J. Biol. Chem., Vol. 263, Issue 3, 1248-1254, 01, 1988
A mammalian mutant cell lacking detectable lanosterol 14 alpha-methyl demethylase activity
HW Chen, DA Leonard, RT Fischer and JM Trzaskos
Medical Products Department, E. I. du Pont de Nemours and Company, Wilmington, Delaware 19898.
A Chinese hamster ovary cell mutant, AR45, was selected for amphotericin B
resistance after treatment with the mutagen ethyl methanesulfonate. The
mutant is a cholesterol auxotroph with a deficiency in cholesterol
biosynthesis. Whole cell experiments demonstrate that the mutant
accumulates the C30 sterols, lanosterol and dihydrolanosterol, under
culture conditions which promote active sterol biosynthesis. Metabolic
studies show that the C29 sterol demethylation product of lanosterol, but
not lanosterol itself, is actively converted to end product cholesterol by
whole cells as well as by microsomal preparations derived from the mutant.
Detectable amounts of several cytochromes can be observed spectrally in the
AR45 demonstrating that it is not a general heme-deficient mutant.
Collectively, these results characterize the AR45 mutant cells as being
lanosterol 14 alpha-methyl demethylase-deficient. The cell line should
prove useful in studying regulation of the demethylase enzyme and the
putative endogenous regulatory oxysterol. It should also be a useful tool
in the molecular cloning and elucidation of genetic properties of the
demethylase.