Gly. This cleavage occurs at a single arginine residue and is an intermediate step in the production of the opiate adrenorphin in vivo. The identity of the product was confirmed by reverse-phase and ion-exchange chromatography. The adrenorphin-Gly-generating enzyme (AGE) was determined by chromatofocusing to have a pI value of 5.2 and bound strongly to a metal-chelate affinity column. After purification by gel-filtration and ion-exchange chromatography AGE was free of contaminating activities, as cleavage of radiolabelled BAM12P generated a single product as judged by reverse-phase and ion-exchange chromatography. The enzyme has a molecular mass of approx. 45 kDa and a pH optimum of 8.6 in Mops, Taps and Hepes buffers, but was inhibited by phosphate buffers. It was inhibited by micromolar concentrations of copper and zinc ions, but not by millimolar concentrations of calcium or manganese ions. The addition of BAM22P, dynorphin 1-13 or dynorphin 1-8 to the incubation mixture inhibited the cleavage of radiolabelled BAM12P. The cleavage was also inhibited by the presence of catecholamines at concentrations similar to those found within the chromaffin granule. This may explain the known effect of reserpine on chromaffin cells of reducing catecholamine levels and simultaneously increasing adrenorphin levels. It may also indicate a function for AGE and adrenorphin as reporters of intragranular conditions.

Review on the Application of Mixed-mode Chromatography for Separation of Structure Isoforms

2018 ◽  
Vol 20 (1) ◽  
pp. 56-60 ◽  
Author(s):  
Tsutomu Arakawa
Keyword(s):  
Ion Exchange ◽  
Mixed Mode ◽  
Ion Exchange Chromatography ◽  
Exchange Chromatography ◽  
Charged State ◽  
Partial Structure ◽  
Reverse Phase ◽  
Oligomer Formation ◽  
Structure Rearrangement ◽  
Disulfide Scrambling

Proteins often generate structure isoforms naturally or artificially due to, for example, different glycosylation, disulfide scrambling, partial structure rearrangement, oligomer formation or chemical modification. The isoform formations are normally accompanied by alterations in charged state or hydrophobicity. Thus, isoforms can be fractionated by reverse-phase, hydrophobic interaction or ion exchange chromatography. We have applied mixed-mode chromatography for fractionation of isoforms for several model proteins and observed that cation exchange Capto MMC and anion exchange Capto adhere columns are effective in separating conformational isoforms and self-associated oligomers.

scholarly journals Isolation of albumin from whole human plasma and fractionation of albumin-depleted plasma

1976 ◽  
Vol 157 (2) ◽  
pp. 301-306 ◽  
Author(s):  
J Travis ◽  
J Bowen ◽  
D Tewksbury ◽  
D Johnson ◽  
R Pannell
Keyword(s):  
Ion Exchange ◽  
Human Plasma ◽  
Plasma Proteins ◽  
Deae Cellulose ◽  
Ion Exchange Chromatography ◽  
Exchange Chromatography ◽  
Affinity Column ◽  
Plasma Albumin ◽  
Cibacron Blue ◽  
Blue Sepharose

The dye Cibacron Blue F-3-GA was conjugated to Sepharose to provide an affinity column for serum albumin. Passage of whole human plasma through a column of Cibacron Blue-Sepharose results in the removal of approx. 98% of the albumin. The latter can be quantitatively recovered by desorption with NaSCN. Albumin-depleted plasma can be readily resolved into discrete fractions by a combination of conventional biochemical techniques. In particular, the resolution of plasma proteins with properties similar to those of native human plasma albumin can readily be accomplished by ion-exchange chromatography of the Sepharose-dye-treated plasma on DEAE-cellulose.

scholarly journals Purification and properties of l-mandelate dehydrogenase and comparison with other membrane-bound dehydrogenases from Acinetobacter calcoaceticus

