The Binding of Atractylate and Carboxy‐atractylate to Mitochondria

Abstract

35S-labelled atractylate and carboxy-atractylate are produced biosynthetically and used for studying the binding of these specific ligands to the ADP, ATP carrier in beef heart mitochondria. The following results are obtained.1. Inhibition of translocation activity goes parallel to the increase of binding by [35S]atractylate. No additional binding is observed after full inhibition of translocation is reached giving evidence that atractylate binds exclusively to the carrier.2. The maximum number of binding sites of both atractylates is about 1.6 pmol/g protein in beef heart mitochondria and decreases on treatment of the membrane by Pi, freezing, ageing, etc. The dissociation constants of the binding are approximately for atractylate Kd = 5 .M and for carboxy-atractylate Kd = M. The mass action plots of the concentration dependence for the binding are nonlinear-convex in particular with carboxy-atractylate and more linear with atractylate. Nonlinearity appears to be caused by some retardation of equilibration in the case of very high affinity binding.3. The binding of atractylate and carboxy-atractylate is relatively fast in intact mitochondria and slower in aged membranes. There is a slower and a faster binding portion.4. The atractylates remove ADP in a nearly 1 : 3 stoichiometry from untreated mitochondria. In aged and Pi-treated membranes the ratio dADP/datractylate approaches 0. Obviously binding of carrier sites to ADP is more sensitive to alterations then that of the atractylates. The assumption is maintained that the binding site for atractylate is identical with that for ADP and ATP.5. Bongkrekate prevents binding of both atractylates. However, when added after, it only removes atractylate but not the carboxy compound because of its different tight binding. The removal of atractylate depends on the synergistic effect of bongkrekate with ADP.6. The binding studies with [35S]atractylate and in particular the interaction with bongkrekate support the reorienting carrier model in which atractylate as an impermeable ligand fixes the binding site of the carrier outside while with bongkrekate the carrier site is turned to the inside.The molecular characterization of the ADP, ATP carrier in the inner mitochondria1 membrane was first begun by studying the binding of labelled ADP and ATP [I]. By the use of atractylate the portion of ADP binding specific for the carrier sites was discriminated from binding to other sites and from the endogenous nucleotides not bound to the carrier. In this way the number of carrier-linked binding sites for ADP and ATP in the mitochondria and the corresponding dissociation constants were determined. Other experiments with atractylate have shed light on the dynamic of the carrier, e.g. changes in the distribution of carrier sites between the two sides of the membrane. The conclusions were corroborated in particular by the use of another inhibitor, bongkrekate, which furnished most striking evidence for the reorientation of carrier sites from the inner to the outer membrane face and thu...

The Binding of Bongkrekate to Mitochondria

“…The facile displacement of isobongkrekate has enabled us to demonstrate in mitochondria the 'm' + 'c' transition, after the 'c' --f 'm' transition had been accomplished earlier with atractylate [17]. All the properties of this transition, its dependence on sufficient amounts of carboxyatractylate and on ADP are in excellent agreement with the reorientation mechanism.…”

“…[35S]Carboxyatractylate and [''Slatractylate were biosynthesized using seedlings of Atractylis gurnniifira, as previously described [17]. The identity and specific activity of these compounds was carefully examined by different assay methods.…”

Estimation of flux control coefficients from inhibitor titrations by non‐linear regression

“…As in the case of antimycin A titration the computer-fitted curve sufficiently covers the experimental points. The estimated dissociation constant Ko = 0.86 nM is somewhat lower than reported earlier (KD = 5-10 nM [15,16]). Since the binding of carboxyatractyloside is weaker than that of antimycin A a remarkable part of the inhibitor is dissociated from the enzyme in the range of Eo and therefore the enzyme is not completely inhibited and needs higher inhibitor concentrations for complete inhibition.…”

“…Carboxyatractyloside binds non-competitively and irreversibly to the mitochondrial adenine nucleotide translocator [15] with an 1:1 stoichiometry [16]. As in the case of antimycin A titration the computer-fitted curve sufficiently covers the experimental points.…”

ATP/ADP antiporter is involved in uncoupling of plant mitochondria induced by low concentrations of palmitate

“…In animal mitochondria CAtr is a non-competitive inhibitor [16] Heat production in plants is commonly linked to cyanide-insensitive respiration (alternative pathway), which does not result in ATP synthesis [18], and can represent a physiological response to low temperatures [19,20]. Practically all the plant mitochondria isolated so far show a residual cyanide-insensitive respiration [18], albeit there are plant cultivars lacking the alternative path, thus suggesting that it is not essential for metabolism [21].…”