A mammalian mutant cell lacking detectable lanosterol 14 alpha-methyl demethylase activity

Area of Science:

• Biochemistry
• Cell Biology
• Genetics

Background:

• Cholesterol biosynthesis is crucial for cellular function.
• Lanosterol 14 alpha-methyl demethylase is a key enzyme in sterol synthesis.
• Understanding enzyme deficiencies can reveal regulatory mechanisms.

Purpose of the Study:

• To characterize a novel Chinese hamster ovary cell mutant, AR45, selected for amphotericin B resistance.
• To investigate the specific metabolic defect in cholesterol biosynthesis within the AR45 mutant.
• To establish the utility of the AR45 cell line for studying sterol demethylase function and regulation.

Main Methods:

• Mutagenesis of Chinese hamster ovary cells using ethyl methanesulfonate.
• Selection of amphotericin B-resistant mutants.
• Analysis of sterol accumulation and metabolic conversion using whole cell and microsomal preparations.
• Spectral analysis of cytochromes to rule out general heme deficiency.

Main Results:

• The AR45 mutant is a cholesterol auxotroph, deficient in cholesterol biosynthesis.
• AR45 cells accumulate C30 sterols, lanosterol and dihydrolanosterol.
• The mutant shows impaired conversion of lanosterol's demethylation product to cholesterol.
• Spectral analysis confirmed the presence of cytochromes, indicating no general heme deficiency.
• The AR45 mutant is deficient in lanosterol 14 alpha-methyl demethylase activity.

Conclusions:

• The AR45 cell line is deficient in lanosterol 14 alpha-methyl demethylase.
• This mutant provides a valuable tool for investigating the regulation of sterol demethylase.
• The cell line will aid in the molecular cloning and genetic analysis of the demethylase enzyme.