Transbilayer movement of phosphatidylserine in erythrocytes. Inhibitors of aminophospholipid transport block the association of photolabeled lipid to its transporter

The ability to cross-link [125I]iodo-azido-phosphatidylserine (125I-N3-PS) to the putative 32-kDa aminophospholipid transporter of human red blood cells (RBC) has been examined by SDS-PAGE. In the absence of transport inhibitors, 125I-N3-PS preferentially labeled the 32-kDa polypeptide, whereas treatment of the cells with pyridyldithioethylamine (PDA), a potent inhibitor of the aminophospholipid translocase, abrogated the association of the probe to this protein. ATP-depletion, low temperature, and diamide or 5,5'-dithiobis(2-nitrobenzoic acid), inhibitors that oxidize an endofacial sulfhydryl distinct from the PDA-sensitive site (Connor, J. and Schroit, A.J. (1990) Biochemistry 29, 37-43), also blocked association of the PS analogue to the protein. Once 125I-N3-PS became associated with the transporter, however, only PDA was able to partially displace it. These data suggest that sulfhydryl reactive reagents inhibit PS transport by blocking the association of PS with its transporter, a process that is also ATP- and temperature-dependent.     

Transbilayer movement of phosphatidylserine in erythrocytes: inhibition of transport and preferential labeling of a 31,000-dalton protein by sulfhydryl reactive reagents

A series of labeled thiolation reagents were synthesized on the basis of the parent structure pyridyldithioethylamine (PDA). These compounds specifically and reversibly inhibit the active intrabilayer transport of phosphatidylserine (PS) in human red blood cells. The binding of PDA to cells can be quantified since the thiol-disulfide exchange reaction yields a chromophore. In addition, the presence of a primary amine makes it amenable to derivatization with a variety of compounds. An iodinated derivative of PDA preferentially labeled a 31,000-dalton red blood cell peptide. The labeled component, which may represent the PS transporter, comigrated with integral membrane protein band 7.     

Association of stomatin (band 7.2b) with Glut1 glucose transporter

Employing a monoclonal antibody directed against the C-terminal peptide of glucose transporter molecule 1 (Glut1), we identified a approximately 30-kDa polypeptide which coimmunoprecipitated with Glut1 from sample of human red blood cells (RBC) membranes. The approximately 30-kDa polypeptide reacted with an antibody directed against stomatin, an integral plasma membrane protein which is also present at a high abundance in the human RBC plasma membrane. Likewise, employing anti-stomatin antibody, we found that Glut1 coimmunoprecipitated with stomatin from samples of RBC membranes. However, neither band 3, which is the most abundant integral membrane protein in the RBC, nor actin coimmunoprecipitated with Glut1, indicating a specific interaction between Glut1 and stomatin. Similar to the results obtained in the RBC, Glut1 and stomatin immunoprecipitated with each other in lysates of Clone 9 cells, a rat liver cell line in which Glut1 is expressed at approximately 1/200 the level present in RBC. Employing conditions that resulted in immunoprecipitation of approximately 10% of Glut1 in RBC membranes led to a approximately 3% coimmunoprecipitation of stomatin. A mixed population of Clone 9 cells stably transfected with a plasmid overexpressing the mouse stomatin exhibited 30 +/- 3% reduction in the basal rate of glucose transport compared to control cells or cells stably transfected with the empty vector. The above results suggest that stomatin is closely associated with Glut1 in the plasma membrane and that overexpression of stomatin results in a depression in the basal rate of glucose transport.     

Aminophospholipid translocation in erythrocytes: evidence for the involvement of a specific transporter and an endofacial protein

