Figure 5.

Separation of octameric and monomeric AO protein by sucrose density centrifugation. Crude extracts (A) prepared from methanol-induced cells producing WT AO, AOΔ16, or AOΔ22 protein or purified, octameric AO (B) isolated from WT cells or cells producing AOΔ22 were subjected to sucrose density centrifugation. The fractions obtained were analyzed by Western blotting using anti-AO antibodies. In crude extracts of WT cells and cells producing AOΔ16, AO protein was predominantly octameric (peak fraction in lane 3), whereas crude extracts prepared from cells producing AOΔ22 contained both AO monomers (peak fraction lane 7) and octamers. Purified, octameric WT AO obtained by gel filtration chromatography remained octameric upon sucrose density centrifugation (B), whereas AO octamers purified from cells synthesizing AOΔ22 partly had dissociated into monomers. Equal volumes of the fractions obtained from each gradient were loaded per lane. From the gradients prepared from AOΔ22 cells larger volumes of each fraction were loaded per lane compared with WT and AOΔ16, to allow detection of the strongly reduced amounts of AO protein in these cells.