Adenine nucleotide translocation of mitochondria. Identification of carrier sites.

Abstract

The present study demonstrates the existence of specific binding sites for ADP and ATP at the mitochondrial membrane which can be attributed to the adenine nucleotide carrier. Parameters of the binding and various other properties of the carrier sites are determined. The differentiation of the carrier sites is based on the assumption that atractyloside removes adenine nucleotides competitively from these sites. For the accurate evaluation and discrimination of the carriers three parallel binding samples are compared: (a) total binding; (b) atractyloside addition before adenine nucleotide, which prevents binding to the carrier and the exchange with endogenous adenine nucleotides; (c) addition of atractyloside after adenine nucleotide, which displaces adenine nucleotide from the carrier but not from the exchanged pool.Total uptake of adenine nucleotides is thus differentiated into three portions: (a) binding to the carrier sites (= specific binding); (b) exchange; (c) binding to unspecific, atractyloside‐insensitive sites. Procedures have been developed for depleting mitochondria of endogenous adenine nucleotides to increase the proportion of the atractyloside‐removable binding in relation to the magnitude of the exchangeable endogenous adenine nucleotide pool. Kinetics of the binding at atractyloside‐removable sites are very rapid compared to “binding” by exchange with endogenous adenine nucleotides, even at 0°. The specificity of the carrier binding, as measured by competitive replacement of [14C]ADP with unlabelled nucleotides, shows binding confined to ATP, ADP, dADP and, to a lesser extent, adenosine tetraphosphate. The high specificity is in agreement with that measured for the adenine nucleotide exchange. From the concentration dependence of the specific adenine nucleotide binding, non‐linear Scatchard plots are obtained consistent with two sites of unequal affinity (Kd′= 0.3 μM; Kd”= 7 μM). The total number of carrier sites is C0/cyt. a= 1.3 (mole/mole). The ratio of high/low affinity types is C′/C”= 1:5. The corresponding values for rat heart are: Kd′= 0.1 μM, Kd”= 4 μM; C0= 2.2; C′/C”= 1:4. With ATP two binding sites of unequal affinity are also obtained. Both Kd′ and Kd” for ATP are 1.5 to 2 times higher than for ADP. ATP binding is not affected by uncouplers. In arsenate depleted rat liver mitochondria a single binding site of high affinity (Kd= 0.5 μM) is found with C0/cyt. a= 1.2 (mole/mole). This is only half the content (per cyt. a) found in rat heart. Some differentiation between specific and unspecific binding is also possible on the basis of SH and histidine reagents. Only the unspecific binding is sensitive to p‐chloromercuribenzoate. The specific binding is selectively sensitive to methylene blue‐catalyzed photo‐oxidation. This fact, and the strong decrease in atractyloside‐removable binding between pH 7.0 and 7.5, suggests the involvement of a histidine group in the specific binding. Sonic particles retain some ability for uptake of adenine nucleotides, but this is no longer atractyloside‐sensitive. The concentration dependence of total adenine nucleotide binding gives non‐linear Scatchard plots which can be fitted by two binding sites with unequal affinities. The possible identity of the atractyloside‐“insensitive” sites in sonic particles with the specific sites in intact mitochondria is discussed. Treatment with detergents also largely removes atractyloside‐sensitive and atractyloside‐removable binding. The occurrence of two sites with different affinities, it is suggested, reflects inner and outer localizations of carriers. High affinity sites may be those which are in contact with residual endogenous adenine nucleotides and therefore apparently saturated early. The models may be discussed in terms of a carrier distribution in a mobile system or in terms of a double‐faced dimer carrier.