1987 ◽  
Vol 248 (3) ◽  
pp. 871-876 ◽  
Author(s):  
M E Hoey ◽  
N Allison ◽  
A J Scott ◽  
C A Fewson
Keyword(s):  
Lactate Dehydrogenase ◽  
Ion Exchange ◽  
Gel Filtration ◽  
Acinetobacter Calcoaceticus ◽  
Ion Exchange Chromatography ◽  
Exchange Chromatography ◽  
Ph Optimum ◽  
Purification And Properties ◽  
Potential Inhibitors ◽  
Triton X

L-Mandelate dehydrogenase was purified from Acinetobacter calcoaceticus by Triton X-100 extraction from a ‘wall + membrane’ fraction, ion-exchange chromatography on DEAE-Sephacel, (NH4)2SO4 fractionation and gel filtration followed by further ion-exchange chromatography. The purified enzyme was partially characterized with respect to its subunit Mr (44,000), pH optimum (7.5), pI value (4.2), substrate specificity and susceptibility to various potential inhibitors including thiol-blocking reagents. FMN was identified as the non-covalently bound cofactor. The properties of L-mandelate dehydrogenase are compared with those of D-mandelate dehydrogenase, D-lactate dehydrogenase and L-lactate dehydrogenase from A. calcoaceticus.

Ion-exchange high-performance liquid-chromatography steps in peptide purifications

1982 ◽  
Vol 2 (10) ◽  
pp. 803-811 ◽  
Author(s):  
Hedvig Von Bahr-Lindstróm ◽  
Ulla Moberg ◽  
Jórgen Sjódahl ◽  
Hans Jórnvall
Keyword(s):  
High Performance Liquid Chromatography ◽  
Liquid Chromatography ◽  
Ion Exchange ◽  
High Speed ◽  
High Performance ◽  
Ion Exchange Chromatography ◽  
Exchange Chromatography ◽  
Reverse Phase ◽  
Complete Purification ◽  
Ion Exchange Hplc

Ion-exchange high-performance liquid chromatography (HPLC; on Ultropac TSK DEAE and CM) is compared with conventional soft-gel ion-exchange chromatography in identical peptide purifications. The results show that separating properties are similar, but as expected, ion-exchange HPLC has a much higher resolving capacity and a higher sensitivity, and allows a considerably shorter total separation time. The same buffer systems as for conventional ion-exchange chromatography can be used, including urea to solubilize large peptides, if care is taken not to exceed the pH limits set by the column matrix. A complete purification scheme by HPLC in the nanomolar range, utilizing exclusion, ion-exchange, and reverse-phase chromatographies, is given with a complex peptide mixture from a digest of a large protein. Similar steps as in conventional soft-gel schemes can be utilized. It is concluded that ion-exchange HPLC is a suitable complement to commonly used reverse-phase HPLC steps and that it permits high speed and sensitivity over wide ranges of peptide sizes and amounts.

Separation and characterization of an α-glucosidase and maltase from Bacillus amyloliquefaciens

1985 ◽  
Vol 31 (8) ◽  
pp. 670-674 ◽  
Author(s):  
William M. Fogarty ◽  
Catherine T. Kelly ◽  
Sunil K. Kadam
Keyword(s):  
Molecular Weight ◽  
Ion Exchange ◽  
Growth Medium ◽  
Isoelectric Point ◽  
Bacillus Amyloliquefaciens ◽  
Ion Exchange Chromatography ◽  
Exchange Chromatography ◽  
Optimum Temperature ◽  
Ph Optimum

A novel α-glucosidase and a maltase were isolated from Bacillus amyloliquefaciens. The formation of both enzymes was induced by trehalose, sucrose, or lactose in the growth medium. Trehalose is by far the most efficient inducer of both systems. The α-glucosidase and maltase were separated and purified by ion-exchange chromatography on DEAE Bio-Gel A. Purified α-glucosidase hydrolysed p-nitrophenyl-α-D-glucoside, isomaltose, and isomaltotriose but sucrose, maltose, or related saccharides were not attacked. β-Glucosides and polymeric glucosides were not degraded. The optimum temperature for α-glucosidase activity was 40 °C and its pH optimum was 5.3. The molecular weight and isoelectric point (pI) of the enzyme were 27 000 and 4.6, respectively. Purified maltase attacked maltose and sucrose, while maltotriose and melezitose were hydrolysed at slower rates and p-nitrophenyl-α-D-glucoside was not degraded. Other properties of the maltase were as follows: optimum temperature for activity, 30 °C; pH optimum, 6.5; molecular weight, 64 000; and pI, 4.7.

