Purification and structural comparisons of the cytosolic and mitochondrial isoenzymes of fumarase from pig liver.
A method has been developed for the purification of cytosolic and mitochondrial isoenzymes of fumarase from total homogenates of pig liver. Separation of the isoenzymes from one another was achieved using chromatofocusing. The isoenzymes were pure as judged by production of single bands on electrophoresis in the presence of sodium dodecyl sulphate; they appeared to have identical or very similar subunit molecular weights. The isoenzymes differed in electrophoretic properties under nondenaturing conditions. One-dimensional peptide maps of fragments produced from the two isoenzymes by chemical cleavage at cysteine residues were identical; maps obtained after digestion with the V8 proteinase from Staphylococcus aureus showed small differences at short times of digestion which could have reflected variations in rates of hydrolysis rather than structural differences. However, two-dimensional peptide maps of digests obtained by treatment of the isoenzymes with trypsin followed by chymotrypsin had 58 peptides in common, but showed two peptides unique to the mitochondrial isoenzyme and five peptides unique to the cytosolic form. Using the dansylation procedure, the mitochondrial isoenzyme was shown to have N-terminal alanine and the cytosolic form to have N-terminal glutamic acid or glutamine. We conclude that the isoenzymes of fumarase are identical over nearly all of their amino acid sequences but differ at their N-termini; the extent of these differences is yet to be established. These results are consistent with the claim (Edwards, Y.H. and Hopkinson,D.A. (1979) Ann. Human Genet. Lond. 42, 303-313) that the isoenzymes are determined at the same genetic locus, but they raise interesting questions about the biosynthesis of the isoenzymes.
D000595:Amino Acid Sequence; D000818:Animals; D002451:Cell Compartmentation; D003600:Cytosol / Q000201:enzymology; D004588:Electrophoresis, Cellulose Acetate; D005649:Fumarate Hydratase / Q000302:isolation & purification*; D007527:Isoenzymes / Q000302:isolation & purification*; D008099:Liver / Q000201:enzymology*; D008930:Mitochondria, Liver / Q000201:enzymology*; D010446:Peptide Fragments / Q000032:analysis; D013552:Swine
D007527:Isoenzymes; D010446:Peptide Fragments; D005649:Fumarate Hydratase