Purification and characterization of dihydrofolate reductase from Walker 256 carcinosarcoma

Publisher Website
Google Scholar
Abstract
Dihydrofolate reductase of [rat] Walker 256 carcinosarcoma was purified to homogeneity by affinity chromatography. The enzyme reduced 28 .mu.mol dihydrofolate (FAH2) .cntdot. min-1 .cntdot. mg protein-1 at 22.degree. C and pH 7.3. Km values with respect to FAH2 and NADPH were 21 and 29 mM, respectively. The IC50 (amount of inhibitor required for 50% loss of enzyme activity) values were 0.2 mM for methotrexate [MTX] and aminopterin and 50 and 67 nM, respectively, for N10-formylfolate and triazinate (NSC-139105). The pH maximum is around pH 7.0 and the isoelectric point is 6.8. This reductase has an apparent MW of 21,500. The N-terminal amino acid is valine and the comparison of the N-terminal 20 residues of this reductase shows very high sequence homology with other mammalian reductases. The enzyme contains 2 cysteine residues and one of these residues is not involved in catalysis. This reductase has 4 tryptophan residues and modification of one of these residues leads to loss of activity. The intrinsic circular dichroic (CD) spectrum of this reductase is very different from the CD spectra of reductase of Escherichia coli B and [mouse leukemia] L1210/MTX. However, the CD spectra of the enzyme-substrate and enzyme-inhibitor complexes are very similar to that of the L1210/MTX enzyme. This suggests that the ligands may be constrained in similar conformation on the 2 enzymes. The fluorescence emission maximum at 314 nM when activated at 286 nM is considerably lower than other mammalian enzymes.