scholarly journals Bovine liver thiol-protein disulphide oxidoreductases. An alternative method for differential purification and resolution of protein disulphide-isomerase and glutathione-insulin transhydrogenase

1980 ◽  
Vol 191 (2) ◽  
pp. 389-393 ◽  
Author(s):  
D A Hillson ◽  
R B Freedman
Keyword(s):  
Ion Exchange ◽  
Rapid Method ◽  
Bovine Liver ◽  
Ion Exchange Chromatography ◽  
Exchange Chromatography ◽  
Protein Disulphide Isomerase ◽  
The Relationship ◽  
Thiol Protein ◽  
Homogeneous Preparation ◽  
Covalent Chromatography

1. Protein disulphide-isomerase (EC 5.3.4.1) and glutathione-insulin transhydrogenase (EC 1.8.4.2) activities in bovine liver were studied in parallel during purification of ‘thiol-protein disulphide oxidoreductase’ by the procedure of Carmichael, Morin & Dixon [(1977) J Biol. Chem. 252, 7163-7167]. The two activities showed no quantitative co-purification and were partially resolved by (NH4)SO4 precipitation, indicating that distinct enzymes are present. 2. Protein disulphide-isomerase was purified by a relatively rapid method involving a combination of the early stages of the Carmichael procedure and covalent chromatography, with a new stepwise elution procedure. Ion-exchange chromatography yields a homogeneous preparation of mol.wt. 57 000. 3. The relationship between protein disulphide-isomerase, glutathione-insulin transhydrogenase and ‘thiol-protein disulphide oxidoreductase’ is discussed.

scholarly journals Resolution of protein disulphide-isomerase and glutathione-insulin transhydrogenase activities by covalent chromatography

1980 ◽  
Vol 191 (2) ◽  
pp. 373-388 ◽  
Author(s):  
D A Hillson ◽  
R B Freedman
Keyword(s):  
Ion Exchange ◽  
Sodium Dodecyl ◽  
Liver Homogenate ◽  
Bovine Liver ◽  
Ion Exchange Chromatography ◽  
Exchange Chromatography ◽  
Major Band ◽  
Protein Disulphide Isomerase ◽  
Reducing Conditions ◽  
Covalent Chromatography

1. Protein disulphide-isomerase (EC 5.3.4.1) and glutathione-insulin transhydrogenase (EC 1.8.4.2) were resolved by covalent chromatography. Both activities, in a partially purified preparation from bovine liver, bind covalently as mixed disulphides to activated thiopropyl-Sepharose 6B, in a new stepwise elution procedure protein disulphide-isomerase is displaced in mildly reducing conditions whereas glutathione-insulin transhydrogenase is only displaced by more extreme reducing conditions. 2. This together with evidence for partial resolution of the two activities by ion-exchange chromatography, conclusively establishes that the two activities are not alternative activities of a single bovine liver enzyme. 3. Protein disulphide-isomerase, partially purified by a published procedure, has now been further purified by covalent chromatography and ion-exchange chromatography. The final material is 560-fold purified relative to a bovine liver homogenate; it has barely detectable glutathione-insulin transhydrogenase activity. 4. The purified protein disulphide-isomerase shows a single major band on sodium dodecyl sulphate/polyacrylamide-gel electrophoresis corresponding to a mol.wt. of 57000. 5. The purified protein disulphide-isomerase has Km values for ‘scrambled’ ribonuclease and dithiothreitol of 23 microgram/ml and 5.4 microM respectively and has a sharp pH optimum at 7.5. The enzyme has a broad thiol-specificity, and several monothiols, at 1mM, can replace dithiothreitol. 6. The purified protein disulphide-isomerase is completely inactivated after incubation with a 2-3 fold molar excess of iodoacetate. The enzyme is also significantly inhibited by low concentrations of Cd2+ ions. These findings strongly suggest the existence of a vicinal dithiol group essential for enzyme activity. 7. When a range of thiols were used as co-substrates for protein disulphide-isomerase activity, the activities were found to co-purify quantitatively, implying the presence of a single protein disulphide-isomerase of broad thiol-specificity. Glutathione-disulphide transhydrogenase activities, assayed with a range of disulphide compounds, did not co-purify quantitatively.

scholarly journals Thiol-protein disulphide oxidoreductases. Differences between protein disulphide-isomerase and glutathione-insulin transhydrogenase activities in ox liver

1976 ◽  
Vol 159 (2) ◽  
pp. 385-393 ◽  
Author(s):  
H C Hawkins ◽  
R B Freedman
Keyword(s):  
High Resolution ◽  
Ion Exchange ◽  
Ion Exchange Chromatography ◽  
Exchange Chromatography ◽  
Heat Denaturation ◽  
Differential Centrifugation ◽  
Protein Disulphide Isomerase ◽  
Final Sample ◽  
Thiol Protein ◽  
Single Enzyme

