SDS–PAGE (10%) and autoradiography
monitoring the reaction
of PhaCCc and sT-PhaCCc with 5 or 50 equiv of
[1-14C]HBCoA. (A) Coomassie-stained gel. Lanes 1–3
are as described for panel B. (B) Autoradiography of the gel in panel
A. Lanes: M, molecular weight standards; lane 1, PhaC (3 μg);
lanes 2 and 3, PhaC (3 μg) reacted with [1-14C]HBCoA
at the indicated substrate:enzyme ratio (S:E). The specific activity
of [1-14C]HBCoA is 2 × 105 cpm/nmol in
lane 2 and 2 × 104 cpm/nmol in lane 3. (C) Coomassie-stained
gel. Lanes 1–3 are as described for panel D. (D) Autoradiography
of the gel in panel C: M, molecular weight standards; lane 1, PhaC
(3 μg); lanes 2 and 3, PhaC (3 μg) reacted with 10 equiv
of sTCoA for 1 min and then chased with 50 and 5 equiv of [1-14C]HBCoA, respectively. The specific activity of [1-14C]HBCoA is 2 × 104 cpm/nmol in lane 2 and 2 ×
105 cpm/nmol in lane 3. W, wells. GF, gel front.