Identification of membrane proteins related to anion transport in Ehrlich ascites tumor cells.
DIDS (4,4'-diisothiocyanostilbene-2,2'-disulfonic acid) can interact covalently with membrane sites, resulting in an inhibition of anion exchange. This inhibition varies from reversible to irreversible, depending on the length of times DIDS interacts with the membranes. During the reversible phase, a kinetic analysis of the nature of its inhibitory effect on C1 self-exchange can be performed. The effect of variations in the chloride concentration on the inhibitory potency of DIDS is consistent with the concept that C1 and DIDS compete for the transport site of the anion exchange system in Ehrlich cells. The value of Ki for DIDS inhibition is approx. 0.5 microM. Since the reversible binding may be specific to the anion recognition site in the transport system, it is likely that the subsequent covalent reaction (which is very slow in the Ehrlich cell) involves a nucleophilic group in the membrane close to the transport site. In this case DIDS can be used as a covalent label for the transport protein. We have synthesized a 3-H labelled DIDS with a high specific activity (0.62 X 10(8) cpm/mumol). Using the labelled DIDS, it has been possible to demonstrate that in low C1 medium, only one membrane protein seems to be labelled, and that the number of binding sites per cell is 7 X 10(7). This value is very close to the known density of binding sites in the red blood cell.
D017878:4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid; D012856:4-Acetamido-4'-isothiocyanatostilbene-2,2'-disulfonic Acid / Q000031:analogs & derivatives / Q000494:pharmacology; D000818:Animals; D027321:Anion Transport Proteins; D001693:Biological Transport, Active / Q000187:drug effects; D002286:Carcinoma, Ehrlich Tumor / Q000378:metabolism*; D002352:Carrier Proteins / Q000302:isolation & purification / Q000378:metabolism*; D002462:Cell Membrane / Q000187:drug effects / Q000378:metabolism / Q000648:ultrastructure; D002712:Chlorides / Q000378:metabolism; D007700:Kinetics; D008565:Membrane Proteins / Q000302:isolation & purification / Q000378:metabolism*; D051379:Mice
D027321:Anion Transport Proteins; D002352:Carrier Proteins; D002712:Chlorides; D008565:Membrane Proteins; D012856:4-Acetamido-4'-isothiocyanatostilbene-2,2'-disulfonic Acid; D017878:4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid