In situ autoradiographic detection of folylpolyglutamate synthetase activity

Journal Article · Sat Nov 01 00:00:00 EST 1986 · Anal. Biochem.; (United States)

DOI:https://doi.org/10.1016/0003-2697(86)90563-4· OSTI ID:5993607

Sussman, D J; Milman, G; Osborne, C; Shane, B

The enzyme folylpolyglutamate synthetase (FPGS) catalyzes the conversion of folate (pteroylmonoglutamate) to the polyglutamate forms (pteroylpolyglutamates) that are required for folate retention by mammalian cells. A rapid in situ autoradiographic assay for FPGS was developed which is based on the folate cofactor requirement of thymidylate synthase. Chinese hamster AUX B1 mutant cells lack FPGS activity and are unable to accumulate folate. As a result, the conversion of (6-/sup 3/H)deoxyuridine to thymidine via the thymidylate synthase reaction is impaired in AUX B1 cells and no detectable label is incorporated into DNA. In contrast, FPGS in wild-type Chinese hamster CHO cells causes folate retention and enables the incorporation of (6-/sup 3/H)deoxyuridine into DNA. Incorporation may be detected by autoradiography of monolayer cultures or of colonies replica plated onto polyester discs. Introduction of Escherichia coli FPGS into AUX B1 cells restores the activity of the thymidylate synthase pathway and demonstrates that the E. coli FPGS enzyme can provide pteroylpolyglutamates which functions in mammalian cells.

• Research Organization: Johns Hopkins Univ., Baltimore, MD
• OSTI ID: 5993607
• Journal Information: Anal. Biochem.; (United States), Vol. 158:2
• Country of Publication: United States
• Language: English

Related Subjects

62 RADIOLOGY AND NUCLEAR MEDICINE
59 BASIC BIOLOGICAL SCIENCES
CHO CELLS
AUTORADIOGRAPHY
BIOLOGICAL PATHWAYS
LIGASES
BIOCHEMISTRY
CARBOXYLIC ACID SALTS
DEOXYURIDINE
DNA
ENZYME ACTIVITY
ESCHERICHIA COLI
FOLIC ACID
GLUTAMIC ACID
MUTANTS
THYMIDINE
TRITIUM COMPOUNDS
AMINO ACIDS
ANIMAL CELLS
ANTIMETABOLITES
AROMATICS
AZAARENES
AZINES
BACTERIA
CARBOXYLIC ACIDS
CHEMISTRY
DRUGS
ENZYMES
HEMATINICS
HEMATOLOGIC AGENTS
HETEROCYCLIC COMPOUNDS
HYDROXY COMPOUNDS
LABELLED COMPOUNDS
MICROORGANISMS
NUCLEIC ACIDS
NUCLEOSIDES
NUCLEOTIDES
ORGANIC ACIDS
ORGANIC COMPOUNDS
ORGANIC NITROGEN COMPOUNDS
PTERIDINES
PYRIMIDINES
RIBOSIDES
URACILS
VITAMIN B GROUP
VITAMINS
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