Тип публикации: статья из журнала
Год издания: 2018
Идентификатор DOI: 10.1016/j.jphotobiol.2018.04.050
Ключевые слова: Bioluminescence, Coelenterazine, Copepod luciferase, Halophilic enzyme, Kinetic cooperativity
Аннотация: The secreted luciferases responsible for light emission of marine copepods have gained popularity for being used in noninvasive imaging of intracellular events. The secreted luciferase of copepod Gaussia princeps is a one subunit protein catalyzing coelenterazine oxidation to emit blue light. It consists of the N-terminal variable Показать полностьюpart that bears a signal peptide for secretion and the C-terminal catalytic domain containing ten highly conserved Cys residues supposing the existence of up to five S-S bonds. Despite wide application of Gaussia luciferase in biomedical research, its biochemical properties are still insufficiently studied due to the general problem of obtaining the proper folded Cys-rich proteins in bacterial cells. Here we report the properties of the proper folded Gaussia luciferase produced in insect cells using baculovirus expression system. This high purity luciferase reveals the highest activity at 15-20 degrees C but retains only similar to 20% activity at 37 degrees C that may hamper its application for in vivo assays. The maximum of bioluminescent activity of GpLuc is found at NaCl concentrations in the range of 1.0-1.5 M and, furthermore, a high NaCl concentration enhances luciferase stability to thermal denaturation, i.e. Gaussia luciferase displays the features characteristic of halophilic enzymes. The studies on bioluminescence kinetics at different coelenterazine concentrations obviously show a positive cooperativity of Gaussia luciferase with coelenterazine (Hill coefficient -1.8 +/- 0.2; K-0.5-2.14 +/- 0.17 mu M). We suggest this effect to be rather due to the so-called kinetic cooperativity conditioned by conformational changes in response to substrate binding than to the presence of two catalytic sites.
Журнал: JOURNAL OF PHOTOCHEMISTRY AND PHOTOBIOLOGY B-BIOLOGY
Выпуск журнала: Vol. 183
Номера страниц: 309-317
ISSN журнала: 10111344
Место издания: LAUSANNE
Издатель: ELSEVIER SCIENCE SA