Interaction of fluorescent adenine nucleotide derivatives with the ADP/ATP carrier in mitochondria. 2. [5-(Dimethylamino)-1-naphthoyl]adenine nucleotides as probes for the transition between c and m states of the ADP/ATP carrier

Abstract

The binding to the ADP/ATP carrier in mitochondrial membranes of the 3'-O-(dimethylamino)naphthoyl (DAN) derivatives of AMP, ADP, and ATP was quantitatively analyzed. The sidedness of the fluorescent type binding to the "m" side only was shown comparing the mitochondrial membranes in various stages of integrity and surface orientation. In particles displacement by bongkrekate (BKA) is direct, whereas in the case of carboxyatractylate (CAT) the requirement for ADP and ATP demonstrates the transition from the "m" to the "c" state. Quantitatively the "physical" binding of [3H]DAN-AMP and fluorescence are well correlated, allowing for a little nonfluorescent binding to the c side. For DAN-AMP KD is 1.6 microM, for DAN-ADP KD is 0.8 microM, and in the Hill plot a straight line with n = 1.25 is obtained. The maximum number of binding sites for [3H]DAN-AMP (1.5 mumol/g of protein) is about equal to the sites found for [3H]BKA if the unspecific binding of both ligands is differentiated by blocking carrier sites with CAT. [3H]CAT binding is somewhat lower in accordance with the limited access of CAT to inverted vesicles. ADP is able to decrease fluorescence only by about 35% at high concentrations (10 mM) whereas GDP has virtually no effect. With ADP, DAN-AMP binding decreases by 30% of the total binding sensitive to BKA. Binding to ATPase is low because of the absence of Mg2+.(ABSTRACT TRUNCATED AT 250 WORDS)

Structural-dynamical properties of the transmembrane segment VI of the mitochondrial oxoglutarate carrier studied by site directed spin-labeling

“…The medium-low accessibility of the N-terminal of the segment and the relatively higher accessibility of the C-terminal part suggests that the channel is partially closed at the matrix side and more opened at the cytoplasmatic side. These results indicate that we are likely probing a carrier conformation resembling the one determined by X-ray diffraction of the AAC in presence of CATR [4] that corresponds to the cstate of the carrier [28]. In the C-terminal region two residues at the end of the segment, T296C and I298C, exhibit a large accessibility to NiEDDA, having a P NiEDDA values of 0.7 and 1.7 respectively.…”

Interaction of fluorescent adenine nucleotide derivatives with the ADP/ATP carrier in mitochondria. 1. Comparison of various 3'-O-ester adenine nucleotide derivatives

“…The interaction with the c state of the carrier is believed to increase going from the AMP to the ATP derivative. However, as discussed in the following paper (Klingenberg et al, 1984), this binding is nonfluorescent. The slow-phase fluorescent increase is visualized to reflect the transition of the non- The surprising finding that the DAN-AMP derivative binds with at least the same efficiency as the DAN-ADP and DAN-ATP derivatives seems at first to argue against binding of the 1,5-DAN-nucleotides to the nucleotide site since the latter had been shown not to accept free AMP.…”

A Fluorescence-Based Method to Measure ADP/ATP Exchange of Recombinant Adenine Nucleotide Translocase in Liposomes

“…The use of fluorescent adenosine nucleotide derivatives suffers from substrate specificity of the ANT-mediated ADP/ATP exchange, since the full structures of the adenosine nucleotides are recognized [23]. Small variations in PN structure are supposed to significantly alter binding and transport of ATP and ADP [24][25][26][27][28][29], and further depend on the configuration of ANT [30].…”