Chemistry and Chemical Biology ETDs

Publication Date

7-6-1977

Abstract

The conversion of farnesyl pyrophosphate to squalene by rat liver microsomes was investigated. The enzyme exhibited an apparent maximum velocity at a substrate concentration of 30 μM. No evidence of substrate inhibition was seen at concentrations up to 65 μM. Phosphatases present in the microsomes or in the 105,000 x g supernatant (S105) did not interfere with squalene synthesis under the experimental conditions used.

s105 slightly enhanced the biosynthesis of squalene at the lowest concentrations of microsomal protein studied. The effect was reversed at higher concentrations of microsomes. A 1O-fold increase in the concentration of s105 did not affect the rate at which squalene was synthesized. Partially purified SCP1 did not activate squalene biosynthesis.

A technique for the preparation of endogenously generated squalene for use as a substrate has been devised. This endogenously generated squalene was firmly bound to the microsomes and was not removed as a soluble squalene:SCP1 complex. Microsomes incubated with cofactors in the absence of SCP1 were minimally active in converting endogenously generated squalene to sterol. Full activity required the addition of partially purified SCP1.

Therefore, a requirement for an activating supernatant protein for the metabolism of intermediates in the cholesterol biosynthetic pathway is first seen with the appearance of the first water-insoluble intermediate, squalene. Activation of the conversion of squalene to sterol (lanosterol) by SCP1 is not an artifact, since partially purified SCP1 activated this reaction even when squalene had been generated from farnesyl pyrophosphate in situ.

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 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 pre-mixed with microsomes and SCP1 for 30 minutes. In addition, microsomes were found to contain a significant amount of squalene (unlabeled) that had been biosynthesized in vivo.

The results suggest that a fraction of the squalene bound to microsomes is present as a squalene:SCP1:microsome complex. SCP1 may facilitate the movement of squalene on or within the microsomal membrane and by this mechanism activate the microsomal conversion of squalene to lanosterol.

Language

English

Document Type

Dissertation

Degree Name

Chemistry

Level of Degree

Doctoral

Department Name

Department of Chemistry and Chemical Biology

First Committee Member (Chair)

Terence Joseph Scallen

Second Committee Member

John Leroy Omdahl

Third Committee Member

Cary Jacks Morrow

Fourth Committee Member

David Lee Vander Jagt

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