The transport of exogenously supplied fluorescent analogues of aminophospholipids from the outer to inner leaflet in red blood cells (RBC) is dependent upon the oxidative status of membrane sulfhydryls. Oxidation of a sulfhydryl on a 32-kDa membrane protein by pyridyldithioethylamine (PDA) has been previously shown [Connor & Schroit (1988) Biochemistry 27, 848-851] to inhibit the transport of NBD-labeled phosphatidylserine (NBD-PS). In the present study, other sulfhydryl oxidants were examined to determine whether additional sites are involved in the transport process. Our results show that diamide inhibits the transport of NBD-PS via a mechanism that is independent of the 32-kDa site. This is shown by the inability of diamide to block labeling of the 32-kDa sulfhydryl with 125I-labeled PDA and to protect against PDA-mediated inhibition of NBD-PS transport. diamide-mediated inhibition, but not PDA-mediated inhibition, could be reversed by reduction with cysteamine or endogenous glutathione. Similarly, treatment of RBC with 5,5'-dithiobis(2-nitrobenzoic acid), which depletes endogenous glutathione and induces oxidation of endofacial proteins [Reglinski et al. (1988) J. Biol. Chem. 263, 12360-12366], inhibited NBD-PS transport in a manner analogous to diamide. Once established, the asymmetric distribution of NBD-PS could not be altered by oxidation of either site. These data indicate that a second site critical to the transport of aminophospholipids resides on the endofacial surface and suggest that the transport of aminophospholipids across the bilayer membrane of RBC depends on a coordinated and complementary process between a cytoskeletal component and the 32-kDa membrane polypeptide; both must be operative for transport to proceed.     

LRIG1 protein in human cells and tissues

We have recently cloned the human LRIG1 gene (formerly LIG1). LRIG1 is a predicted integral cell-surface protein showing similarities to Kekkon-1, the Drosophila melanogaster epidermal growth-factor-receptor antagonist. A specific peptide antibody, LRIG1-151, was raised in rabbits and used to study the LRIG1 protein. LRIG1 migrated in denaturing polyacrylamide gel electrophoresis under reducing conditions as two species with apparent molecular weights of 143 kDa and 134 kDa, and as two fragments corresponding to an N-terminal 111-kDa species and a C-terminal 32-kDa species. Under non-reducing conditions, both apparent monomers and apparent higher molecular weight complexes were evident. Immunoblotting analysis of cell-surface-biotinylated lysates and confocal microscopy revealed that LRIG1 was localized to the cell surface in ZR-75 cells expressing endogenous LRIG1 and in COS-7 cells expressing a synthetic LRIG1-GFP fusion protein. Immunohistochemical analysis of normal human tissues showed staining for LRIG1 in epithelia in various organs, scattered neurons, and muscles. Immunoblotting demonstrated LRIG1 protein in tissue lysates from normal human prostate, mammary epithelial cells, ileum, stomach, lung, and cerebral cortex. These results demonstrate that LRIG1 is an integral cell-surface membrane protein that is expressed by specific cells in various human tissues and that its 143-kDa form might be cleaved into 111-kDa and 32-kDa fragments.     

The plasma membrane calcium pump: regulation by a soluble Ca2+ binding protein

The Ca2+ pump of the plasma membrane of human red blood cells is associated with the activity of a (Ca2+ + Mg2+)-ATPase. Both the ATPase and the pump are stimulated above basal activities by calmodulin, an ubiquitous Ca2+-binding protein. Calmodulin isolated from human red blood cells was shown to be equipotent and equieffective with that isolated from beef brain. Half-maximal activation of ATPase (isolated red blood cell membranes, 37 C) and transport (inside-out red blood cell membrane vesicles, 25 C) were obtained with 2.5 and 4.4 nM calmodulin, respectively. Ca2+ dependence of Ca2+ transport was measured in the absence and in the presence of 50 nM calmodulin. At all Ca2+ concentrations above 2 X 10(-7) M Ca2+, the rate of transport was greater in the presence of calmodulin. The results implicate calmodulin in the regulation of the plasma membrane Ca2+ pump, but the mechanism(s) remain to be elucidated.     

The red blood cell glucose transporter presents multiple, nucleotide-sensitive sugar exit sites.

