The rate of endogenous respiration of Saccharomyces cerevisiae cells incubated at 0 degrees C under aerobic conditions in the absence of exogenous substrates decreased exponentially with a half-period of about 5 h when measured at 30 degrees C. This was associated with an indirectly shown decrease in the level of oxaloacetate in the mitochondria in situ. The initial concentration of oxaloacetate significantly decreased the activity of succinate dehydrogenase. The rate of cell respiration in the presence of acetate and other exogenous substrates producing acetyl-CoA in mitochondria also decreased, whereas the respiration rate on succinate increased. These changes were accompanied by an at least threefold increase in the L-malate concentration in the cells within 24 h. It is suggested that the increase in the L-malate level in the cells and the concurrent decrease in the oxaloacetate level in the mitochondria should be associated with a deceleration at 0 degrees C of the transport of endogenous respiration substrates from the cytosol into the mitochondria. This deceleration is likely to be caused by a high Arrhenius activation energy specific for transporters. The physiological significance of L-malate in regulation of the S. cerevisiae cell respiration is discussed.
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.
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.
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