کد مقاله | کد نشریه | سال انتشار | مقاله انگلیسی | نسخه تمام متن |
---|---|---|---|---|
8326461 | 1539976 | 2007 | 9 صفحه PDF | دانلود رایگان |
عنوان انگلیسی مقاله ISI
Expression of functional human glutaminase in baculovirus system: Affinity purification, kinetic and molecular characterization
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کلمات کلیدی
MPPIMACGIPLGAKGARecombinant protein expression - بیان پروتئین نوترکیبMitochondria - میتوکندریاNucleus - هستهmitochondrial processing peptidase - پردازش پپتیداز میتوکندریimmobilized metal affinity chromatography - کروماتوگرافی وابسته به فلز متمرکزglutamate - گلوتاماتglutamine - گلوتامینphosphate-activated glutaminase - گلوتامیناز فعال فسفات
موضوعات مرتبط
علوم زیستی و بیوفناوری
بیوشیمی، ژنتیک و زیست شناسی مولکولی
زیست شیمی
پیش نمایش صفحه اول مقاله

چکیده انگلیسی
Glutaminase catalyzes the hydrolysis of glutamine yielding stoichiometric amounts of glutamate plus ammonium ions. In mammals, there are two different genes encoding for glutaminase, known as liver (L) and kidney (K) types. The human L-type isoform expressed in baculovirus yielded functional recombinant enzyme in Sf9 insect cells. A novel affinity chromatography method, based on its specific interaction with a PDZ protein, was developed for purification. Kinetic constants were determined for the purified human isozyme, which showed an allosteric behaviour for glutamine, with a Hill index of 2.7 and S0.5 values of 32 and 64Â mM for high and low Pi concentrations, respectively. Whereas the protein showed a low Pi dependence typical for L-type glutaminases, the enzyme was unexpectedly inhibited by glutamate, a kinetic characteristic exclusive of K-type isozymes, and was slightly activated by ammonia, unlike the classical liver enzymes which show an absolute dependence on ammonia. Subcellular fractionation demonstrates that recombinant human glutaminase was targeted to both mitochondria and nucleus, and in both locations the protein was catalytically active. This is the first report of the expression of a functional L-type mammalian glutaminase enzyme. The study also provides a simple and efficient method for affinity purification of the recombinant enzyme. Moreover, the data imply that this human enzyme may represent a new isoform different from classical kidney and liver isozymes.
ناشر
Database: Elsevier - ScienceDirect (ساینس دایرکت)
Journal: The International Journal of Biochemistry & Cell Biology - Volume 39, Issue 4, 2007, Pages 765-773
Journal: The International Journal of Biochemistry & Cell Biology - Volume 39, Issue 4, 2007, Pages 765-773
نویسندگان
José A. Campos-Sandoval, Amada R. López de la Oliva, Carolina Lobo, Juan A. Segura, José M. Matés, Francisco J. Alonso, Javier Márquez,