At any instant, the human erythrocyte sugar transporter presents at least one sugar export site but multiple sugar import sites. The present study asks whether the transporter also presents more than one sugar exit site. We approached this question by analysis of binding of [3H]cytochalasin B (an export conformer ligand) to the human erythrocyte sugar transporter and by analysis of cytochalasin B modulation of human red blood cell sugar uptake. Phloretin-inhibitable cytochalasin B binding to human red blood cells, to human red blood cell integral membrane proteins, and to purified human red blood cell glucose transport protein (GluT1) displays positive cooperativity at very low cytochalasin B levels. Cooperativity between sites and K(d(app)) for cytochalasin B binding are reduced in the presence of intracellular ATP. Red cell sugar uptake at subsaturating sugar levels is inhibited by high concentrations of cytochalasin B but is stimulated by lower (<20 nM) concentrations. Increasing concentrations of the e1 ligand forskolin also first stimulate then inhibit sugar uptake. Cytochalasin D (a cytochalasin B analogue that does not interact with GluT1) is without effect on sugar transport over the same concentration range. Cytochalasin B and ATP binding are synergistic. ATP (but not AMP) enhances [3H]cytochalasin B photoincorporation into GluT1 while cytochalasin B (but not cytochalasin D) enhances [gamma-32P]azidoATP photoincorporation into GluT1. We propose that the red blood cell glucose transporter is a cooperative tetramer of GluT1 proteins in which each protein presents a translocation pathway that alternates between uptake (e2) and export (e1) states but where, at any instant, two subunits must present uptake (e2) and two subunits must present exit (e1) states.     

Alterations in pyridoxal 5'-phosphate inhibition of human erythrocyte anion transport associated with osmotic hemolysis and resealing

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.     

The 1A protein of respiratory syncytial virus is an integral membrane protein present as multiple, structurally distinct species.

The respiratory syncytial virus (RSV) 1A protein was previously identified as a 7.5-kilodalton (kDa) nonglycosylated species that, on the basis of its predicted sequence determined from the sequence of its mRNA, contains a hydrophobic central domain that was suggestive of membrane interaction. Here, four major, structurally distinct intracellular species of the 1A protein were identified in cells infected by RSV or by a recombinant vaccinia virus expressing the 1A gene. The four species of 1A were: (i) the previously described, nonglycosylated 7.5-kDa species that appeared to be the full-length, unmodified 1A protein; (ii) a nonglycosylated 4.8-kDa species that was carboxy-coterminal with the 7.5-kDa species and might be generated by translational initiation at the second AUG in the sequence; (iii) a 13- to 15-kDa species that contained one or two N-linked carbohydrate side chains of the high-mannose type; and (iv) a 21- to 30-kDa glycosylated species that appeared to be generated from the 13- to 15-kDa species by further modification of the N-linked carbohydrate. All four forms of the 1A protein were synthesized and processed on intracellular membranes, and several lines of biochemical evidence showed that all four species were integral membrane proteins. Thus, the 1A protein is a third RSV integral membrane protein and is present as such in both glycosylated and nonglycosylated forms. With the use of antiserum raised against a synthetic peptide representing the C terminus of the 1A protein, indirect immunofluorescence showed that the 1A protein was expressed at the cell surface. Antibody-antigen complexes formed at the surface of intact infected cells were immunoprecipitated, showing that the 7.5-kDa, 13- to 15-kDa, and 21- to 30-kDa, but not the 4.8-kDa, species, were accessible to extracellular antibodies. Thus, the 1A protein is a candidate to be a viral surface antigen. The small size, gene map location integral membrane association, and cell surface expression of the 1A protein strongly suggested that it is a counterpart to the SH protein that has been described for simian virus type 5. We suggest that, in the future, the RSV 1A protein be given the same designation, namely, SH.     

Presenilin 1 is required for maturation and cell surface accumulation of nicastrin

Proteolytic processing of amyloid precursor protein generates beta-amyloid (Abeta) peptides that are deposited in senile plaques in brains of aged individuals and patients with Alzheimer's disease. Presenilins (PS1 and PS2) facilitate the final step in Abeta production, the intramembranous gamma-secretase cleavage of amyloid precursor protein. Biochemical and pharmacological evidence support a catalytic or accessory role for PS1 in gamma-secretase cleavage, as well as a regulatory role in select membrane protein trafficking. In this report, we demonstrate that PS1 is required for maturation and cell surface accumulation of nicastrin, an integral component of the multimeric gamma-secretase complex. Using kinetic labeling studies we show that in PS1(-/-)/PS2(-/-) cells nicastrin fails to reach the medial Golgi compartment, and as a consequence, is incompletely glycosylated. Stable expression of human PS1 restores these deficiencies in PS1(-/-) fibroblasts. Moreover, membrane fractionation studies show co-localization of PS1 fragments with mature nicastrin. These results indicate a novel chaperone-type role for PS1 and PS2 in facilitating nicastrin maturation and transport in the early biosynthetic compartments. Our findings are consistent with PS1 influencing gamma-secretase processing at multiple steps, including maturation and intracellular trafficking of substrates and component(s) of the gamma-secretase complex.     

