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 00:00:00 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), Vol. 265:28; ISSN 0021-9258
• Country of Publication: United States
• Language: English

Related Subjects

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