Physicochemical, Catalytic, and Immunochemical Properties of Fumarases Crystallized Separately from Mitochondrial and Cytosolic Fractions of Rat Liver1

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Abstract
Fumarases located in the cytosolic and mitochondrial fractions were separately purified and crystallized from rat liver. Polyacrylamide gel electrophoresis of these two crystallized fumarases in the presence of dodecyl sulfate gave a single protein band in a position corresponding to a molecular weight of about 49, 000 in each case. A single protein band was also obtained by acidic polyacrylamide gel electrophoresis of the cytosolic or mitochondrial fumarase in the presence of 8 M urea. In both experiments, the subunits of these two fumarases migrated to the same position, indicating that both native enzymes are composed of subunits which could not be differentiated from each other by these procedures. The molecular weight of both fumarases was estimated to be 200, 000 by sucrose density gradient centrifugation and molecular sieve chromatography with Sephadex G-200. A subunit cross-linking experiment using glutaraldehyde revealed that both native enzymes were composed of four identical subunits. The amino acid composition of the cytosolic furnarase was shown to be very similar to that of the mitochondrial one by amino acid analysis. From the data obtained by amino acid analysis, the molecular weights of both native enzymes were calculated to be about 195, 000. Kinetic constants such as the apparent Km, the optimal pH and the specific activity of the cytosolic fumarase were quite similar to those of the mitochondrial enzyme. Furthermore, these two fumarases could not be differentiated from each other by immunochemical techniques, since a single precipitin band was formed between the rabbit antiserum against the cytosolic fumarase and the cytosolic or mitochon-drial fumarase. In addition, these precipitin bands fused completely with each other on the Ouchterlony double immunodiffusion test. These results may suggest that the fumarases located in the cytosolic and mito-chondrial fractions of the rat liver cell may be products of the same nuclear gene, and also that the structures of these two mature fumarases are very similar, if not identical.