Evidence That Maize Acetyl-Coenzyme A Carboxylase Does Not Function Solely as a Homodimer

Acetyl-coenzyme A carboxylase (EC 6.4.1.2; ACCase) was purified from etiolated maize coleoptiles using cyclohexanedione affinity and DyeMatrex gel orange A, followed by anion-exchange chromatography. Purification yielded ACCase240, which was bound to orange A dye (purified 68×), and ACCase220, having no affinity for orange A dye (purified 79×). ACCase220 contained two biotinylated polypeptides with molecular mass of 220 and 85 kDa. The 85-kDa protein was separated from the 220-kDa protein and had ACCase activity. ACCase220 was composed of seven proteins as determined by native PAGE. Two-dimensional electrophoresis (native/SDS PAGE) of ACCase220 revealed that these seven native proteins share constituent subunits. These subunits of ACCase220 were cross-linked using dithiobis(succinimidylpropionate), and unassociated polypeptides were removed by YM100 ultrafiltration. Reduction of cross-linked ACCase resulted in the reappearance of multiple polypeptides originally observed by SDS PAGE. On the basis of these results, we provide the first evidence that gramineae ACCase does not function solely as a homodimer.