Folate-dependent enzymes in cultured Chinese hamster ovary cells: induction of 5-methyltetrahydrofolate homocysteine cobalamin methyltransferase by folate and methionine
The effects of media vitamin B/sub 12/(CNB/sub 12/), L-methionine, folic acid, dl-5-methyltetrahydrofolate (5-MeH/sub 4/folate), homocysteine, and other nutrients on four one-carbon enzymes in cultured Chinese hamster ovary (CHO) cells were examined. Excess 10 mM methionine elevates the amount of B/sub 12/ methyltransferase 1.8--2.3-fold at media folate concentrations of 0.2--2.0 ..mu..M. Conversely, excess 100 ..mu..M folic acid increases the amount of B/sub 12/ holoenzyme by 2.4--3.0-fold when the medium contains 0.01--0.1 mM methionine. These increases in B/sub 12/ methyltransferase promoted by 100 ..mu..M media folate and 10mM methionine are inhibited by cycloheximide. 5-MeH/sub 4/folate will support growth and induce methyltransferase synthesis more efficiently than folic acid. Upon transfer to methionine-free media, wild-type CHO cells will survive and can be repeatedly subcultured in the absence of exogenous methionine, provided it is supplemented with 1.0 ..mu..M CNB/sub 12/, 0.1 mM homocysteine, and 100 ..mu..m folic acid or 10 ..mu..m dl-5-MeH/sub 4/folate. No growth occurs if homocysteine is omitted, but a requirement for added CNB/sub 12/ does not become evident until the cells have undergone at least two or three divisions. Survival upon transfer from 0.1 mM methionine-containing to methionine-free media is dependent upon the B/sub 12/ holomethyltransferase content of the cells used as an inoculum. Inoculum cells must have been previously grown in media supplemented with 1.0 ..mu..m CNB/sub 12/ to stabilize and convert apo- to holomethyltransferase, and 100 ..mu..M folate (or 10 ..mu..M dl-5-MeH/sub 4/folate) to induce maximal enzyme-protein synthesis. Transfer to methionine-deficient medium does not result in more than a 20-25 percent increase in the cellular B/sub 12/ enzyme content over the level already induced by 100 ..mu..M folate in 0.1 mM methionine-supplemented media.
- Research Organization:
- Univ. of California, Livermore
- OSTI ID:
- 7355003
- Journal Information:
- Arch. Biochem. Biophys.; (United States), Vol. 171
- Country of Publication:
- United States
- Language:
- English
Similar Records
The folate-binding module of Thermus thermophilus cobalamin-dependent methionine synthase displays a distinct variation of the classical TIM barrel: a TIM barrel with a `twist'
The role of methionine in the intracellular accumulation and function of folates
Related Subjects
ANIMAL CELLS
ENZYMES
CYCLOHEXIMIDE
BIOLOGICAL EFFECTS
BIOSYNTHESIS
FOLIC ACID
METHIONINE
TRANSFERASES
VITAMIN B-12
CELL CULTURES
HAMSTERS
NUTRIENTS
AMINO ACIDS
ANIMALS
ANTIBIOTICS
CARBOXYLIC ACIDS
DRUGS
FUNGICIDES
HETEROCYCLIC COMPOUNDS
HYDROXY COMPOUNDS
LIPOTROPIC FACTORS
MAMMALS
ORGANIC ACIDS
ORGANIC COMPOUNDS
ORGANIC NITROGEN COMPOUNDS
ORGANIC SULFUR COMPOUNDS
PESTICIDES
PTERIDINES
RODENTS
SYNTHESIS
VERTEBRATES
VITAMIN B GROUP
VITAMINS
550200* - Biochemistry