Folate-dependent enzymes in cultured Chinese hamster ovary cells: Induction of 5-methyltetrahydrofolate homocysteine cobalamin methyltransferase by folate and methionine

Abstract

The effects of media vitamin B 12(CNB 12), l-methionine, folic acid, dl-5-methyltetrahydrofolate (5-MeH 4folate), homocysteine, and other nutrients on four one-carbon enzymes in cultured Chinese hamster ovary (CHO) cells were examined. Excess 10 m m methionine elevates the amount of B 12 methyltransferase 1.8 – 2.3-fold at media folate concentrations of 0.2 – 2.0 μ m. Conversely, excess 100 μ m folic acid increases the amount of B 12 holoenzyme by 2.4 – 3.0-fold when the medium contains 0.01 – 0.1 m m methionine. These increases in B 12 methyltransferase promoted by 100 μ m media folate and 10 m m methionine are inhibited by cycloheximide. 5-MeH 4folate 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 μ m CNB 12, 0.1 m m homocysteine, and 100 μ m folic acid or 10 μ m dl-5-MeH 4folate. No growth occurs if homocysteine is omitted, but a requirement for added CNB 12 does not become evident until the cells have undergone at least two or three divisions. Survival upon transfer from 0.1 m m methionine-containing to methionine-free media is dependent upon the B 12 holomethyltransferase content of the cells used as an inoculum. Inoculum cells must have been previously grown in media supplemented with 1.0 μ m CNB 12 to stabilize and convert apo- to holomethyltransferase, and 100 μ m folate (or 10 μ m dl-5-MeH 4folate) to induce maximal enzyme-protein synthesis. Transfer to methionine-deficient medium does not result in more than a 20–25% increase in the cellular B 12 enzyme content over the level already induced by 100 μ m folate in 0.1 m m methionine-supplemented media. A mutant auxotroph CHO AUXB1 with a triple growth requirement for glycine + adenosine + thymidine (McBurney, M. W., and Whitmore, G. F. (1974) Cell, 2, 173) cannot survive in media lacking exogenous methionine. High concentrations of media folic acid or dl-5-MeH 4folate fail to induce elevated amounts of B 12 methyltransferase in this mutant. Excess 10 m m medium methionine does, however, elevate its B 12 enzyme as in the parent CHO cells. An additional mutant AUXB3 that requires glycine + adenosine (McBurney, M. W., and Whitmore, G. F. (1974) Cell, 2, 173) barely survives in methionine-deficient media. It has a folate-induced B 12 enzyme level intermediate between wild-type CHO cells and AUXB1. The level of B 12 methyltransferase induced by high media folate concentrations is a critical determinant of CHO cell survival in methionine-free media.