The amino acid conjugate formed by the interaction of the anion transport inhibitor 4,4'-diisothiocyano-2,2'-stilbenedisulfonic acid (DIDS) with band 3 protein from human red blood cell membranes.

The specific anion transport inhibitor 4,4'-diisothiocyano-2,2'-stilbenedisulfonic acid (DIDS) and its reduced analog (H2DIDS), when irreversibly bound to band 3 protein of the red blood cell membrane, form amino acid conjugates through interaction with the epsilon-amino group of a particular lysine residue. The specific residue is located in a transmembrane segment of band 3 protein and appears to be a close neighbor of the transport site.

The location of a disulfonic stilbene binding site in band 3, the anion transport protein of the red blood cell membrane.

The binding site for 4,4'-diisothiocyano-2,2'-stilbenedi sulfonic acid, a specific, potent, irreversible inhibitor of anion transport in red blood cells is located in a 15 000 dalton transmembrane segment of band 3, produced by chymotrypsin treatment of ghosts stripped of extrinsic proteins. The segment was cleaved into three fragments of 7000 daltons by CNBr. The C-terminus of the segment is located in the 7000 daltons by the N-terminus in one of the 4000 dalton fragment; the N-terminus in one of the 4000 dalton fragments; and the binding site for 4,4'-diisothiocyano-2,2'-stilbenedisulfonic acid in the middle 4000 dalton fragment. The latter was cleaved by N-bromosuccinimide into two fragments of 2000 daltons. The binding site for 4,4'-diisothiocyano-2,2'-stilbenedisulfonic acid was located on the fragment containing the newly formed N-terminus. It is concluded that the binding site is located about 9000 daltons from the C-terminus (at the outside face of the membrane) and 6000 daltons from the N-terminus (at the cytoplasmic face). In view of the existing evidence that the binding site may be located near the outside face of the membrane, it is suggested that the 15 000 dalton segment is folded, so that it crosses the bilayer three times.

Pepsin cleavage of band 3 produces its membrane-crossing domains.

After prolonged treatment of red-cell ghosts with pepsin followed by SDS-urea-acrylamide gel electrophoresis of the membrane peptide fraction, a heavily stained band representing peptides of about 4 kDa (with traces of higher molecular weights) was found. If the cells were first labelled with the disulfonic stilbene, DIDS (4,4'-diisothiocyano-2,2'-stilbenedisulfonic acid) or with N-ethylmaleimide, probes that react with specific sites in Band 3 the anion transport protein, both agents were largely located in the 4 kDA band. With less intensive pepsin treatment, Stained bands of about 17, 12 and 8 kDa were also visible, and DIDS labelling was associated with these higher molecular weight peptides. The 4 kDa band apparently contains at least five or six different peptides. A single peptide containing the DIDS-binding site was separated from others in the band by ion-exchange chromatography. The location of the DIDS-peptide in the primary structure of Band 3 was determined by matching the known location of DIDS and of a methionine residue cleavable by cyanogen bromide. It is concluded that two additional 4 kDA peptides are labelled with N-ethylmaleimide. Because the location of the N-ethylmaleimide-binding sites are known, these two peptides could also be mapped in the primary structure of Band 3. The findings are consistent with the suggestion that pepsin can digest those portions of Band 3 (and probably of other intrinsic peptides) that are exposed on either side of the membrane, leaving only those domains that cross the bilayer. For Band 3, the data are consistent with a structure containing five crossing strands per monomer, each crossing strand being about 4 kDa.

The sulfhydryl groups of the 35,000-dalton C-terminal segment of band 3 are located in a 9000-dalton fragment produced by chymotrypsin treatment of red cell ghosts.

Five sulfhydryl groups of band 3, the anion-transport protein of the red blood cell membrane, can be labeled by N-ethylmaleimide (NEM). Two of these are located in a 35,000-dalton, C-terminal segment produced by chymotrypsin treatment of cells. Extensive treatment of unsealed ghosts with chymotrypsin results in the disappearance of the 35,000-dalton segment, but its two NEM-binding sites area preserved in a 9000-dalton peptide. The latter must therefore be a proteolytic product of the larger segment. Labeling of sulfhydryl groups of band 3 by an impermeant analog of NEM occurs in inside-out, but not in right-side-out vesicles derived from red cell ghosts, supporting the conclusion that NEM-reactive sulfhydryl groups, including those in the 35,000- and 9000-dalton segments, are exposed at the cytoplasmic face of the membrane. These findings support the conclusion that the 35,000-dalton segment crosses the bilayer, and suggest that the 9000-dalton segment may be a membrane-crossing portion of the 35,000-dalton segment.

Effect of cholesterol upon the conformation of band 3 and its transmembrane fragment.

Vesicles enriched in the anion transport protein band 3 and its transmembrane domain were prepared, and the cysteine residues were labelled with an extrinsic fluorescent probe, monobromobimane. Fluorescence energy transfer was demonstrated between intrinsic tryptophans and monobromobimane, and an average interchromophoric distance, Rav, was defined. Rav values and fluorescence emission wavelengths were used to monitor the conformation of band 3 and its transmembrane domain as a function of cholesterol content. The vesicles were treated with ovolecithin liposomes to reduce the cholesterol concentration, and there was an increase in Rav from 17.25 to 20.70 A (1 A = 0.1 nm) in intact band 3. A somewhat smaller increase in Rav for the transmembrane domain was observed (18.03-19.04). The tryptophan fluorescence emission wavelength was also blue shifted in the cholesterol-depleted preparations relative to the untreated samples. Combining the effects of cholesterol depletion upon Rav and the fluorescence emission maxima, it is suggested that the conformation of band 3 is influenced by the level of cholesterol in the bilayer.