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Studies of the ADP/ATP carrier of mitochondria with fluorescent ADP analogue formycin diphosphate
Abstract:
The ADP/ATP carrier was studied by a fluorescent substrate, formycin diphosphate which is the only fluorescent ADP analogue to bind. Its low quantum yield, short decay time and spectral overlap with tryptophan has as yet prevented its wider use. By incorporating fluorescent acceptors of formycin diphosphate fluorescence, anthracene-maleimide and vinylanthracene, into the membrane, these difficulties were circumvented. Only bound formycin diphosphate transfers energy to the probes so that the secondary emission of these probes is a measure for membrane-bound formycin diphosphate. The fluorescent transfer is inhibited by ADP, bongkrekate and carboxyatractylate whether added before or after incubation of formycin diphosphate showing that only binding to the adenine nucleotide carrier is measured. It also shows directly that the earlier demonstrated ADP fixation by bongkrekate is indeed a displacement into the matrix. The fluorescence decay time of the bound formycin diphosphate is measured as 1.95 ns compared to 0.95 ns of the free formycin diphosphate, indicating that formycin diphosphate is bound at the carrier in a non-polar environment. The depolarization decay time was found to be larger than 15 ns, indicating that carrier-bound formycin diphosphate is immobile within this time period.
Insights
This study quantifies ADP/ATP carrier binding using fluorescent formycin diphosphate. Novel energy transfer methods overcome limitations, revealing non-polar binding and immobility of the carrier.
Area of Science:
- Biochemistry
- Molecular Biology
- Membrane Transport
Background:
- The ADP/ATP carrier is crucial for cellular energy transfer.
- Formycin diphosphate is a fluorescent ADP analog, but its use is limited by low quantum yield and spectral properties.
- Previous methods struggled to accurately quantify carrier-bound formycin diphosphate.
Purpose of the Study:
- To develop a reliable method for studying ADP/ATP carrier binding using formycin diphosphate.
- To investigate the binding environment and dynamics of formycin diphosphate on the carrier.
- To confirm the mechanism of action for inhibitors like bongkrekate.
Main Methods:
- Incorporation of fluorescent energy acceptors (anthracene-maleimide, vinylanthracene) into membranes.
- Utilizing Förster Resonance Energy Transfer (FRET) to detect bound formycin diphosphate.
- Measuring fluorescence decay times and depolarization decay times of formycin diphosphate.
- Assessing the effect of ADP, bongkrekate, and carboxyatractylate on fluorescence signals.
Main Results:
- Energy transfer from formycin diphosphate to membrane-bound acceptors accurately measures bound ligand.
- Fluorescent transfer is specifically inhibited by ADP, bongkrekate, and carboxyatractylate, confirming measurement of adenine nucleotide carrier binding.
- Bongkrekate's action is shown to be a displacement into the matrix.
- Bound formycin diphosphate exhibits a longer fluorescence decay time (1.95 ns vs. 0.95 ns) and a depolarization decay time >15 ns, indicating a non-polar environment and immobility.
Conclusions:
- A novel FRET-based assay allows precise quantification of ADP/ATP carrier-bound formycin diphosphate.
- Formycin diphosphate binds to the carrier in a non-polar environment and is immobile on the timescale of the measurement.
- This method provides direct evidence for inhibitor mechanisms and the carrier's binding site characteristics.