Subunit and peptide compositions of yeast phosphoglucose isomerase isoenzymes.

Three isoenzymes of yeast phosphoglucose isomerase were studied by physical and chemical methods to establish their subunit properties and to identify the structural differences among them which give rise to their heterogeneous chromatographic behavior. Equilibrium sedimentation ultracentrifugation in 6 M guanidine hydrochloride and polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate showed all three isoenzymes to be dimers composed of subunits of equal size. (The latter technique was found to give rise to multiple band artifacts if special precautions were not taken to avoid interference from possible protease contaminations.) The subunit molecular weights of each isoenzyme species were determined to be 60,000, i.e. one-half of the molecular weight of 120,000 of the native, undissociated isoenzymes (KEYPE, T. D., NAKAGAWA, Y., AND NOLTJUNN, E. A. (1974) J. Biol. Chem. 249, 46174624). Equilibrium binding studies with the competitive inhibitor pyridoxal 5’phosphate indicated preferential binding at a single site per monomer and yielded a dissociation constant of 0.4 mM, in reasonable agreement with a kinetically derived Ki of 0.22 mIib. Treatment with carboxypeptidase established methionine as the COOH-terminal residue for both subunits common to all three isoenzymes. Several attempts to identify any NH?terminal residues yielded negative results, suggesting that they might be blocked. Extensive peptide-mapping studies, made possible through use of a high sensitivity, miniscale thin layer method, yielded fully resolved and excellently visible maps from 25 to 50 pg of peptide mixture and permitted identification of all but 1 to 3 of the theoretically expected number of 50 tryptic peptides. The data showed, moreover, that within each isoenzyme the two subunits are not only equal in size but also identical in their polypeptide sequence. Finally, the maps allowed unequivocal identification of differences in peptide composition between the individual isoenzymes. These were concluded to cause their differences in over-all net charge and, consequently, in their different chromatographic properties.