Sterol-mediated regulation of mevalonic acid synthesis. Accumulation of 4-carboxysterols as the predominant sterols synthesized in a Chinese hamster ovary cell cholesterol auxotroph (mutant 215)

Journal Article · Fri Oct 05 04:00:00 EDT 1990 · Journal of Biological Chemistry; (USA)

OSTI ID:6272792

Plemenitas, A; Havel, C M; Watson, J A ^[1]

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1. Univ. of California, San Francisco (USA)

Chinese hamster ovary-215 (CHO-215) mutant cells are auxotrophic for cholesterol. Berry and Chang (Berry, D. J., and Chang, T. Y. (1982) Biochemistry 21, 573-580) suggested that the metabolic lesion was at the level of 4-methyl sterol oxidation. However, the observed cellular accumulation of lanosterol was not consistent with a defect at this metabolic site. With the use of a novel Silica Sep Pak sterol separation procedure, we demonstrated that 60-80% of the acetonesoluble lipid radioactivity in (5-3H)mevalonate-labeled CHO-215 cells was incorporated into acidic sterols. 7(8),Cholesten-4 beta-methyl,4 alpha-carboxy,3 beta-ol was the dominant end product. In addition to this acidic sterol, 7(8),24-cholestadien,4 beta-methyl,4 alpha-carboxy,3 beta-ol and 7(8),24-cholestadien,4 alpha-carboxy,3 beta-ol were also isolated. Incubation of cell-free extracts with (3H)7(8)-cholesten-4 beta-methyl, 4 alpha-carboxy,3 beta-ol and pyridine nucleotides confirmed that CHO-215 4-carboxysterol decarboxylase activity was less than 1% of that for wild type cells. Thus, a correspondence between decreased 4-carboxysterol decarboxylase activity and the spectrum of accumulated sterol products by intact CHO-215 cells was demonstrated. No detectable cholesterol was synthesized by CHO-215 cells. 3H-Product accumulation studies demonstrated that 7(8),24-cholestadien, 4 beta-methyl,4 alpha-carboxy,3 beta-ol increased prior to its subsequent saturation at the delta 24 carbon. Furthermore, the steady state ratio for delta 24-saturated acidic sterols/unsaturated acidic sterols was dependent on media cholesterol source and amount. Finally, the accumulated acidic sterol(s) were not regulatory signal molecules for the modulation of 3-hydroxy-3-methyl-glutaryl coenzyme. A reductase activity in response to cholesterol availability.

• OSTI ID: 6272792
• Journal Information: Journal of Biological Chemistry; (USA), Journal Name: Journal of Biological Chemistry; (USA) Vol. 265:28; ISSN JBCHA; ISSN 0021-9258
• Country of Publication: United States
• Language: English

Related Subjects

550201* -- Biochemistry-- Tracer Techniques
59 BASIC BIOLOGICAL SCIENCES
ANIMAL CELLS
BIOCHEMICAL REACTION KINETICS
BIOSYNTHESIS
CARBON-CARBON LYASES
CARBOXY-LYASES
CARBOXYLIC ACIDS
CHO CELLS
DECARBOXYLASES
ENZYME ACTIVITY
ENZYMES
HOMEOSTASIS
HYDROGEN COMPOUNDS
HYDROXY ACIDS
HYDROXY COMPOUNDS
ISOTOPE APPLICATIONS
KINETICS
LYASES
MASS SPECTRA
MEVALONIC ACID
ORGANIC ACIDS
ORGANIC COMPOUNDS
REACTION KINETICS
SPECTRA
STEROIDS
STEROLS
SYNTHESIS
TRACER TECHNIQUES
TRITIUM COMPOUNDS