Major Intrinsic Membrane Protein Research Articles

The N-terminal 11 amino acids of human erythrocyte band 3 are critical for aldolase binding and protein phosphorylation: implications for band 3 function

The 911 amino acid band 3 (SLC4A1) is the major intrinsic membrane protein of red cells and is the principal Cl-/HCO3- exchanger. The N-terminal cytoplasmic domain of band 3 anchors the spectrin-based membrane skeleton to the lipid bilayer through its interaction with ankyrin and also binds glycolytic enzymes and hemoglobin. We identified a son of a consanguineous marriage with severe anemia in association with marked deficiency of band 3 (12% +/- 4% of normal). Direct nucleotide sequencing of SLC4A1 gene demonstrated a single base substitution (T --> C) at position + 2 in the donor splice site of intron 2, resulting in the generation of a novel mutant protein. Biochemical characterization of the mutant protein showed that it lacked the first 11 N-terminal amino acids (band 3 Neapolis). The expression of the mutant protein resulted in the complete absence of membrane-bound aldolase, and the mutant band 3 could not be tyrosine phosphorylated. The ability of the malarial parasite P falciparum to invade these red cells was significantly decreased. The identification of a novel band 3 mutant and its structural and functional characterization enabled us to identify pivotal roles for the 11 N-terminal amino acids in several protein functions and, in turn, in red-cell physiology.

Characterization of upstream sequences of the LIM2 gene that bind developmentally regulated and lens-specific proteins*

Abstract During lens development, lens epithelial cells differentiate into fiber cells. To date, four major lens fiber cell intrinsic membrane proteins (MIP) ranging in size from 70 kD to 19 kD have been characterized. The second most abundant lens fiber cell intrinsic membrane protein is MP19. This protein probably is involved with lens cell communication and relates with cataractogenesis. The aim of this research is to characterize upstream sequences of the MP19 (also called LIM2) gene that bind developmentally regulated and lens-specific proteins. We have used the gel mobility assays and corresponding competition experiments to identify and characterize cis elements within approximately 500 bases of LIM2 upstream sequences. Our studies locate the positions of some cis elements, including a “CA” repeat, a methylation Hha I island, an FnuD II site, an Ap1 and an Ap2 consensus sequences, and identify some specific cis elements which relate to lens-specific transcription of LIM2. Our experiments also preli...

New Insights into the Function of N-Terminal 11 Amino Acids of Band 3 from Structural and Functional Study of a Naturally Occuring Band 3 Variant.

The 911 amino acid human erythroid AE1 (eAE1) Cl-/HCO3- exchanger SLC4A1 (usually called band 3) is the major intrinsic membrane protein of red cells. The N-terminal cytoplasmic domain of AE1 represents the anchoring site for membrane-associated proteins such as ankyrin, protein 4.2, protein 4.1, glycolytic enzymes (including aldolase and glyceraldeyde-3-phosphate dehydrogenase (GAPDH) and hemoglobin. We identified marked band 3 deficiency in the second son of a consanguineous marriage with a life-threatening nonimmune hemolytic anemia. The patient was transfusion-dependent prior to splenectomy. SDS-PAGE and immunoblotting analysis of the proband red cell membrane proteins showed approximately 12±4% of band 3 and protein 4.2 compared to controls. Direct nucleotide sequence of SLC4A1 gene showed a single base substitution (T->C) at position +2 in the donor splice site of intron 2 (Band 3 Neapolis). Functionally, the mutation causes an altered splicing with the consequent formation of two different mature mRNAs, one including intron 2 and one skipping exon 2. While intron 2 retention leads to premature translation termination, exon 2 skipping causes the loss of the normal start site of eAE1 protein translation. The purification of mutant band 3 and its characterization by MALDI mass spectrometry demonstrated the lack of the first 11 amino acids due to the usage of second in frame start site. Real-time RT-PCR analyses of reticulocyte mRNA showed a marked decrement in band 3 transcription accounting for protein deficiency. The lack of the 11 N-terminal amino acids resulted in complete absence of membrane bound aldolase while other glycolitic enzymes (for example GAPDH) were membrane bound. Syk tyrosine kinase recognized the truncated band 3 as a substrate in vitro. In spite of this ability to be phosphorylated by Syk and to recruit Lyn tyrosine kinase in vitro, we were unable to demonstrate Tyr-phosphorylation of mutant band 3 in intact erythrocytes following stimulation by oxidative stress. This finding implies a requirement for the 11 N-terminal amino acids for the sequential Tyr-phosphorylation of band 3 in intact red cell membranes. The mutant band 3 was largely present in the high molecular weight aggregate fraction (about 5.2 fold higher than control), indicating its increased tendency to cluster in the membrane. The spontaneous clustering of truncated band 3 strongly suggests that the negatively charged N-terminal domain may regulate oligomeric state of band 3 in the membrane. Biophysical characterization showed that band 3 deficiency resulted in decreased cohesion between lipid bilyer and spectrin based membrane skeleton accounting for membrane loss. The structural and functional characterization of the naturally occuring mutant band 3 has enabled us to identify a significant role for the 11 N-terminal amino acids in band 3 function and in red cell membrane physiology.

