Figure 7.
FIG. 7. ThDP inhibits the phosphorylation of wild-type but
not mutant E1b. A, the reaction mixture contained human apoE1b
protein, lipoylated E2b, and maltose-binding protein-tagged rat
BCKD kinase in the absence and presence of increasing ThDP
concentrations. The phosphorylation reaction was initiated by
adding 0.4 mM [ -32P]ATP and was
incubated at 25 °C for 1 min. The reaction mixtures were
separated by SDS-PAGE. 32P incorporation into the subunit
of E1b proteins was quantified by PhosphorImaging. The
PhosphorImage counts in wild-type and each mutant E1b in the
absence of ThDP was set as 100% with respect to the
corresponding E1b protein. B, PhosphorImaging of 32P
incorporation into the subunit of the S302A-
E1b
mutant and E1b double mutants containing the S302A- mutation
and a second mutation in the hydrogen-bonding network. The
phosphorylation was carried out in the absence of ThDP.
The above figure is reprinted
by permission from the ASBMB:
J Biol Chem
(2004,
279,
32968-32978)
copyright 2004.
-32P]ATP and was
incubated at 25 °C for 1 min. The reaction mixtures were
separated by SDS-PAGE. 32P incorporation into the 
subunit
of E1b proteins was quantified by PhosphorImaging. The
PhosphorImage counts in wild-type and each mutant E1b in the
absence of ThDP was set as 100% with respect to the
corresponding E1b protein. B, PhosphorImaging of 32P
incorporation into the