Purification of a human plasma membrane glycoprotein from human red blood cells by affinity chromatography using a monoclonal antibody

Using the monoclonal antibody LICR-LON-Fib75.1 coupled to Sepharose as an affinity chromatography column, a membrane glycoprotein with an apparent molecular weight of 18,000 on sodium dodecyl sulfate-polyacrylamide gels has been purified from human red blood cells. The purified protein contained 25% carbohydrate by weight, the predominant sugars being galactose, mannose, and glucosamine. Amino acid analysis indicated that the protein was relatively rich in aspartate, glutamate, valine, and leucine and had a low proline and methionine content. The molecule could be removed from intact red blood cells by trypsin and could be labeled with iodine by lactoperoxidase-catalyzed cell surface iodination of red blood cells. The protein could also be labeled using the lipidsoluble photoactivatable reagent 3-(trifluoromethyl)-3-(m-[125I]iodophenyl) diazirine) and partitioned into the lower phase of the phase-separable detergent Triton X-114. During size-exclusion chromatography in different detergents alterations were observed in the apparent molecular weight of the protein. These results suggest that this Fib75.1-binding protein is an external red blood cell membrane glycoprotein which is capable of binding detergent. Proteins with a similar molecular weight have also been isolated from two human tumor cell lines by immunoprecipitation with this monoclonal antibody.     

Assembly and targeting of peripheral and integral membrane subunits of the yeast vacuolar H(+)-ATPase.

Previous purification and characterization of the yeast vacuolar proton-translocating ATPase (H(+)-ATPase) have indicated that it is a multisubunit complex consisting of both integral and peripheral membrane subunits (Uchida, E., Ohsumi, Y., and Anraku, Y. (1985) J. Biol. Chem. 260, 1090-1095; Kane, P. M., Yamashiro, C. T., and Stevens, T. H. (1989) J. Biol. Chem. 264, 19236-19244). We have obtained monoclonal antibodies recognizing the 42- and 100-kDa polypeptides that were co-purified with vacuolar ATPase activity. Using these antibodies we provide further evidence that the 42-kDa polypeptide, a peripheral membrane protein, and the 100-kDa polypeptide, an integral membrane protein, are genuine subunits of the yeast vacuolar H(+)-ATPase. The synthesis, assembly, and targeting of three of the peripheral subunits (the 69-, 60-, and 42-kDa subunits) and two of the integral membrane subunits (the 100- and 17-kDa subunits) were examined in mutant yeast cells containing chromosomal deletions in the TFP1, VAT2, or VMA3 genes, which encode the 69-, 60-, and 17-kDa subunits, respectively. The steady-state levels of the various subunits in whole cell lysates and purified vacuolar membranes were assessed by Western blotting, and the intracellular localization of the 60- and 100-kDa subunits was also examined by immunofluorescence microscopy. The results suggest that the assembly and/or the vacuolar targeting of the peripheral subunits of the yeast vacuolar H(+)-ATPase depend on the presence of all three of the 69-, 60-, and 17-kDa subunits. The 100-kDa subunit can be transported to the vacuole independently of the peripheral membrane subunits as long as the 17-kDa subunit is present; but in the absence of the 17-kDa subunit, the 100-kDa subunit appears to be both unstable and incompetent for transport to the vacuole.     

A putative receptor for Venezuelan equine encephalitis virus from mosquito cells.

We have identified a cellular protein from a continuous mosquito cell line (C6/36) that appears to play a significant role in the attachment of Venezuelan equine encephalitis (VEE) virus to these cells. VEE virus bound to a 32-kDa polypeptide present in the C6/36 plasma membrane fraction, and binding to this polypeptide was dose dependent and saturable and competed with homologous and heterologous alphaviruses. These observations suggest that this polypeptide binds virus via a receptor-ligand interaction. The 32-kDa polypeptide was expressed on the surfaces of C6/36 cells, and monoclonal antibodies directed against either this cell polypeptide or the VEE virus E2 glycoprotein, which is thought to be the viral attachment protein, interfered with virus attachment. Collectively, these data provide evidence suggesting that the 32-kDa polypeptide serves as a receptor for VEE virus infection of cells. We have characterized this cell polypeptide as a laminin-binding protein on the basis of its ability to interact directly with laminin as well as its immunologic cross-reactivity with the high-affinity human laminin receptor.     

