[Detection of ligand-induced conformation changes in lactate dehydrogenase by using fluorescent probes].
The binding of ANS to apolactate dehydrogenase (apo-LDH) is accompanied by a 300-fold increase in dye fluorescence with a shift of the emission maximum from 515 to 479 nm, as well as by quenching of intrinsic protein fluorescence. A tetrameric LDH molecule has 6.4 +/- 1.6 non-interacting dye-binding sites with an association constant equal to (4.3 +/- 1.6) X 10(3) M-1. NAD+ added at saturating concentrations does not alter the number of ANS binding sites or the association constant value. The formation of binary LDH.NAD+, LDH.NADH, LDH.AMP and LDH.pyruvate complexes causes the quenching of fluorescence of the enzyme-bound ANS. The extent of quenching observed at ligand saturating concentrations differs for each ligand. Pyruvate added to the binary LDH.AMP complex exerts no effect on the fluorescence of protein-bound ANS; this indicates that the binding of AMP causes some alterations in the microenvironment of the substrate-binding site. Nicotinamide mononucleotide (NMN) can act as a coenzyme in the LDH-catalyzed reaction. AMP added together with NMN displays an inhibitory effect. The cationic (auramine O) and anionic (ANS) fluorescent probes bound to LDH exhibit different responses to conformational changes accompanying the transition from the apoenzyme to the LDH X NAD-pyruvate complex.
D000817:Anilino Naphthalenesulfonates; D000818:Animals; D001051:Apoenzymes / Q000032:analysis; D001576:Benzophenoneidum; D001665:Binding Sites; D005456:Fluorescent Dyes; D066298:In Vitro Techniques; D007700:Kinetics; D007770:L-Lactate Dehydrogenase / Q000032:analysis*; D008024:Ligands; D009132:Muscles / Q000201:enzymology; D011487:Protein Conformation; D013552:Swine
D000817:Anilino Naphthalenesulfonates; D001051:Apoenzymes; D005456:Fluorescent Dyes; D008024:Ligands; D001576:Benzophenoneidum; D007770:L-Lactate Dehydrogenase
Obnaruzhenie vyzyvaemykh ligandami konformatsionnykh izmeneniĭ laktatdegidrogenazy s pomoshch'iu fluorestsentnykh zondov.