Figure 5: Experiments to confirm α2β4 stoichiometry of two-subunit PCs.
From: A distinct holoenzyme organization for two-subunit pyruvate carboxylase
(a) Gel filtration profiles for two-subunit PCs (Mf, M. flagellates; Pa, P. aeruginosa; Pf, P. fluorescens; Td, T. denitrificans) and the single-chain SaPC. (b) Coomassie-stained SDS–PAGE gel with purified two-subunit PCs from different species. Two lanes were run for each protein, with twice as much protein loaded in the first lane. The band for the α subunit in this lane is roughly the same as that for the β subunit in the second lane, consistent with the 1:2 stoichiometry. (c) SDS–PAGE gel with known amounts of α and β subunits and their mixtures, indicating that the purified holoenzyme has 1:2 stoichiometry. The β subunit in the MfPC holoenzyme sample is slightly smaller than the β subunit sample alone as it does contain a His tag. (d) Size distribution of PCs in solution at a concentration of 0.5 mg ml−1, based on the best-fit results by the continuous size distribution analysis. Two-subunit PCs (MfPC wild-type and deletion mutant, and PaPC) showed a similar major species of S=11.5, while single-chain SaPC showed a tetrameric species of S=14.8. (e) Gel filtration profiles from mixing experiments of different ratios of MfPC α and β subunits. The vertical dashed line indicates the elution volume of the complex that is formed on mixing. (f) Coomassie-stained SDS–PAGE gels of peak fractions collected from two different mixing experiments (F5 and F6 indicates collected fractions 5 and 6, respectively, indicated in e).
