Figure 3: Time-dependence of PDC inactivation by PDK3.
From: Global view of cognate kinase activation by the human pyruvate dehydrogenase complex
(Top) PDK3 (3.0 μg, 0.22 μM) in 20 mMTris·HCl (pH 7.4) supplemented with 0.1 M KCl, 5.0 mM MgCl2 and 2.0 mM DTT was incubated with E1 (80 μg, 3.5 μM) in the presence of either L2S (0.8 μg, 0.22 μM) (line 1, 
); (line 2, ◽) L1L2S (1.2 μg, 0.22 μM) (line 2, ◽) or E2·E3BP (2.0 μg, 0.22 μM) (line 3, 
). Phosphorylation was initiated by ATP (2.0 mM) at 30 °C. (Middle) PDK3 (1.0 μg, 0.12 μM) in 20 mMTris·HCl (pH 7.4) supplemented with 0.1 M KCl, 5.0 mM MgCl2 and 2.0 mM DTT was first pre-incubated for 1 hour at 4 °C with either E2·E3BP (45 μg, 7.55 μM), (line 6, 
); L1L2S (10 μg, 2.7 μM) (line 5, 
); L2S (5.0 μg, 2.12 μM), (line 4, ◽); L1 (50 μg, 42 μM, (line 3, 
); L3S’ (10 μg, 4.0 μM) (line 2, 
) or with no source of the lipoyl domain (line 1, ●). Phosphorylation was initiated by ATP (0.1 mM) at 30 °C. (Bottom) PDK3 (1.0 μg, 0.12 μM) in 20 mMTris-HCl (pH 7.4) supplemented with 0.1 M KCl, 5.0 mM MgCl2 and 2.0 mM DTT was pre-incubated for 1 hour at 4 °C with either E2-Catalytic domain (1.0 μg, 0.33 μM) plus L1L2S (1.0 μg, 0.27 μM) (line 4, ♦); L1L2S (10 μg, 2.7 μM) (line 3, 
); E2-Catalytic domain (45 μg, 4.8 μM) (2, ○) or with no lipoyl domain source (1, ●). Phosphorylation reaction was initiated by ATP (0.1 mM) at 30 °C.
