GST formation

Phase 1 clinical trials of the natural polyphenol quercetin (33) against several types of cancer have shown that this compound is well tolerated when administered at a dose of 70 mg/kg by i.v. bolus [63,64]. Further studies revealed that 33 inhibits GST Pl-1 completely after 2 h at a concentration of 25 pM however, addition of GSH partially restores activity. HPLC and LC-MS studies indicate that 33 inhibits GST Pl-1 through the formation of a covalent yet reversible bond with GST Pl-1 cysteine residue at position 47. 

Glutathione-5-transferase (GST, human) HA and NH2O—Et inhibit GST in the mM range. Inhibition is linked to lipid peroxidation and to formation of active oxygen radicals 55... 

In protein microarrays, capture molecules need to be immobilized in a functional state on a solid support. In principle, the format of the assay system does not limit the choice of appropriate surface chemistry. The same immobilization procedure can be applied for both planar and bead-based systems. Proteins can be immobilized on various surfaces (Fig. 1) (12). Two-dimensional polystyrene, polylysine, aminosilane, or aldehyde, epoxy- or thiol group-coated surfaces can be used to immobilize proteins via noncovalent or covalent attachment (13,14). Three-dimensional supports like nitrocellulose or hydrogel-coated surfaces enable the immobilization of the proteins in a network structure. Larger quantities of proteins can be immobilized and kept in a functional state. Affinity binding reagents such as protein A, G, and L can be used to immobilize antibodies (15), streptavidin is used for biotinylated proteins (16), chelate for His-tagged proteins (17, 18), anti-GST antibodies for GST fusion proteins (19), and oligonucleotides for cDNA or mRNA-protein hybrids (20). 

Although in many cases the formation of the GSH conjugate protects cellular proteins from electrophilic attack, not all GSH conjugations with xenobiotics lead to detoxification. For example, in the case of haloalkanes, GST-catalyzed conjugation with GSH can lead to the formation of the highly electrophilic episulfonium ion [35], which can covalently bind DNA and cause mutations. 

Human liver preparations metabolize ethylene dibromide to water-soluble and irreversibly protein- and DNA-bound metabolites by both cytochrome P450 and glutathione S-transferase (GST) enzymes (Wiersma et al., 1986). DNA adduct formation occurs also in isolated human hepatocytes (Cmarik et al., 1990). 

In rodents and humans, ethylene dibromide is metabolized both by cytochrome P450 and GST enzymes the latter seem to be responsible for DNA adduct formation. In rodents, covalently bound radioactivity has been detected in the epithelial lining of a number of organs. 

The last two free-energy contributions involve only the surfactant heads. The steric contribution, gst, accounts for steric interactions between the surfactant heads at the micellar interface. This contribution can be responsible for synergism in mixed micelle formation if the heads of the two-surfactant types have different sizes, as illustrated by the large grey heads and small black heads ii Figure 12.14. The steric contribution depends only on the size of the surfactant heads. 

Glutathione S-Transferases (GSTs) and Mercapturic Acid Formation... 

Recently, active recombinant a-LTX has been generated using bacteria in which both thioredoxin reductase and glutathione reductase are inactivated to improve the formation of disulphide bonds in expressed proteins (Li et al. 2005). The toxin is expressed as a fusion with glutathione-S-transferase (GST), which is used for affinity purification of the recombinant toxin and can be subsequently removed by selective proteolysis. Considering the relative ease of generating recombinant proteins in bacteria, this approach will facilitate structure-function studies of a-LTX. 

R R Alkene c=c r R CYP, GST Epoxidation, glutathione adduct formation 0 Amide R-C-NH-R Amidase (esterase)... 

In this experiment, you will purify the C-terminal domain of the enzyme glutathione-S-transferase (GST) from an E. coli cell extract by exploiting the affinity that the enzyme has for its natural substrate, glutathione. Although the chromatography experiment used in the purification will not be performed in the column format (see Experiment 2), the batch wash format used here could easily be adapted for use in the column format. 

Figure 13.8. GST may enhance the toxicity of xenobiotics. Ethylene dibromide is a substrate of GST (theta and others) to be converted to l-bromo-2-.S -glutathionyl ethane, which facilitates spontaneous formation of the episulfonium ion. The episulfonium ion attacks the N7 position of guanine and causes DNA adduct formation. GST pi Ilel05 to Val polymorphism, causing increased GSTpi activity, appears to correlate with higher testicular and bladder cancer. This may also be involved in GSTpi-mediated activation of unidentified xenobiotics. (Adapted from Henderson et al. Proc. Natl. Acad. Sci. USA 95, 5275-5280,1998.)...

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