Protein oxidation thiolation

If cellular redox state, determined by the glutathione status of the heart, plays a role in the modulation of ion transporter activity in cardiac tissue, it is important to identify possible mechanisms by which these effects are mediated. Protein S-,thiolation is a process that was originally used to describe the formation of adducts of proteins with low molecular thiols such as glutathione (Miller etal., 1990). In view of the significant alterations of cardiac glutathione status (GSH and GSSG) and ion-transporter activity during oxidant stress, the process of S-thiolation may be responsible for modifications of protein structure and function. 

G21. Grant, C. M., Quinn, K. A., and Dawes, I. W., Differential protein S-thiolation of glyceraldehyde-3-phosphate dehydrogenase isoenzymes influences sensitivity to oxidative stress. Mol. Cell Biol. 19, 2650-2656 (1999). 

P3. Padgett, C. M., and Whorton, A. R., Cellular responses to nitric oxide Role of protein S-thiolation/dethiolation. Arch. Biochem. Biophys. 358,232—242 (1998). 

Seres, T., Ravichandran, V., Moriguchi, T., Rokutan, K., Thomas, J. A., and Johnston, R. B. J., Protein S-thiolation and dethiolation during the respiratory burst in human monocytes. A reversible post-translational modification with potential for buffering the effects of oxidant stress. J. Immunol 156, 1973-1980 (1996). 

Another interesting aspect of MT reactivity is its abihty to react with radical species. Thus, it has been shown that mammalian M 7-MT (M = Zn° and/or Cd ) and yeast Cu7-MT are efficient scavengers of free radicals such as hydroxyl (OH ), superoxide (02 ), or nitric oxide (NO). In all cases, the free radical attack occurs at the metal-bound thiolates, leading to protein oxidation and/or modification and... 

Although we have discussed the low oxidation state of the early atmosphere and its implications for the form of sulfur in organisms, today this chemistry is largely restricted to anoxic environments. Here sulfur is available as hydrogen sulhde and can be incorporated into amino acids such as cysteine and methionine and then into proteins. The thiolate group RS of cysteine of the thioether of methionine can act as bases or ligands for transition metals such as iron, zinc, molybdenum, and copper. 

Protection from any poisonous metal ions liberated from their sulfides by oxidation by 02 was secured by the use of strong chelating agents in the cytoplasm, most of which are proteins, or small molecules, thiolates, which were connected to exit pumps or to chemical metabolic tricks for metal ion neutralisation (sequestration). The genes that code for these proteins are usually to be found on plasmids in the cytoplasm of the bacterial cells (Section 5.15). Bacteria adapt very quickly to... 

Purify the thiolated protein from unreacted Traut s reagent by gel filtration using your buffer of choice (i.e., 20mM sodium phosphate, 0.15M NaCl, ImM EDTA, pH 7.2). The addition of EDTA to this buffer helps to prevent oxidation of the sulfhydryl groups and the resultant disulfide formation. After purification, use the thiolated protein immediately... 

Purify the thiolated protein from excess DTT by dialysis or gel filtration using 50 mM sodium phosphate, 0.15 M NaCl, ImM EDTA, pH 7.2. The modified protein should be used immediately in a conjugation reaction to prevent sulfhydryl oxidation and formation of disulfide crosslinks. 

Peroxynitrite reacts with heme proteins such as prostacycline synthase (PGI2), microperoxidase, and the heme thiolate protein P450 to form a ferryl nitrogen dioxide complex as an intermediate [120]. Peroxynitrite also reacts with acetaldehyde with the rate constant of 680 1 mol 1 s" 1 forming a hypothetical adduct, which is decomposed into acetate, formate, and methyl radicals [121]. The oxidation of NADH and NADPH by peroxynitrite most certainly occurs by free radical mechanism [122,123], Kirsch and de Groot [122] concluded that peroxynitrite oxidized NADH by a one-electron transfer mechanism to form NAD and superoxide ... 

Metallothioneins (MT) are unique 7-kDa proteins containing 20 cysteine molecules bounded to seven zinc atoms, which form two clusters with bridging or terminal cysteine thiolates. A main function of MT is to serve as a source for the distribution of zinc in cells, and this function is connected with the MT redox activity, which is responsible for the regulation of binding and release of zinc. It has been shown that the release of zinc is stimulated by MT oxidation in the reaction with glutathione disulfide or other biological disulfides [334]. MT redox properties led to a suggestion that MT may possesses antioxidant activity. The mechanism of MT antioxidant activity is of a special interest in connection with the possible antioxidant effects of zinc. (Zinc can be substituted in MT by some other metals such as copper or cadmium, but Ca MT and Cu MT exhibit manly prooxidant activity.)... 

This enzyme [EC 1.14.15.3], also known as alkane 1-monooxygenase, lauric acid ca-hydroxylase, fatty acid hydroxylate fatty acids in the [Pg.47]

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