کد مقاله | کد نشریه | سال انتشار | مقاله انگلیسی | نسخه تمام متن |
---|---|---|---|---|
10537696 | 962814 | 2005 | 9 صفحه PDF | دانلود رایگان |
عنوان انگلیسی مقاله ISI
Bacterial expression, folding, purification and characterization of soluble NTPDase5 (CD39L4) ecto-nucleotidase
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کلمات کلیدی
SECNTAIPTGDMEMDTTAcrSNTPDasesTBSMWCOKLHMOPS3-[N-morpholino]propanesulfonic acid - 3- [N-مورفولینو] پروپان سولفونیک اسیدDulbecco's modified Eagle Medium - Eagle Medium اصلاح شده Dulbecconitrilotriacetic acid - اسید نیتریلوتوریکاتیکisopropyl-β-d-thiogalactopyranoside - ایزوپروپیل-ب-دی-تیوگالکتوپیرانوزیدProtein refolding - بازخورد پروتئینBacterial expression - بیان باکتریاییTris-buffered saline - تریس بافر شورEnzymatic characterization - تعریف آنزیمیdithiothreitol - دیتیوتریتولinorganic phosphate - فسفات معدنیnucleoside triphosphate diphosphohydrolase - نوکلئوزید تری فسفات دی فسفو هیدرولازPositive cooperativity - همکاری مثبتMolecular Weight Cut Off - وزن مولکولی کاهش یافته استSize exclusion chromatography - کروماتوگرافی اندازهای طردیkeyhole limpet hemocyanin - کلم هول limpet هموسیانین
موضوعات مرتبط
مهندسی و علوم پایه
شیمی
شیمی آنالیزی یا شیمی تجزیه
پیش نمایش صفحه اول مقاله
چکیده انگلیسی
The ecto-nucleoside triphosphate diphosphohydrolases (eNTPDases) are a family of enzymes that control the levels of extracellular nucleotides, thereby modulating purinergically controlled physiological processes. Six of the eight known NTPDases are membrane-bound enzymes; only NTPDase 5 and 6 can be released as soluble enzymes. Here we report the first bacterial expression and refolding of soluble human NTPDase5 from inclusion bodies. The results show that NTPDase5 requires the presence of divalent cations (Mg2+ or Ca2+) for activity. Positive cooperativity with respect to hydrolysis of its preferred substrates (GDP, IDP and UDP) is observed, and this positive cooperativity is attenuated in the presence of nucleoside monophosphate products (e.g., GMP and AMP). In addition, comparing the biochemical properties of wild-type NTPDase5 and those of a mutant NTPDase5 (C15S, which lacks the single, non-conserved cysteine residue), also expressed in bacteria, suggests that Cys15 is not essential for either proper refolding or enzymatic activity (indicating this residue is not involved in a disulfide bond). Moreover, the substrate profile of bacterially expressed NTPDase5, as well as the C15S mutant, was determined to be similar to that of full-length, membrane-bound and soluble NTPDase5 expressed in mammalian COS cells.
ناشر
Database: Elsevier - ScienceDirect (ساینس دایرکت)
Journal: Biochimica et Biophysica Acta (BBA) - Proteins and Proteomics - Volume 1747, Issue 2, 14 March 2005, Pages 251-259
Journal: Biochimica et Biophysica Acta (BBA) - Proteins and Proteomics - Volume 1747, Issue 2, 14 March 2005, Pages 251-259
نویسندگان
Deirdre M. Murphy-Piedmonte, Patrick A. Crawford, Terence L. Kirley,