scholarly journals Altered chromatographic behaviour of mitochondrial ADP/ATP translocase induced by stabilization of the protein by binding of 6′-O-fluorescein-atractyloside

2003 ◽  
Vol 376 (3) ◽  
pp. 757-763 ◽  
Author(s):  
Vernon R. SMITH ◽  
Ian M. FEARNLEY ◽  
John E. WALKER
Keyword(s):  
Specific Inhibitor ◽  
Binding Constants ◽  
Ion Exchange Chromatography ◽  
Exchange Chromatography ◽  
Mitochondrial Carrier ◽  
Chromatographic Behaviour ◽  
Dodecyl Maltoside ◽  
Mitochondrial Carrier Family ◽  
Purification Protocol ◽  
Major Conformation

Atractyloside (ATR) is a high-affinity specific inhibitor of the mitochondrial ADP/ATP translocase (AAT). The binding of a fluorescent derivative, 6´-O-fluorescein-ATR (FATR), to mitochondria has been characterized. The binding constants obtained are in agreement with previously published values for ATR, demonstrating that FATR is a suitable probe of the AAT. AAT inhibited by FATR (FATR-AAT) was solubilized in dodecyl maltoside and purified by two separate ion-exchange chromatography steps at different pHs, which allowed FATR-AAT to be purified to homogeneity. The presence of the bound fluorescent probe enabled the inhibited AAT to be distinguished from the unliganded protein during chromatography, as they were markedly different in their chromatographic behaviour. The purified FATR-AAT was dimeric and in a single major conformation containing 1 mole FATR per mole of AAT dimer. In contrast, uninhibited AAT was monomeric and conformationally unstable. Use of the fluorescent ATR derivative in the development of the protocol enabled the stable dimeric AAT to be monitored directly and purified more effectively. The purification protocol was repeated using non-derivatized ATR, and highly pure AAT was obtained that was devoid of other members of the mitochondrial carrier family.

scholarly journals Multi-element ion-exchange chromatography and high-precision MC-ICP-MS isotope analysis of Mg and Ti from sub-mm-sized meteorite inclusions

2018 ◽  
Vol 33 (4) ◽  
pp. 613-628 ◽  
Author(s):  
K. K. Larsen ◽  
D. Wielandt ◽  
M. Bizzarro
Keyword(s):  
Ion Exchange ◽  
High Precision ◽  
Isotope Analysis ◽  
Ion Exchange Chromatography ◽  
Exchange Chromatography ◽  
Chromatographic Purification ◽  
Icp Ms ◽  
Purification Protocol

We present a refined multi-element (Mg, Ca, Ti, V, Cr, Fe, Ni, Zr, Mo, Ru, Hf and W) ion chromatographic purification protocol applied to high-precision isotope analysis of Mg and Ti using MC-ICP-MS.

Degradation of endogenous heptadecapeptide gastrin by endopeptidase 24.11 in the pig

1987 ◽  
Vol 253 (1) ◽  
pp. G33-G39
Author(s):  
D. M. Power ◽  
N. Bunnett ◽  
A. J. Turner ◽  
R. Dimaline
Keyword(s):  
Specific Inhibitor ◽  
Ion Exchange Chromatography ◽  
Exchange Chromatography ◽  
Antral Mucosa ◽  
Venous Outflow ◽  
Endopeptidase 24.11 ◽  
Specific Antisera ◽  
Hydrolysis Of

Hydrolysis of heptadecapeptide gastrin (G-17) by endopeptidase 24.11 (EC 3.4.24.11) was studied in vivo and in vitro in the pig. Ion exchange chromatography and radioimmunoassay with three region-specific antisera were used to identify the products of porcine G-17 degradation. Incubation of antral extracts with pure endopeptidase 24.11 resulted in a substantial loss of intact G-17: 80% C-terminal immunoreactivity was lost in 60 min. This hydrolysis was completelyinhibited by phosphoramidon, which is a specific inhibitor of endopeptidase 24.11. In antral extracts G-17 accounted forgreater than 95% of total C-terminal immunoreactivity, compared with less than 60% C-terminal immunoreactivity in the gastric venous outflow; shorter C-terminal forms comprised the major part of the remaining immunoreactivity. After infusion of phosphoramidon, the concentration of intact G-17 was increased, and there was a corresponding reduction in the concentration of other C-terminal immunoreactive fragments. We conclude that endopeptidase 24.11 degrades G-17 in vitro and in vivo and may be responsible for the generation of C-terminal fragments from G-17 after secretion from the porcine antral mucosa.

Studies on an Inhibitor of Plasminogen Activation in Human Serum

1973 ◽  
Vol 30 (02) ◽  
pp. 414-424 ◽  
Author(s):  
Ulla Hedner
Keyword(s):  
Ion Exchange ◽  
Human Serum ◽  
Antithrombin Iii ◽  
Gel Filtration ◽  
Ion Exchange Chromatography ◽  
Exchange Chromatography ◽  
Specific Adsorption ◽  
Partial Purification ◽  
Plasminogen Activation ◽  
Preparative Electrophoresis

SummaryA procedure is described for partial purification of an inhibitor of the activation of plasminogen by urokinase and streptokinase. The method involves specific adsorption of contammants, ion-exchange chromatography on DEAE-Sephadex, gel filtration on Sephadex G-200 and preparative electrophoresis. The inhibitor fraction contained no antiplasmin, no plasminogen, no α1-antitrypsin, no antithrombin-III and was shown not to be α2 M or inter-α-inhibitor. It contained traces of prothrombin and cerulo-plasmin. An antiserum against the inhibitor fraction capable of neutralising the inhibitor in serum was raised in rabbits.

Preparation and Characterization of NH2-Terminal Fibrinogen Bβ Fragments from N-DSK of Human Fibrinogen

1984 ◽  
Vol 51 (01) ◽  
pp. 016-021 ◽  
Author(s):  
S Birken ◽  
G Agosto ◽  
B Lahiri ◽  
R Canfield
Keyword(s):  
High Performance ◽  
Gel Filtration ◽  
Ion Exchange Chromatography ◽  
Exchange Chromatography ◽  
Reverse Phase ◽  
Human Fibrinogen ◽  
Human Volunteers ◽  
Cnbr Cleavage ◽  
Two Peaks

SummaryIn order to investigate the early release of NH2-terminal plasmic fragments from the Bβ chain of fibrinogen, substantial quantities of Bβ 1-42 and Bβ 1-21 are required as immunogens, as radioimmunoassay standards and for infusion into human volunteers to determine the half-lives of these peptides. Towards this end methods that employ selective proteolytic cleavage of these fragments from fibrinogen have been developed. Both the N-DSK fragment, produced by CNBr cleavage of fibrinogen, and Bβ 1-118 were employed as substrates for plasmin with the finding of higher yields from N-DSK. Bβ 1-42 and Bβ 1-21 were purified by gel filtration and ion-exchange chromatography on SP-Sephadex using volatile buffers. When the purified preparation of Bβ 1-42 was chromatographed on reverse-phase high performance liquid chromatography, two peaks of identical amino acid composition were separated, presumably due either to pyroglutamate or to amide differences.

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.

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