Dec 2, 1975

Purification and properties of Escherichia coli dihydrofolate reductase

Biochemistry
D P BaccanariJ J Burchall

Abstract

Dihydrofolate reductase has been purified 40-fold to apparent homogeneity from a trimethoprim-resistant strain of Escherichia coli (RT 500) using a procedure that includes methotrexate affinity column chromatography. Determinations of the molecular weight of the enzyme based on its amino acid composition, sedimentation velocity, and sodium dodecyl sulfate gel electrophoresis gave values of 17680, 17470 and 18300, respectively. An aggregated form of the enzyme with a low specific activity can be separated from the monomer by gel filtration; treatment of the aggregate with mercaptoethanol or dithiothreitol results in an increase in enzymic activity and a regeneration of the monomer. Also, multiple molecular forms of the monomer have been detected by polyacrylamide gel electrophoresis. The unresolved enzyme exhibits two pH optima (pH 4.5 and pH 7.0) with dihydrofolate as a substrate. Highest activities are observed in buffers containing large organic cations. In 100 mM imidazolium chloride (pH 7), the specific activity is 47 mumol of dihydrofolate reduced per min per mg at 30 degrees. Folic acid also serves as a substrate with a single pH optimum of pH 4.5. At this pH the Km for folate is 16 muM, and the Vmax is 1/1000 of the rate...Continue Reading

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Mentioned in this Paper

Macromolecular Compounds
Alkalescens-Dispar Group
Escherichia Coli Infections
Slow-K
Protein Conformation
Tetrahydrofolate Dehydrogenase
Sodium Chloride, (24)NaCl
Methotrexate, (DL)-Isomer
Hydrogen-Ion Concentration
Osmolality

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