The progress of the purification was followed by electron microscopy (Figure 4-lA and B), SDS-PAGE (Figure 4-2), and assay of B12-dependent diol dehydratase (Table 4-1), an enzyme previously shown to be associated with these organelles (Bobik et al. 1999, Havemann et al. 2002).

Electron Microscopy

      Electron microscopy showed that the lysis by sonication effectively released many polyhedral organelles. Prolonged treatment (30 min.) of cells with lysozyme and EDTA prior to sonication produced extracts in which polyhedra were better separated from membrane fragments and amorphous debris. Sonication gave a higher yield of purified polyhedra than did lysis of cells using the French press. In crude lysates generated by sonication, polyhedra appeared to be bound to the outside of membrane vesicles whereas in lysates generated using a French Press, they were encased within membrane vesicles, which apparently interfered with purification. In addition, it was important to exclude MgC12 from the lysis buffer otherwise the polyhedral organelles pelleted with the unbroken cells and cell debris in the initial centrifugation step.

      After sonication and removal of cell debris by centrifugation, soluble extracts were treated with the proprietary detergent, B-PER II, sodium chloride, and magnesium chloride. This removed the majority of lipid vesicles and separated the polyhedra from the remaining vesicles and glycogen rosettes. The subsequent 48,000 x g centrifugation yielded a pellet highly enriched in polyhedra and separated from soluble proteins, while the final low-speed and sucrose density gradient centrifugation steps removed the remaining high molecular weight contaminants.

      Electron microscopy indicated that the purified organelles were nearly homogenous