Purification, immunoassay, and tissue distribution of rat C1-tetrahydrofolate synthase.
C1-tetrahydrofolate synthase (C1-THF synthase), a eukaryotic trifunctional enzyme, catalyzes three sequential folate-mediated one-carbon interconversions. These three reactions supply the activated one-carbon units required in the metabolism of purines, thymidylate, and several amino acids. In order to study the regulation of C1-THF synthase expression in mammals, we have purified the enzyme to homogeneity from rat liver, raised polyclonal antisera to it in rabbits, and developed a sensitive solid-phase immunoassay for the enzyme. The enzyme was purified approximately 600-fold to a specific activity of 24.6 U/mg protein based on 10-formyl-THF synthetase activity. Western blot analysis indicated that the antisera is specific for one protein in crude liver extracts which comigrates with purified C1-THF synthase. Using the solid-phase immunoassay, as little as 200 pg of immunoreacting protein can be detected in tissue homogenates. Several rat tissues were examined for the three C1-THF synthase enzymatic activities and immunoreactive protein. The results indicated that the level of C1-THF synthase is regulated in a tissue-specific manner. Enzyme assays revealed that certain tissues differ by more than 100-fold in enzyme activity, with liver and kidney containing the highest levels, and lung and muscle the lowest. However, immunoassay of these same tissues indicated only a 10-fold difference in C1-THF synthase concentration. This apparent masking of enzyme activity was observed in all tissues, but to varying degrees. These results emphasize the advantages of an immunoassay in studying the regulation of C1-THF synthase.
Cheek
W D
WD
Clayton Foundation Biochemical Institute, Department of Chemistry, University of Texas, Austin 78712.
Appling
D R
DR
D000619:Aminohydrolases / Q000276:immunology / Q000302:isolation & purification*; D000818:Animals; D000906:Antibodies / Q000276:immunology; D000918:Antibody Specificity; D001921:Brain / Q000201:enzymology; D005574:Formate-Tetrahydrofolate Ligase / Q000276:immunology / Q000302:isolation & purification*; D007118:Immunoassay; D007668:Kidney / Q000201:enzymology; D008025:Ligases / Q000302:isolation & purification*; D008099:Liver / Q000201:enzymology*; D008168:Lung / Q000201:enzymology; D008297:Male; D008754:Methylenetetrahydrofolate Dehydrogenase (NADP) / Q000276:immunology / Q000302:isolation & purification*; D009097:Multienzyme Complexes / Q000276:immunology / Q000302:isolation & purification*; D009206:Myocardium / Q000201:enzymology; D010088:Oxidoreductases / Q000302:isolation & purification*; D051381:Rats; D011919:Rats, Inbred Strains; D013194:Staining and Labeling
D000906:Antibodies; D009097:Multienzyme Complexes; C021836:formyl-methenyl-methylenetetrahydrofolate synthetase; D010088:Oxidoreductases; D008754:Methylenetetrahydrofolate Dehydrogenase (NADP); D000619:Aminohydrolases; D008025:Ligases; D005574:Formate-Tetrahydrofolate Ligase