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) catalyzing the reaction of 1,3-diphosphoglycerate synthesis in human erythrocytes to form complexes with enzymes which use this metabolite as substrate (3-phosphoglycerate kinase (3-PGK) or 2,3-diphosphoglycerate mutase (2,3-DPGM)) was studied. It was found that highly active 2,3-DPGM can be extracted from human erythrocyte hemolysates in a complex with GAPDH adsorbed on Sepharose-bound anti-GAPDH antibodies at pH 6.5, the molar ratio being one 2,3-GPGM subunit per subunit of GAPDH. No complexation 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 x 2,3-DPGM and GAPGH x 3-PGK complexes were detected. The Kd values of the complexes determined with purified enzyme preparations were in the range 2.40-2.48 microM for both the GAPDH x 2,3-DPGM and GAPGH x 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 adsorbed on the specific antibodies covalently bound to Sepharose was used in the titration experiments, the Kd for the GAPDH x 2,3-DPGM complex was found to be 0.54 microM, and the Kd for the GAPDH x 3-PGK complex was 0.49 microM. The concentration of 2,3-diphosphoglycerate determined 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 pH 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 was twice as large as that found after incubation at pH 6.5. The results are interpreted on the hypothesis that specific protein-protein interactions between GAPDH and 2,3-DPGM or between GAPDH and 3-PGK may play a role in determining the fate of 1,3-diphosphoglycerate produced in the GAPDH-catalyzed reaction.
Fokina
K V
KV
A. N. Belozersky Institute of Physico-Chemical Biology, Moscow State University, Russia.
Dainyak
M B
MB
Nagradova
N K
NK
Muronetz
V I
VI
D019794:2,3-Diphosphoglycerate; D000911:Antibodies, Monoclonal; D001731:Bisphosphoglycerate Mutase / Q000378:metabolism*; D002846:Chromatography, Affinity; D019281:Dimerization; D004163:Diphosphoglyceric Acids / Q000032:analysis / Q000378:metabolism*; D004912:Erythrocytes / Q000201:enzymology*; D005947:Glucose / Q000378:metabolism; D005987:Glyceraldehyde-3-Phosphate Dehydrogenases / Q000276:immunology / Q000378:metabolism*; D005988:Glyceric Acids / Q000032:analysis; D006019:Glycolysis / Q000502:physiology; D006801:Humans; D006863:Hydrogen-Ion Concentration; D009097:Multienzyme Complexes / Q000378:metabolism; D010735:Phosphoglycerate Kinase / Q000378:metabolism*; D011485:Protein Binding
D000911:Antibodies, Monoclonal; D004163:Diphosphoglyceric Acids; D005988:Glyceric Acids; D009097:Multienzyme Complexes; D019794:2,3-Diphosphoglycerate; C015891:glycerate 1,3-biphosphate; C005156:3-phosphoglycerate; D005987:Glyceraldehyde-3-Phosphate Dehydrogenases; D010735:Phosphoglycerate Kinase; D001731:Bisphosphoglycerate Mutase; D005947:Glucose