scholarly journals Oxidative refolding of recombinant prochymosin

1999 ◽  
Vol 340 (1) ◽  
pp. 345-351 ◽  
Author(s):  
Chongjuan WEI ◽  
Bin TANG ◽  
Yuying ZHANG ◽  
Kaiyu YANG
Keyword(s):  
Inclusion Bodies ◽  
Tertiary Structure ◽  
Reduced Form ◽  
Thiol Groups ◽  
Terminal Domain ◽  
Free Thiol ◽  
Rate Limiting Step ◽  
Oxidative Refolding ◽  
Disulphide Bonds ◽  
Two Stages

The disulphide-coupled refolding of recombinant prochymosin from Escherichia coli inclusion bodies was investigated. Prochymosin solubilized from inclusion bodies is endowed with free thiol groups and disulphide bonds. This partially reduced form undergoes renaturation more efficiently than the fully reduced form, suggesting that some native structural elements existing in inclusion bodies and remaining after denaturation function as nuclei to initiate correct refolding. This assumption is supported by the finding that in the solubilized prochymosin molecule the cysteine residues located in the N-terminal domain of the protein are not incorrectly paired with the other cysteines in the C-terminal domain. Addition of GSH/GSSG into the refolding system facilitates disulphide rearrangement and thus enhances renaturation, especially for the fully reduced prochymosin. Based on the results described in this and previous papers [Tang, Zhang and Yang (1994) Biochem. J. 301, 17-20], a model to depict the refolding process of prochymosin is proposed. Briefly, the refolding process of prochymosin consists of two stages: the formation and rearrangement of disulphide bonds occurs at the first stage in a pH 11 buffer, whereas the formation and adjustment of tertiary structure leading to the native conformation takes place at the second stage at pH 8. The pH 11 conditions help polypeptides to refold in such a way as to favour the formation of native disulphide bonds. Disulphide rearrangement, the rate-limiting step during refolding, can be achieved by thiol/disulphide exchange initiated by free thiol groups present in the prochymosin polypeptide, GSH/GSSG or protein disulphide isomerase.

scholarly journals Thiol groups of normal immunoglobulin G

1974 ◽  
Vol 137 (2) ◽  
pp. 281-289 ◽  
Author(s):  
Barbara M. Buchwald ◽  
G. E. Connell
Keyword(s):  
Heavy Chain ◽  
Immunoglobulin G ◽  
Specific Radioactivity ◽  
Reduced Form ◽  
Light Chains ◽  
Thiol Groups ◽  
Myeloma Proteins ◽  
Disulphide Bonds ◽  
Sh Group ◽  
Normal Human

Normal human immunoglobulin G (IgG) was treated with radioactive N-ethylmaleimide in the absence of a reducing agent but in the presence of a denaturing solvent. The sites of reaction were determined by isolation of the radioactive peptides from thermolytic digests of the heavy and light chains. Six radioactive peptides were purified from the digest of the light chain and ten from that of the heavy chain. When sequenced, all the peptides were identical with peptides that would be predicted for the half-cystine residues involved in disulphide bonds. The specific radioactivity of the peptides indicated that the proportion of the half-cystine residues in the reduced form varied from 0.57 to 2.54%. These results indicate that the disulphide bonds of IgG are not completely oxidized. The estimate of 0.2mol of SH group/mol of IgG (Cecil & Stevenson, 1965; Luks & Connell, 1968) can be accounted for if 0.8% of every half-cystine residue of the intrachain bonds was in the reduced form. Variable cysteine residues as have been described in several myeloma proteins must occur extremely infrequently among the immunoglobulins.

Photodegradation of hydrophobic disperse dyes and dye-bath effluents by silicadodecatungstate (SiW12O404-/5-) nanoparticles

2004 ◽  
Vol 49 (4) ◽  
pp. 171-176 ◽  
Author(s):  
I. Arslan-Alaton ◽  
S. Dogruel
Keyword(s):  
Chemical Oxidation ◽  
Reduced Form ◽  
Disperse Dyes ◽  
Power Ultrasound ◽  
Substrate Complex ◽  
Rate Limiting Step ◽  
Uv Photodegradation ◽  
The One ◽  
Rate Limiting ◽  
Decolorization Kinetics

