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.

Abstract

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.