 BackResearch Article ScienceAsia 24 (1998): 275-286 |doi: 10.2306/scienceasia1513-1874.1998.24.275 CHROMATOGRAPHIC DIFFERENTIATION OF THE MITOCHONDRIAL AND CYTOSOLIC FUMARASES OF BAKER'S YEASTDAWAN SHIMBHUa, SHOWN DOONANbABSTRACT: A new method, designed to avoid the use of harsh conditions, has been developed for separation and purification of the isoenzymes of fumarase from pig liver. Peptide maps using high performance liquid chromatography (HPLC) of tryptic digests of the two isoenzymes had most of the peptides in common, but also showed a small number of differences. These results supported the hypothesis, advanced by O'hare and Doonan1, that the isoenzymes differ in a small region of their N-termini but have identical C-terminal sequences. It proved not to be possible to confirm this hypothesis by direct N-terminal sequence analysis since the N-terminus of the cytosolic isoenzyme was found to be blocked. A similar result was obtained for the single enzyme from erythrocytes, a purification procedure for which was also developed. Purification of the cytosolic and mitochondrial fumarases of yeast was also achieved. Unlike the mammalian isoenzymes, those from yeast were found to have different subunit molecular weights. Peptide mapping of digests of the two isoenzymes with trypsin and with S. aureus protease was carried out. Sets of peptides obtained from the two isoenzymes were mostly identical as judged by retention times on HPLC but some significant differences were observed particularly in the digests with S. aureus protease. Amino acid compositions of the two isoenzymes from yeast were also very similar to those of the mammalian forms. It is argued that the yeast isoenzymes may prove to be the more useful pair for establishing the details of the structural relationships between cytosolic and mitochondrial fumarases. a Department of Biochemistry, Faculty of Science, Naresuan University, Phitsanuloke, Thailand. Received November 15, 1998
|