Polyoxometalate (POM) silicadodecatungstic acid has been applied as a photochemical catalyst for the degradation of ®SETAPERS Black WNSP, a disperse dyestuff preparation widely used to dye polyester and polyamide fabrics. It could be demonstrated that the disperse dyestuff was photo-reduced by SiW12O405-, the one electron-reduced form of POM, as evidenced by Heteropolyblue (HPB) formation. For completion of the photochemical redox cycle, isopropanol (IsOH) was required. Acetone (Ac) served solely as an effective solute and photosensitizer; however this effect was suppressed in the presence of POM. Threshold (0.087 mM) and optimum (0.375 mM) POM concentrations existed and decolorization kinetics were inhibited upon the addition of dye auxiliary chemicals. Increasing the dyestuff concentration from 50 mg/L to 150 mg/L did not affect initial decolorization kinetics revealing that not the formation of the excited [POM-Substrate]* complex, but its reduction to HPB was the rate limiting step. POM-mediated, IsOH-assisted UV-photodegradation of disperse dyes and dye-baths is by far more effective than applying other, more well known chemical oxidation methods (O3, H2O2/UV, Power Ultrasound). Key to the action of POM redox catalysts is the feature that particularly heteropoly tungstates undergo facile re-oxidation to their original state, thus allowing regeneration of the photocatalyst, a feature that may become critical for real-scale application.

Oxidative protein folding in the mammalian endoplasmic reticulum

2004 ◽  
Vol 32 (5) ◽  
pp. 655-658 ◽  
Author(s):  
C.E. Jessop ◽  
S. Chakravarthi ◽  
R.H. Watkins ◽  
N.J. Bulleid
Keyword(s):  
Endoplasmic Reticulum ◽  
Mammalian Cells ◽  
Redox State ◽  
Structure And Function ◽  
Reduced Form ◽  
Secretory Proteins ◽  
Oxidative Protein Folding ◽  
Disulphide Bonds ◽  
And Function ◽  
Substrate Proteins

Native disulphide bonds are essential for the structure and function of many membrane and secretory proteins. Disulphide bonds are formed, reduced and isomerized in the endoplasmic reticulum of mammalian cells by a family of oxidoreductases, which includes protein disulphide isomerase (PDI), ERp57, ERp72, P5 and PDIR. This review will discuss how these enzymes are maintained in either an oxidized redox state that allows them to form disulphide bonds in substrate proteins or a reduced form that allows them to perform isomerization and reduction reactions, how these opposing pathways may co-exist within the same compartment and why so many oxidoreductases exist when PDI alone can perform all three of these functions.

THE PYRUVIC PHOSPHOFERASE OF ERYTHROCYTES: I. PROPERTIES OF THE ENZYME AND ITS ACTIVITY IN ERYTHROCYTES OF VARIOUS SPECIES

1955 ◽  
Vol 33 (1) ◽  
pp. 38-45 ◽  
Author(s):  
P. F. Solvonuk ◽  
H. R. Collier
Keyword(s):  
Mammalian Species ◽  
Thiol Groups ◽  
Newborn Rats ◽  
Free Thiol ◽  
The Mean ◽  
Very High

Mammalian erythrocytes contain a pyruvic phosphoferase (PPFase) which is activated by K+ and Mg++ and inhibited by Na+ and Ca++. The K+ can be replaced by Rb+ or NH4+, and the Mg++ can be partially replaced by Mn++ or Co++ as activators of the enzyme. The PPFase apparently requires free thiol groups for its activity, as it is completely inhibited by 10−4 M p-chloromercuribenzoate and this inhibition is partially reversed by glutathione. The mean PPFase of the erythrocytes of six mammalian species was determined and found to be in the following order of decreasing activity: man, rat, dog, rabbit, cat, ox. The erythrocytes of chicks and of newborn rats showed a very high PPFase activity.

scholarly journals Selective reduction of a disulphide bridge in hen ovotransferrin

1985 ◽  
Vol 228 (3) ◽  
pp. 661-665 ◽  
Author(s):  
J Williams ◽  
K Moreton ◽  
A D Goodearl
Keyword(s):  
Selective Reduction ◽  
Iron Binding ◽  
Thiol Groups ◽  
Native Protein ◽  
Brief Treatment ◽  
Disulphide Bridge ◽  
Binding Properties ◽  
Terminal Domain ◽  
Fluorescent Derivative

Brief treatment of iron-saturated hen ovotransferrin with dithiothreitol selectively cleaves the disulphide bridge between residues 478 and 671, which is in the C-terminal domain of the protein. The reduced alkylated protein is less stable than the native protein, and its iron-binding properties are different. A fluorescent derivative was prepared by coupling N-iodoacetyl-N'-(5-sulpho-1-naphthyl)ethylenediamine to the thiol groups.

