mersalyl and malonic-acid

The mitochondrial inner membrane anion channel catalyzes the electrophoretic transport of a wide variety of anions and is inhibited by matrix divalent cations and protons. In this paper, evidence is provided that mersalyl and p-chloromercuribenzene-sulfonate each interact with this uniporter at two distinct sites. Binding to site 1 causes a shift in the pH dependence of transport, characterized by a decrease in the pIC50 for protons from about 7.8 to about 7.3, and leads to substantial stimulation of transport in the physiological pH range. This effect is not reversed by addition of thiols such as thioglycolate. Binding of mersalyl and p-chloromercuribenzenesulfonate to site 2 inhibits the transport of most anions including Pi, citrate, malonate, sulfate and ferrocyanide. The transport of Cl- is inhibited about 60% by mersalyl, but is not inhibited by p-chloromercuribenzenesulfonate. These data suggest that inhibition is a steric effect dependent on the size of the anion and the size of the R group of the mercurial. This inhibition is reversed by thioglycolate. Dose/response curves indicate that mersalyl binds to site 1 as the dose increased from 7 to 13 nmol/mg, whereas it binds to site 2 as the dose is increased from 10 to 18 nmol/mg. Thus, at certain pH values both stimulatory and inhibitory phases can be seen in the same dose/response curve. It is suggested that these sites may contain thiol groups and that physiological regulators may exist which can effect changes in activity of the inner membrane anion uniporter similar to those exerted by mercurials.

Topics: 4-Chloromercuribenzenesulfonate; Animals; Binding Sites; Chloride Channels; Chlorides; Dose-Response Relationship, Drug; Hydrogen-Ion Concentration; Intracellular Membranes; Ion Channels; Kinetics; Malonates; Membrane Proteins; Mersalyl; Mitochondria, Liver; Mitochondrial Swelling; Organomercury Compounds; Phenylmercury Compounds; Phosphates; Rats; Sulfates; Thioglycolates

The nature of the inhibition of the dicarboxylate carrier by compounds reacting with SH groups has been investigated. (1) Mersalyl and p-hydroxymercuribenzoate increase the Km without changing the V of malonate/Pi exchange, when they are added simultaneously with the dicarboxylate. If, on the other hand, the mitochondria are preincubated with SH reagents prior to the addition of malonate, the mersalyl inhibition of malonate/Pi exchange becomes predominantly non-competitive with respect to malonate. (2) In the case of Pi/Pi exchange, catalyzed by the dicarboxylate carrier, the mersalyl inhibition is competitive with respect to Pi (as indicated by Lineweaver-Burk plots), even when mersalyl is added before the substrate. Dixon plots of the rate of Pi uptake against mersalyl concentration are, however, non-linear, suggesting that the inhibition is partially competitive. (3) Dicarboxylates and dicarboxylate analogous protect against SH reagent inhibition of both dicarboxylate and Pi uptake via the dicarboxylate carrier. The protectors are effective when added before, or together with the SH reagents, but do not reverse the inhibition once it has been established. Protection by substrate analogues progressively decreases, as the time of incubation with the SH reagent increases. (4) The presence of Pi does not protect against the SH reagent inhibition of the Pi uptake. (5) The rate of SH reagent inhibition of the dicarboxylate carrier is competively inhibited by dicarboxylates. (6) It is concluded that SH reagents bind at or near the dicarboxylate specific binding site and distant from the Pi binding site. As a result of this reaction these inhibitors prevent dicarboxylate binding directly and decrease the affinity for Pi by an indirect conformational change.

Topics: Animals; Dicarboxylic Acid Transporters; Dicarboxylic Acids; Enzyme Inhibitors; Malonates; Mersalyl; Mitochondria, Liver; Rats; Sulfhydryl Compounds; Sulfhydryl Reagents

Topics: Diuretics; Iodoacetates; Maleates; Malonates; Mercuric Chloride; Mercury; Mersalyl; Organomercury Compounds; Phlorhizin