A STUDY ON THE COMPLEXES BETWEEN HUMAN ERYTHROCYTE ENZYMES PARTICIPATING IN THE CONVERSIONS OF 1,3-DIPHOSPHOGLYCERATE

The ability of D-glyceraldehyde-3-phosphate dehydrogenase (GAPDH) cata lyzing the reaction of 1,3-diphosphoglycerate synthesis in human eryth rocytes to form complexes with enzymes which use this metabolite as su bstrate (3-phosphoglycerate kinase (3-PGK) or 2,3-diphosphoglycerate m utase (2,3-DPGM)) was studied. It was found that highly active 2,3-DPG M can be extracted from human erythrocyte hemolysates in a complex wit h GAPDH adsorbed on Sepharose-bound anti-GAPDH antibodies at pH 6.5, t he molar ratio being one 2,3-GPGM subunit per subunit of GAPDH. No com plexation was, however, detected at pH 8.0. The opposite was true for the interaction between GAPDH and 3-PGK, which could be observed at pH 8.0. In experiments carried out at pH 7.4, both GAPDH . 2,3-DPGM and GAPGH . 3-PGK complexes were detected. The K-d values of the complexes determined with purified enzyme preparations were in the range 2.40-2 .48 mu M for both the GAPDH . 2,3-DPGM and GAPGH . 3-PGK enzyme pairs, when titrations of GAPDH covalently bound to CNBr-activated Sepharose were performed by the soluble 2,3-DPGM or 3-PGK. If, however, GAPDH a dsorbed on the specific antibodies covalently bound to Sepharose was u sed in the titration experiments, the K-d for the GAPDH . 2,3-DPGM com plex was found to be 0.54 mu M, and the K-d for the GAPDH . 3-PGK comp lex was 0.49 mu M. The concentration of 2,3-diphosphoglycerate determi ned after 1 h of incubation of erythrocytes in the presence of glucose was found to increase 1.5-fold if the incubation was carried out at p H 6.5, but did not change upon incubation at pH 8.0. On the other hand , the concentration of 3-phosphoglycerate after incubation at pH 8.0 w as twice as large as that found after incubation at pH 6.5. The result s are interpreted on the hypothesis that specific protein-protein inte ractions between GAPDH and 2,3-DPGM or between GAPDH and 3-PGK may pla y a role in determining the fate of 1,3-diphosphoglycerate produced in the GAPDH-catalyzed reaction. (C) 1997 Academic Press.