Analysis of integral membrane protein contributions to the deformability and stability of the human erythrocyte membrane.

Three major hypotheses have been proposed to explain the role of membrane-spanning proteins in establishing/maintaining membrane stability. These hypotheses ascribe the essential contribution of integral membrane proteins to (i) their ability to anchor the membrane skeleton to the lipid bilayer, (ii) their capacity to bind and stabilize membrane lipids, and (iii) their ability to influence and regulate local membrane curvature. In an effort to test these hypotheses in greater detail, we have modified both the membrane skeletal and lipid binding interactions of band 3 (the major membrane-spanning and skeletal binding protein of the human erythrocyte membrane) and have examined the impact of these modifications on erythrocyte membrane morphology, deformability, and stability. The desired changes in membrane skeletal and protein-lipid interactions were induced by 1) reaction of the cells with 4,4'-diisothiocyanostilbene-2,2'-disulfonate (DIDS), an inhibitor of band 3-mediated anion transport that dissociates band 3 into dimers (increasing its surface area in contact with lipid) and severs band 3 linkages to the membrane skeleton; 2) a fragment of ankyrin that ruptures the same ankyrin-band 3 bridge to the membrane skeleton, but drives the band 3 subunit equilibrium toward the tetramer (i.e. decreasing the band 3 surface area in contact with lipid); and 3) an antibody to the ankyrin-binding site on band 3 that promotes the same changes in band 3 skeletal and lipid interactions as the ankyrin fragment. We observed that although DIDS induced echinocytic morphological changes in the treated erythrocytes, it had little impact on either membrane deformability or stability. In contrast, resealing of either the ankyrin fragment or anti-band 3 IgG into erythrocytes caused spontaneous membrane fragmentation and loss of deformability/stability. Because these and other new observations cannot all be reconciled with any single hypothesis on membrane stability, we suggest that more than one hypothesis may be operative and provide an explanation of how each might individually contribute to net membrane stability.

South-east asian ovalocytic (SAO) erythrocytes have a cold sensitive cation leak: implications for in vitro studies on stored SAO red cells.

South-east Asian ovalocytosis (SAO) results from the heterozygous presence of an abnormal band 3, which causes several alterations in the properties of the erythrocytes. Although earlier studies suggested that SAO erythrocytes are refractory to invasion in vitro by the malarial parasite Plasmodium falciparum, a more recent study showed that fresh SAO cells were invaded by the parasites, but became resistant to invasion on storage because intracellular ATP was depleted more rapidly than normal. Here we show that SAO red cells are much more leaky to sodium and potassium than normal red cells when stored in the cold. This leak was much less marked when the cells were stored at 25 or 37 degreesC. Incubation for 3.5 h at 37 degreesC of cold-stored SAO red cells did not restore sodium and potassium to normal levels, probably because the depleted ATP level in cold-stored SAO red cells is further reduced with incubation at 37 degreesC. The increased leakiness of SAO red cells is non-specific and extends to calcium ions, taurine, mannitol and sucrose. These results suggest that SAO red cells undergo a structural change on cooling. Since many of the reports describing altered properties of SAO red cells have used cells which have been stored in the cold, these results need re-evaluation using never-chilled SAO red cells to assess whether the cells have the same abnormal properties under in vivo conditions.

Effect of band 3 subunit equilibrium on the kinetics and affinity of ankyrin binding to erythrocyte membrane vesicles.

The membrane-spanning protein, band 3, anchors the spectrin-based membrane skeleton to the lipid bilayer via the bridging protein, ankyrin. To understand how band 3 subunit stoichiometry influences this membrane-skeletal junction, we have induced changes in the band 3 association equilibrium and assayed the kinetics and equilibrium properties of ankyrin binding. We observe that band 3 oligomers convert slowly to dimers and ultimately monomers following removal of ankyrin. Addition of excess ankyrin back to these membranes enriched in dissociated band 3 then shifts band 3 almost entirely to tetramers, confirming that the tetrameric form of band 3 constitutes the preferred oligomeric state of ankyrin binding. 4, 4'-Diisothiocyanostilbene-2,2'-disulfonic acid (DIDS) labeling of band 3, which is shown to shift most of the band 3 population to dimers, eliminates the majority of ankyrin-binding sites on the membrane and greatly reduces retention of band 3 in detergent-extracted membrane skeletons. Furthermore, DIDS- modified membranes lack all low affinity ankyrin-binding sites and roughly half of all high affinity sites. Since labeled membranes lack the rapid kinetic phase of ankyrin binding and exhibit only half of the normal amplitude of the slow kinetic phase, it can be concluded that the rapid phase of ankyrin association involves low affinity sites and the slow phase involves high affinity sites. A model accounting for these data and most previous data on ankyrin-band 3 interactions is provided.

