Interaction between D-glyceraldehyde-3-phosphate dehydrogenase and 3-phosphoglycerate kinase labeled by fluorescein-5'-isothiocyanate: evidence that the dye participates in the interaction.
An interaction of rabbit muscle D-glyceraldehyde-3-phosphate dehydrogenase and 3-phosphoglycerate kinase labeled with FITC was studied by following the changes in fluorescence intensity of the bound dye. The association between the two enzymes was found to be a rather slow process characterized by a second order rate constant of 1.1 +/- 0.2.10(3) M-1 s-1, the KD of the complex between apoenzymes being 3.2.10(-7) M. The stability of the complex increased upon increase of temperature and ionic strength of the medium, suggesting a hydrophobic character of association. The ligands which bind at the active centers of the two enzymes (NAD+, ATP, 3-phosphoglycerate) weakened the bienzyme association. Unlabeled 3-phosphoglycerate kinase was unable to displace the FITC-labeled enzyme from the complex. Taken together, the results indicate that interaction between D-glyceraldehyde-3-phosphate dehydrogenase and 3-phosphoglycerate kinase labeled by FITC is assisted by the dye, which may bind at nucleotide-binding sites of GPDH. No interaction was observed between the FITC-labeled 3-phosphoglycerate kinase and lactate dehydrogenase, which suggests that protein-protein interaction at specific "recognition" sites may be a prerequisite for the complex formation.
Sukhodolets
M V
MV
A.N. Belozersky Laboratory of Molecular Biology and Bioorganic Chemistry, Moscow State University, USSR.
Muronetz
V I
VI
Nagradova
N K
NK
D000818:Animals; D016650:Fluorescein-5-isothiocyanate; D005452:Fluoresceins; D005987:Glyceraldehyde-3-Phosphate Dehydrogenases / Q000378:metabolism*; D007700:Kinetics; D009132:Muscles / Q000201:enzymology; D009994:Osmolar Concentration; D010735:Phosphoglycerate Kinase / Q000378:metabolism*; D011817:Rabbits; D013050:Spectrometry, Fluorescence; D013696:Temperature; D013861:Thiocyanates
D005452:Fluoresceins; D013861:Thiocyanates; D005987:Glyceraldehyde-3-Phosphate Dehydrogenases; D010735:Phosphoglycerate Kinase; D016650:Fluorescein-5-isothiocyanate