Detection of S-Nitrosation and S-Glutathionylation of the Human Branched-Chain Aminotransferase Proteins

The human branched-chain aminotransferase (hBCAT) enzymes play an integral role in brain glutamate and branched-chain amino acid (BCAA) metabolism. Optimal hBCAT activity is dependent on the oxidation state of their redox reactive thiols, where post-translational modification by nitric oxide (NO) an...

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Bibliographic Details
Published inMethods in molecular biology (Clifton, N.J.) Vol. 1990; p. 71
Main Authors Forshaw, Thomas E, Conway, Myra E
Format Journal Article
LanguageEnglish
Published United States 01.01.2019
Subjects
Brain - enzymology
Cytosol - enzymology
Glutathione - metabolism
Humans
Mutation
Nitric Oxide - metabolism
Nitrosation
Oxidation-Reduction
Transaminases - chemistry
Transaminases - genetics
Transaminases - metabolism
Redox
CXXC motif
Mass spectrometry
Nitric oxide
Glutathione
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Summary:The human branched-chain aminotransferase (hBCAT) enzymes play an integral role in brain glutamate and branched-chain amino acid (BCAA) metabolism. Optimal hBCAT activity is dependent on the oxidation state of their redox reactive thiols, where post-translational modification by nitric oxide (NO) and glutathione results in reversible inhibition. Incubation of the cytosolic isoform (hBCATc) with S-nitrosating agents was found to inhibit in both a time and dose dependent manner through formation of a mixture of products including cysteine-nitric oxide (SNO) and S-glutathionylation. Mechanistic details of these redox interactions were studied using labeling with fluorescein-5-maleimide and confirmed via mass spectrometry and Western blot analysis. Though the mitochondrial isoform (hBCATm) was inhibited by nitrosating agents adduct formation could only be observed by DTNB titration as neither SNO, S-glutathionylation or disulfide bond formation could be detected. These studies revealed that the two isoforms of hBCAT, namely hBCATc and hBCATm, were differently regulated by S-nitrosation or S-glutathionylation pointing to distinct functional/mechanistic responses to GSNO modification. Detection of these adducts is essential for studies into the effect of NO on cells and the redox proteome which can offer insight into several pathological states and normal functioning of the cell.
ISSN:1940-6029
DOI:10.1007/978-1-4939-9463-2_6