Separation of intact pyruvate dehydrogenase complex using blue native agarose gel electrophoresis

We show that the blue native gel polyacrylamide electrophoresis system (BN- PAGE) can be applied to pyruvate dehydrogenase complex (PDC). BN-PAGE has b een used extensively to study the multisubunit enzymes of oxidative phospho rylation, as nondenaturing separation in the first dimension maintains holo enzyme integrity. However, the standard protocol was inappropriate for PDC as, at 10 MDa, it is approximately ten times larger than the largest respir atory chain enzyme complex. Therefore, agarose was substituted for polyacry lamide. Moreover, a substantial decrease in salt concentration was necessar y to prevent dissociation of PDC. As with standard BN-PAGE, immunoblots of second-dimensional sodium dodecyl sulfate-PAGE (SDS-PAGE) provided more det ailed information on specific subunits and subcomplexes. The method was app lied to human heart mitochondrial fragments, control cultured human cells, rho(0) cells that lack mitochondrial DNA, and two cell lines derived from p atients with PDC deficiency. The PDC deficient cell lines showed a clear co rrelation between amount of PDC holoenzyme and disease severity. In cells l acking mitochondrial DNA, synthesis and assembly of all PDC subunits (all n uclearly encoded) appeared normal, suggesting that respiratory function has no regulatory role in PDC biogenesis. Blue native agarose gel electrophore sis coupled with standard second-dimensional SDS-PAGE provides a new tool t o be used in conjunction with biochemical assays and immunoblots of one-dim ensional SDS-PAGE to further elucidate the nature of PDC in normal and dise ase states. Furthermore, other cellular protein complexes of 1 MDa or more can be analysed by this method.