1. Protein disulphide-isomerase and glutathione-insulin transhydrogenase activities were assayed in parallel through a conventional purification of protein disulphide-isomerase from ox liver. 2. Throughout a series of purification steps (differential centrifugation, acetone extraction, (NH4)2SO4 precipitation and ion-exchange chromatography), the two activities appeared in the same fractions but were purified to different extents. 3. The final sample was 143-fold purified in protein disulphide-isomerase but only 10-fold purified in glutathione-insulin transhydrogenase; nevertheless the two activities in this preparation were not resolved by high-resolution isoelectric focusing and both showed pI4.65. 4. In a partially purified preparation containing both activities, glutathione-insulin transhydrogenase was far more sensitive to heat denaturation than was protein disulphide-isomerase; conversely protein disulphide-isomerase was more sensitive to inactivation by deoxycholate. 5. The data are inconsistent with a single enzyme being responsible for all the protein disulphide-isomerase and glutathione-insulin transhydrogenase activity of ox liver. It is suggested that several similiar thiol-protein disulphide oxidoreductases of overlapping specificities may better account for the data.

The relationship between Candida species charge density and chitosan activity evaluated by ion-exchange chromatography

2011 ◽  
Vol 879 (31) ◽  
pp. 3749-3751 ◽  
Author(s):  
A. Palmeira-de-Oliveira ◽  
L.A. Passarinha ◽  
C. Gaspar ◽  
R. Palmeira-de-Oliveira ◽  
B. Sarmento ◽  
...  
Keyword(s):  
Ion Exchange ◽  
Charge Density ◽  
Candida Species ◽  
Ion Exchange Chromatography ◽  
Exchange Chromatography ◽  
The Relationship

THE RELATIONSHIP OF RADIOIMMUNOASSAY TO BIOASSAY: IN VITRO STUDIES WITH SYNTHETIC LYSINE VASOPRESSIN IN AQUEOUS SOLUTION INACTIVATED BY HEAT

1978 ◽  
Vol 88 (3) ◽  
pp. 465-473 ◽  
Author(s):  
Hanne Løve Lembøl
Keyword(s):  
Aqueous Solution ◽  
Ion Exchange ◽  
Arginine Vasopressin ◽  
Ion Exchange Chromatography ◽  
Exchange Chromatography ◽  
Lysine Vasopressin ◽  
Relationship Of ◽  
The Relationship ◽  
Immunological Heterogeneity

ABSTRACT The relationship of radioimmunoassay to pressor assay and antidiuretic assay was investigated in a simple in vitro system of synthetic lysine vasopressin in aqueous solution inactivated by heating at 100°C for 9, 18, 27, 36, 54 and 72 h. An apparent dissociation between radioimmunoassay and bioassay was demonstrated, with biological activity being lost more rapidly than immunological activity. The half-times were 32 h for radioimmunoassay, 23 h for antidiuretic assay and 22 h for pressor assay. However, ion-exchange chromatography showed immunological heterogeneity but biological homogeneity of the lysine vasopressin used, and indicated that the presence of impurities in the vasopressin might to some extent explain the discrepancy between assay results. Synthetic arginine vasopressin and arginine vasopressin of pituitary origin showed a similar immunological heterogeneity by ion-exchange chromatography.

scholarly journals A simple procedure for the isolation of protein disulphide-isomerase

1987 ◽  
Vol 247 (1) ◽  
pp. 237-239 ◽  
Author(s):  
J Koivu ◽  
R Myllylä ◽  
K I Kivirikko
Keyword(s):  
Ion Exchange ◽  
Affinity Chromatography ◽  
Simple Procedure ◽  
Ion Exchange Chromatography ◽  
Exchange Chromatography ◽  
Beta Subunit ◽  
Protein Disulphide Isomerase ◽  
Step Procedure ◽  
Beta 2

A two-step procedure is described for the purification of protein disulphide-isomerase (PDI). This procedure is based on the previous finding that the beta-subunit of the prolyl 4-hydroxylase tetramer (alpha 2 beta 2) is identical with PDI [Koivu, Myllylä, Helaakoski, Pihlajaniemi, Tasanen & Kivirikko (1987) J. Biol. Chem. 262, 6447-6449; Pihlajaniemi, Helaakoski, Tasanen, Myllylä, Huhtala, Koivu & Kivirikko (1987) EMBO J. 6, 643-649]. The procedure involves purification of the prolyl 4-hydroxylase tetramer by a simple affinity chromatography and subsequent isolation of the beta-subunit from the dissociated tetramer by ion-exchange chromatography.

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.

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