Abstract

Further unique effects of bongkrekate on the binding of ADP to the inner mitochondrial membrane are described in addition to a preceding report by us and explained so as to demonstrate the reorientation of a mobile carrier across the membrane.1. The dependence of the bongkrekate‐induced increase of ADP binding is nonlinear with respect to the bongkrekate concentration. Conscquently the apparent Kd for ADP decreases nonlinearly with bongkrekate concentration. The nonlinearity is kinetic in nature and is not explained by regulatory (allosteric) effects. The nonlinearity is diminished at lower pH.2. The binding of bongkrekate appears to be dependent on ADP indicating a reciprocal enhancement effect between ADP and bongkrekate binding. As a consequence the binding of high‐affinity nucleotide (such as ADP) is relatively more increased by bongkrekate the binding of nucleotides with less affinity such as dADP. This specificity preference for ADP is also increased by bongkrekate as compared to other analogues such as formycin diphosphate, and the imidodiphosphate and methylene diphosphate derivations of adenylate. The counteraction of bongkrekate and atractylate on ADP binding also depends on the cooperation between ADP and bongkrekate.3. An apparent irreversibility of the bongkrekate‐induced ADP binding is demonstrated by several effects. In the course of time bongkrekate + ADP supersede the counteraction of atractylate, even at higher atractylate concentration. Also carboxy‐atractylate, with still higher affinity than atractylate, cannot abolish the bongkrekate. [14C]ADP once fixed with bongkrekate at low pH cannot be removed by exchange with excess ADP or atractylate even at pH 8, although at this pH bongkrekate is unable to induce a binding increase.4. The results can be explained by the “reorientation mechanism” of the bongkrekate effect by which the ADP · carrier complex is translocated inside and all carriers become trapped inside by bongkrekate as the immobile carrier · bongkrekate complex. The reciprocal enhancement of bongkrekate and ADP binding is explained by the fact, that ADP is required to move the carrier inside where bongkrekate can bind to the carrier. The irreversibility of the bongkrekate effect results from the shift of the carrier inside and the resulting removal of the carrier out of the equilibrium with external ligands such as atractylate or ADP. The results thus produce the most direct evidence so far available on the molecular level, for the existence of a mobile metabolite carrier.

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