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Na2SeO3 could affect the anion flux of Band 3 of inside-out erythrocyte membrane vesicles (IOVs). Such effect was believed to be based on the interaction of SH groups of Band 3 with Na2SeO3. This effect could be eliminated when the cytoplasmic domain of Band 3 was proteolytically removed by trypsin. This suggested that SH groups in the cytoplasmic domain were involved in such interaction. Measurement of the pH dependence of intrinsic fluorescence intensity provided evidence that conformational changes of Band 3 occurred as a consequence of interaction with selenite. KI quenching of intrinsic fluorescence of Band 3 could also show that there was a conformational change in the cytoplasmic domain of Band 3 after reaction with Na2SeO3. Such conformational change in turn could be transmitted to the membrane domain of Band 3 monitored by quenching of intrinsic fluorescence of Band 3 using hypocrellin B (HB) (a photosensitive pigment obtained from a parasitic fungus growing in Yunnan, China). It is suggested that the cytoplasmic domain of Band 3 is not necessary for its anion flux, but is essential for the regulation (e.g., by Se) of its active site located at the membrane domain, and hence, it may provide evidence of communication between the cytoplasmic domain and the membrane domain of Band 3.

The role of a transmembrane Ca2+ gradient in anion transport by Band 3 of human resealed erythrocyte ghosts has been studied. The results show that a transmembrane Ca2+ gradient is essential for the conformation of erythrocyte Band 3 with higher anion transport activity. The dissipation of the transmembrane Ca2+ gradient by the ionophore A23187 inhibits the anion transport activity. The extent of this inhibition approaches 90% as the Ca2+ concentration on both sides of the ghost membrane is increased to 1.0 mM and half-maximum inhibition is observed at 0.25 mM Ca2+. Addition of ATP (0.4 mM) to the resealing medium can partly reestablish the transmembrane Ca2+ gradient by activation of Ca2+-ATPase and alleviate the inhibition to some extent. N-ethylmaleimide, an inhibitor of erythrocyte Ca2+-ATPase, prevents such restoration. Electron micrographs reveal that numerous larger intramembranous particles can be observed on the P-faces of freeze-fractured resealed ghosts in the absence of a transmembrane Ca2+ gradient.

Zn2+ could inhibit the anion transport activity of spectrin-stripped inside-out human erythrocyte membrane vesicles (IOVs). Removal of the cytoplasmic domain from Band 3 by trypsin could eliminate Zn2+ inhibition. The location of a Zn(2+)-binding site was confirmed by atomic absorbance spectrometry. The results of time-resolved fluorescence and intrinsic fluorescence quenching by KI and hypocrellin B (a photosensitive pigment obtained from a parasitic fungus growing in Yunnan, China) showed that the cytoplasmic domain is necessary for the Zn(2+)-induced conformational changes of the whole molecule as well as the membrane domain of Band 3. It is suggested that Zn2+ induced a conformational change in the cytoplasmic domain of Band 3, which in turn was transmitted to the membrane domain, resulting in an inhibition of activity of Band 3. Such long-range conformational changes may imply that the cytoplasmic domain is poised to function as a cytosolic arm in order to modulate the structure of the membrane domain of Band 3.

The transport activity of Band 3 of spectrin-stripped inside-out erythrocyte membrane vesicles (IOVs) or resealed ghosts was enhanced in the presence of trace amounts of Na2SeO3 (0.2-0.5 p.p.m.); however, at higher concentrations of Na2SeO3 (4.0 p.p.m.), an inverse result was obtained. Reassociation of spectrin with IOVs has no effect either on the transport activity of Band 3 or on the enhancement of its activity by Na2SeO3. Sulfhydryl reagents (p-chloromercuribenzoic acid and N-ethylmaleimide) could also inhibit Band 3 activity and eliminate the selenium effect. It is suggested that SH groups are involved in anion transport of Band 3 and that the selenium effect is based on the interaction of SH groups of Band 3 with Na2SeO3.

It has been shown that Se can markedly prevent the dissociation of spectrin from erythrocyte ghosts. We now report that the transformation of incubated spectrin oligomers to its tetramers and dimers was obviously decreased in the presence of trace amounts (0.5-2.0 p.p.m.) of Na2SeO3. The spectrin tetramers and dimers are in a reversible equilibrium and Se could alter this equilibrium in favour of tetramers. This Se effect is concentration dependent and an inverse result was obtained with higher Na2SeO3 concentrations (greater than 4.0 p.p.m.). We suggest that the equilibrium state of the spectrin tetramer-dimer may be governed by a conformation adjustment induced by Se and the difference in conformation in the presence of low and high Na2SeO3 concentration may lead to an alteration of the spectrin tetramer-dimer balance.

Na2SeO3 supplementation in the dialysis medium could obviously prevent the dissociation of spectrin from the erythrocyte membranes. Such Se effect could be eliminated by pretreatment of erythrocyte membranes with a SH-blocking reagent, iodoacetamide(IAA) or addition of a SH-compound, dithio-threitol. The fluorescence intensity of erythrocyte membranes labelled with the fluorescent probe N-(3-pyrenyl)-maleimide decreased with increasing Na2SeO3 concentration used for pretreatment of ghosts. 31P-NMR spectra of erythrocyte membrane dialyzed in the presence or absence of Na2SeO3 concentration showed a difference in chemical shift anisotropy (delta sigma) between these two samples. These data suggest that the stabilization effect is based on changes in lipid-protein interaction and conformation of membrane skeletal components induced by reaction of their SH groups with Na2SeO3.

Na2SeO3 supplementation in the ageing medium could protect aged erythrocyte ghosts from decreases in lipid fluidity, Na, K-ATPase activity, and sensitivity to ouabain. Results also showed that Se could obviously prevent the dissociation of spectrin from the erythrocyte membrane. Furthermore, Se could markedly promote the reassociation of spectrin with the spectrin-stripped inside out membrane vesicles(IOVs) of erythrocytes. The protective action of Se on biomembranes is generally interpreted in terms of the activity of Se-containing glutathione peroxidase (GSHPx). However, since GSHPx is mainly distributed in the cytoplasm of erythrocytes, the stabilizing effect of Se on erythrocyte membranes might not be related to the activity of this enzyme.

The content of binding-Se in erythrocyte membrane may affect its structure and function. Decrease in Se content will lead to a lower activity of Na, K-ATPase and lipid fluidity in ghosts, and a marked increase in Na, K-ATPase activity as well as lipid fluidity of erythrocyte membrane was observed by supplementation of low concentration of Na2SeO3 (0.03-0.3 ppm Se), in vitro. This effect was obvious only when the ghosts were incubated with Se at 0-4 degrees C for at least half an hour prior to assay.

During the ageing of erythrocyte membrane, the spectrin content, Na,K-ATPase activity as well as the lipid fluidity are obviously decreased. However, supplementation of a trace amount of Na2SeO3 in the medium could prevent the dissociation of spectrin from membrane and delay the changes of Na,K-ATPase activity and lipid fluidity. The effectiveness is proportional to Se concentration within the range of 0.1-1.0 ppm. Similar effect of supplementation of Se on the intact erythrocytes during ageing has been also observed. The protective action of Se on biomembranes is generally interpreted in terms of the activity of Se-containing glutathione peroxidase (GSHPx). However, GSHPx mainly distributes in the cytoplasma of erythrocytes, therefore it seems that the protective action of supplemented Se on the isolated erythrocyte membrane might not be related to the activity of GSHPx.