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. 2021 Sep 22;24(10):103153. doi: 10.1016/j.isci.2021.103153

Figure 2.

Figure 2

ECSIT deflavination and loss of ACAD9 dehydrogenation activity and restoration of dehydrogenation activity by FAD

(A) UV-VIS spectra of VLCAD and ACAD9 before and after after incubation with ECSIT. Black and blue lines show VLCAD and ACAD9 (20 μM protein concentration) after overnight dialysis in 100 mM Tris, pH 7.4, 100 mM NaCl, 10% glycerol. Spectra of VLCAD or ACAD9 (20 μM protein concentration) mixed with 20 μM ECSIT in 100 mM Tris, pH 7.4, 100 mM NaCl, 10% glycerol and dialyzed in the same buffer overnight are shown in red (VLCAD + ECSIT) and green (ACAD9+ECSIT). The inset shows the same spectra with an expanded scale.

(B and C) Dehydrogenation activity, as % of maximum activity, after incubation of ACAD9 with increasing concentrations of (B) ECSIT, or (C) C-ECSIT. The dashed lines show biphasic decreases in the dehydrogenation activity with an inflection point at an ECSIT:ACAD9 ratio near 0.4:1. Values are expressed as mean ± SD.

(D) Restoration of dehydrogenation activity by addition of FAD to HPLC-purified ACAD9/ECSIT binary (red) or ACAD9/ECSIT/NDUFAF1 ternary (blue) complexes. The concentration of each complex was 0.1 μM. Numbers in parentheses indicate the molar ratio of FAD:complex. Activity is expressed as % of maximum activity. Values are expressed as mean ± SD.