Abstract
We studied human 101F6 protein to clarify its physiological function as a ferric reductase and its relationship to tumor suppression activity. We found for the first time that purified 101F6 both in detergent micelle state and in phospholipid bilayer nanodisc state has an authentic ferric reductase activity by single turnover kinetic analyses. The kinetic analysis on the ferrous heme oxidation of reduced 101F6 upon the addition of a ferric substrate, ferric ammonium citrate (FAC), showed concentration-dependent accelerations of its reaction with reasonable values of KM and Vmax. We further verified the authenticity of the ferric reductase activity of 101F6 using nitroso-PSAP as a Fe2+-specific colorimetric chelator. 101F6 in nanodisc state showed higher efficiency for FAC than in detergent micelle state.
Highlights
Human 101F6 gene in chromosome 3p21.3 was predicted to have a tumor suppression activity [1,2]
We studied human 101F6 protein to clarify its physiological function as a ferric reductase and its relationship to tumor suppression activity
The nanodisc mixtures were injected onto an Size exclusion chromatography (SEC) column (SuperdexTM 200 10/300 GL column connected with an AKTA Pure chromatography system; GE Healthcare)
Summary
Human 101F6 gene in chromosome 3p21.3 was predicted to have a tumor suppression activity [1,2]. The growth of lung cancer cells was inhibited upon the forced expression of the 101F6 gene via the induction of apoptosis and/or autophagy [2,3]. We and other groups have conducted detailed studies on the heterologously expressed 101F6 at the molecular level [5,6,7], but its physiological functions are still obscured. The use of β-OG and DDM, can affect the stability of 101F6 and may interfere with its biochemical and biophysical measurements. To avoid these problems, we tried for the first time the reconstitution of 101F6 into phospholipid bilayer nanodisc
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