Structural modeling identified the tRNA-binding domain of Utp8p, an essential nucleolar component of the nuclear tRNA export machinery of Saccharomyces cerevisiae

2009 ◽  
Vol 87 (2) ◽  
pp. 431-443 ◽  
Author(s):  
Andrew T. McGuire ◽  
Robert A.B. Keates ◽  
Stephanie Cook ◽  
Dev Mangroo
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Binding Site ◽  
Structure Prediction ◽  
Positive Charge ◽  
Tertiary Structure ◽  
Secondary Structure Prediction ◽  
Fold Recognition ◽  
Terminal Domain ◽  
Fold Type ◽  
Trna Binding

Utp8p is an essential 80 kDa intranuclear tRNA chaperone that transports tRNAs from the nucleolus to the nuclear tRNA export receptors in Saccharomyces cerevisiae . To help understand the mechanism of Utp8p function, predictive tools were used to derive a partial model of the tertiary structure of Utp8p. Secondary structure prediction, supported by circular dichroism measurements, indicated that Utp8p is divided into 2 domains: the N-terminal beta sheet and the C-terminal alpha helical domain. Tertiary structure prediction was more challenging, because the amino acid sequence of Utp8p is not directly homologous to any known protein structure. The tertiary structures predicted by threading and fold recognition had generally modest scores, but for the C-terminal domain, threading and fold recognition consistently pointed to an alpha–alpha superhelix. Because of the sequence diversity of this fold type, no single structural template was an ideal fit to the Utp8p sequence. Instead, a composite template was constructed from 3 different alpha–alpha superhelix structures that gave the best matches to different portions of the C-terminal domain sequence. In the resulting model, the most conserved sequences grouped in a tight cluster of positive charges on a protein that is otherwise predominantly negative, suggesting that the positive-charge cleft may be the tRNA-binding site. Mutations of conserved positive residues in the proposed binding site resulted in a reduction in the affinity of Utp8p for tRNA both in vivo and in vitro. Models were also derived for the 10 fungal homologues of Utp8p, and the localization of the positive charges on the conserved surface was found in all cases. Taken together, these data suggest that the positive-charge cleft of the C-terminal domain of Utp8p is involved in tRNA-binding.

scholarly journals Purification and properties of two glyoxylate reductases from a species of Pseudomonas

1966 ◽  
Vol 101 (3) ◽  
pp. 781-791 ◽  
Author(s):  
LN Cartwright ◽  
RP Hullin
Keyword(s):  
Equilibrium Constants ◽  
Maximal Activity ◽  
High Order ◽  
Thiol Groups ◽  
Molecular Weights ◽  
Free Thiol ◽  
Purification And Properties ◽  
Keto Acids ◽  
Michaelis Constants

1. Two enzymes that catalyse the reduction of glyoxylate to glycollate have been separated and purified from a species of Pseudomonas. Their molecular weights were estimated as 180000. 2. Reduced nicotinamide nucleotides act as the hydrogen donators for the enzymes. The NADH-linked enzyme is entirely specific for its coenzyme but the NADPH-linked reductase shows some affinity towards NADH. 3. Both enzymes convert hydroxypyruvate into glycerate. 4. The glyoxylate reductases show maximal activity at pH6.0-6.8, are inhibited by keto acids and are strongly dependent on free thiol groups for activity. 5. The Michaelis constants for glyoxylate and hydroxypyruvate were found to be of a high order. 6. The reversibility of the reaction has been demonstrated for both glyoxylate reductases and the equilibrium constants were determined. 7. The reduction of glyoxylate and hydroxypyruvate is not stimulated by anions.

scholarly journals Thiol-dependent metallo-endopeptidase characteristics of Pz-peptidase in rat and rabbit

1990 ◽  
Vol 267 (2) ◽  
pp. 531-533 ◽  
Author(s):  
U Tisljar ◽  
A J Barrett
Keyword(s):  
Catalytic Activity ◽  
Rabbit Muscle ◽  
Thiol Groups ◽  
Rat Testis ◽  
Free Thiol

Pz-peptidase was purified from rat testis and rabbit muscle. Zinc was detectable in the rat enzyme. The activity of the enzyme from both species was slowly but completely abolished by EDTA and restored by Zn2+. Free thiol groups were also important for the catalytic activity of rat Pz-peptidase, as previously reported for the rabbit enzyme. We conclude that in both species Pz-peptidase has the characteristics of a thiol-dependent metallo-endopeptidase.

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