Electrically silent hydrogen ion fluxes across a planar bilayer lipid membrane (BLM) induced by an addition of dicarboxylic (DC) acids at one side of BLM are monitored by measuring pH changes in the unstirred layers near the BLM surface via recording protonophore-dependent potentials. Two groups of DC acids are studied: (1) 2-n-alkylmalonic acids with an alkyl chain of different length which carry both carboxylic groups at one terminus of the hydrocarbon chain (alpha,alpha-DC acids); and (2) dicarboxylic acids of different linear chain length having carboxylic groups at the opposite ends of the hydrocarbon chain (alpha,omega-DC acids). It is shown that the pH optimum of hydrogen ion fluxes for the DC acids is shifted considerably to acidic pH values compared to monocarboxylic acids and is located near pH 5. For both types of DC acids at pH&z. Lt;5, the total transport is limited by diffusion of the anionic forms of the acids across the unstirred layers, while at pH&z.Gt;5 the transport is limited by diffusion of the neutral form across the membrane. The fluxes of alpha,alpha-DC acids are similar to those of alpha,omega-DC acids provided that the acids have the similar number of carbon atoms, the fluxes grow with the increase in the chain length of the alkyl radical.
2‐n‐Alkylmalonates with various length of the alkyl residue have been used to study the topography of the active center of the dicarboxylate transporter in intact rat liver mitochondria. Measurements of the Ki
values of these competitive inhibitors suggest that in the transporter there is a large hydrophobic region at least 1.7 nm in size, containing a polar domain (ca. 0.5 nm) and situated close to a substrate‐binding site. These zones are assumed to be involved in the mechanism of dicarboxylate transport.
Transport of succinate into Saccharomyces cerevisiae cells was determined using the endogenous coupled mitochondrial succinate oxidase system. The dependence of succinate oxidation rate on the substrate concentration was a curve with saturation. At neutral pH the K(m) value of the mitochondrial "succinate oxidase" was fivefold less than that of the cellular "succinate oxidase". O-Palmitoyl-L-malate, not penetrating across the plasma membrane, completely inhibited cell respiration in the presence of succinate but not glucose or pyruvate. The linear inhibition in Dixon plots indicates that the rate of succinate oxidation is limited by its transport across the plasmalemma. O-Palmitoyl-L-malate and L-malate were competitive inhibitors (the K(i) values were 6.6 +/- 1.3 microM and 17.5 +/- 1.1 mM, respectively). The rate of succinate transport was also competitively inhibited by the malonate derivative 2-undecyl malonate (K(i) = 7.8 +/- 1.2 microM) but not phosphate. Succinate transport across the plasma membrane of S. cerevisiae is not coupled with proton transport, but sodium ions are necessary. The plasma membrane of S. cerevisiae is established to have a carrier catalyzing the transport of dicarboxylates (succinate and possibly L-malate and malonate).
Earlier it has been demonstrated that the active site (substrate-binding site + active site channel) of rat liver mitochondrial dicarboxylate transporter is characterized by rather complex topography. Probing the active site with 2-monoalkylmalonates revealed the existence of internal and external lipophilic areas separated by a polar region. A two substrate-binding site model of the transporter has been supposed. The correctness of this model has been evaluated by probing the active site with O-acyl-L-malates differing from 2-monoalkylmalonates by 0.23 nm longer distance from the anion groups to the aliphatic chain. Changes in the polar group of the probe did not prevent its binding and showed the same variable lipophilicity pattern for the transporter channel. Probing with alpha,omega-alkylene dimalonates did not reveal the second substrate-binding site at the active site. The substrate-binding site did not show any differences in affinity to O-acyl-derivatives of L-malate and D-malate, except L-malate binds more effectively than D-malate. This suggests involvement of the L-malate hydroxyl group in substrate binding and stereospecific behavior of the transporter substrate-binding site. A modified one substrate-binding site model of the dicarboxylate transporter is discussed.
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