Methotrexate-resistant Chinese hamster ovary cells contain a dihydrofolate reductase with an altered affinity for methotrexate.
Previous reports [Flintoff, W. F., Davidson, S. V., & Siminovitch, L. (1976) Somatic Cell Genet. 2, 245--261; Gupta, R. S., Flintoff, W. F., & Siminovitch, L. (1977) Can. J. Biochem. 55, 445--452] described a series of Chinese hamster ovary cells that were resistant to the cytotoxic action of methotrexate and contained a dihydrofolate reductase that was less sensitive to inhibition by the drug than wild-type enzyme. In this study, binding of labeled methotrexate to the reductase--NADPH complex and separation of free and bound drug by filtration through Sephadex G--25 have been used to demonstrate that clonal isolates of these resistant cells contain a dihydrofolate reductase varying between 2.5- and 6-fold lower in affinity for the drug than the wild-type enzyme. The apparent dissociation constant for the wild-type enzyme is 0.5 x 10(-9) M. Using two-dimensional polyacrylamide gel electrophoresis, 11 independently selected resistant isolates have been shown to contain a reductase with a similar overall net charge as the wild-type enzyme. Reductase purified from either wild-type or resistant cells contains two components after isoelectric focusing in polyacrylamide gels. The major component represents about 90% of the total protein and has a pI of about 8.0. The minor component representing about 10% of the reductase protein has a pI between 7.2 and 7.6.
D000818:Animals; D002460:Cell Line; D006224:Cricetinae; D003412:Cricetulus; D004351:Drug Resistance; D005260:Female; D007700:Kinetics; D008727:Methotrexate / Q000494:pharmacology*; D009154:Mutation; D010053:Ovary; D011485:Protein Binding; D013762:Tetrahydrofolate Dehydrogenase / Q000378:metabolism*
D013762:Tetrahydrofolate Dehydrogenase; D008727:Methotrexate