4-acetamido-4--isothiocyanatostilbene-2-2--disulfonic-acid and benzoylamido-4--aminostilbene-2-2--disulfonate

Treatment of human erythrocytes with the membrane-impermeant carbodiimide 1-ethyl-3-[3-(trimethylammonio)propyl]carbodiimide (ETC) in citrate-buffered sucrose leads to irreversible inhibition of phosphate-chloride exchange. The level of transport inhibition produced was dependent on the concentration of citrate present during treatment, with a maximum of approx. 60% inhibition. [14C]Citric acid was incorporated into Band 3 (Mr = 95,000) in proportion to the level of transport inhibition, reaching a maximum stoichiometry of 0.7 mol citrate per mol Band 3. The citrate label was localized to a 17 kDa transmembrane fragment of the Band 3 polypeptide. Citrate incorporation was prevented by the transport inhibitors 4,4'-diisothiocyano- and 4,4'-dinitrostilbene-2,2'-disulfonate. ETC plus citrate treatment also dramatically reduced the covalent labeling of Band 3 by [3H]4,4'-diisothiocyano-2,2'-dihydrostilbene disulfonate (3H2DIDS). Noncovalent binding of stilbene disulfonates to modified Band 3 was retained, but with reduced affinity. We propose that the inhibition of anion exchange in this case is due to carbodiimide-activated citrate modification of a lysine residue in the stilbenedisulfonate binding site, forming a citrate-lysine adduct that has altered transport function. The evidence is consistent with the hypothesis that the modified residue may be Lys a, the lysine residue involved in the covalent reaction with H2DIDS. Treatment of erythrocytes with ETC in the absence of citrate resulted in inhibition of anion exchange that reversed upon prolonged incubation. This reversal was prevented by treatment in the presence of hydrophobic nucleophiles, including phenylalanine ethyl ester. Thus, inhibition of anion exchange by ETC in the absence of citrate appears to involve modification of a protein carboxyl residue(s) such that both the carbodiimide- and the nucleophile-adduct result in inhibition.

Topics: 4-Acetamido-4'-isothiocyanatostilbene-2,2'-disulfonic Acid; 4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid; Anion Exchange Protein 1, Erythrocyte; Anions; Carbodiimides; Citrates; Erythrocyte Membrane; Ethyldimethylaminopropyl Carbodiimide; Humans; In Vitro Techniques; Ion Channels; Stilbenes

Canine renal brush border membrane proteins that bind stilbenedisulfonate inhibitors of anion exchange were identified by affinity chromatography. A 130-kDa integral membrane glycoprotein from brush border membrane was shown to bind specifically to 4-acetamido-4'-isothiocyanostilbene-2,2'-disulfonate immobilized on Affi-Gel 102 resin. The bound protein could be eluted effectively with 1 mM 4-benzamido-4'-aminostilbene-2,2'-disulfonate (BADS). The 130-kDa protein did not bind to the affinity resin in the presence of 1 mM BADS or when the solubilized extract was covalently labeled with 4,4'-diisothiocyanostilbene-2,2'-disulfonate (DIDS). This protein was labeled with [3H]H2DIDS, and the labeling was prevented by BADS. The 130-kDa protein did not cross-react with antibody raised against human or dog erythrocyte Band 3 protein. The 130-kDa protein was accessible to proteinase K and chymotrypsin digestion in vesicles but not to trypsin. The 130-kDa protein was sensitive to endo-beta-N-acetylglucosaminidase F treatment both in the solubilized state and in brush border membrane vesicles showing that it was a glycoprotein and that the carbohydrate was on the exterior of the vesicles. This glycoprotein was resistant to endo-beta-N-acetylglucosaminidase H treatment suggesting a complex-type carbohydrate structure. The protein bound concanavalin A, wheat germ agglutinin, and Ricinus communis lectins, and it could be purified using wheat germ agglutinin-agarose.

