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S-Nitrosoglutathione Inactivation of the Mitochondrial and Cytosolic BCAT Proteins: S-nitrosation and S-thiolation

Coles, Steven, Easton, P., Sharrod, H., Hutson, S.M., Hancock, J.T., Patel, V.B. and Conway, M.E. (2009) S-Nitrosoglutathione Inactivation of the Mitochondrial and Cytosolic BCAT Proteins: S-nitrosation and S-thiolation. Biochemistry, 48 (3). pp. 645-656. ISSN Print: 0006-2960 Online: 1520-4995

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Abstract

Specific proteins with reactive thiol(ate) groups are susceptible to nitric oxide (NO) modification, which can result in S-nitrosation, S-thiolation, or disulfide bond formation. In the present study the effect of NO modification on the functionality of human mitochondrial and cytosolic branched-chain aminotransferases (hBCATm and hBCATc, respectively) was investigated. Here, the NO reactive agents, S-nitrosoglutathione (GSNO), S-nitroso-N-acetyl-dl-penacillamine, and sodium nitroprusside, inactivated both isoforms in a dose-dependent manner. Furthermore, low concentrations of GSNO caused a time-dependent loss in BCAT activity (50 ± 3% and 77 ± 2% for hBCATc and hBCATm, respectively) correlating with the loss of four and one to two thiol groups, respectively, confirming the thiols as targets for NO modification. Analysis of GSNO-modified hBCATc by quadrupole time-of-flight mass spectrometry identified a major peak containing three NO adducts and a minor peak equivalent to two NO adducts and one glutathione (GSH) molecule, the latter confirmed by Western blot analysis. Moreover, prolonged exposure or increased levels of GSNO caused increased S-glutathionylation and partial dimerization of hBCATc, suggesting a possible shift from regulation by NO to one of adaptation during nitrosated stress. Although GSNO inactivated hBCATm, neither S-nitrosation, S-glutathionylation, nor dimerization could be detected, suggesting differential mechanisms of regulation through NO between isoforms in the mitochondria and cytosol. Reversal of GSNO-modified hBCAT using GSH alone was only partial, and complete reactivation was only possible using the glutaredoxin/GSH system (97 ± 4% and 91 ± 3% for hBCATc and hBCATm, respectively), implicating the importance of a full physiological redox system for activation/inactivation. To conclude, these results clearly demonstrate distinct functional/mechanistic responses to GSNO modification between BCAT isoforms and offer intriguing comparisons between the BCAT proteins and the respective cytosolic and mitochondrial hTrx and hGrx proteins.

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Uncontrolled Keywords: S-Nitrosoglutathione, inactivation, mitochondrial, cytosolic, BCAT proteins, S-nitrosation, S-thiolation
Subjects: Q Science > QD Chemistry
Divisions: Academic Departments > Institute of Science and the Environment
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Depositing User: Steven Coles
Date Deposited: 15 Sep 2017 09:08
Last Modified: 15 Sep 2017 10:24
URI: https://eprints.worc.ac.uk/id/eprint/5885

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