Multiple forms of rat-liver dihydropteridine reductase identified by their differing isoelectric points.
Purified rat-liver dihydropteridine reductase is homogeneous by gel filtration (Mr approximately 51,000), sodium dodecyl sulfate-polyacrylamide gel electrophoresis (Mr approximately 25,500), and native polyacrylamide gel electrophoresis, suggesting that the enzyme is composed of two identical subunits. However, analysis by isoelectric focusing has revealed three enzyme forms with approximate isoelectric points of 6.5, 5.9, and 5.7 (designated forms, I, II, and III, respectively). The three forms, isolated in 65% yield by preparative chromatofocusing, are stable in 0.05 M phosphate buffer, pH 6.8, containing 1 mM beta-mercaptoethanol and exhibit similar kinetic constants when the catalytic activities of the isolated forms are compared with quinonoid dihydrobiopterin as substrate. All forms generate complexes with the enzymatic cofactor NADH which are also detectable by IEF. When examined further by IEF under denaturing conditions in 6 M urea the enzyme demonstrates a differing subunit composition for its three forms. Two distinct subunits, designated alpha and beta, can be identified, and additional evidence suggests that the native enzyme forms I, II, and III represent the three differing dimeric combinations alpha alpha (form I), alpha beta (form II), and beta beta (form III).
D000818:Animals; D004093:Dihydropteridine Reductase / Q000302:isolation & purification / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D007525:Isoelectric Focusing; D007526:Isoelectric Point; D007700:Kinetics; D008099:Liver / Q000201:enzymology*; D046911:Macromolecular Substances; D008623:Mercaptoethanol / Q000494:pharmacology; D009243:NAD / Q000378:metabolism; D009247:NADH, NADPH Oxidoreductases / Q000302:isolation & purification / Q000378:metabolism; D011489:Protein Denaturation; D051381:Rats; D013050:Spectrometry, Fluorescence
D046911:Macromolecular Substances; D009243:NAD; D008623:Mercaptoethanol; D004093:Dihydropteridine Reductase; D009247:NADH, NADPH Oxidoreductases