Topography of the active site of the Saccharomyces cerevisiae plasmalemmal dicarboxylate transporter studied using lipophilic derivatives of its substrates.

2-Alkylmalonates and O-acyl-L-malates have been found to competitively inhibit the dicarboxylate transporter of Saccharomyces cerevisiae cells, and the substrate derivatives chosen did not penetrate across the plasmalemma under the experiment conditions. Probing of the active site of this transporter has revealed a large lipophilic area stretching between the 0.72 to 2.5 nm from the substrate-binding site. Itaconate inhibited the transport fivefold more effectively than L-malate. This suggests the existence of a hydrophobic region immediately near the dicarboxylate-binding site (to 0.72 nm). The yeast plasmalemmal transporter was different from the rat liver mitochondrial dicarboxylate transporter. An area with variable lipophilicity adjoining the substrate-binding site has been revealed in the latter by a similar method. This area is mainly hydrophobic at distances up to 1.76 nm from the binding site and is separated by a hydrophilic region from 0.38 to 0.88 nm. Fumarate but not maleate competitively inhibited succinate transport into the S. cerevisiae cells. It is suggested that the plasmalemmal transporter binds the substrate in the trans-conformation. The prospects of the proposed approach for scanning lipophilic profiles of channels of different transporters are discussed.

Properties of yeast Saccharomyces cerevisiae plasma membrane dicarboxylate transporter.

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).

Study of active site topography of rat liver mitochondrial dicarboxylate transporter using lipophilic substrate derivatives.

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.

Permeation of dicarboxylic acids with different terminal position of two carboxylic groups through planar bilayer lipid membranes.

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.

The anion-carrier mediated uncoupling effect of dicarboxylic fatty acids in liver mitochondria depends on the position of the second carboxyl group.

Effects of dicarboxylic fatty acids with varying positions of the carboxyl groups on respiration and membrane potential of liver mitochondria were studied. Tetradecylmalonic acid (a fatty acid with two carboxyl groups in the alpha-position) efficiently uncoupled oxidative phosphorylation similarly to palmitic acid with the same number of carbon atoms. Similarly to the uncoupling by palmitic acid, the coupling effects of carboxyatractylate and glutamate changed reciprocally with changes in pH of the incubation medium: on increasing the pH from 7.0 to 7.8, the coupling effect of carboxyatractylate increased and that of glutamate decreased. A dicarboxylic fatty acid with the second carboxyl at the end of the alkyl chain in the omega-position (alpha, omega-tetradecyldicarboxylic acid) stimulated respiration of the mitochondria at a significantly higher concentration than myristic acid with the same number of carbon atoms, but unlike the latter nearly failed to decrease the transmembrane potential DeltaPsi. Neither carboxyatractylate nor glutamate inhibited the respiration stimulated by this dicarboxylic fatty acid.

[A method of determining the specific volume of liposomes with Turnbull blue]. / Metod opredeleniia udel'nogo ob''ema liposom s pomoshch'iu Turnbulevoisini.

A method for measuring the specific volume of liposomes by using ferricyanide as a maker is proposed. Ferricyanide is determined by the formation of Turnbull's blue. Advantages of the use of this reaction are an eightfold increase in the sensitivity and the possibility to determine the marker compound in the spectral region (730 nm) free of any considerable interference of natural compounds. Results of the use of this method for measuring the specific volume of liposomes prepared by ultrasonic treatment of a lipid suspension are reported.

[Uncoupling effect of dicarbonic analogs of stearic and palmitic acids in heart mitochondria]. / Razobshchaiushchee deistvie dikarbonovykh analogov stearinovoi i pal'mitinovoi kislot v mitokhondriiakh serdtsa.

The uncoupling effect of 2-hexadecylmalonic acid (HDMA) and 2-tetradecylmalonic acid (TDMA), the dicarboxylic derivatives of stearic and palmitic acids, have been studied in heart mitochondria. The effects of these compounds and related fatty acids on the induced by ascorbate oxidation with PMS (or TMPD) in the presence of oligomycin, phosphate and rotenone have been compared. It was found that both HDMA and TDMA uncoupled mitochondria with a similar efficiency as did the corresponding fatty acids, while the uncoupling effect of low concentrations of HDMA and TDMA were almost completely suppressed by carboxyatractylate.

Probing the active center of the mitochondrial dicarboxylate transporter.

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.

[Characteristics of the interaction between 2-alkylmalonates and the substrate-binding site of the dicarboxylate carrier of rat liver mitochondria]. / Osobennosti vzaimodeistviia 2-alkilmalonatov s tsentrom sviazyvaniia substratov dikarboksilatnogo perenoschika mitokhondrii pecheni krys.

The effect of thirteen synthetic 2-n-alkyl derivatives of malonic acid on the rate of the mitochondrial succinate oxidase reaction controlled by the dicarboxylate carrier, was studied. These compounds were shown to act as competitive inhibitors of succinate transport and could interact with the carrier according to the 1:1 stoichiometry. The affinity of the inhibitor for the carrier increased in parallel with the increase in the alkyl residue length. This dependence was impaired only in the case of pentyl-, hexyl- and heptylmalonates having close values of inhibition constants. The data obtained suggest that the polar site and two hydrophobic regions are located in the vicinity of the carrier active site. The possible organization of the carrier substrate-binding site is discussed.