Studies on the conversion of enzymatically generated, microsome-bound squalene to sterol.

We have previously demonstrated that sterol carrier protein1 (SCP1) is required for the conversion of squalene to lanosterol by rat liver microsomes (Srikantaiah, M. V., Hansbury, E., Loughran, E. D., and Scallen, T. J. (1976) J. Biol. Chem. 251, 5496-5505). These studies, however, had the disadvantage that the highly waterinsoluble substrate squalene was added exogenously to incubations in small quantities of an organic solvent mixture (dioxane:propylene glycol, 2:l). Using the .techniques described here and in the accompanying article (Gavey, K. L., and Scallen, T. J. (1978) J. Biol. Chem. 253, 5470-5475) radioactive farnesyl pyrophosphate was used to enzymatically generate squalene in situ in the microsomal membranes. This preparation has the advantage that no agent, e.g. organic solvent or detergent, is required for the addition of the substrate squalene. This endogenously generated squalene was firmly bound to the microsomes. Microsomes incubated with cofactors in the absence of SCPl were only minimally active in converting endogenously generated squalene to sterol. A series of experiments demonstrated that SCPl was required to activate the conversion of squalene to lanosterol, even when squalene was biosynthesized in situ bound to the microsomal membranes. Therefore, a requirement for a specific supernatant protein, e.g. SCP1, for the activation of microsomal enzymes in cholesterol biosynthesis is first seen only after the formation of squalene, the first water-insoluble intermediate in the pathway. The nature of the interaction between SCP1, squalene, and microsomes was investigated, using both endogenously generated squalene and exogenously added squalene as substrates. Incubations begun with the addition of exogenous squalene exhibited a lag phase before the formation of radioactively labeled sterol was observed. This phenomenon was not evident when endogenously generated squalene was converted to sterol; nor was a lag phase evident when radioactively labeled squalene was premixed anaerobically with microsomes and SCPl for 30 min prior to incubation in an atmosphere of oxygen. In addition, microsomes were found to contain a significant amount of endogenous unlabeled squalene that had been biosynthesized in vivo. The results suggest that a fraction of the squalene bound to microsomes is present as a squalenea SCPl *microsome complex. SCPl may facilitate the