48] Fluorescent nucleotide analogs as active site probes for the ADP/ATP carrier and the uncoupling protein

Abstract

Publisher Summary This chapter focuses on the fluorescent nucleotide analogs as active site probes for the adenosine diphosphate (ADP)/ adenosine triphosphate (ATP) carrier and the uncoupling protein. The 1,5- dimethylaminonaphthoyl (DAN) derivatives DAN-adenine monophosphate (AMP), DAN-ADP, and DAN-ATP have identical fluorescence properties. Their fluorescence is virtually quenched in aqueous solution, whereas a high fluorescence yield is obtained in nonaqueous media, such as dioxane and ethanol. The DAN-nucleotides added to rat liver mitochondria do not give a marked fluorescence. In beef heart mitochondria, they exhibit a relatively small signal which is fully enhanced in submitochondrial (sonic) particles. Also breaking the mitochondria by freezing-thawing or by detergents enhances the fluorescence response. The DAN-nucleotides bind also to the soluble and partially purified ADP/ATP carrier (AAC) with a fluorescence maximum at a slightly longer wavelength, 519 Nm. In the solubilized AAC, the full fluorescence response to addition of DAN-AMP is observed only in the presence of ADP or ATP. This is interpreted to show that the AAC is, from the start, partially in the m-state and partially in the c-state. Only when the substrates for the ADP–ATP transport are provided the transition between the c- and m-state can be accomplished since the unloaded carrier cannot undergo this transition. Thus the carrier population in the c-state is largely converted into the m-state under the pulling force of the DAN-AMP binding.