Complete reductive dehalogenation of brominated biphenyls by anaerobic microorganisms in sediment.

We sought to determine whether microorganisms from the polychlorinated biphenyl (PCB)-contaminated sediment in Woods Pond (Lenox, Mass.) could dehalogenate brominated biphenyls. The PCB dechlorination specificities for the microorganisms in this sediment have been well characterized. This allowed us to compare the dehalogenation specificities for brominated biphenyls and chlorinated biphenyls within a single sediment. Anaerobic sediment microcosms were incubated separately at 25 degrees C with 16 different mono- to tetrabrominated biphenyls (350 muM) and disodium malate (10 mM). Samples were extracted and analyzed by gas chromatography with an electron capture detector and a mass spectrometer detector at various times for up to 54 weeks. All of the tested brominated biphenyls were dehalogenated. For most congeners, including 2,6-dibromobiphenyl (26-BB) and 24-25-BB, the dehalogenation began within 1 to 2 weeks. However, for 246-BB and 2-2-BB, debromination was first observed at 7 and 14 weeks, respectively. Most intermediate products did not persist, but when 2-2-BB was produced as a dehalogenation product, it persisted for at least 15 weeks before it was dehalogenated to 2-BB and then to biphenyl. The dehalogenation specificities for brominated and chlorinated biphenyls were similar: meta and para substituents were generally removed first, and ortho substituents were more recalcitrant. However, the brominated biphenyls were better dehalogenation substrates than the chlorinated biphenyls. All of the tested bromobiphenyls, including those with ortho and unflanked meta and para substituents, were ultimately dehalogenated to biphenyl, whereas their chlorinated counterparts either were not dehalogenation substrates or were only partially dehalogenated. Our data suggest that PCB-dechlorinating microorganisms may be able to dehalogenate brominated biphenyls and may exhibit a relaxed specificity for these substrates.

Calorimetric evidence for allosteric subunit interactions associated with inhibitor binding to band 3 transporter.

A calorimetric endotherm occurring at 68 degrees C (the C-transition) has been assigned previously to the integral domain of band 3 and was shown to be shifted to 78 degrees C after covalent binding of 4,4'-diisothiocyanostilbene-2,2'-disulfonate (DIDS). In this study, we correlate the fractional appearance of the shifted C-transition with the fraction of DIDS bound to the band 3 monomer population. Our results show a distinctly nonlinear correlation plot with the appearance of the shifted C-transition lagging behind DIDS labeling of the band 3 monomer population. The lag suggests that both monomers of a band 3 dimer must be labeled by DIDS in order for the shifted C-transition to appear at 78 degrees C, implying that the thermal unfolding of the integral domain of band 3 is modulated by allosteric interactions between subunits. This is the first in situ structural evidence supporting ligand-mediated subunit interactions within a "carrier"-type transporter protein oligomer.

Structural and functional characterization of band 3 from Southeast Asian ovalocytes.

To determine why deletion of the nine amino acids joining the membrane and cytoplasmic domains of band 3 from Southeast Asian ovalocytes (SAO) renders the erythrocytes rigid, we compared the structural and functional properties of SAO and normal band 3. Calorimetric data, inhibitor binding studies, and anion transport assays all reveal that the membrane-spanning domain of SAO band 3 is denatured, while proteolysis studies and circular dichroism spectroscopy suggest the mutant domain retains much secondary structure. It is concluded that the transmembrane helices of SAO band 3 are dissociated and randomized but not unfolded. The cytoplasmic domain of SAO band 3 was shown to be structurally and functionally normal based on (i) calorimetric properties, (ii) native conformational change, (iii) ability to form an intersubunit disulfide bond, (iv) affinity and capacity for binding ankyrin and protein 4.1, and (v) kinetics of association with ankyrin. However, both normal and mutant isoforms of band 3 in SAO cells were found to adhere nonspecifically to the spectrin skeleton. Further, when SAO cells were osmotically swollen, the detergent extractability of band 3 became normal. We propose that much of band 3 is nonspecifically entrapped in the spectrin network in SAO cells and that this nonspecific adhesion may be responsible for the rigidity of the SAO erythrocyte.

Reductive ortho and meta Dechlorination of a Polychlorinated Biphenyl Congener by Anaerobic Microorganisms.

We used gas chromatography-mass spectrometry to study the metabolic fate of 2,3,5,6-tetrachlorobiphenyl (2356-CB) (350 muM) incubated with unacclimated methanogenic pond sediment. The 2356-CB was dechlorinated to 25-CB (21%), 26-CB (63%), and 236-CB (16%) in 37 weeks. This is the first experimental demonstration of ortho dechlorination of a polychlorinated biphenyl by anaerobic microorganisms.