scholarly journals An examination of potential matrices for the affinity chromatography of NADP+-linked dehydrogenases with special reference to 6-phosphogluconate dehydrogenase

1977 ◽  
Vol 161 (3) ◽  
pp. 561-568 ◽  
Author(s):  
P Griffiths ◽  
A Houlton ◽  
M J Adams ◽  
M J Harvey ◽  
P D G Dean
Keyword(s):  
Affinity Chromatography ◽  
Large Scale ◽  
Pyridoxal Phosphate ◽  
Specific Activity ◽  
Systematic Investigation ◽  
Inorganic Pyrophosphate ◽  
Phosphogluconate Dehydrogenase ◽  
Sheep Liver ◽  
Effects Of Ph ◽  
Careful Manipulation

1. 6-phosphogluconate dehydrogenase from sheep liver has been purified 350-fold by affinity chromatography with a final specific activity of 18 micronmol of NADP+/reduced min per mg of protein and an overall yield of greater than 40%. 2. A systematic investigation of potential ligands has been carried out: these included 6-phosphogluconate and NADP+, pyridoxal phosphate and several immobilized nucleotides. The results indicate that NADP+ is the most suitable ligand for the purification of 6-phosphogluconate dehydrogenase. 3. The effects of pH and alternative eluents have been examined in relation to the parameters known to affect the desorption phase of affinity chromatography; careful manipulation of the elution conditions permitted the separation of glucose 6-phosphate dehydrogenase, glutathione reductase and 6-phosphogluconate dehydrogenase from sheep liver on NADP+-Sepharose 4B. 4. A large-scale purification scheme for 6-phosphogluconate dehydrogenase is presented that uses the competitive inhibitors inorganic pyrophosphate and citrate as specific eluents.

LARGE SCALE PREPARATION OF VON WILLEBRAND FACTOR BY AFFINITY CHROMATOGRAPHY

1987 ◽  
Author(s):  
J Newman ◽  
D Farb
Keyword(s):  
Factor Viii ◽  
Affinity Chromatography ◽  
Von Willebrand Factor ◽  
Large Scale ◽  
Specific Activity ◽  
Protein A ◽  
Sephadex Column ◽  
Scale Preparation ◽  
Von Willebrand ◽  
Willebrand Factor

Treatment of bleeding in von Willebrand's disease usually consists of the infusion of cryoprecipitate or plasma. DDAVP is effective in some patients. Commercial concentrates for the treatment of Hemophilia A are ineffective as a source of von Willebrand Factor (vWF) replacement in von Willibrand's disease presumedly because of the absence of higher molecular weight forms of vWF protein. A vWF concentrate obtained during the course of preparation of an affinity purified Factor VIII may provide an alternative therapeutic agent without impacting the available Factor VIII supplies. The process used for the preparation of a highly purified Factor VIII concentrate (MonoclateTM, Armour Pharmaceutical Co.) from cryoprecipitate includes an affinity chromatography step which separates vWF/Factor VIII complex from other proteins in cryoprecipitate using an anti-vWF monoclonal antibody (C. A. Fulcher & T. S. Zimmerman, 1982). Factor VIII is then dissociated from the vWF remaining on the column and is eluted immediately by 3M sodium thiocyanante (NaSCN) as a step in the regeneration of the column. Unless the NaSCN is rapidly removed from the elutriant, the vWF activity as measured by platelet agglutination is destroyed. We have taken the NaSCN eluate and processed it immediately over an in-line G-10 or G-25 Sephadex column which removes the NaSCN while the vWF is eluted with a buffered isotonic solution. Alternatively, the vWF has been precipitated from the NaSCN by ammonium sulfate or polyethylene glycol. VWF prepared by any of these methods retains platelet agglutinating activity and has a distribution of vWF multimers similar to those of vWF in normal plasma.The potency of vWF prepared from cryoprecipitate by this process is 20 units/ml and the specific activity is 20 units/mg. In several fractionations of kilogram amounts of cryoprecipitate, vWF was isolated and subjected to lyophilization and heating at 68° for 30 hours without loss of bioactivity. Drug development in progress suggest that the combined effect of affinity chromatography and exposure to NaSCN may negate the need for a heating procedure to reduce the risk of viral transmittance.

Quinonoid dihydropterin reductase from beef liver

1977 ◽  
Vol 55 (11) ◽  
pp. 1145-1152 ◽  
Author(s):  
Karl K. Korri ◽  
Diana Chippel ◽  
Michael M. Chauvin ◽  
Anne Tirpak ◽  
K. G. Scrimgeour
Keyword(s):  
Molecular Weight ◽  
Affinity Chromatography ◽  
Specific Activity ◽  
Subunit Composition ◽  
Beef Liver ◽  
Enzyme Preparations ◽  
Ligand Affinity ◽  
Sheep Liver ◽  
Isolation Techniques ◽  
Reaction Proceeds

Quinonoid dihydropterin reductase has been purified from beef liver. This enzyme has been shown to be indistinguishable from the reductase of sheep liver in molecular weight, subunit composition, and terminal residues. Both beef and sheep liver reductases possess acyl isoleucine as the N-terminal residue. Use of improved isolation techniques, including general ligand affinity chromatography, has yielded enzyme preparations of much higher specific activity than previously reported. Affinity chromatography experiments also suggest that the enzymic reaction proceeds by a compulsory ordered mechanism.

scholarly journals The chemical mechanism of sheep liver 6-phosphogluconate dehydrogenase. A Schiff-base intermediate is not involved

1986 ◽  
Vol 234 (3) ◽  
pp. 671-677 ◽  
Author(s):  
C M Topham ◽  
K Dalziel
Keyword(s):  
Schiff Base ◽  
Metal Ions ◽  
Large Scale ◽  
Chemical Mechanism ◽  
Heavy Isotope ◽  
Phosphogluconate Dehydrogenase ◽  
Sheep Liver ◽  
Base Mechanism

[2-18O]Ribulose 5-phosphate was prepared and shown to be converted enzymically by 6-phosphogluconate dehydrogenase from sheep liver into 6-phosphogluconate with complete retention of the heavy isotope. This finding unequivocally excludes the possibility of a Schiff-base mechanism for the enzyme. The involvement of metal ions has already been excluded, and other possible mechanisms are discussed. The enzyme was purified by an improved large-scale procedure, which is briefly described.

In vivo Production of Soluble Inorganic Pyrophosphatases in Two Strains of Vibrio cholerae

1970 ◽  
Vol 24 (1) ◽  
pp. 38-41
Author(s):  
Taslima Taher Lina ◽  
Mohammad Ilias
Keyword(s):  
Vibrio Cholerae ◽  
Specific Activity ◽  
Experimental Conditions ◽  
Environmental Strain ◽  
Cell Extracts ◽  
Atcc Strain ◽  
The Family ◽  
Effects Of Ph ◽  
Vibrio Cholerae O1

The in vivo production of soluble inorganic pyrophosphatases (PPases) was investigated in two strains, namely, Vibrio cholerae EM 004 (environmental strain) and Vibrio cholerae O1 757 (ATCC strain). V. cholerae is known to contain both family I and family II PPase coding sequences. The production of family I and family II PPases were determined by measuring the enzyme activity in cell extracts. The effects of pH, temperature, salinity of the growth medium on the production of soluble PPases were studied. In case of family I PPase, V. cholerae EM 004 gave the highest specific activity at pH 9.0, with 2% NaCl + 0.011% NaF and at 37°C. The strain V. cholerae O1 757 gave the highest specific activity at pH 9.0, with media containing 0% NaCl and at 37°C. On the other hand, under all the conditions family II PPase did not give any significant specific activity, suggesting that the family II PPase was not produced in vivo in either strains of V. cholerae under different experimental conditions. Keywords: Vibrio cholerae, Pyrophosphatases (PPases), Specific activityDOI: http://dx.doi.org/10.3329/bjm.v24i1.1235 Bangladesh J Microbiol, Volume 24, Number 1, June 2007, pp 38-41

Large-scale production of citrate synthase from a cloned gene

1982 ◽  
Vol 60 (12) ◽  
pp. 1143-1147 ◽  
Author(s):  
Harry W. Duckworth ◽  
Alexander W. Bell
Keyword(s):  
Escherichia Coli ◽  
Large Scale ◽  
Citrate Synthase ◽  
Specific Activity ◽  
Tetracycline Resistance ◽  
Scale Production ◽  
Ampicillin Resistance ◽  
Colicin E1 ◽  
Large Scale Production ◽  
Cloned Gene

