Reculation of folylpolyglutamate synthetase in extracts of H35 hepatoma cells

Conference · Fri May 01 04:00:00 EDT 1987 · Federation Proceedings, Federation of American Societies for Experimental Biology; (USA)

OSTI ID:5084532

Johnson, T B; Galivan, J; Nair, M G ^[1]

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1. New York State Health Labs., Albany (USA)

Folylpolyglutamate synthetase (FPGS) in extracts of H35 hepatoma cells was assayed using 250 {mu}M methotrexate (MTX) as the substrate under conditions where ({sup 3}H)glutamate incorporation was linear with respect to time and rotein concentration. Extracts from confluent cultures with reduced cellular folates exhibited nearly 1.7-fold higher FPGS specific activity than extracts of control cultures (600 pmol/hr/mg). Extracts of rapidly dividing cells (72 hrs) showed nearly a 2.3-fold increase. The addition of reduced exogenous folates such as folinic acid and 5-methyltetrahydrofolate (20 {mu}M, 24 hrs) to confluent cultures of folate-depleted cells typically lowered the FPGS activity in the resultant extracts by 40%, while a 42-hour exclusion of methionine from the media reduced the activity by half. The combination of methionine exclusion and folate addition for 42 hrs resulted in 75% lower FPGS activity vs extracts of control cultures of folate-depleted cells. These data suggest that the change sin the glutamylation rate of MTX in whole cells due to culture conditions such as folate restriction, reduced folate addition, methionine exclusion, and growth state are at least in part a consequence of alterations in FPGS activity. Using MTX or N{sup 10}-propargyl-5,8-dideazafolic acid (CB3717) as the starting substrate under appropriate assay conditions, FPGS from extracts catalyzed the formation of similar polyglutamate products as seen in analogous whole cell experiments.

• OSTI ID: 5084532
• Report Number(s): CONF-870644--
• Conference Information: Journal Name: Federation Proceedings, Federation of American Societies for Experimental Biology; (USA) Journal Volume: 46:6
• Country of Publication: United States
• Language: English

Related Subjects

550201* -- Biochemistry-- Tracer Techniques
59 BASIC BIOLOGICAL SCIENCES
AMINO ACIDS
ANIMAL CELLS
AROMATICS
AZAARENES
BIOCHEMICAL REACTION KINETICS
CARBOXYLIC ACIDS
CITROVORUM FACTOR
DRUGS
ENZYME ACTIVITY
ENZYMES
FOLIC ACID
GLUTAMIC ACID
HEMATINICS
HEMATOLOGIC AGENTS
HETEROCYCLIC COMPOUNDS
HYDROGEN COMPOUNDS
HYDROXY COMPOUNDS
ISOTOPE APPLICATIONS
KINETICS
LIGASES
LIPOTROPIC FACTORS
LIVER CELLS
METHIONINE
ORGANIC ACIDS
ORGANIC COMPOUNDS
ORGANIC NITROGEN COMPOUNDS
ORGANIC SULFUR COMPOUNDS
PTERIDINES
REACTION KINETICS
RESPONSE MODIFYING FACTORS
SOMATIC CELLS
TRACER TECHNIQUES
TRITIUM COMPOUNDS
TUMOR CELLS
VITAMIN B GROUP
VITAMINS