Isolation and characterization of dihydrofolic acid reductase from methotrexate-sensitive and -resistant human cell lines.
Dihydrofolic acid reductase has been purified by affinity chromatography to apparent homogeneity from the human HeLa BU-25 cell line and from two methotrexate-resistant variants, one deriving from HeLa BU-25 and the other from the human VA2-B cell line. The purified enzymes from the three sources have been characterized in their physical and enzymatic properties. They were not found to differ significantly as concerns their electrophoretic mobility in polyacrylamide gels under a variety of conditions, their specific dihydrofolic acid reductase and folic acid reductase activities, their Km values for folic acid and TPNH, their sensitivity to methotrexate, and the pH dependence of their folic acid reductase activity. The human dihydrofolic acid reductase has an apparent molecular weight of 21,000 to 22,000, a Km for folic acid of 6.1 to 7.6 X 10(-6) M and a Km for TPNH of 1.6 to 1.7 X 10(-4) M, turnover numbers of about 500 and 65 mol/min/mol of enzyme for the dihydrofolic acid reductase and the folic acid reductase activity, respectively. The values of the above mentioned physical and kinetic parameters are comparable to those reported for the dihydrofolic acid reductase from other animal cell systems. The dihydrofolic acid reductase content of the two-resistant cell lines is at least 200-fold higher than that of the methotrexate-sensitive HeLa BU-25 cell line. The available evidence indicates that this increased dihydrofolic acid reductase content results from a hyperproduction of an enzyme identical or similar to that of the sensitive cells, presumably due to a selective dihydrofolic acid reductase gene amplification, as previously reported for other cell lines of rodent origin.
D004351:Drug Resistance; D006367:HeLa Cells / Q000201:enzymology; D006801:Humans; D007700:Kinetics; D008727:Methotrexate / Q000494:pharmacology*; D009154:Mutation; D013762:Tetrahydrofolate Dehydrogenase / Q000302:isolation & purification* / Q000378:metabolism; D013937:Thymidine Kinase / Q000172:deficiency
D013762:Tetrahydrofolate Dehydrogenase; D013937:Thymidine Kinase; D008727:Methotrexate