Topics: 4-Acetamido-4'-isothiocyanatostilbene-2,2'-disulfonic Acid; 4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid; Amino Acids; Animals; Cross Reactions; Dogs; Electrophoresis, Polyacrylamide Gel; Glycoproteins; Kidney; Microvilli; Molecular Weight; Stilbenes; Time Factors

The effect of temperature and chemical modification on the interaction of the human erythrocyte Band 3 protein (the anion transport protein) with 4-acetamido-4'-isothiocyanostilbene 2,2'-disulfonate (SITS; Ki = 10 microM)-Affi-Gel 102 resin was studied. Band 3 binds to the affinity resin in two states; weakly bound, which is eluted by 1 mM 4-benzamido-4'-aminostilbene 2,2'-disulfonate (BADS; Ki = 2 microM), and strongly bound, which is eluted only under denaturing conditions by 1% lithium dodecyl sulfate (LDS). At 4 degrees C, most of band 3 was present initially in the weakly bound form and very little in the strongly bound form. With longer incubations at 4 degrees C, the weakly bound form was slowly converted to the strongly bound form. At 37 degrees C, most of Band 3 was rapidly converted to the strongly bound form, with some Band 3 still remaining in the weakly bound form. Band 3 dimers, labelled with 4,4'-diisothiocyanostilbene 2,2'-disulfonate (DIDS) in one monomer, did bind to immobilized SITS but did not become tightly bound upon incubation at 37 degrees C. Since the covalent attachment of DIDS to one monomer prevented the adjacent monomer from becoming tightly bound to immobilized SITS ligand, this observation suggests that the inhibitor-binding sites of the two adjacent monomers must be interacting with each other. When the inhibitor site of Band 3 was selectively modified by citrate in the presence of 1-ethyl-3-(3-azonia-4,4-dimethylpentyl)carbodiimide (EAC), Band 3 bound to the resin was more easily eluted by BADS, suggesting reduced affinity for immobilized SITS. However, citrate-modified Band 3 did become tightly bound upon incubation at 37 degrees C.

Topics: 4-Acetamido-4'-isothiocyanatostilbene-2,2'-disulfonic Acid; 4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid; Anion Exchange Protein 1, Erythrocyte; Erythrocyte Membrane; Humans; Sodium Dodecyl Sulfate; Stilbenes; Temperature

Affinity chromatography of Band 3 was performed using a series of affinity matrices synthesized with various inhibitor ligands and spacer arms. Hydrophilic spacer arms greater than four atoms in length were essential for Band 3 binding. An affinity resin prepared by reacting 4-acetamido-4'-isothiocyanostilbene-2,2'-disulfonate (Ki = 10 microM) with Affi-Gel 102 was found to be the most effective resin of the series tested. Solubilized proteins from human erythrocyte membranes were incubated with the affinity resin, and pure Band 3 was recovered by eluting with 4-benzamido-4'-aminostilbene-2,2'-disulfonate (BADS; Ki = 2 microM). Band 3 bound to the resin specifically in its stilbene disulfonate binding site, and optimal binding was achieved at pH 8 and at high ionic strength. At 4 degrees C, up to 80% of the bound Band 3 could be eluted by 1 mM BADS, whereas the remainder could be eluted under denaturing conditions using 1% lithium dodecyl sulfate. At 22 or 37 degrees C, the amount of BADS-elutable Band 3 was reduced with a concomitant increase of Band 3 in the lithium dodecyl sulfate elute. Thus, for successful affinity chromatography, the experiment must be carried out rapidly at 4 degrees C. This procedure was also used to purify the Band 3 protein from mouse, horse, pig, and chicken erythrocytes.

Topics: 4-Acetamido-4'-isothiocyanatostilbene-2,2'-disulfonic Acid; 4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid; Anion Exchange Protein 1, Erythrocyte; Chromatography, Affinity; Electrophoresis, Polyacrylamide Gel; Erythrocyte Membrane; Humans; Stilbenes; Temperature