4-acetamido-4--isothiocyanatostilbene-2-2--disulfonic-acid and Hemolysis

Hyphal cells of Candida albicans bind to human hemoglobin, but not yeast cells. The amount of hemoglobin receptor is significantly higher in hyphal cells than on yeast cells. Only the hyphal cells of C. albicans use hemoglobin as a source of iron. The culture supernatant of C. albicans promoted the disruption of human red blood cells (RBC). Hemolytic activity was detected in a sugar-rich fraction (about 200 kDa) purified by Sephacryl S-100 chromatography. As the hemolytic activity was adsorbed by concanavalin A (Con A)-Sepharose, the hemolytic factor might be a mannoprotein. The activity was inactivated by periodate oxidation, indicating that the sugar moiety of the mannoprotein plays an important role in hemolysis. 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 RBC. The binding of mannan to RBC was demonstrated by flow cytometric analysis and was inhibited by the addition of the band 3 protein inhibitor, 4,4'-diisothiocyanato-stilbene-2,2'-disulfonic acid (DIDS). The hemolysis caused by mannan is inhibited by DIDS, 4-acetamido-4'-isothiocyanato-stilbene-2,2'-disulfonic acid, and Bis (sulfosuccinimidyl) suberate, but not by pyridoxal-5'-phosphate. A new platinum derivative of the form H[Pt(IV) (Hdigly)Cl2(OH)2] (Hdigly = glycylglycine) has candidacidal activity 10-fold lower than that of cisplatin.

Topics: 4-Acetamido-4'-isothiocyanatostilbene-2,2'-disulfonic Acid; Anion Exchange Protein 1, Erythrocyte; Antifungal Agents; Candida albicans; Drug Design; Drug Resistance, Fungal; Erythrocytes; Fungal Proteins; Hemoglobins; Hemolysis; Humans; Membrane Glycoproteins; Platinum Compounds; Protein Binding

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

Effects of anion transport inhibitors such as 4,4'-diisothiocyanostilbene-2,2'-disulfonate (DIDS) and 4-acetamido-4'-isothiocyanostilbene-2,2'-disulfonate on hemolysis of human erythrocytes at 200 MPa were examined by changing intracellular pH (7.2-7.9). These inhibitors suppressed the hemolysis at neutral pH but enhanced it at alkaline pH. However, such an enhancement was suppressed by cross-linking of membrane proteins using diamide. From the near-UV CD spectra of band 3 and the relation between hemolysis and anion transport in intact or trypsin-treated erythrocytes, it was found that such hemolytic properties were characterized by the binding of inhibitors to band 3. In addition, spectrin detachment from the erythrocyte membrane by high pressure was considerably suppressed by DIDS treatment at neutral pH, but not by DIDS labeling at alkaline pH. These results suggest that the interaction of the cytoplasmic domain of band 3 with the cytoskeleton, which is induced by the binding of ligands to the exofacial domain of band 3, is dependent on the intracellular pH, i.e., the linking is tightened at neutral pH but relaxed at alkaline pH.

Topics: 4-Acetamido-4'-isothiocyanatostilbene-2,2'-disulfonic Acid; 4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid; Anion Exchange Protein 1, Erythrocyte; Buffers; Circular Dichroism; Erythrocyte Membrane; Hemolysis; Humans; Hydrogen-Ion Concentration; Ion Transport; Osmolar Concentration; Phosphates; Pressure; Protein Conformation; Spectrin; Trypsin; Ultraviolet Rays

The relationship between erythrocyte shape and the critical cell volume was investigated. Agents able to increase the critical cell volume induced three main stable shapes of erythrocytes: discocytic, stomatocytic, and echinocytic. The absence of correlation between shape and critical cell volume under isoosmotic conditions suggests that relative differences between the surface areas of the inner and the outer leaflet of the cell membrane do not influence the critical volume of a cell.

