Kinetic analysis of the inhibition of anion transport in sarcoplasmic reticulum vesicles by a disulfonic stilbene derivative. Measurement of the change in chloride-diffusion potential by using a fluorescent cyanine dye.
The characterization of the anion transport system in sarcoplasmic reticulum (SR) vesicles was performed by kinetic analysis of the reversible inhibition of gluconate efflux by a disulfonic stilbene derivative; 4-acetamido-4'-isothiocyano-stilbene-2,2'-disulfonate (SITS). The gluconate efflux in SR vesicles was measured by following the change in Cl- ion-diffusion potential due to the mutual diffusion of Cl- and gluconate- using a potential probe; 3,3'-dipropylthiadicarbocyanine iodide (diS-C3-(5)). The main results are as follows. (1) Gluconate efflux was increased by intravesicular gluconate- and was decreased by both extravesicular gluconate- and extravesicular Cl-. (2) Gluconate efflux was depressed when membrane potential became inside-negative. (3) SITS reacted with the site of the anion transport system from the outside of the vesicles and one molecule of SITS inhibited each transport unit. (4) Extravesicular Cl- competed with SITS at the common site of the anion transport system. The dissociation constants for Cl- and SITS are 70 mM and 0.048 microM, respectively. (5) The inhibition of gluconate efflux by SITS was noncompetitive with both intravesicular gluconate and extravesicular gluconate. These results suggest that the anion transport system in SR vesicles can be described by a carrier-mediated transport model in which anions are transported by a mobile transport site accompanied by a net negative charge.
D012856:4-Acetamido-4'-isothiocyanatostilbene-2,2'-disulfonic Acid / Q000494:pharmacology*; D000818:Animals; D000838:Anions; D052160:Benzothiazoles; D001692:Biological Transport / Q000187:drug effects; D002232:Carbocyanines; D002712:Chlorides / Q000378:metabolism*; D004058:Diffusion; D004305:Dose-Response Relationship, Drug; D005456:Fluorescent Dyes; D005942:Gluconates / Q000378:metabolism*; D007700:Kinetics; D008564:Membrane Potentials; D011804:Quinolines; D011817:Rabbits; D012519:Sarcoplasmic Reticulum / Q000378:metabolism*; D013267:Stilbenes / Q000494:pharmacology*
D000838:Anions; D052160:Benzothiazoles; D002232:Carbocyanines; D002712:Chlorides; D005456:Fluorescent Dyes; D005942:Gluconates; D011804:Quinolines; D013267:Stilbenes; D012856:4-Acetamido-4'-isothiocyanatostilbene-2,2'-disulfonic Acid; C012944:3,3'-dipropyl-2,2'-thiadicarbocyanine