Figure 5.

Effect of Denaturation on the ³²P-Labeling of Succinyl-CoA Synthetase. After incubation with ³²P, succinyl-CoA synthetase was run using 1D and subsequent 2D BN-PAGE on purified succinyl-CoA synthetase or total porcine heart mitochondria (A) or was precipitated with methanol (B) or trichloro-acetic acid (C). The insert to the left of Panel A shows that the bacterial succinyl-CoA synthetase complex has a molecular weight of ∼140 kDa, consistent with previous reports (59). Proteins were first separated by either molecular weight with BN-PAGE (A) or isoelectric focusing point (B-C) over a non-linear pH range of 3-11, and then by molecular weight from ∼150kD to 10kD. In all panels, an arrow is used to indicate the ³²P-binding to succinyl-CoA synthetase following SDS gel electrophoresis, where BSCSα represents purified bacterial succinyl-CoA synthetase, α-subunit and SCSα represents porcine heart mitochondrial succinyl-CoA synthetase, α-subunit.