کد مقاله کد نشریه سال انتشار مقاله انگلیسی نسخه تمام متن
1957042 1057873 2005 10 صفحه PDF دانلود رایگان
عنوان انگلیسی مقاله ISI
Use of Helper Enzymes for ADP Removal in Infrared Spectroscopic Experiments: Application to Ca2+-ATPase
موضوعات مرتبط
علوم زیستی و بیوفناوری بیوشیمی، ژنتیک و زیست شناسی مولکولی زیست شیمی
پیش نمایش صفحه اول مقاله
Use of Helper Enzymes for ADP Removal in Infrared Spectroscopic Experiments: Application to Ca2+-ATPase
چکیده انگلیسی

Adenylate kinase (AdK) and apyrase were employed as helper enzymes to remove ADP in infrared spectroscopic experiments that study the sarcoplasmic reticulum Ca2+-ATPase. The infrared absorbance changes of their enzymatic reactions were characterized and used to monitor enzyme activity. AdK transforms ADP to ATP and AMP, whereas apyrase consumes ATP and ADP to generate AMP and inorganic phosphate. The benefits of using them as helper enzymes are severalfold: i), both remove ADP generated after ATP hydrolysis by ATPase, which enables repeat of ATP-release experiments several times with the same sample without interference by ADP; ii), AdK helps maintain the presence of ATP for a longer time by regenerating 50% of the initial ATP; iii), apyrase generates free Pi, which can help stabilize the ADP-insensitive phosphoenzyme (E2P); and iv), apyrase can be used to monitor ADP dissociation from transient enzyme intermediates with relatively high affinity to ADP, as shown here for ADP dissociation from the ADP-sensitive phosphoenzyme intermediate (Ca2E1P). The respective infrared spectra indicate that ADP dissociation relaxes the closed conformation immediately after phosphorylation partially back toward the open conformation of Ca2E1 but does not trigger the transition to E2P. The helper enzyme approach can be extended to study other nucleotide-dependent proteins.

ناشر
Database: Elsevier - ScienceDirect (ساینس دایرکت)
Journal: - Volume 88, Issue 5, May 2005, Pages 3615–3624
نویسندگان
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