Reconstitution of inhibitor binding properties of the isolated adenosine 5'-diphosphate,adenosine 5'-triphosphate carrier-linked binding protein.
We studied the binding of carboxyatractylate (CAT) and bongkrekate (BKA) to the solubilized ATP,ADP carrier-linked binding protein, which had been incorporated into liposomes. After solubilization with 3-lauramido-N,N-dimethylpropylamine oxide the binding protein had largely lost it affinity and binding capacity for both CAT and BKA. On incorporation into phospholipid vesicles, CAT binding was restored to a considerable extent (3.5 mumol of CAT/g of protein), reaching the original affinity as observed in mitochondria (Kd = 10(-8) M). With high amounts of CAT and under the influence of ADP the binding can be increased to 6.8 mumol of CAT/g of protein, indicating a movement of binding sites in the liposomal membrane. The binding of BKA was also reconstituted with high affinity (Kd = 8 X 10(-8) M) and to the same extent (6.4 mumol of BKA/g of protein). As in the case of intact mitochondria, this reconstituted binding depends on the presence of ADP. This dependence on ADP has an apparent Km = 7 muM, similar to the carrier affinity for ADP in intact mitochondria. The reorientation model of Klingenberg for the ADP,ATP carrier implicating an ADP-catalyzed transition between the CAT binding form (c state) and BKA binding form (m state) in the inner mitochondrial membrane has been confirmed in this reconstituted system.[1]References
- Reconstitution of inhibitor binding properties of the isolated adenosine 5'-diphosphate,adenosine 5'-triphosphate carrier-linked binding protein. Krämer, R., Klingenberg, M. Biochemistry (1977) [Pubmed]
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