Isolation of σ-(L-α-aminoadipyl)-L-cysteinyl-D-valine from culture broths by covalent chromatography

2007 ◽  
Vol 50 (4) ◽  
pp. 523-533 ◽  
Author(s):  
Colin D. Orford ◽  
Maxwell W. Adlard ◽  
David Perry
Keyword(s):  
Covalent Chromatography

scholarly journals A convenient method of preparation of high-activity urease from Canavalia ensiformis by covalent chromatography and an investigation of its thiol groups with 2,2'-dipyridyl disulphide as a thiol titrant and reactivity probe

1976 ◽  
Vol 159 (2) ◽  
pp. 245-257 ◽  
Author(s):  
R Norris ◽  
K Brocklehurst
Keyword(s):  
Convenient Method ◽  
Specific Activity ◽  
Class Ii ◽  
High Reactivity ◽  
Class I ◽  
Thiol Groups ◽  
Class Iii ◽  
Active Centre ◽  
Class Ib ◽  
Covalent Chromatography

1. A convenient method of preparation of jack-bean urease (EC3.5.1.5) involving covalent chromatography by thiol-disulphide interchange is described. 2. Urease thus prepared has specific activity comparable with the highest value yet reported (44.5 ± 1.47 kat/kg, Km = 3.32 ± 0.05 mM; kcat. = 2.15 × 104 ± 0.05 × 104s-1 at pH7.0 and 38°C). 3. Titration of the urease thiol groups with 2,2'-dipyridyl disulphide (2-Py-S-S-2-Py) and application of the method of Tsou Chen-Lu [(1962) Sci. Sin.11, 1535-1558] suggests that the urease molecule (assumed to have mol.wt. 483000 and ε280 = 2.84 × 105 litre·mol-1-cm-1) contains 24 inessential thiol groups of relatively high reactivity (class-I), six ‘essential’ thiol groups of low reactivity (class-II) and 54 buried thiol groups (class-III) which are exposed in 6M-guanidinium chloride. 4. The reaction of the class-I thiol groups with 2-Py-S-S-2-Py was studied in the pH range 6-11 at 25°C(I = 0.1 mol/l) by stopped-flow spectrophotometry, and the analogous reaction of the class-II thiol groups by conventional spectrophotometry. 5. The class-I thiol groups consist of at least two sub-classes whose reactions with 2-Py-S-S-2-Py are characterized by (a) pKa = 9.1, k = 1.56 × 104M-1·s-1 and (b) pKa = 8.1, k = 8.05 × 102M-1·s-1 respectively. The reaction of the class-II thiol groups is characterized by pKa = 9.15 and k = 1.60 × 102M-1·s-1. 6. At pH values 7-8 the class-I thiol groups consist of approx. 50% class-Ia groups and 50% class-Ib groups. The ratio class Ia/class Ib decreases as the pH is raised according to a pKa value ≥ approx. 9.5, and at high pH the class-I thiol groups consist of at most 25% class-Ia groups and at least 75% class-Ib groups. 7. The reactivity of the class-II thiol groups towards 2-Py-S-S-2-Py is insensitive to the nature of the group used to block the class-I thiols. 8. All the ‘essential’ thiol groups in urease appear to be eeactive only as uncomplicated thiolate ions. The implications of this for the active-centre chemistry of urease relative to that of the thiol proteinases are discussed.

Preparation of bovine mercaptalbumin by means of covalent chromatography

FEBS Letters ◽  
1974 ◽  
Vol 42 (2) ◽  
pp. 183-186 ◽  
Author(s):  
Jan Carlsson ◽  
Anders Svenson
Keyword(s):  
Covalent Chromatography

Covalent Chromatography, A Powerful Tool in Sequence Studies of Membrane Proteins

1987 ◽  
pp. 189-209 ◽  
Author(s):  
Yuri A. Ovchinnikov ◽  
Najmutin G. Abdulaev ◽  
Alexander S. Bogachuk ◽  
Carol A. Morris
Keyword(s):  
Membrane Proteins ◽  
Covalent Chromatography

scholarly journals Uncoupling protein 1 binds one nucleotide per monomer and is stabilized by tightly bound cardiolipin

