Journal
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
Volume 315, Issue 2, Pages 502-508Publisher
ACADEMIC PRESS INC ELSEVIER SCIENCE
DOI: 10.1016/j.bbrc.2004.01.082
Keywords
acetylcholinesterase; H2O2; human skin
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Previously it has been demonstrated that the human epidermis synthesises and degrades acetylcholine and expresses both muscarinic and nicotinic receptors. These cholinergic systems have been implicated in the development of the epidermal calcium gradient and differentiation in normal healthy skin. In vitiligo severe oxidative stress occurs in the epidermis of these patients with accumulation of H2O2 in the 10(-3) M range together with a decrease in catalase expression/activity due to deactivation of the enzyme active site. It was also shown that the entire recycling of the essential cofactor (6R)-L-erythro-5,6,7,8-tetrahydrobiopterin via pterin-4a-carbinolamine dehydratase (PCD) and dihydropteridine reductase (DHPR) is affected by H2O2 oxidation of Trp/Met residues in the enzyme structure leading to deactivation of these proteins. Using fluorescence immunchistochemistry we now show that epidermal H2O2 in vitiligo patients yields also almost absent epidermal acetyleholinesterase (AchE). A kinetic analysis using pure recombinant human AchE revealed that low concentrations of H2O2 (10(-6) M) activate this enzyme by increasing the V-max > 2-fold, meanwhile high concentrations of H2O2 (10(-3) M) inhibit the enzyme with a significant decrease in V-max. This result was confirmed by fluorescence excitation spectroscopy following the Trp fluorescence at lambda(max) 280nm. Molecular modelling based on the established 3D structure of human AchE supported that H2O2-mediated oxidation of Trp(432), Trp(435), and Met(436) moves and disorients the active site His(440) of the enzyme, leading to deactivation of the protein. To our knowledge these results identified for the first time H2O2 regulation of AchE. Moreover, it was shown that H2O2-mediated oxidation of AchE contributes significantly to the well-established oxidative stress in vitiligo. (C) 2004 Elsevier Inc. All rights reserved.
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