title: Reversible inactivation of dihydrolipoamide dehydrogenase by Angeli's salt.
abstract: Dihydrolipoamide dehydrogenase (DLDH) is a key component of 3 mitochondrial α-keto acid dehydrogenase complexes including pyruvate dehydrogenase complex, α-ketoglutarate dehydrogenase complex, and branched chain amino acid dehydrogenase complex. It is a pyridine-dependent disulfide oxidoreductase that is very sensitive to oxidative modifications by reactive nitrogen species (RNS) and reactive oxygen species (ROS). The objective of this study was to investigate the mechanisms of DLDH modification by RNS derived from Angeli's salt. Studies were conducted using isolated rat brain mitochondria that were incubated with varying concentrations of Angeli's salt followed by spectrophotometric enzyme assays, blue native gel analysis, and 2-dimensional gel-based proteomic approaches. Results show that DLDH could be inactivated by Angeli's salt in a concentration dependent manner and the inactivation was a targeting rather than a random process as peroxynitrite did not show any detectable inhibitory effect on the enzyme's activity under the same experimental conditions. Since Angeli's salt can readily decompose at physiological pH to yield nitroxyl anion (HNO) and nitric oxide, further studies were conducted to determine the actual RNS that was responsible for DLDH inactivation. Results indicate that it was HNO that exerted the effect of Angeli's salt on DLDH. Finally, two-dimensional Western blot analysis indicates that DLDH inactivation by Angeli's salt was accompanied by formation of protein s-nitrosothiols, suggesting that s-nitrosylation is likely the cause of loss in enzyme's activity. Taken together, the present study provides insights into mechanisms of DLDH inactivation induced by HNO derived from Angeli's salt.
authors.affiliation: Department of Pharmacology and Neuroscience and Institute for Aging and Alzheimer's Disease Research, University of North Texas Health Science Center, Fort Worth, Texas.
authors.forename: Liang-Jun
authors.initials: LJ
authors.lastname: Yan
authors.forename: Li
authors.initials: L
authors.lastname: Liu
authors.forename: Michael J
authors.initials: MJ
authors.lastname: Forster