4-acetamido-4--isothiocyanatostilbene-2-2--disulfonic-acid and bis(sulfosuccinimidyl)suberate

The culture supernatant of Candida albicans promoted the disruption of human red blood cells (RBCs). The haemolytic activity was detected in a sugar-rich fraction (about 200 kDa) from Sephacryl S-100 chromatography. As the haemolytic activity was adsorbed by concanavalin A-Sepharose, the haemolytic factor may be a mannoprotein. The activity was inactivated by periodate oxidation, indicating that the sugar moiety of the mannoprotein played an important role in the haemolysis. The structure of the sugar moiety of the mannoprotein was identified as a cell-wall mannan by 1H-NMR analysis, and purified C. albicans mannan promoted the disruption of RBCs. The binding of mannan to RBCs was demonstrated by flow cytometric analysis and was inhibited by the addition of band 3 protein inhibitor, 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid (DIDS). The haemolysis caused by mannan was inhibited by DIDS, SITS (4-acetamido-4'-isothiocyanatostilbene-2,2'-disulfonic acid) and bis(sulfosuccinimidyl) suberate, but not by pyridoxal 5-phosphate. These results indicated that a mannoprotein released from C. albicans bound to the band 3 protein on RBCs, thereby promoting their disruption.

Topics: 4-Acetamido-4'-isothiocyanatostilbene-2,2'-disulfonic Acid; 4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid; Anions; Biological Transport; Candida albicans; Cell Wall; Erythrocytes; Hemolysis; Hydrogen; Magnetic Resonance Spectroscopy; Mannans; Membrane Glycoproteins; Succinimides

Treatment of intact human erythrocytes with bis(sulfosuccinimidyl)suberate converted band 3 to two species with lower electrophoretic mobility in sodium dodecyl sulfate (SDS). The presence of the noncovalent anion transport inhibitor, 4,4'-dinitrostilbene-2,2'-disulfonate, promoted the lowest mobility form, while a closely related analogue, 4,4'-diisothiocyano-2,2'-stilbenedisulfonate, did not. Ferguson analysis of the electrophoretic behavior of the two slowly migrating bands strongly suggested that they represented dimers and tetramers of band 3. Increasing the temperature of the SDS solution to greater than 60 degrees C quantitatively converted the tetrameric species to the dimeric form. We conclude that band 3 can be intermonomerically cross-linked by bis(sulfosuccinimidyl)suberate as covalent dimers within two alternate quaternary forms in a manner modulated by the ligand occupying the intramonomeric stilbenedisulfonate site. In one form, band 3 covalent dimers are noncovalently associated as a SDS-resistant tetramer, while in the other form, covalent dimers are not so associated. There is no obvious relationship between ligand stereochemistry and the resulting quaternary form, suggesting that the two forms reflect alternate allosterically modulated porter quaternary structures. The significance of these two quaternary states to the transport or the ankyrin binding functions of band 3 is unknown.

Topics: 4-Acetamido-4'-isothiocyanatostilbene-2,2'-disulfonic Acid; 4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid; Anion Exchange Protein 1, Erythrocyte; Cross-Linking Reagents; Humans; Kinetics; Ligands; Macromolecular Substances; Stilbenes; Succinimides; Thermodynamics

The extracellular lysine residues in the human erythrocyte anion transport protein (band 3) have been investigated using chemical modification with the impermeant homobifunctional active ester bis(sulfosuccinimidyl)-suberate (BSSS). This agent forms covalent intra- and intermolecular cross-links in human band 3 in intact cells (Staros and Kakkad. 1983. J. Membr. Biol. 74:247). We have found that the intermolecular cross-link has no detectable effect on the anion transport function of band 3. The intramolecular cross-link, however, causes major changes in the characteristics of the anion transport. These functional alterations are caused by the modification of lysine residues at the stilbene disulfonate binding site. BSSS pretreatment at pH 7.4 irreversibly inhibits Cl-Br exchange by at least 90% when the transport is assayed at extracellular pH above 8. In the same BSSS-pretreated cells, however, the Cl-Br exchange rate is activated by lowering the pH of the flux medium (intracellular pH fixed at 7). The flux is maximal at pH 5-6; a further lowering of the extracellular pH inhibits the anion exchange. This acid-activated Cl-Br exchange in the BSSS-treated cells is mediated by band 3, as indicated by phenylglyoxal and phloretin inhibition of the flux. Thus, the BSSS pretreatment has little effect on the maximal Cl-Br exchange flux catalyzed by band 3, but it shifts the alkaline branch of its extracellular pH dependence by approximately 5 pH units. BSSS also eliminates the self-inhibition of Cl-halide exchange by high extracellular Br or I concentrations. These results indicate that the BSSS-modified lysines do not participate directly in anion translocation, but that one of the lysines normally provides a positive charge that is necessary for substrate anion binding. This positive charge is removed by the BSSS treatment but can be replaced by lowering the extracellular pH. The results also provide insight regarding the halide selectivity of the maximal rate of chloride-halide exchange: the native selectivity (Br much greater than I) is nearly abolished by BSSS treatment, which suggests that the selectivity results from the very strong binding of iodide to an outward-facing modifier site.

Topics: 4-Acetamido-4'-isothiocyanatostilbene-2,2'-disulfonic Acid; 4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid; Carrier Proteins; Chemical Phenomena; Chemistry; Cross-Linking Reagents; Erythrocyte Membrane; Humans; Hydrogen-Ion Concentration; In Vitro Techniques; Lysine; Membrane Proteins; Peptide Fragments; Succinimides

Subunit interactions in the band 3 protein of the human red blood cell membrane have been examined by a combination of cross-linking, chemical labeling, and in situ proteolysis. In agreement with Staros (Staros, J. V. (1982) Biochemistry 21, 3950-3955), we find that the membrane-impermeant active ester bis(sulfosuccinimidyl) suberate (BSSS) cross-links band 3 in intact cells to a dimer, with no formation of higher oligomer. Combined cross-linking of the outer surface with BSSS and the cytoplasmic domain with Cu2+/o-phenanthroline does not produce significant covalent tetramer of band 3 (beyond that produced by Cu2+/o-phenanthroline alone). Therefore, the membrane domains and cytoplasmic domains of the same pair of subunits are cross-linked to each other. 4,4'-Diisothiocyanodihydrostilbene-2,2'-disulfonate (H2DIDS) is known to form a covalent cross-link between complementary chymotryptic fragments (Mr 60,000 and 35,000). Edman degradation of band 3 from H2DIDS/chymotrypsin-treated cells shows that the H2DIDS cross-link is between fragments of the same subunit. In contrast, BSSS forms both intramolecular and intermolecular cross-links between complementary chymotryptic fragments. No intermolecular cross-links between two 35,000-dalton or two 60,000-dalton fragments are detectable. We have localized one end of the BSSS intermolecular cross-link to within 4 residues of the exofacial chymotrypsin cleavage site. The polypeptide sequence on each side of the site suggests that hydrophobic membrane-crossing segments emerge at the cell surface near the site of intermolecular cross-linking. This is the first detailed information available on the regions of the band 3 primary structure near the interface between subunits.

Topics: 4-Acetamido-4'-isothiocyanatostilbene-2,2'-disulfonic Acid; 4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid; Anion Exchange Protein 1, Erythrocyte; Chymotrypsin; Humans; Macromolecular Substances; Molecular Weight; Peptide Fragments; Protein Conformation; Stilbenes; Succinimides