2015 ◽  
Vol 112 (22) ◽  
pp. 6973-6978 ◽  
Author(s):  
Yang Lee ◽  
Chrissie Willers ◽  
Edmund R. S. Kunji ◽  
Paul G. Crichton
Keyword(s):  
Adipose Tissue ◽  
Brown Adipose Tissue ◽  
Uncoupling Protein ◽  
Size Exclusion ◽  
Uncoupling Protein 1 ◽  
Mitochondrial Carriers ◽  
Mitochondrial Inner Membrane ◽  
Brown Adipose ◽  
Integral Role ◽  
Covalent Chromatography

Uncoupling protein 1 (UCP1) catalyzes fatty acid-activated, purine nucleotide-sensitive proton leak across the mitochondrial inner membrane of brown adipose tissue to produce heat, and could help combat obesity and metabolic disease in humans. Studies over the last 30 years conclude that the protein is a dimer, binding one nucleotide molecule per two proteins, and unlike the related mitochondrial ADP/ATP carrier, does not bind cardiolipin. Here, we have developed novel methods to purify milligram amounts of UCP1 from native sources by using covalent chromatography that, unlike past methods, allows the protein to be prepared in defined conditions, free of excess detergent and lipid. Assessment of purified preparations by TLC reveal that UCP1 retains tightly bound cardiolipin, with a lipid phosphorus content equating to three molecules per protein, like the ADP/ATP carrier. Cardiolipin stabilizes UCP1, as demonstrated by reconstitution experiments and thermostability assays, indicating that the lipid has an integral role in the functioning of the protein, similar to other mitochondrial carriers. Furthermore, we find that UCP1 is not dimeric but monomeric, as indicated by size exclusion analysis, and has a ligand titration profile in isothermal calorimetric measurements that clearly shows that one nucleotide binds per monomer. These findings reveal the fundamental composition of UCP1, which is essential for understanding the mechanism of the protein. Our assessment of the properties of UCP1 indicate that it is not unique among mitochondrial carriers and so is likely to use a common exchange mechanism in its primary function in brown adipose tissue mitochondria.

scholarly journals Differences in the interaction of the catalytic groups of the active centres of actinidin and papain Rapid purification of fully active actinidin by covalent chromatography and characterization of its active centre by use of two-protonic-state reactivity probes

1981 ◽  
Vol 197 (3) ◽  
pp. 739-746 ◽  
Author(s):  
K Brocklehurst ◽  
B S Baines ◽  
J P Malthouse
Keyword(s):  
Cysteine Proteinase ◽  
Thiol Group ◽  
Interactive System ◽  
Active Centre ◽  
Ph Values ◽  
Hydrophobic Binding ◽  
Rapid Purification ◽  
Active Centres ◽  
Covalent Chromatography

1. A rapid method of isolation of fully active actinidin, the cysteine proteinase from Actinidia chinensis (Chinese gooseberry or kiwifruit), by covalent chromatography, was devised. 2. The active centre of actinidin was investigated by using n-propyl 2-pyridyl disulphide, 4-(N-aminoethyl 2′-pyridyl disulphide)-7-nitrobenzo-2-oxa-1,3-diazole and 4-chloro-7-nitrobenzofurazan as reactivity probes. 3. The presence in actinidin in weakly acidic media of an interactive system containing a nucleophilic sulphur atom was demonstrated. 4. The pKa values (3.1 and 9.6) that characterize this interactive system are more widely separated than those that characterize the interactive active centre systems of ficin (EC 3.4.22.3) and papain (EC 3.4.22.2) (3.8 and 8.6, and 3.9 and 8.8 respectively). 5. Actinidin was shown to resemble ficin rather than papain in (i) the disposition of the active-centre imidazole group with respect to hydrophobic binding areas, and (ii) the inability of the active-centre aspartic acid carboxy group to influence the reactivity of the active-centre thiol group at pH values of about 4. 6. The implications of the results for one-state and two-state mechanisms for cysteine-proteinase catalysis are discussed.

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