This chapter describes methods pertinent to the assay of the reactions in squalene synthesis. The enzyme may be solubilized and depleted of membrane lipids, with loss of catalytic activities. When reconstituted with the appropriate lipids, normal catalytic behavior can be restored. Methods for solubilization, delipidation, and reconstitution are given. Squalene synthetases are associated with the subcellular membranes in mammalian liver and in yeast. Although the enzyme and its products might initially seem to be of only moderate interest, pursuit of the reaction mechanisms and the underlying enzymology in very fundamental ways has stimulated a remarkable chapter in biochemical research. This has included the systematic resolution of all of the stereochemical transformations in the reactions between mevalonate and squalene. The rate of squalene synthesis seems more closely correlated with the rate of the condensation reaction than the amount of presqualene pyrophosphate present. The substrate “delivered” from the active site of the first reaction may be preferred over presqualene pyrophosphate accumulated in the membranes. Although these interpretations remain speculative, they are instructive in considering conditions for assay of the reactions and for solubilization and reconstitution of the enzyme.
ASJC Scopus subject areas
- Molecular Biology