Stoichiometry and dissociation constants for interaction of tetracaine with mitochondrial adenosinetriphosphatase as determined by fluorescence.
The stoichiometry and dissociation constants for the interaction of tetracaine with chloroform-released ATPase prepared from beef heart mitochondria were determined from the enhancement of tetracaine fluorescence intensity that occurs upon binding. There is a single class of approximately 60 thermodynamically equivalent binding sites on ATPase for tetracaine; these have a microscopic dissociation constant of 4.9 X 10(-4) M at 25 degrees C under solvent conditions that are similar to those used for enzyme assay. Analysis of enzyme kinetic data according to a partial noncompetitive scheme gave an inhibitor constant for tetracaine of 4.8 X 10(-4) M. The numerical agreement between the dissociation constant and the inhibitor constant shows that the filling of the same class of sites is probably responsible for both the enzyme inhibition and the fluorescence enhancement. The sites are hydrophobic, as evidenced by the blue shift and the magnitude of the fluorescence enhancement that occur upon binding.
D000251:Adenosine Triphosphatases / Q000378:metabolism*; D000818:Animals; D002417:Cattle; D002725:Chloroform; D007700:Kinetics; D008433:Mathematics; D008929:Mitochondria, Heart / Q000201:enzymology*; D011485:Protein Binding; D013050:Spectrometry, Fluorescence; D013056:Spectrophotometry, Ultraviolet; D013367:Submitochondrial Particles / Q000201:enzymology; D013748:Tetracaine / Q000378:metabolism*
D013748:Tetracaine; D002725:Chloroform; D000251:Adenosine Triphosphatases