PMID: 1204646Dec 1, 1975Paper

Dihydrofolate reductase from bovine liver. Enzymatic and structural properties

European Journal of Biochemistry
H Baumann, K J Wilson

Abstract

Dihydrofolate reductase from bovine liver has been purified 5000-fold employing conventional techniques and methotrexate/aminohexyl/Sepharose affinity chromatography. Electrophoresis of the isolated enzyme on polyacrylamide gels resulted in the separation of two enzymatically active protein components which were not interconvertible by treatment with dihydrofolate and/or the coenzyme. The two forms, present in a ratio of 20:1, were found by isoelectric focusing to have isoelectric points of 7.15 and 5.94. They had identical specific activities toward dihydrofolate (26.1-27.0 U/mg) and folate (1.3-2.2 U/mg), and had identical molecular weights (23500) and amino acid compositions. Due to the small quantity of the acidic form and the similarity of the two forms, the amino-terminal sequence (19 residues) was determined on a mixture of carboxymethylated reductase. The single sulfhydryl group of the enzyme can be modified by several sulfhydryl reagents in the native enzyme without loss of activity. Modification of the same residue occurs in the denaturated state and partially inhibits renaturation to the fully acitve enzyme. One disulfide bridge was detected by reduction and alkylation. The cleavage of this bond did not effect the en...Continue Reading

References

Mar 5, 1971·Biochemical and Biophysical Research Communications·R B DunlapF M Huennekens
Nov 30, 1971·Annals of the New York Academy of Sciences·F M HuennekensG P Mell
Jan 1, 1974·Methods in Enzymology·B T Kaufman
Nov 21, 1972·Biochemistry·M A HermodsonK A Walsh
Aug 6, 1971·Biochemical and Biophysical Research Communications·B T Kaufman, J V Pierce
Feb 19, 1971·Biochemical and Biophysical Research Communications·B Alpert, R Banerjee
Oct 10, 1968·Biochemical and Biophysical Research Communications·G P MellF M Huennekens
May 1, 1959·Archives of Biochemistry and Biophysics·G L ELLMAN
Dec 28, 1964·Annals of the New York Academy of Sciences·L ORNSTEIN

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Citations

Jan 1, 1980·Advances in Enzyme Regulation·G H Hitchings, S L Smith
Sep 1, 1986·Journal of Pharmacological Methods·A K Yap, D K Luscombe
Feb 15, 1977·FEBS Letters·D Stone, A W Phillips
Mar 23, 2005·Cancer Research·Enma Navarro-PeránJosé Neptuno Rodríguez-López

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