title: Characterization of glucose-6-phosphate dehydrogenase isozymes from human and pig brain.
abstract: Homogenates of human and pig brain in 10 mM Tris-HCl, pH 8.0 were centrifuged at 25,400 x g for 1 h. The supernatants were electrophoresed in polyacrylamide gels were stained for glucose-6-phosphate dehydrogenase (EC 1.1.1.49) activity. Five distinct bands were visible. Isozymes corresponding to two of those bands were purified from human and pig brain. The isozymes were electrophoretically homogeneous. The native proteins, Mr, 220,000, dissociated in sodium dodecyl sulphate-polyacrylamide gels into a 57,000 Mr subunit. Therefore, the native isozymes are tetramers. None of the isozymes required additional metal ions for activity. At 1 mM concentration Mg2+ and Ca2+, independently or together, activated the isozymes 1.5-fold. The isozymes were NADP(+)-specific. Kmapp values of the G6PD isozymes were similar for NADP+ (6-8 microM), but different for G6P (56-180 microM). The specific activities of the isozymes varied from 50 to 210 units per mg of protein. All isozymes were inhibited by NADPH. The inhibition was competitive with respect to NADP+ and non-competitive with respect to G6P. NADH did not affect any of the isozymes. ATP inhibited the isozymes competitively with respect to G6P and non-competitively with respect to NADP+. Palmitoyl-CoA dissociated the active tetramers into enzymatically inactive dimeric forms. This treatment also abolished the 6-phosphogluconate activity of the isozyme II from both sources. High performance liquid chromatography peptide maps of the tryptic digest and amino acid analyses of the isozymes showed extensive homologies between the corresponding isozymes from the two species. Interestingly, only the isozyme II in human and pig brain was active with 6-phosphogluconate as a substrate (Kmapp = 864 and 279 microM). The specific activities of the isozyme II with 6-phosphogluconate (14 and 48 unit per mg of protein for human and pig brain isozyme II, respectively) was four times less than those with G6P. It is therefore suggested that isozyme II is a bifunctional enzyme.
authors: Department of Biochemistry, University of Tennessee, Knoxville 37996.
authors: S W
authors: SW
authors: Cho
authors: J G
authors: JG
authors: Joshi
mesh_terms: D000596:Amino Acids / Q000378:metabolism; D000818:Animals; D001921:Brain / Q000201:enzymology*; D005954:Glucosephosphate Dehydrogenase / Q000378:metabolism*; D006801:Humans; D007527:Isoenzymes / Q000378:metabolism*; D010449:Peptide Mapping; D013045:Species Specificity; D013552:Swine / Q000378:metabolism*; D014018:Tissue Distribution
chemical_list: D000596:Amino Acids; D007527:Isoenzymes; D005954:Glucosephosphate Dehydrogenase