Delta endotoxin is a potent inhibitor of the (Na,K)-ATPase

A 68-kDa protein, delta endotoxin, produced by Bacillus thuringiensis ssp. Kurstaki inhibits ion transport, (Na,K)-ATPase, and K+-p-nitrophenylphosphatase activity catalyzed by the Na+ pump. The Ki for inhibition of the K+-p-nitrophenylphosphatase activity of purified dog kidney (Na,K)-ATPase was approximately 0.37 microM. Delta endotoxin had a similar Ki for inhibition of (Na,K)-ATPase activity when assayed at low Na+ concentration (10 mM) but the inhibition was reversed when high concentrations of Na+ (100 mM NaCl) were added to the assay. Phosphorylation of the active site aspartyl residue with 32PO3-4 was also blocked by delta endotoxin. Ouabain-sensitive 86Rb+ uptake into intact human red blood cells was not inhibited by externally added toxin; however, strophanthidin-inhibitable 22Na+ uptake into inside-out vesicles from red blood cells was completely blocked by delta endotoxin (Ki = 0.73 microM). These data suggest that delta endotoxin must enter the cell before it can inhibit the Na+ pump.     

Bidirectional transbilayer movement of phospholipid analogs in human red blood cells. Evidence for an ATP-dependent and protein-mediated process.

The transbilayer movement of fluorescent and isotopically labeled analogs of phosphatidylserine (PS), phosphatidylethanolamine (PE), and phosphatidylcholine (PC) from the outer to the inner leaflet (flip) and from the inner to the outer leaflet (flop) of human red blood cells (RBC) was examined. The inward movement of 1-oleoyl-2-(N-4-nitrobenzo-2-oxa-1,3-diazole-aminocaproyl)- (C6-NBD-), 1-oleoyl-2-(N-(3-(3-[125I]iodo-4-hydroxyphenyl)propionyl)aminocaproyl)- (C6-125I-), or 1-oleoyl-2-(N-(3-3-[125I]iodo-4-azido-phenyl)propionyl)aminocaproyl- (C6-125I-N3-) analogs of PC and PE were relatively slow. In contrast, all analogs of PS and PE analogs containing aminododecanoic acid (C12 lipids) were rapidly transported to the cell's inner leaflet. Analysis of 125I-N3 lipids cross-linked to membrane proteins revealed labeling of 32-kDa Rh polypeptides that was dependent on the lipid's capacity to be transported to the inner leaflet but was independent of lipid species. To investigate whether lipids could also be transported from the inner to the outer leaflet, lipid probes residing exclusively in the inner leaflet were monitored for their appearance in the outer leaflet. Lipid movement could not be detected at 0 degrees C. At 37 degrees C, however, approximately 70% of the PC, 40% of the PE, and 15% of the PS redistributed to the cells outer leaflet, thereby attaining their normal asymmetric distribution. Continuous incubation in the presence of bovine serum albumin depleted the cells of the analogs (t1/2 approximately 1.5 h) in a manner that was independent of lipid species. Similar to the inward movement of aminophospholipids, the outward movement of PC, PE, and PS was ATP-dependent and could be blocked by oxidation of membrane sulfhydryls and by the histidine reagent bromophenacyl bromide. Evidence is presented which suggests that the outward movement of lipids is an intrinsic property of the cells unrelated to compensatory mechanisms due to an imbalance in lipid distribution.     

Is an intact cytoskeleton required for red cell urea and water transport?

