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The cells were grown for several weeks in their stated culture medium until the time of assay.Between and cells were washed three times in HBSS and suspended to. A total of mL dialyzed fetal calf serum was added to each culture.mL icecold phosphatebuffered saline was added to each well and mL was removed for cell counting.The remaining cells were washed twice in icecold PBS, and then mL icecold TCA was added for minutes.After hours at C in CO, the reaction was stopped with icecold PBS and the cells prepared for counting as for the incorporation of propionic acid incorporated per cells per hour.Additional vitamin B prevented this cell death and restored the growth rate. High concentrations of methyltetrahydrofolate did not reverse the cytotoxic effects of cyanocobalamin. The results were identical. The cells grew normally for approximately days, after which growth ceased and the cells died.Additional vitamin B   supplements maintained viability and allowed continued growth did not reverse the cytotoxicity of the inhibitor.When the cells were cultured with the additional concentrations of vitamin B, tetrahydrofolate incorporation was stimulated, and this difference was lost.In an attempt to determine whether some of the incorporation of tetrahydrofolate might be taking place via a pathway other than <a href="http://www.targetmol.com/compound/Chlorpheniramine-maleate"></a> methionine synthase, the uptake of label was also determined in the presence of excess methionine in the assay system.Although methionine synthase remains active in vivo in the presence of excess methionine, the dilution of the labeled end product by the excess of exogenous unlabeled methionine should greatly reduce the incorporation of label into trichloroacetic acidprecipitable macromolecules, unless the newly synthesized methionine is channeled directly into cellular methylation reactions or protein synthesis, rather than through a general metabolic pool.Cyanocobalamin is unlikely to be a direct inhibitor of methionine synthase, because it is an efcient cofactor for the apoenzyme in a cellfree system.It more probably affects some other aspect of the intracellular metabolism of vitamin B, such as uptake, transport, or reduction.Small concentrations of methionine prevented cytotoxicity, and allowed the cells to proliferate at a reduced rate.Methionine at concentrations of mmolL or more protected cells completely from the lethal effects of cyanocobalamin. The cells divided at a normal rate for at least months and showed no morphologic evidence of megaloblastosis.This indicates that methionine deciency is responsible for the early cytotoxic effects of cyanocobalamin and that, when the methionine concentration in the culture medium exceeds approximately mmolL, the remethylation of homocysteine is unimportant for cell growth.Because HL cells grew normally for an indenite period in the presence of cyanocobalamin when provided with sufcient methionine, it seems reasonable to conclude that they did not develop intracellular folate deciency secondary to the inhibition of vitamin B.Nonspecic cytotoxicity is excluded by the lack of growth inhibition in the presence of methionine and because the effects are reversed by vitamin B.This phenomenon has not previously been observed and, if conrmed, is worthy of further study.There was substantial incorporation of label from tetrahydrofolate when the assay system contained excess methionine.One explanation for this nding might be that the uptake occurred via a pathway other than methionine synthase, although channeling of the newly synthesized methionine directly into cellular methylation reactions or protein synthesis cannot be excluded.

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