Subunit and catalytic component stoichiometries and solution behavior of
an in vitro reconstituted PDC. A, in vitro assembly of
the human pyruvate dehydrogenase complex. The E2p/E3BP core (0.45 nmol) in a
potassium phosphate buffer, pH 7.5, was mixed with E3 (17.5 nmol) in a final
volume of 2.6 ml (lane 1); the same sample of E2p/E3BP core was mixed
with E1p (32 nmol) (lane 2); or the same E2p/E3BP core sample was
mixed with E3 (17.5 nmol) and E1p (32 nmol) (lane 3). The protein
mixtures were passed through a Sephacryl S-400 column to remove the excess
unbound E3 or E1p. The stoichiometries of protein subunits were estimated by
both Coomassie Blue staining intensity directly and by comparing to the
standard curves obtained with individual protein standards. The subunit
stoichiometry of E2p:E3BP in the 60-meric E2p/E3BP core is 40:20. The molar
ratio of E2p/E3BP 60-mer:E1p heterotetramer:E3 dimer in the reconstituted PDC
is 1:37:18 based on the staining intensity method normalized for the E1β
subunit by comparison with the standard curve for this subunit. B, SV
studies on PDC and its complexes with E3 and E1p. The solid line
shows the c(s) distribution of PDC core alone, and the
dashed line shows the distribution when a saturating concentration of
E3 is added, and the dotted line shows the distribution when a
saturating concentration of E1p is added. The large peaks on the left
side of the distributions are because of excess E3 or E1p. C,
ITC measurements of E3 binding to the reconstituted PDC core. The top
panel is the thermogram that results from titrating the E3 dimer into a
solution of E2p/E3BP. The solid varying line depicts enthalpy
changes, and the flat line is the base line, as calculated by ORIGIN
7.0. The squares in the bottom panel are the integrated
heats from each injection as derived from the data in the top panel,
and the solid line is the fit to these data using a model that
considers only a single type of binding sites. The molar ratio for E3
dimer:E2p/E3BP core is 18.8:1, based on the 40/20 E2p/E3BP model.