Substrate-induced modifications of the intrinsic fluorescence of the isolated adenine nucleotide carrier protein: demonstration of distinct conformational states

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Abstract
The effects of ATP or ADP and the specific inhibitors carboxyatractyloside (CATR) and bongkrekic acid (BA) on the conformation of the isolated adenine nucleotide (AdN) carrier protein were studied by fluorescence spectroscopy. The addition of ATP to the AdN carrier resulted in a rapid fluorescence increase of the tryptophanyl residue(s) at 355 nm, which leveled up in < 1 s at 22.degree. C. Among the natural nucleotides, only ATP and ADP were effective. At 10.degree. C or below, the kinetics of the fluorescence increased induced by ATP were biphasic, consisting of a rapid phase of < 1 s, followed by a slower phase that lasted for a few seconds and had virtually the same amplitude as the rapid one. Both phases were abolished when CATR was added prior to ATP or fully reversed when CATR was added after the fluorescence response to ATP had been elicited. The number of CATR binding sites present on the carrier protein was determined by CATR specific inhibition of the ATP-induced increase in intrinsic fluorescence. Addition of BA prior to or together with ATP nearly doubled the amplitude of the ATP-induced fluorescence signal. At 10.degree. C or below, the fluorescence response to ATP in the presence of BA could also be decomposed into rapid and slow phases. The amplitude of the rapid phase was not modified in the presence of BA, but the amplitude of the slow phase was .apprx. 3 times higher than that of the rapid phase. The same results were obtained when ATP was replaced by ADP. In the absence of ATP, CATR was found to induce per se a modification of the intrinsic fluorescence that differed from that induced by ATP by its excitation and emission spectra. These results are discussed on the basis of a minimal model where the AdN carrier is supposed to exist in 2 native conformations, the CATR and BA conformations that are trapped and stabilized by CATR and BA, respectively; interconversion between the 2 conformations is triggered by ATP or ADP. On the basis of several observations that point to a tetrameric organization of the AdN carrier protein, it is equally possible that the transition between the CATR and BA conformations is multiphasic and may proceed with sequential modification of each subunit of the tetramer.

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