Starting with a colicin E1 resistance recombinant plasmid which contains gltA, the gene for citrate synthase in Escherichia coli, we have constructed an ampicillin-resistance plasmid containing the gltA region as a 2.9-kilobase-pair insert in the tetracycline-resistance region of pBR322. Escherichia coli HB101 harbouring this plasmid, when grown on rich medium containing ampicillin, contains citrate synthase as about 8% of its soluble protein. The enzyme has been purified from this rich source and is identical to the chromosomal enzyme prepared previously in every property tested, except for specific activity, which is 64 U∙mg−1 as compared with 45–50 U∙mg−1 previously obtained. The N-terminal sequences of both enzymes are reported, and they are identical up to residue 16 at least. The overall yield of pure enzyme, starting with the cells grown in 15 L of medium, is 600–800 mg.

scholarly journals The nature of carbon dioxide substrate and equilibrium constant of the 6-phosphogluconate dehydrogenase reaction

1969 ◽  
Vol 115 (4) ◽  
pp. 633-638 ◽  
Author(s):  
R. H. Villet ◽  
K. Dalziel
Keyword(s):  
Carbon Dioxide ◽  
Free Energy ◽  
Equilibrium Constant ◽  
Heat Of Reaction ◽  
Phosphogluconate Dehydrogenase ◽  
Sheep Liver ◽  
Dehydrogenase Reaction ◽  
Reductive Carboxylation ◽  
Primary Substrate

1. It was shown that dissolved CO2 and not HCO3− or H2CO3 is the primary substrate for reductive carboxylation with 6-phosphogluconate dehydrogenase from sheep liver. 2. The equilibrium constant of the reaction was measured in solutions of various ionic strengths and at several temperatures, and the free energy and heat of reaction were determined.

Cloning, Expression, Purification, and Characterization of the 6-Phosphogluconate Dehydrogenase from Sheep Liver

1998 ◽  
Vol 13 (2) ◽  
pp. 251-258 ◽  
Author(s):  
Lilian Chooback ◽  
Nancy E. Price ◽  
William E. Karsten ◽  
John Nelson ◽  
Paula Sundstrom ◽  
...  
Keyword(s):  
Purification And Characterization ◽  
Phosphogluconate Dehydrogenase ◽  
Sheep Liver

scholarly journals A Novel Purification Procedure for Active Recombinant Human DPP4 and the Inability of DPP4 to Bind SARS-CoV-2

2020 ◽  
Author(s):  
Cecy Xi ◽  
Arianna Arianna Di Fazio ◽  
Naveed Nadvi ◽  
Karishma Patel ◽  
Michelle Xiang ◽  
...  
Keyword(s):  
Affinity Chromatography ◽  
High Purity ◽  
Specific Activity ◽  
Specific Binding ◽  
Exchange Chromatography ◽  
Cell Surface Receptor ◽  
Spike Protein ◽  
High Yield ◽  
Purification Procedure ◽  
Dipeptidyl Peptidase 4

Proteases catalyse irreversible posttranslational modifications that often alter a biological function of the substrate. The protease dipeptidyl peptidase 4 (DPP4) is a pharmacological target in type 2 diabetes therapy primarily because it inactivates glucagon-like protein-1. DPP4 also has roles in steatosis, insulin resistance, cancers and inflammatory and fibrotic diseases. In addition, DPP4 binds to the spike protein of MERS virus, causing it to be the human cell surface receptor for that virus. DPP4 has been identified as a potential binding target of SARS-CoV-2 spike protein, so this question requires experimental investigation. Understanding protein structure and function requires reliable protocols for production and purification. We developed such strategies for baculovirus generated soluble recombinant human DPP4 (residues 29-766) produced in insect cells. Purification used differential ammonium sulfate precipitation, hydrophobic interaction chromatography, dye affinity chromatography in series with immobilised metal affinity chromatography, and ion exchange chromatography. The binding affinities of DPP4 to the SARS-CoV-2 full-length spike protein and its receptor binding domain (RBD) were measured using surface plasmon resonance. This optimised DPP4 purification procedure yielded 1 to 1.8 mg of pure fully active soluble DPP4 protein per litre of insect cell culture with specific activity >30 U/mg, indicative of high purity. No specific binding between DPP4 and CoV-2 spike protein was detected. In summary, a procedure for high purity high yield soluble human DPP4 was achieved and used to show that, unlike MERS, SARS-CoV-2 does not bind human DPP4.

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