Effect Of SH Reagents Research Articles

Structure/function relationships of the human mitochondrial ornithine/citrulline carrier by Cys site-directed mutagenesis. Relevance to mercury toxicity

The effect of SH reagents on the human mitochondrial ornithine/citrulline carrier (hORC) was studied. Site-directed Cys mutants were employed to gain information on structure/function relationships. The substitutions of each Cys by Ala did not alter the hORC activity measured as [3H]ornithine/ornithine antiport in proteoliposomes. N‑ethylmaleimide inhibited the transport of WT with IC50 of 149 μM. C51A, C50A and C132A showed a much higher IC50. MTSEA and MTSET also inhibited the WT with IC50 of 0.40 μM and 1.60 μM, respectively. C51A and C132A showed much higher IC50 values for both reagents. The triple mutant C50/51/132A showed an IC50 for the three reagents that was higher than that of the single mutants. The data strongly suggests that C132, C50 and C51 are involved in inhibition of hORC. Inhibition of WT and mutants by CuPhenanthroline, an S-S forming reagent, suggested that C132 may form disulfides with C50 or C51, impairing the transporter function. The structure/function relationships information deriving from the inhibition studies, were corroborated by the homology structural model of the transporter. The effect of HgCl2 and methyl mercury was also tested on hORC in the light of their capacity to bind thiol residues. Both reagents potently inhibit the transporter.

Stimulation by pro-apoptotic valinomycin of cytosolic NADH/cytochrome c electron transport pathway—Effect of SH reagents

Intrinsic and extrinsic apoptosis are both characterised by the presence of cytochrome c (cyto-c) in the cytosol. We present data on the extra-mitochondrial NADH oxidation catalysed by exogenous (cytosolic) cyto-c, as a possible answer to the paradox of apoptosis being an energy-dependent program but characterized by the impairment of the respiratory chain. The reduction of molecular oxygen induced by the cytosolic NADH/cyto-c pathway is coupled to the generation of an electrochemical proton gradient available for ATP synthesis. Original findings show that SH reagents inhibit the NADH/cyto-c system with a conformational change mechanism. The mitochondrial integrity-test of sulfite oxidase unequivocally demonstrates that this enzyme (120kDa) can be released outside but exogenous cyto-c (12.5kDa) does not permeate into mitochondria. Valinomycin at 2nM stimulates both the energy-dependent reversible mitochondrial swelling and the NADH/cyto-c oxidation pathway. The pro-apoptotic activity of valinomycin, as well as to the dissipation of membrane potential, can be also ascribed to the increased activity of the NADH/cyto-c oxidation pathway useful as an additional source of energy for apoptosis. It can be speculated that the activation of the NADH/cyto-c system coupled to valinomycin-induced mitochondrial osmotic swelling may represent a strategy to activate apoptosis in confined solid tumours.

Conformation-dependent accessibility of Cys-136 and Cys-155 of the mitochondrial rat carnitine/acylcarnitine carrier to membrane-impermeable SH reagents

During substrate translocation mitochondrial carriers cycle between the cytoplasmic-state (c-state) with substrate-binding site open to the intermembrane space and matrix-state (m-state) with the binding site open to the mitochondrial matrix. Here, the accessibility of Cys-58, Cys-136 and Cys-155 of the rat mitochondrial carnitine/acylcarnitine carrier (CAC) to membrane-impermeable SH reagents was examined as a function of the conformational state. Reconstituted mutant CACs containing the combinations Cys-58/Cys-136, Cys-58/Cys-155, and Cys-136/Cys-155 transport carnitine with a ping-pong mechanism like the wild-type, since increasing substrate concentrations on one side of the membrane decreased the apparent affinity for the substrate on the other side. In view of this mechanism, the effect of SH reagents on the transport activity of mutant CACs was tested by varying the substrate concentration inside or outside the proteoliposomes, keeping the substrate concentration on the opposite side constant. The reagents MTSES, MTSEA and fluorescein-5-maleimide did not affect the carnitine/carnitine exchange activity of the mutant carrier with only Cys-58 in contrast to mutant carriers with Cys-58/Cys-136, Cys-58/Cys-155 or Cys-136/Cys-155. In the latter, the inhibitory effect of the reagents was more pronounced when the intraliposomal carnitine concentration was increased, favouring the m-state of the carrier, whereas the effect was less when the concentration of carnitine was increased in the external compartment of the proteoliposomes, favouring the c-state. Moreover, the mutant carrier proteins with Cys-136/Cys-155, Cys-58/Cys-136 or Cys-58/Cys-155 were more fluorescent when extracted from fluorescein-5-maleimide-treated proteoliposomes containing 15 mM internal carnitine as compared to 2.5 mM. These results are discussed in terms of conformational changes of the carrier occurring during substrate translocation.

Studies on Gastro Intestinal Absorption of Mercury Compounds. I. Effect of Sulfhydryl Reagents on Rat Small Intestinal Absorption of Mercuric Chloride.

