Energy-dependent Enhancement of Aurovertin Fluorescence

Quantitative measurements of aurovertin binding to submitochondrial particles confirm previous conclusions that the fluorescence responses of aurovertin formed in a complex with soluble and membrane-bound mitochondrial ATPase were a reflection of similar alterations in the two forms of the enzyme and add support to the conclusion that aurovertin functions as a reporter molecule for conformational change.The rate and magnitude of the energy-dependent fluorescence enhancement of aurovertin was strongly influenced by the composition of the reaction mixture, particularly by the presence of adenine nucleotides and Mg2f.At high concentrations of aurovertin (5 PM), addition of succinate to submitochondrial particles led to a 2-fold increase in both binding affinity and fluorescence increment without changing the number of binding sites or the amount of aurovertin bound.The fast phase of the two-phase energized fluorescence response, revealed by rapid flow studies at pH 6.6, fitted first order kinetics with a half-time in the range 34 to 47 ms.The characteristics of the energized response were identical in submitochondrial particles and in intact mitochondria.Inhibition of ATPase activity by the protein inhibitor of the enzyme was associated with partial quenching of fluorescence, masking of one of the two binding sites for aurovertin on the soluble ATPase, a Z-fold increase in the binding tinity for the probe, and a reduction of 30% in the fluorescence increment.It was concluded that dissociation of ATP and Mg2+ during the energized response reflected, at least in part, conformational changes in the ATPase which were reported by changes in aurovertin fluorescence.A procedure is presented for isolation of aurovertin.Aurovertin is an antibiotic, isolated from the mold Calcarisporium arbuscula (I), which is virtually nonfluorescent in water or organic solvents but exhibits considerable fluorescence in the presence of mitochondrial ATPase (F# (2-4) or in solvents of