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Methylenetetrahydrofolate Reductase in Cultured Human Cells. II. Genetic and Biochemical Studies of Methylenetetrahydrofolate Reductase Deficiency

Summary: Methylenetetrahydrofolate (methylene-H4PteGlu) reductase activities in extracts of both normal and reductase-deficient cells were low and quite variable during the logarithmic phase of growth. Higher activities were detected reproducibly when the cultures were confluent. Methylene-H4PteGlu reductase activities in extracts of fibroblasts from the parents of patient CP were about half of the level observed in normal control subjects. In fibroblasts from patient CP, the activity was 20% of normal whereas, from patients LM and BM, the activities were 19% and 14% of normal, respectively. When incubated at 55° in a solution containing all the components of the standard reaction mixture except the 5-methyl-H4PteGlu substrate, the reductase activity in extracts of fibroblasts from two unrelated normal control subjects decreased to 31% and 22%, respectively, of the initial values after 30 min of incubation. In contrast, the reductase in extracts of cells patient CP was rapidly and exponentially inactivated at 55°. The reductase activity in extracts from patients LM and BM, the sisters, decreased to 22% and 38%, respectively, of the initial values. In repeated experiments the heat inactivation of reductase activity in extracts of cells from LM and BM closely resembled the normal control subjects in total extent and time course of inactivation. The reductase activity in extracts from WMa, a fourth, unreleated patient was also completely inactivated but somewhat less rapidly than with patient CP. These results provide strong evidence that the reductase deficiency in these three families is due to different alleles. The data suggest that in CP and WMa there is a mutationally induced structural defect in the aporeductase as the basis for the observed alteration in thermostability, presumably reflecting reduced ability to bind the FAD cofactor.Speculation: The observation that methylene-H4PteGlu reductase activity in crude fibroblast extracts from two of the three families studied shows reduced thermal stability as well as decreased activity raises the possibility that this enzyme is unusually susceptible to mutationally induced alterations in cofactor binding.