Topics: 4-Acetamido-4'-isothiocyanatostilbene-2,2'-disulfonic Acid; 4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid; Animals; Arsenates; Biophysical Phenomena; Biophysics; Cattle; Cell Size; Erythrocytes; Glycolysis; Hemolysis; Hot Temperature; In Vitro Techniques; Phosphates; Vanadates

The kinetics of hemolysis of erythrocytes in glycerol-containing media was studied spectrophotometrically. The hemolytic process starts by a rapid process, obeying a first order rate law, which is followed by a slow change in absorbance. The kinetics of hemolysis may be described by (a) the maximum absorption, Emax, due to cellular expansion, (b) the rate constant, k, of the fast process and (c) the final absorption at its end, Einf and the ratio Einf/Emax. At pH 6.85 in normal human cells, k = 0.72 min-1 while in hereditary spherocytosis cells, k = 1.06 min-1, iron deficiency k = 0.52 and beta-thalassemia minor k = 0.36 min-1. The percentages of Einf/Emax were 35.3 in control cells, while they were 9.8, 50.0 and 88.3 in spherocytosis, iron deficiency and thalassemia, respectively. Thus these kinetic parameters may help to distinguish and understand the above mentioned erythrocyte disorders. At physiological pH (7.4-7.2), no hemolysis was detected in the medium used. When the pH decreased, hemolysis occurred, its rate increasing gradually until pH 6.3. On further acidosis, the hemolytic rate slowed down again. Addition of DIDS to the whole blood prior to the test inhibits hemolysis. Similar effect of DIDS was noted in washed cells; this effect was partially reversed by albumin. These results suggest that a process involving band 3 affects the rate and degree of glycerol-induced hemolysis of normal red blood cells.

Topics: 4-Acetamido-4'-isothiocyanatostilbene-2,2'-disulfonic Acid; 4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid; Culture Media; Erythrocytes, Abnormal; Glycerol; Hemolysis; Humans; Hydrogen-Ion Concentration; Kinetics; Spectrophotometry, Ultraviolet

Topics: 4-Acetamido-4'-isothiocyanatostilbene-2,2'-disulfonic Acid; 4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid; Animals; Anion Exchange Protein 1, Erythrocyte; Cell Fusion; Cell Line; Cell Membrane; Cells, Cultured; Cholesterol; Erythrocyte Membrane; Hemolysis; Humans; Liposomes; Parainfluenza Virus 1, Human; Receptors, Cell Surface; Receptors, Virus; Spectrometry, Fluorescence; Viral Fusion Proteins

The effect of DIDS on osmotic properties of bovine erythrocytes was studied. The isoosmotic volume, the amount of intracellular solutes, and the osmotically non-active volume were not influenced by DIDS. An increase of osmotic fragility of erythrocytes upon DIDS treatment was evident and identical both in NaCl and in NaCl + KCl hypotonic solutions. These results suggest that the critical cell volume decreases. The link between the effect of DIDS on the membrane skeleton extractability and on the osmotic fragility was postulated.

Topics: 4-Acetamido-4'-isothiocyanatostilbene-2,2'-disulfonic Acid; 4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid; Animals; Anions; Bicarbonates; Cattle; Chlorides; Erythrocytes; Hemolysis; In Vitro Techniques; Kinetics; Osmolar Concentration; Osmotic Fragility; Stilbenes

When pyridoxal 5'-phosphate (PLP) is covalently bound to band 3 protein in intact red blood cells and those cells are subjected to the osmotic hemolysis and resealing process, a significant reduction in the original PLP anion transport inhibitory potency occurs. We show that partial deinhibition is not due to the development of a second anion transport pathway in resealed ghosts. Rather, partial deinhibition arises from a hemolysis-induced conformational change in CH17 (17-kDa integral chymotryptic domain of band 3). This change causes the extracellular exposure of new transport inhibitory sites. Exposure of the new sites leads to a 2-fold increase in PLP labeling of CH17 in resealed ghosts compared with CH17 in intact red cells. The hemolysis and resealing process has no effect on the labeling of CH35 (35-kDa integral chymotryptic fragment of band 3). Double-labeling studies show restoration of transport inhibitory potency to near red cell levels when the newly exposed CH17 sites are labeled with PLP in resealed ghosts. The results support the view that CH17 contains PLP transport inhibitory sites. They show that a major conformational change occurs in band 3 with hemolysis.

Topics: 4-Acetamido-4'-isothiocyanatostilbene-2,2'-disulfonic Acid; 4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid; Anion Exchange Protein 1, Erythrocyte; Anions; Biological Transport; Dithionite; Erythrocyte Membrane; Erythrocytes; Hemolysis; Humans; Kinetics; Protein Conformation; Pyridoxal Phosphate

