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Molecular Pharmacology, Vol 3, 359-369, Copyright © 1967 by the American Society for Pharmacology and Experimental Therapeutics

Dihydrofolate Reductase from Trypanosoma equiperdum

I. Isolation, Partial Purification, and Properties

JULIAN J. JAFFE 1 and JOHN J. MCCORMACK JR. 1

1 Department of Pharmacology, University of Vermont, College of Medicine, Burlington, Vermont 05401

Dihydrofolate reductase was isolated from acetone powder extracts of T. equiperdum. Folic acid was not an effective substrate; indeed, it was a weak inhibitor, the concentration required for 50% inhibition being 3 x 10-5 M. NADPH was a much more efficient hydrogen donor than NADH. Michaelis constants of 4.3 x 10-6 M for dihydrofolate and 1.2 x 10-5 M for NADPH were obtained. Trypanosomal dihydrofolate reductase showed a broad peak of maximal activity between pH 6.0 and 7.7, with activity sharply decreasing above and below those values. Activity increased with increasing temperature within the range of 20°-40°, but fell off sharply above 40°. 4-Amino analogs of folic acid are potent inhibitors of trypanosomal dihydrofolate reductase, the concentration for 50% inhibition by methotrexate, for example, being 2 x 10-10 M. The trypanosomal reductase differs from those of mammalian and bacterial origin with respect to susceptibility to inhibition by a number of 2,4-diaminoheterocyclic compounds.

A substance (or substances) was isolated from homogenates of T. equiperdum which possessed spectrophotofluorometric properties closely resembling those of 2-amino-4-hydroxypteridines and which effectively replaced folic acid as a growth-promoting factor for Lactobacillus casei.

Note:
ACKNOWLEDGMENTS The authors wish to express their gratitude to Dr. George Hitchings for his encouragement and for providing us with the diaminoheterocyclic compounds we used in these studies. We also wish to thank Dr. James Burchall for assaying our "pteridine fraction" in his L. casei system. This work was supported by Grant No. CA-08114-03 from the National Cancer Institute, United States Public Health Service, Bethesda, Maryland.

Submitted on February 16, 1967






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