Dihydrofolate reductase from a resistant subline of the L1210 lymphoma. Purification by affinity chromatography and ultraviolet difference spectrophotometric and circular dichroic studies

Abstract

Dihydrofolate reductase has been isolated in 80% yield and purity >99% from a methotrexate-resistant subline of the LI 210 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%.

Interaction of methotrexate, folates, and pyridine nucleotides with dihydrofolate reductase: calorimetric and spectroscopic binding studies.

“…The formation of binary and ternary complexes of dihydrofolate reductase with cofactors, substrates, and drugs has been examined by a wide variety of techniques including UV absorption (11)(12)(13), fluorescence (14,15), circular dichroism (16), and NMR spectroscopy (17,18). In general, these studies confirmed the tightness of binding and, in addition, suggested the possibility of conformational changes in the enzyme associated with the binding process.…”

The charge state of trimethoprim bound to Lactobacillus casei dihydrofolate reductase

“…There appear to be two major differences between the binding of methotrexate and that of folate: methotrexate is protonated at N1 when bound to the enzyme [2][3][4][5][6][7][8][9][10], while folate is apparently not, and the pteridine ring of methotrexate binds to the enzyme in an orientation which differs by a rotation of about 180 ° about its long axis from that adopted by folate [11][12][13][14]. The recent determination of the crystal structure of the enzyme-trimethoprim complex [15] shows that the orientation of the 2,4-diaminopyrimidine ring in the binding site is very similar to that of the analogous part of methotrexate [11,16].…”

Inhibition of Multiplication of Mouse Carcinoma FM3A Cells by Methotrexate Encapsulated in Liposomes That Contain Hematoporphyrin Derivative in the Membrane

“…Since the concentration of CF was 10 mM in REV which were sized to 0.4-11m by a Unipore filter just before application to cells, 1.5x [10][11][12][13][14][15][16][17][18] we were able to calculate the minimum number of REV bound and delivered to a cell. Under the experimental conditions specified in Table I, the amount of CF delivered by REV containing HPD during an 1-hr incubation and retained in each cell during a 16-hr incubation in the REV -free medium was approximately 1.5 x 10-18 mol per cell.…”

“…16 l The inhibition leads to the starvation of thymidine triphosphate which is required for DNA synthesis, resulting in the arrest of chromosomal replication and cell proliferation. 17 l We encapsulated MTX in REV containing HPD as descdbed for the preparation of CFcontaining REV.…”