The aqueous leak induced in the human erythrocyte membrane by crosslinking of spectrin via disulfide bridges formed in the presence of diamide (Deuticke, B., Poser, B., Lütkemeier, P. and Haest, C.W.M. (1983) Biochim. Biophys. Acta 731, 196-210) was further characterized with respect to its ion selectivity by means of (a) measurements of cell volume changes or hemolysis, (b) determination of membrane potentials and (c) analysis of potential-driven ion fluxes. The leak turned out to be slightly cation-selective (PK:PCl approximately equal to 4:1). It discriminates mono- from divalent ions (PNa:PMg greater than 100:1, PCl:PSO4 greater than 10:1) and to a much lesser extent monovalent ions among each other. The selectivities for monovalent ions follow the sequence of free solution mobilities, increasing in the order Li+ less than or equal to Na+ less than K+ less than or equal to Rb+ less than Cs+ and F- less than Cl- less than Br- less than I-. Polyatomic anions also fit into that order. Quantitatively, the ratios of permeabilities of the leak are larger than those of the ion mobilities in free solution. The ion permeability of the leak is concentration-independent up to at least 150 mM. The ion milieu, however, has marked effects on leak permeability, most pronounced for chaotropic ions (guanidinium, nitrate, thiocyanate), which increase leak fluxes of charged and uncharged solutes. The results support the view that, besides geometric constraints, weak coulombic or dipolar interactions between penetrating ions and structural elements of the leak determine permselectivity.

Topics: 4-Acetamido-4'-isothiocyanatostilbene-2,2'-disulfonic Acid; 4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid; Cell Membrane Permeability; Erythrocyte Membrane; Hemolysis; Humans; Magnesium; Mannitol; Membrane Potentials; Membrane Proteins; Potassium Chloride; Sodium Chloride; Time Factors

The rate of volume changes of human red blood cells in the presence of Tris-HCl is pH-dependent. At 37 degrees C, t 1/2 is 25-30 min at pH 7.4 and 10-20 min at pH 8.4. Hemolysis in Tris-HCl is delayed by H2DIDS but is promoted by low concentrations of bicarbonate. This bicarbonate effect has been reversed by inhibiting carbonic anhydrase with acetazolamide. K+ loss of red blood cells is increased at 37 degrees C in isotonic NaCl solutions containing in addition Tris-HCl. This Tris effect is enhanced from pH 6.4 to 8.4. At pH 8.4 K+ loss is stimulated about 3-fold by addition of 160 mM Tris-HCl. The onset of the Tris effect is delayed at pH 7.4 and below, but not at pH 8.4. Such a delay is absent after preincubation of the cells with Tris-HCl. After binding H2DIDS to red cells, no Tris-dependent increase of K+ loss has been observed. K+ loss of reconstituted red cell ghosts with equal internal and external chloride concentrations remained unaffected by Tris-HCl added to the external solution. In ghosts containing sucrose for isotonicity instead of choline chloride K+ loss is smaller but is stimulated by Tris-HCl approaching the rate in those ghosts with equal internal and external chloride concentrations. The transfer of Tris-HCl into red blood cells depends on the pH and on the chloride shift. As there is evidence that Tris-HCl raises the intracellular pH and reduces the Donnan potential at the membrane, K+ loss of red cells may be increased following an intracellular buffer interaction of hemoglobin and Tris-HCl.

Topics: 4-Acetamido-4'-isothiocyanatostilbene-2,2'-disulfonic Acid; 4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid; Bicarbonates; Erythrocyte Membrane; Erythrocytes; Hemolysis; Humans; In Vitro Techniques; Osmolar Concentration; Potassium; Time Factors; Tromethamine

Hexavalent chromium (Cr(VI)) anion gradually penetrated into bovine erythrocytes and bound with cytoplasmic components. Its penetration was strongly inhibited by the NH2-reactive agent, 4-acetamido-4'-isothiocyano-stilbene-2,2'-disulfonic acid (SITS) and the SH-reactive agent, N-ethylmaleimide (NEM). Gel filtration showed that the intracellular component that bound to chromium was hemoglobin. The binding affinity of Cr(VI) to hemoglobin in the absence of glutathione in vitro was found to be much less than in intact erythrocytes. However, in the presence of glutathione, the binding affinity of Cr(VI) to hemoglobin became much higher. This indicates that reduction of hemoglobin or Cr(VI) by glutathione is involved in the binding. Cr(VI) interacted only weakly with the membrane and did not cause hemolysis of bovine erythrocytes, unlike heavy metals such as Hg2+.

Topics: 4-Acetamido-4'-isothiocyanatostilbene-2,2'-disulfonic Acid; Animals; Cattle; Chromates; Chromatography, Gel; Cytoplasm; Erythrocyte Membrane; Erythrocytes; Ethylmaleimide; Glutathione; Hemoglobins; Hemolysis; Ion Channels; Potassium Compounds