Folate-dependent enzymes in cultured Chinese hamster cells: Folylpolyglutamate synthetase and its absence in mutants auxotrophic for glycine + adenosine + thymidine☆

摘要: Abstract Dialyzed sonicates from Chinese hamster ovary (CHO) and V-79 lung cells catalyze the addition of l -[U- 14 C]glutamate to tetrahydrofolate (H 4 PteGlu). Catalysis is optimal between pH 8.5 10.2 dependent on Mg 2+ a purine nucleotide triphosphate. Cobalamins do not stimulate system even when are grown in absence cyanocobalamin (CN-Cbl). Incubations with dl -H -[G- 3 H]PteGlu + show that product routinely assayed by DEAE-cellulose chromatography tetrahydropteroyldiglutamate PteGluGlu). Higher reduced folylpolyglutamates formed standard assay level PteGlu decreased 100 μ m 1–5 . Using either dialyzed extracts or 25-fold purified enzyme fraction, dATP 1.6 times more effective than ATP. The folyl specificity for diglutamate synthesis H > -homofolate 5-formyl-H 5-MeH PteGlu. -5-MeH only about 15% as active Extracts CHO mutant AUXB1 (requiring glycine adenosine thymidine) ght-1 hypoxanthine have PteGluGlu synthetase activity. also inactive other three compounds cited above Twelve out 16 revertant clones were isolated media lacking thymidine contained 44–66% wild-type Both parent catalyzed conversion tetrahydropteroyl triglutamate higher glutamyl conjugates. again lacked these catalytic properties. In contrast, revertants 50% activity displayed proportionate ability synthesize polyglutamyl From our findings published genetic data, we conclude cultured single can successively add at least glutamates Loss its function certain mutants responsible their triple auxotrophy.