Identification of the anion exchange protein of Ehrlich cells: a kinetic analysis of the inhibitory effects of 4,4'-diisothiocyano-2,2'-stilbene-disulfonic acid (DIDS) and labeling of membrane proteins with 3H-DIDS.
In Ehrlich ascites tumor cells 4,4'-diisothiocyano-2,2'-stilbene-disulfonic acid (DIDS) inhibits the chloride exchange both reversibly and irreversibly. The reversible inhibition is practically instantaneous and of a competitive nature with Ki about 2 microM at zero chloride concentration. This is succeeded by a slow irreversible binding of DIDS to the transporter, with a chloride dependence suggesting binding to the same site as for reversible DIDS binding/inhibition. To identify the membrane protein involved in anion exchange, cells were labeled with 3H-DIDS. Incubation of cells for 10 min with 25 microM DIDS at pH 8.2 leads to more than 95% inhibition of the DIDS-sensitive chloride exchange flux when the chloride concentration is low (15 mM). This condition was used for the 3H-DIDS-labeling experiments. After incubation the cells were disrupted, the membranes isolated and solubilized, and the proteins separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis. The distribution of the 3H-activity in the gel showed only one major peak, which could be related to protein with a mol wt of about 30,000 Daltons. The number of transport sites was estimated at about 400,000 per cell, and from the DIDS-sensitive chloride flux under steady-state conditions we calculate a turnover number of 340 ions per sec per site.
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; D001665:Binding Sites; D002286:Carcinoma, Ehrlich Tumor / Q000378:metabolism*; D002352:Carrier Proteins / Q000037:antagonists & inhibitors* / Q000378:metabolism; D002712:Chlorides / Q000378:metabolism*; D005260:Female; D007474:Ion Exchange; D007700:Kinetics; D051379:Mice; D008970:Molecular Weight; D009363:Neoplasm Proteins / Q000037:antagonists & inhibitors / Q000378:metabolism; D013267:Stilbenes / Q000494:pharmacology*
D027321:Anion Transport Proteins; D002352:Carrier Proteins; D002712:Chlorides; D009363:Neoplasm Proteins; D013267:Stilbenes; D012856:4-Acetamido-4'-isothiocyanatostilbene-2,2'-disulfonic Acid; D017878:4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid