کد مقاله کد نشریه سال انتشار مقاله انگلیسی نسخه تمام متن
7556642 1491292 2018 21 صفحه PDF دانلود رایگان
عنوان انگلیسی مقاله ISI
Development of a mass spectrometry-based tryptophan 2, 3-dioxygenase assay using liver cytosol from multiple species
ترجمه فارسی عنوان
تریپتوفان 2، 3-دیوکسیریناز با استفاده از سی تیوسول کبدی از گونه های مختلف
موضوعات مرتبط
مهندسی و علوم پایه شیمی شیمی آنالیزی یا شیمی تجزیه
چکیده انگلیسی
A novel and rapid method to determine the potency of inhibitors for tryptophan 2, 3-dioxygenase (TDO2) activities in human and preclinical species was successfully developed and validated utilizing LC-MS/MS. Previously reported TDO2 activity assays are resource intensive, requiring cloning and overexpression of TDO2. Here, we demonstrated that liver cytosol contained sufficient active TDO2 for evaluating the potency of TDO2 inhibitors across multiple species. TDO2 expression in human cytosol was estimated by LC-MS/MS to be 41 pmoL/mg cytosolic protein, with similar levels in dogs and monkeys, whereas mice and rats had 9.6 and 5.0-fold greater expression, respectively. Reaction conditions for TDO2-mediated conversion of l-tryptophan to kynurenine were optimized. Marked differences in kinetic parameters and inhibition potency were observed in TDO2 across species, with different Km values in dog (0.055 mM), monkey (0.070 mM), human (0.19 mM), mouse (0.32 mM) and rat (0.36 mM). Subsequently, IC50 values were determined for a series of TDO2 inhibitors in liver cytosol of five species, and good agreement with the literature values was observed for human enzyme. Taken together, these data indicate that TDO2 inhibition can be rapidly determined in readily available hepatic cytosol to assess potential species differences in potency.
ناشر
Database: Elsevier - ScienceDirect (ساینس دایرکت)
Journal: Analytical Biochemistry - Volume 556, 1 September 2018, Pages 85-90
نویسندگان
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