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Research ArticleArticle

Purification and Properties of Folate Reductase, the Cellular Receptor of Amethopterin

SIGMUND F. ZAKRZEWSKI, MAIRE T. HAKALA and CHARLES A. NICHOL
Molecular Pharmacology September 1966, 2 (5) 423-431;
SIGMUND F. ZAKRZEWSKI
Department of Experimental Therapeutics, Roswell Park Memorial Institute, New York State Department of Health, Buffalo, New York 14203
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MAIRE T. HAKALA
Department of Experimental Therapeutics, Roswell Park Memorial Institute, New York State Department of Health, Buffalo, New York 14203
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CHARLES A. NICHOL
Department of Experimental Therapeutics, Roswell Park Memorial Institute, New York State Department of Health, Buffalo, New York 14203
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Abstract

Folate reductase has been purified from an amethopterin-resistant subline of cultured Sarcoma-180 cells. The molecular weight of this enzyme was estimated to be 21,000. The specific activity of the purified reductase was 3.1 x 10-8 mole of the enzyme per milligram of protein, and on this basis the preparation was calculated to be 65% pure. During free boundary electrophoresis, however, the enzyme migrates as a single peak toward the anode at pH 8.7. Both folate and dihydrofolate are substrates for this reductase, dihydrofolate being reduced much faster than folate at 30°. The Michaelis constant (Km) and the turnover number with dihydrofolate as substrate are independent of pH between pH 4.5 and 6.3. When folate is the substrate, both Km and the turnover number decrease with increasing pH. The enthalpy of activation is +14.7 kcal for dihydrofolate and +5.7 kcal for folate. The properties of the reductase of Sarcoma-180 are compared with those of other preparations of this enzyme from different sources.

ACKNOWLEDGMENT The authors are indebted to Dr. Peter Stelos for performing the free boundary electrophoresis and to Mrs. Jadwiga Drobniak and Mrs. Marcia Berggren for their capable technical assistance. This investigation was supported in part by research grants (CA-02906 and CA-04175) from the National Cancer Institute of the United States Public Health Service.

  • Copyright ©, 1966, by Academic Press Inc.

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Molecular Pharmacology
Vol. 2, Issue 5
1 Sep 1966
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Research ArticleArticle

Purification and Properties of Folate Reductase, the Cellular Receptor of Amethopterin

SIGMUND F. ZAKRZEWSKI, MAIRE T. HAKALA and CHARLES A. NICHOL
Molecular Pharmacology September 1, 1966, 2 (5) 423-431;

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Research ArticleArticle

Purification and Properties of Folate Reductase, the Cellular Receptor of Amethopterin

SIGMUND F. ZAKRZEWSKI, MAIRE T. HAKALA and CHARLES A. NICHOL
Molecular Pharmacology September 1, 1966, 2 (5) 423-431;
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