The effect of SH reagents on the absorption of mercuric chloride through the rat small intestine was investigated by the in situ recirculation method. The concentrations of mercury in the perfusion solutions, intestine, blood, kidney and liver were determined at each time intervals. Without SH reagents, the mercury transfer into the blood increased slightly with the time proceeds and mercury was absorbed through the intestine. The addition of L-cysteine, glutathione, 3-mercapto-1, 2-propanediol, dimercaptosuccinic acid, D (-)-penicillamine, diethyldithiocarbamate (DDTC), pyridoxine-5-thiol and thiola did not promote the mercury absorption, nor decreased the mercury content in the perfusion solutions. On the other hand, 2, 3-dimercapto-1-propanol (BAL) and 2-mercaptoethanol (MET) distinctly increased the mercury concentration in the blood, and then promoted the absorption of mercury through the intestine. To investigate the mechanism of the mercury absorption, the effect of SH compounds on the partition coefficients of mercuric chloride between octanol and water was determined. BAL and MET greatly increased the partition coefficient, but most of the other SH reagents except DDTC rather decreased it. Therefore we conclude that the lipophilic chelate formation with SH reagents is the main mechanism for the promotion of mercury absorption through the intestine.

Calcium release from isolated sarcoplasmic reticulum due to 4,4'-dithiodipyridine.

The effects of SH reagents on Ca2+ release from sarcoplasmic reticulum (SR) vesicles were examined by the tracer method using 45Ca2+. Among the various SH reagents tested, 4,4'-dithiodipyridine (PDS) was found to induce Ca2+ release most specifically from the heavy fraction of SR vesicles. Further, the following results were obtained. (i) PDS bound covalently to proteins in the SR membrane and induced Ca2+ release. (ii) The Ca2+ release was further enhanced by ATP and caffeine, but inhibited by procaine, ruthenium red and various divalent cations. (iii) PDS enhanced the Ca2+ release in the whole range of Ca2+ concentrations tested. (iv) Choline permeability was also enhanced by PDS. Further, the electrical conductance of the Ca2+-induced Ca2+ release channels was studied by incorporating them into lipid bilayers and it was found that PDS increased the probability of opening of the channels. These results suggest that PDS binds to certain SH groups of the Ca2+-induced Ca2+ release channels in the SR membrane and thus induces Ca2+ release.

Dual effect of N-ethylmaleimide on Cl? transport across the thin ascending limb of Henle's loop

Effects of SH reagents on Cl- transport were studied in the isolated hamster thin ascending limb of Henle's loop (TAL) perfused in vitro. Parachloromercuribenzene sulfonate (PCMBS) at 10(-4) M in the bath decreased the relative permeability for Cl-/Na+ (PCl/PNa), as determined by the transmural diffusion voltage (VT) generated under a NaCl concentration gradient, from 2.71 +/- 0.16 to 1.11 +/- 0.09 (P less than 0.001). The effect of PCMBS was prevented by the pretreatment with 10(-3) M dithiothreitol (DTT). N-Ethylmaleimide (NEM) at 10(-3) M in the bath exhibited a dual action on Cl- permeability of the TAL: It inhibited the Cl- permeability in fresh preparations, whereas it stimulated the Cl- permeability in the preparations pretreated with SH reagents including NEM, maleimide and PCMBS. The inhibitory effect was irreversible but the stimulatory effect was reversible. Both responses were prevented by DTT. Since dextran-maleimide did not show any inhibitory effect on PCl/PNa, the SH site responsible for the inhibition may be located inside of the cell. The stimulatory effect of NEM on PCl/PNa was markedly reduced when bath pH was reduced to 5.8. On the other hand, when the bathing fluid was made nominally Ca2+ free, the stimulatory effect of NEM was unaffected, although the basal level of PCl/PNa was reduced These observations suggest that the conductive Cl- pathway in the TAL is either stimulated or inhibited by modifying two distinct SH sites. The site of modulation by proton binding may exist distally to these SH sites. The regulatory mechanism involving Ca2+ may be independent of the SH regulatory sites.

Effect of SH reagents on atractyloside binding to mitochondria and ADP translocation. Potentiation by ADP and its prevention by uncoupler FCCP

Effect of SH reagents on atractyloside binding to mitochondria and ADP translocation. Potentiation by ADP and its prevention by uncoupler FCCP

Substrate Specificity and Some Properties of Crystalline Mold Maltase

The substrate specificity of crystalline mold maltase was investigated.The enzyme acts upon various α-heteroglucosides or saccharides. Aryl-α-glucosides were hydrolyzed much faster than alkyl-α-glucosides. The enzyme acts on the maltose derivatives whose reducing groups have been masked. But among glucosylfructoses turanose, maltulose and isomaltulose were attacked with a slow rate while the enzyme was quite inert to sucrose. Malto- and isomalto-oligosaccharides were also hydrolyzed and the enzyme ceased its action at seven to eight units of hexose in both series of oligosaccharides.The opt. pH range of Takamaltase was 4.2~4.6 and opt. temp., 50~55°C. Cu++ and Hg++ strongly inhibited the enzyme activity but other metal ions tested had no effects. It is suggested that the enzyme is not a sulfhydryl enzyme because of the lack of effects of SH-reagents on the activity.