Quantitative Amino Acid Analysis of Feedstuff Hydrolysates by Reverse Phase Liquid Chromatography and Conventional Ion-Exchange Chromatography

1984 ◽  
Vol 67 (5) ◽  
pp. 1024-1026
Author(s):  
Robert G Elkin
Keyword(s):  
Liquid Chromatography ◽  
Amino Acid ◽  
Ion Exchange ◽  
Soybean Protein ◽  
Amino Acid Content ◽  
Ion Exchange Chromatography ◽  
Exchange Chromatography ◽  
Reverse Phase ◽  
Reverse Phase Liquid Chromatography ◽  
Phase Liquid

Abstract Corn, soybean meal, and isolated soybean protein samples were acidhydrolyzed and analyzed for amino acid content by reverse phase liquid chromatography (LC) and by conventional ion-exchange chromatography (IEC) using an amino acid analyzer. The former method employed pre-column derivatization with orthophthalaldehyde (OPTA)/ethanethiol and fluorescence detection. In the LC procedure, glycine and threonine were not resolved, and proline and cyst(e)ine were not detected. In general, amino acid values obtained by LC and IEC compared closely within and across feedstuffs, and both agreed well with published amino acid composition data. The notable exceptions were aspartic acid, glutamic acid, and alanine. Results of this study suggest that reverse phase LC with pre-column OPTA derivatization can be applied to accurately measure primary amino acids in individual feedstuffs.

UDP-D-Glucose: Flavonol 3 -0 -and 7-O-Glucosyl Transferases from Young Leaves of Paederia scandens var. mairei

1993 ◽  
Vol 48 (7-8) ◽  
pp. 563-569 ◽  
Author(s):  
Nariyuki Ishikura ◽  
Zhi-qing Yang ◽  
Susumu Teramoto
Keyword(s):  
Ion Exchange ◽  
Ammonium Sulfate ◽  
Isoelectric Point ◽  
Ion Exchange Chromatography ◽  
Exchange Chromatography ◽  
The Other ◽  
Hydroxyl Group ◽  
Ph Optimum ◽  
Young Leaves ◽  
Paederia Scandens

Two different O-glucosyl transferases (F3GT and F7GT ) catalyzing the transfer of ᴅ-glucose from UDP-ᴅ-glucose to the 3 and 7 positions of flavonol, respectively, were isolated from the young leaves of Paederia scandens var. mairei. F3GT and F7GT, which were recovered in about a 1:1 ratio in the activity, were purified by about 140- and 136-fold, respectively, by precipitation with ammonium sulfate followed by ion exchange chromatography and chromatofocusing. F3GT and F7GT both had a pH optimum of 7.5 in Tris-HCl buffer, and an Mr of 43 kDa. Neither F3GT nor F7GT had a Mg2+ requirement. Both were inhibited by each 1 mᴍ of Zn2+, Cu2+, N-ethylmaleimide and p-chloromercuribenzoate, and both were stimulated by 14 mᴍ 2-M E . F3GT and F7GT were different from each other in having an isoelectric point (pI) at pH 5.12 and 4.50, respectively. F3GT mediated the transfer of D-glucose exclusively to the 3-hydroxyl group of kaempferol and some flavonols, but neither the 7-O-glucosides nor the 3-O-glucosides of their flavonols were able to accept D-glucose. On the other hand, F7GT mediated the transfer of D-glucose exclusively to the 7-hydroxyl group of kaempferol and some flavonols, and in addition, the 3-O -glucosides of kaempferol and quercetin were able to accept D-glucose thoughn less efficiently. Consequently, the possibility of sequential steps of 3-O- and then 7-O-glucosylations of flavonols to give the 3,7-di-O-glucoside was discussed.

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