Inhibition of mitochondrial carnitine/acylcarnitine transporter by H₂O₂: Molecular mechanism and possible implication in pathophysiology

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• 作者：Annamaria Tonazzi^a ; ^b ; ¹ ; ^{a.tonazzi@ibbe.cnr.it} ; Lara Console^b ; ^c ; ¹ ; Cesare Indiveri^a ; ^c ; ^{indiveri@unical.it}
• 关键词：DTE ; dithioerythritol ; NEM ; N-ethylmaleimide ; Pipes ; 1 ; 4-piperazinediethanesulfonic acid ; Hepes ; 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid ; CACT ; carnitine/acylcarnitine translocase ; WT ; wild-type ; ANC ; adenine nucleotide carrier ; NAC ; N-acetyl-l
• 刊名：Chemico-Biological Interactions
• 出版年：2013
• 出版时间：25 April, 2013
• 年：2013
• 卷：203
• 期：2
• 页码：423-429
• 全文大小：1125 K

文摘

H₂O₂ inhibits the [³H]carnitine/carnitine antiport catalysed by the mitochondrial carnitine/acylcarnitine transporter reconstituted in proteoliposomes. The inhibition was reversed by dithioerythritol, N-acetylcysteine and l-cysteine. Inhibition time-dependence revealed a faster and a slower reaction stages with orders of reaction of 1.0 and 1.9, respectively. Inhibition was tested on mutants in which one or more of the six Cys residues had been substituted with Ser or with Val. The four replacement mutant C23S/C58S/C89S/C283S containing C136 and C155 was inhibited as the wild-type. Mutants C23V/C58V/C155V/C89S/C283S and C23V/C58V/C136V/C89S/C283S containing only C136 or C155, respectively, were inhibited at a much lower extent respect to the wild-type, while the mutant C136S/C155S in which the two Cys were substituted and the C-less protein were virtually insensitive to inhibition. DTE reversed the inhibition of the H₂O₂ sensitive proteins except that in the case of the mutants containing only C136 or C155 after long time of incubation with H₂O₂. The IC₅₀ values obtained by dose-response curves of H₂O₂ inhibition were 0.17 mM for the wild-type, 0.39 mM for the four replacement mutant containing C136 and C155, 2.23 or 1.8 mM in the five replacement mutants containing the single C136 or C155, respectively. Carnitine and acetylcarnitine protected the protein from the inhibition by H₂O₂. Inhibition kinetics showed a competitive behaviour of H₂O₂ respect to carnitine. All the data concur to demonstrate that H₂O₂ interacts with C136 and C155 and completely inactivates the transporter by inducing the formation of a disulphide.