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Interaction of Polyglutamyl Derivatives of Methotrexate, 10-Deazaaminopterin, and Dihydrofolate with Dihydrofolate Reductase¹

Department of Biochemistry and Pharmacology, Tufts University Health Science Campus, Boston, Massachusetts 02111 [P. K., R. L. K., Y. G.]; Department of Biochemistry, University of South Alabama, Mobile, Alabama 36688 [M. G. N., C. M. B.]; and Laboratory of Nutrition and Endocrinology, National Institute of Arthritis, Diabetes, Digestive and Kidney Diseases, NIH, Bethesda, Maryland 20205 [B. T. K.]

Polyglutamyl derivatives of methotrexate (MTX) and 10-deazaaminopterin (10-DAM) containing a total of one through six glutamate residues (Glu residues) were tested as inhibitors of dihydrofolate reductase (DHFR) derived from sheep, chicken, and beef liver. The ability of dihydropteroylpentaglutamate to antagonize the inhibitory activity of these analogues was also studied. The most striking effects were seen with sheep liver DHFR, where polyglutamylation of MTX causes stepwise decreases in the concentration required for 50% inhibition (IC₅₀) with each additional Glu residue until MTX with a total of six Glu residues has an IC₅₀ value that of MTX. With 10-DAM the pattern is more complex. The IC₅₀ values increase with addition of Glu residues until a maximum is reached with 10-DAM having a total of three Glu residues which has a value twice that of 10-DAM. 10-DAM with a total of four Glu residues and 10-DAM with a total of five Glu residues have progressively lower IC₅₀ values, the latter being equipotent with 10-DAM. With dihydropteroylpentaglutamate as substrate instead of dihydrofolate, the IC₅₀ values are increased 2- to 5-fold for both MTX and 10-DAM derivatives. The results obtained with chicken liver and beef liver DHFR are generally similar to those described for the sheep liver enzyme, but the effects of polyglutamylation are less pronounced. The addition of 0.2 M KCl to the assay system reduces the differences in inhibitory potency of the polyglutamyl derivatives with all three enzymes tested. We conclude that polyglutamylation can alter the interaction of folate analogues and dihydrofolate with DHFR.

¹ Supported by Grants CA 10914 (R. L. K.) and CA 32687 and CA 27101 (M. G. N.) from the National Cancer Institute.

² To whom requests for reprints should be addressed.

Received 1/10/86. Revised 5/20/86. Accepted 6/20/86.