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Isolation and Characterization of Dihydrofolic Acid Reductase from Methotrexate-sensitive and -resistant Human Cell Lines

Morandi, Carlo and Attardi, Giuseppe (1981) Isolation and Characterization of Dihydrofolic Acid Reductase from Methotrexate-sensitive and -resistant Human Cell Lines. Journal of Biological Chemistry, 256 (19). pp. 10169-10175. ISSN 0021-9258. http://resolver.caltech.edu/CaltechAUTHORS:20120718-075748821

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Abstract

Dihydrofolic acid reductase has been purified by affinity chromatography to apparent homogeneity from the human HeLa BU-25 cell line and from two methotrexate-resistant variants, one deriving from HeLa BU-25 and the other from the human VA_2-B cell line. The purified enzymes from the three sources have been characterized in their physical and enzymatic properties. They were not found to differ significantly as concerns their electrophoretic mobility in polyacrylamide gels under a variety of conditions, their specific dihydrofolic acid reductase and folic acid reductase activities, their K_m values for folic acid and TPNH, their sensitivity to methotrexate, and the pH dependence of their folic acid reductase activity. The human dihydrofolic acid reductase has an apparent molecular weight of 21,000 to 22,000, a K_m for folic acid of 6.1 to 7.6 X 10^(-6) M and a K_m for TPNH of 1.6 to 1.7 X 10^(-4) M, turnover numbers of about 500 and 65 mol/min/mol of enzyme for the dihydrofolic acid reductase and the folic acid reductase activity, respectively. The values of the above mentioned physical and kinetic parameters are comparable to those reported for the dihydrofolic acid reductase from other animal cell systems. The dihydrofolic acid reductase content of the two-resistant cell lines is at least 200-fold higher than that of the methotrexate-sensitive HeLa BU-25 cell line. The available evidence indicates that this increased dihydrofolic acid reductase content results from a hyperproduction of an enzyme identical or similar to that of the sensitive cells, presumably due to a selective dihydrofolic acid reductase gene amplification, as previously reported for other cell lines of rodent origin.


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Additional Information:© 1981 by American Society for Biochemistry and Molecular Biology. Received for publication, April 27, 1981. These investigations have been supported by Public Health Service grant GM-11726 from the National Institutes of Health. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. The HeLa BU-25 cell line was generously provided by Dr. Saul Kit. We are very grateful to Drs. M. Forbes and S. Kushner of Lederle Laboratories for kindly supplying us with L methotrexate. The technical assistance of Benneta Keely and Arger Drew is gratefully acknowledged.
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NIH Public Health Service GrantGM-11726
Record Number:CaltechAUTHORS:20120718-075748821
Persistent URL:http://resolver.caltech.edu/CaltechAUTHORS:20120718-075748821
Official Citation:Isolation and characterization of dihydrofolic acid reductase from methotrexate-sensitive and -resistant human cell lines. C Morandi and G Attardi J. Biol. Chem. 1981 256: 10169-10175
Usage Policy:No commercial reproduction, distribution, display or performance rights in this work are provided.
ID Code:32539
Collection:CaltechAUTHORS
Deposited By: Ruth Sustaita
Deposited On:18 Jul 2012 16:21
Last Modified:18 Jul 2012 16:21

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