Role of Hsp90 and ATP in modulating apyrase activity and firefly luciferase kinetic
Maria A. Kirillova
Elena N. Esimbekova
Valentina A. Kratasyuk
Институт фундаментальной биологии и биотехнологии
Journal Name:International Journal of Biological Macromolecules
Journal Quartile in Scopus:Q1
Journal Quartile in Web of Science:Q1
Bibliographic Citation:Maria A. Kirillova. Role of Hsp90 and ATP in modulating apyrase activity and firefly luciferase kinetic [Текст] / Maria A. Kirillova, Ranjan Rajeev, Elena N. Esimbekova, Valentina A. Kratasyuk // International Journal of Biological Macromolecules. — 2019. — Т. 131. — С. 691-696
Текст статьи не публикуется в открытом доступе в соответствии с политикой журнала.
The present manuscript describes a novel bioassay consisting of apyrase and heat shock protein 90 (Hsp90) without additional co-chaperone supplementation; intended for high-throughput screening of anti-cancer drugs and prognosis of stress. In this regard, Hsp90 and adenosine 5'-triphosphate (ATP) mediated firefly luciferase (FLuc) kinetics was investigated using apyrase and FLuc as client proteins. Bioluminescent assay containing Hsp90, ATP, and apyrase led to complete loss of luminescence at 50ºC which indicates the protective role of Hsp90 against thermal denaturation. Similarly, the assay sample comprising Hsp90, ATP, and FLuc showed 2 fold increments in luminescence than their counterparts. Introduction of bovine serum albumin (BSA) to the pre-incubated assay mixture led to an initial rise in the luminescence (28 %) in comparison to the sample containing Hsp90, ATP and FLuc. Therefore, FLuc based HTS assays are not suitable for clinical samples which may contain stabilizing agents. However, thermally denatured FLuc and apyrase could not regain their active conformation even when Hsp90 and ATP were introduced in the assay system. This observation justifies the role of Hsp90 to be protective rather than a reparation agent when acts without co-chaperones.