E A Nigg

Labeling of human erythrocyte membranes with eosin probes used for protein diffusion measurements : inhibition of anion transport and photo-oxidative inactivation of acetylcholinesterase

The binding of eosin-isothiocyanate (eosin-NCS) and iodoacetamido-eosin (IA-eosin) to band 3 proteins in the membrane of human erythrocytes is characterized by studying the effect of these probes on the anion transport system. Although the unbrominated fluorescein precursors do not affect anion transport, both eosin labels are strong inhibitors of sulphate exchange in intact erythrocytes. 50% inhibition is obtained by binding 4.7 . 10(5) or 6.0 . 10(5) molecules/cell for eosin-NCS and IA-eosin, respectively. Both eosin probes are irreversibly bound and occupy common binding sites with 4,4-diisothiocyano-1,2-diphenyl-ethane-2,2disulfonic acid (H2DIDS), although other sites are labeled as well. The inhibition of anion transport is light independent and can therefore not be attributed to a photosensitizing action of the eosin probes. Both eosin derivatives, however, inactivate acetylcholinesterase upon illumination of air-equilibrated samples of hemoglobin-free labeled ghosts. The inactivation of the enzyme is accompanied by the formation of protein aggregates as visualized by polyacrylamide gel electrophoresis. These effects are not observed when intact erythrocytes are illuminated in the presence of eosin probes suggesting a protective effect of hemoglobin during the labeling procedure. Protection of ghosts from photo-oxidation is achieved by displacing air with argon. These results are discussed in relation to the use of these and similar probes to measure protein diffusion in membranes.

Cytoskeletal organization, vinculin-phosphorylation, and fibronectin expression in transformed fibroblasts with different cell morphologies

Neoplastic transformation of fibroblasts results in widely different cell morphologies. We have attempted to correlate cell morphology with cytoskeletal organization and fibronectin expression in murine and avian fibroblasts transformed by a diverse group of viral and chemical agents. The distribution of vinculin, alpha-actinin, actin, and surface fibronectin was studied, and, where appropriate, also the extent of phosphotyrosine modification of vinculin. Irrespective of the transforming agent we found that increased cell rounding was generally correlated with a reduction in vinculin-containing focal adhesions, a dissolution of microfilament bundles, and a reduction of extracellular fibronectin. In contrast, spindle-shaped fibroblasts expressed relatively high levels of surface fibronectin. Reorganization of vinculin, actin, and alpha-actinin into rosette-like structures was observed in polygonal or rounded cells transformed by viruses encoding tyrosine kinases, but was not seen in fibroblasts transformed by agents without associated tyrosine kinase activity or in spindle-shaped cells. No correlation was found between the extent of phosphotyrosine modification of vinculin and the extent of cell rounding. Irrespective of cell morphology, the extent of tyrosine phosphorylation of vinculin was high in all cells transformed by viruses carrying the src gene, but low in those transformed by viruses expressing the fps gene. Our results indicate that the morphology of a transformed cell is determined by a combination of several factors which are affected to different extents by different transforming agents.

Rotational diffusion of band 3 proteins in membranes from En(a—) and neuraminidase-treated normal human erythrocytes

Recent experiments have demonstrated an association between band 3 and glycophorin A in the human eythrocyte membrane (Nigg, E.A., Bron, C., Girardet, M. and Cherry, R.J. (1980) Biochemistry 19, 1887-1893). Here, the influence of sialoglycoproteins on the rotational diffusion of band 3 in the human erythrocyte membrane was investigated by studying membranes from En(a-) and neuraminidase-treated erythrocytres. Rotational diffusion was measured by observing flash-induced transient dichroism of eosin-labeled band 3. Although erythrocytes of the rare phenotype En(a-) lack the major sialoglycoprotein, glycophorin A, no significant difference in band 3 rotation at pH 7.4 and 37 degrees C could be detected between En(a-) and normal erythrocyte membranes. Band 3 rotation at pH 7.4 was also insensitive to the enzymatic removal of sialic acid from the surface of normal erythrocytes. Moreover, the existence of an essentially similar temperature-dependent equilibrium between band 3 proteins with different mobilities was observed in normal, En(a-) and neuraminidase-treated erythrocytes. From these results it is concluded that glycophorin A contributes less than 15% to the cross-sectional diameter of the band 3-glycophorin A complex in the plane of the normal membrane. The rotation of the complex at pH 7.4 is not significantly influenced by either steric or electrostatic interactions involving the oligosaccharide moiety of glycophorin A.

Influence of influenza and Sendai virus on the rotational mobility of Band 3 proteins in human erythrocyte membranes.

Abstract The effect of influenza and Sendai viruses on the rotational mobility of band 3 proteins in the human erythrocyte membrane was measured using the technique of flash photolysis. Both viruses produced a marked reduction of band 3 rotation. In the case of Sendai virus, loss of mobility correlated with aggregation of intramembrane particles seen in freeze-fracture electron microscopy following fusion of viral envelopes with the plasma membrane. No such aggregation is seen with influenza virus and it is concluded that immobilization is associated with cross-linking of the virus receptor, glycophorin A, by the adsorbed virus particle. This result is consistent with the existence of a structural association between glycophorin A and band 3.