Abstract:
:Luciferin-regenerating enzyme (LRE) contributes to in vitro recycling of D-luciferin. In this study, reinvestigation of the luciferase-based LRE assay is reported. Here, using quick change site-directed mutagenesis seven T-LRE (Lampyris turkestanicusLRE) mutants were constructed and the most functional mutant of T-LRE (T(69)R) was selected for this research and the effects of D- and L-cysteine on T(69)R T-LRE-luciferase-coupled assay are examined. Our results demonstrate that bioluminescent signal of T(69)R T-LRE-luciferase-coupled assay increases and then reach equilibrium state in the presence of 5 mm D-cysteine. In addition, results reveal that 5 mm D- and L-cysteine in the absence of T(69)R T-LRE cause a significant increase in bioluminescence intensity of luciferase over a long time as well as decrease in decay rate. Based on activity measurements, far-UV CD analysis, ANS fluorescence and DLS (Dynamic light scattering) results, D-cysteine increases the activity of luciferase due to weak redox potential, antiaggregatory effects, induction of changes in conformational structure and kinetics properties. In conclusion, in spite of previous reports on the effect of LRE on luciferase bioluminescent intensity, the majority of increase in luciferase light output and time-course originate from the direct effects of D-cysteine on structure and activity of firefly luciferase.
journal_name
Photochem Photobioljournal_title
Photochemistry and photobiologyauthors
Hemmati R,Hosseinkhani S,Sajedi RH,Azad T,Tashakor A,Bakhtiari N,Ataei Fdoi
10.1111/php.12430subject
Has Abstractpub_date
2015-07-01 00:00:00pages
828-36issue
4eissn
0031-8655issn
1751-1097journal_volume
91pub_type
杂志文章abstract::In this work, the changes in isoflavone levels and the expression of genes involved in their biosynthesis were studied in two Astragalus by UPLC-MS and real-time PCR after 10 days of UV-B treatment (λmax = 313 nm, 804 J m-2 ). Isoflavones were significantly induced by UV-B irradiation. The influence might be activate...
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