Purification and characterization of dihydrofolate reductase from soybean seedlings.
Dihydrofolate reductase (DHFR; EC 1.5.1.3) was purified to homogeneity from soybean seedlings by affinity chromatography on methotrexate-aminohexyl Sepharose, gel filtration on Ultrogel AcA-54, and Blue Sepharose chromatography. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the enzyme gave a single protein band corresponding to a molecular weight of 22,000. The enzyme is not a 140,000 Da heteropolymer as reported by others. Amino acid sequence-specific antibodies to intact human DHFR and also antibodies to CNBr-generated fragments of human DHFR bound to the plant enzyme on Western blots and cross-reacted significantly in immunoassays, indicating the presence of sequence homology between the two enzymes. The plant and human enzymes migrated similarly on nondenaturing polyacrylamide electrophoretic gels as monitored by activity staining with a tetrazolium dye. The specific activity of the plant enzyme was 15 units/mg protein, with a pH optimum of 7.4. Km values of the enzyme for dihydrofolate and NADPH were 17 and 30 microM, respectively. Unlike other eukaryotic enzymes, the plant enzyme showed no activation with organic mercurials and was inhibited by urea and KCl. The affinity of the enzyme for folate was relatively low (I50 = 130 microM) while methotrexate bound very tightly (KD less than 10(-10) M). Binding of pyrimethamine to the plant enzyme was weaker, while trimethoprim binding was stronger than to vertebrate DHFR. Trimetrexate, a very potent inhibitor of the human and bacterial enzymes showed weak binding to the plant enzyme. However, certain 2,4-diaminoquinazoline derivatives were very potent inhibitors of the plant DHFR. Thus, the plant DHFR, while showing similarity to the vertebrate and bacterial enzymes in terms of molecular weight and immunological cross-reactivity, can be distinguished from them by its kinetic properties and interaction with organic mercurials, urea, KCl and several antifolates.
D000596:Amino Acids / Q000032:analysis; D000906:Antibodies; D000936:Antigen-Antibody Complex; D003429:Cross Reactions; D005493:Folic Acid Antagonists / Q000494:pharmacology; D006801:Humans; D007700:Kinetics; D007780:Lacticaseibacillus casei / Q000201:enzymology; D046911:Macromolecular Substances; D008970:Molecular Weight; D010944:Plants / Q000201:enzymology*; D013025:Glycine max; D013045:Species Specificity; D013762:Tetrahydrofolate Dehydrogenase / Q000302:isolation & purification* / Q000378:metabolism
D000596:Amino Acids; D000906:Antibodies; D000936:Antigen-Antibody Complex; D005493:Folic Acid Antagonists; D046911:Macromolecular Substances; D013762:Tetrahydrofolate Dehydrogenase