In order to determine the membrane protein(s) responsible for urea and water transport across the human red cell membrane, we planned to reconstitute purified membrane proteins into phosphatidylcholine vesicles. In preparatory experiments, we reconstituted a mixture of all of the red cell integral membrane proteins into phosphatidylcholine vesicles, but found that p-chloromercuribenzenesulfonate (pCMBS), which normally inhibits osmotic water permeability by approximately 90%, has no effect on this preparation. The preparation was also unable to transport urea at the high rates found in red cells, though glucose transport was normal. White ghosts, washed free of hemoglobin and resealed, also did not preserve normal urea and pCMBS-inhibitable water transport. One-step ghosts, prepared in Hepes buffer in a single-step procedure, without washing, retained normal urea and pCMBS-inhibitable water transport. Perturbations of the cytoskeleton in one-step ghosts, by removal of tropomyosin, or by severing the ankyrin link which binds band 3 to spectrin, caused the loss of urea and pCMBS-inhibitable water transport. These experiments suggest that an unperturbed cytoskeleton may be required for normal urea and pCMBS-inhibitable water transport. They also show that the pCMBS inhibition of water transport is dissociable from the water transport process and suggest a linkage between the pCMBS water transport inhibition site and the urea transport protein.     

Review. Leaky Cl--HCO3- exchangers: cation fluxes via modified AE1

The abundant membrane protein AE1 normally functions as an obligate anion exchanger, with classical carrier properties, in human red blood cells. Recently, four single point mutations of hAE1 have been identified that have lost the anion exchange function, and act as non-selective monovalent cation channels, as shown in both red cell flux and oocyte expression studies. The red cell transport function shows a paradoxical temperature dependence, and is associated with spherocytic and stomatocytic red cell defects, and haemolytic anaemias. Other forms of AE1, including the native AE1 in trout red cells, and the human mutation R760Q show both channel-like and anion exchange properties. The present results point to membrane domains 9 and 10 being important in the functional modification of AE1 activity.     

Mutant presenilin (A260V) affects Rab8 in PC12D cell

Most familial early-onset Alzheimer's disease (FAD) is caused by mutations in the presenilin-1 (PS1) gene. Abeta is derived from amyloid precursor protein (APP) and an increased concentration of Abeta 42 is widely believed to be a pathological hallmark of abnormal PS function. Therefore, the interaction between PS1 and APP is a central theme in attempts to clarify the molecular mechanism of AD. To examine the effect of PS1 mutations on APP metabolism, we made PC12D cell lines that express human PS1 or mutant PS1 (A260V). In PC12D cells expressing the PS1A260V mutant, we found that Rab8, a GTPase involved in transport from the trans-Golgi network (TGN) to the plasma membrane (PM), was significantly reduced in PC12D cells expressing the A260V mutant and that APP C-terminal fragment (CTF), the direct precursor of Abeta, accumulated in the heavy membrane fraction including membrane vesicles involved in TGN-to-PM transport. Furthermore, the total intracellular Abeta production was reduced in these cells. Combined together, we have observed that PS1 mutation disturbs membrane vesicle transport, resulting in prolonged residence of APP CTF during TGN-to-PM transport pathway. Therefore, it is highly likely that reduction of Abeta is closely related to the retention of APP CTF during TGN-to-PM transport.     

Involvement of Rh blood group polypeptides in the maintenance of aminophospholipid asymmetry

The human erythrocyte (RBC) Rh blood group system consists of a complex of distinct integral membrane polypeptides with physical properties common to the aminophospholipid transporter responsible for the transbilayer movement of phosphatidylserine (PS) in RBC. To assess the involvement of Rh polypeptides in PS translocation, the aminophospholipid translocase was labeled with a photoactivatable PS analogue, 125I-azido-PS, and with an inhibitor of PS transport, 125I-labeled 2-(2-pyridyldithio)ethylamine. The ability of monoclonal Rh antibodies to immunoprecipitate the labeled transporter was determined. Immunoprecipitated Rh polypeptides were found to be labeled with the aminophospholipid translocase markers, suggesting that Rh proteins are involved in the transbilayer movement of PS.     

Kinetic independence between red cell anion exchange and urea transport

Urea equilibrium exchange fluxes were measured in human red cells under conditions which recruit the anion transporter into an outward-facing or an inward-facing state (with respect to the anion transport site). Regardless of these conditions, urea transport always occurred at the same rate: 41 +/- 2 mol.(kg cell solids.min)-1 with 1.5 M urea at 0 degrees C. These data suggest that the pathway on the band-3 protein which mediates anion transport is kinetically uncoupled from urea transport and is probably not involved in the transport of urea across the red cell membrane.