Abstract
A rapid three-step procedure utilizing heat treatment, ammonium sulfate fractionation, and affinity chromatography on Matrex gel Orange A purified fumarase (EC 4.2.1.2) 632-fold with an 18% yield from crude extracts of Euglena gracilis var. bacillaris. The apparent molecular weight of the native enzyme was 120,000 as determined by gel filtration on Sephacryl S-300. The preparation was over 95% pure, and the subunit molecular weight was 60,000 as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, indicating that the enzyme is a dimer composed of two identical subunits. The pH optimum for E. gracilis fumarase was 8.4. The Km values for malate and fumarate were 1.4 and 0.031 mM, respectively. Preparative two-dimensional gel electrophoresis was used to further purify the enzyme for antibody production. On Ouchterlony double-immunodiffusion gels, the antifumarase serum gave a sharp precipitin line against total E. gracilis protein and purified E. gracilis fumarase. It did not cross-react with purified pig heart fumarase. On immunoblots of purified E. gracilis fumarase and crude cell extracts of E. gracilis, the antibody recognized a single polypeptide with a molecular weight of approximately 60,000, indicating that the antibody is monospecific. This polypeptide was found in E. gracilis mitochondria. The antibody cross-reacted with an Escherichia coli protein whose molecular weight was approximately 60,000, the reported molecular weight of the fumA gene product of E. coli, but it failed to cross-react with proteins found in crude mouse cell extracts, Bacillus subtilis extracts, or purified pig heart fumarase.