Isolation of a mammalian glycosylation mutant with altered dolichol metabolism

Conference · Thu May 01 04:00:00 EDT 1986 · Fed. Proc., Fed. Am. Soc. Exp. Biol.; (United States)

OSTI ID:7246097

Stoll, J; Krag, S S

Mutagenized B4-2-1 Chinese hamster ovary cells grown on polyester replicas were screened for a reduction in the incorporation of (2-/sup 3/H)mannose into TCA-precipitable material. Mutant F2A8 incorporated 3 to 5-fold less (/sup 3/H)mannose into protein and oligosaccharide-lipid than B4-2-1 although levels of GDP-(/sup 3/H)mannose were comparable. G protein of vesicular stomatitis virus grown in the mutant migrated slightly faster during SDS-PAGE suggesting altered glycosylation; the other viral proteins appeared normal. Glucosylphosphoryldolichol (GPD) synthase and GlcNAc-1-P transferase activities in the presence of saturating exogenous dolichyl phosphate were similar in the two cell types. Using endogenous dolichyl phosphate, the GPD synthase activity of the mutant had one-third the activity of the parent, suggesting a lower pool of dolichyl phosphate. In order to examine the identity and amount of dolichol pools, cells were labelled for twenty-four hours with (2-/sup 3/H)mevalonic acid in the presence of serum and an inhibitor of HMG-CoA reductase, mevinolin. While mutant and parental lines had similar amounts of (/sup 3/H)cholesterol and (/sup 3/H)ubiquinone, the (/sup 3/H)polyisoprenol levels were five to ten-fold higher in the mutant. Using C/sub 18/ reverse phase hplc, the number of isoprene units in the polyisoprenol isolated from F2A8 was found to range from 17 to 19, as would be expected for dolichol. This polyisoprenol coeluted on silica hplc with unsaturated polyisoprene, not dolichol.

• Research Organization: Johns Hopkins Univ., Baltimore, MD
• OSTI ID: 7246097
• Report Number(s): CONF-8606151-
• Conference Information: Journal Name: Fed. Proc., Fed. Am. Soc. Exp. Biol.; (United States) Journal Volume: 45:6
• Country of Publication: United States
• Language: English

Related Subjects

550501* -- Metabolism-- Tracer Techniques
59 BASIC BIOLOGICAL SCIENCES
ALDEHYDES
ANIMAL CELLS
AROMATICS
BENZOQUINONES
CARBOHYDRATES
CARBOXYLIC ACIDS
CHO CELLS
CHOLESTEROL
CHROMATOGRAPHY
COENZYMES
ENZYME ACTIVITY
ENZYME INHIBITORS
ENZYMES
HEXOSES
HYDROXY ACIDS
HYDROXY COMPOUNDS
ISOTOPE APPLICATIONS
LABELLED COMPOUNDS
LIGASES
LIPIDS
LIQUID COLUMN CHROMATOGRAPHY
MANNOSE
METABOLISM
MEVALONIC ACID
MONOSACCHARIDES
MUTANTS
OLIGOSACCHARIDES
ORGANIC ACIDS
ORGANIC COMPOUNDS
ORGANIC OXYGEN COMPOUNDS
QUINONES
SACCHARIDES
SCREENING
SEPARATION PROCESSES
STEROIDS
STEROLS
TRACER TECHNIQUES
TRANSFERASES
TRITIUM COMPOUNDS
UBIQUINONE