TY - JOUR
T1 - Dihydrofolate Reductase from a Resistant Subline of the L1210 Lymphoma. Purification by Affinity Chromatography and Ultraviolet Difference Spectrophotometric and Circular Dichroic Studies
AU - Gupta, Sagar V.
AU - Greenfield, Norma J.
AU - Poe, Martin
AU - Makulu, David R.
AU - Williams, Myra N.
AU - Moroson, Barbara A.
AU - Bertino, Joseph R.
PY - 1977/7/1
Y1 - 1977/7/1
N2 - Dihydrofolate reductase has been isolated in 80% yield and purity >99% from a methotrexate-resistant subline of the L1210 lymphoma (L1210/MTX) by the use of affinity chromatography. Purity has been established by titrating enzymic activity and protein fluorescence with a stoichiometric inhibitor methotrexate (MTX) and confirmed by polyacrylamide gel electrophoresis. The apparent molecular weight calculated from filtration through a Sephadex G-100 column is 21 000 ± 3%. This reductase contains four tryptophan residues per molecule. The binary complexes of enzyme with folate, dihydrofolate, MTX, NAD+, and NADPH resulted in a changed ultraviolet absorption spectra compared to the unmixed components. The changes observed upon binding of dihydrofolate, MTX, and NADPH in the range 240 to 400 nm in the spectra were similar to those reported for the reductase of Escherichia coli (Poe, M., Greenfield, N. J., Hirschfield, J. M., and Hoogsteen, K. (1974a), Cancer Biochem. Biophys. 1, 7; Poe, M., Greenfield, N. J., and Williams, N. (1974b), J. Biol. Chem. 249, 2710). This indicates that the chemical environment of the bound substrate, inhibitor, and coenzyme may be the same in the two enzymes. Absorbance changes at 280 and 290 nm suggest that one (or more) tryptophan residues are perturbed upon binding of these ligands. The intrinsic circular dichroic (CD) spectra of dihydrofolate reductase from L1210/MTX and E. coli B (Greenfield, N. J., Williams. M. N., Poe, M., and Hoogsteen, K. (1972), Biochemistry 11, 4706) are very different. Both the “backbone” ellipticity and aromatic region are quite distinctive. However, the CD of the enzyme-substrate and inhibitor complexes as well as enzyme-MTX-NADPH ternary complexes exhibit some homologies as compared with the E. coli B enzyme. This suggests that the ligands may be constrained in similar conformation on the two enzymes.
AB - Dihydrofolate reductase has been isolated in 80% yield and purity >99% from a methotrexate-resistant subline of the L1210 lymphoma (L1210/MTX) by the use of affinity chromatography. Purity has been established by titrating enzymic activity and protein fluorescence with a stoichiometric inhibitor methotrexate (MTX) and confirmed by polyacrylamide gel electrophoresis. The apparent molecular weight calculated from filtration through a Sephadex G-100 column is 21 000 ± 3%. This reductase contains four tryptophan residues per molecule. The binary complexes of enzyme with folate, dihydrofolate, MTX, NAD+, and NADPH resulted in a changed ultraviolet absorption spectra compared to the unmixed components. The changes observed upon binding of dihydrofolate, MTX, and NADPH in the range 240 to 400 nm in the spectra were similar to those reported for the reductase of Escherichia coli (Poe, M., Greenfield, N. J., Hirschfield, J. M., and Hoogsteen, K. (1974a), Cancer Biochem. Biophys. 1, 7; Poe, M., Greenfield, N. J., and Williams, N. (1974b), J. Biol. Chem. 249, 2710). This indicates that the chemical environment of the bound substrate, inhibitor, and coenzyme may be the same in the two enzymes. Absorbance changes at 280 and 290 nm suggest that one (or more) tryptophan residues are perturbed upon binding of these ligands. The intrinsic circular dichroic (CD) spectra of dihydrofolate reductase from L1210/MTX and E. coli B (Greenfield, N. J., Williams. M. N., Poe, M., and Hoogsteen, K. (1972), Biochemistry 11, 4706) are very different. Both the “backbone” ellipticity and aromatic region are quite distinctive. However, the CD of the enzyme-substrate and inhibitor complexes as well as enzyme-MTX-NADPH ternary complexes exhibit some homologies as compared with the E. coli B enzyme. This suggests that the ligands may be constrained in similar conformation on the two enzymes.
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U2 - 10.1021/bi00633a005
DO - 10.1021/bi00633a005
M3 - Article
C2 - 19040
AN - SCOPUS:0017648237
SN - 0006-2960
VL - 16
SP - 3073
EP - 3079
JO - Biochemistry
JF - Biochemistry
IS - 14
ER -