Myelin: Delivery by raft.

Recent results suggest that membrane proteins are delivered to the myelin sheath of an oligodendrocyte on rafts with a distinctive lipid composition. The major intrinsic membrane protein of myelin, proteolipid protein, interacts with rafts in oligodendrocytes but not with the different rafts found in other cell types.

Water-selective and multifunctional aquaporins from Lotus japonicus nodules.

By using reverse transcriptase-polymerase chain reaction, two cDNAs were isolated that encode major intrinsic membrane proteins (MIPs) that are expressed in nitrogen-fixing root nodules of Lotus japonicus. Lotus intrinsic membrane protein 1 (LIMP 1) is expressed at high levels in both nodule and root tissues and shows highest sequence similarity to members of the tonoplast intrinsic protein (TIP) subfamily of plant MIPs. Functional analysis of LIMP 1 by expression in Xenopus laevis oocytes show that it is a water-specific aquaporin. In contrast, LIMP 2 shows the highest sequence similarity to soybean nodulin 26 (67.8% amino acid sequence identity). LIMP 2 is also a nodulin, showing expression only in mature nitrogen fixing nodules of L. japonicus. LIMP 2 is a multifunctional aquaglyceroporin, and displays the ability to flux both water as well as glycerol upon expression in Xenopus oocytes. Additionally, the carboxyl terminal region of LIMP 2 has a conserved phosphorylation motif that is phosphorylated by a calmodulin-like domain protein kinase. Overall, the data show that L. japonicus nodules contain two structurally and functionally distinct MIP proteins: one (LIMP 2) which appears to be the nodulin 26 ortholog of L. japonicus and another (LIMP 1) which appears to be a member of the TIP subfamily.

MRNAs encoding aquaporins are present during murine preimplantation development.

The present study was conducted to investigate the mechanisms underlying fluid movement across the trophectoderm during blastocyst formation by determining whether aquaporins (AQPs) are expressed during early mammalian development. AQPs belong to a family of major intrinsic membrane proteins and function as molecular water channels that allow water to flow rapidly across plasma membranes in the direction of osmotic gradients. Ten different AQPs have been identified to date. Murine preimplantation stage embryos were flushed from the oviducts and uteri of superovulated CD1 mice. Reverse transcription-polymerase chain reaction (RT-PCR) methods employing primer sets designed to amplify conserved sequences of AQPs (1-9) were applied to murine embryo cDNA samples. PCR reactions were conducted for up to 40 cycles involving denaturation of DNA hybrids at 95 degrees C, primer annealing at 52-60 degrees C and extension at 72 degrees C. PCR products were separated on 2% agarose gels and were stained with ethidium bromide. AQP PCR product identity was confirmed by sequence analysis. mRNAs encoding AQPs 1, 3, 5, 6, 7, and 9 were detected in murine embryos from the one-cell stage up to the blastocyst stage. AQP 8 mRNAs were not detected in early cleavage stages but were present in morula and blastocyst stage embryos. The results were confirmed in experimental replicates applied to separate embryo pools of each embryo stage. These results demonstrate that transcripts encoding seven AQP gene products are detectable during murine preimplantation development. These findings predict that AQPs may function as conduits for trophectoderm fluid transport during blastocyst formation.

Highly purified thermo-stable oxygen-evolving photosystem II core complex from the thermophilic cyanobacterium Synechococcus elongatus having His-tagged CP43.

The carboxyl terminus of the CP43 subunit of photosystem II (PSII) in the thermophilic cyanobacterium, Synechococcus elongatus, was genetically tagged with six consecutive histidine residues to create a metal binding site on the PSII supramolecular complex. The histidine-tagging enabled rapid isolation of an intact cyanobacterial PSII core complex from dodecyl maltoside-solubilized thylakoids by a simple one-step Ni(2+)-affinity column chromatography. The isolated core complex was in a dimeric form with a molecular mass of about 580 kDa, consisting of five major intrinsic membrane proteins (CP47, CP43, D1, D2 and cytochrome b-559), three extrinsic proteins (33 kDa, 12 kDa, and cytochrome c-550), and a few low molecular mass membrane proteins, and evolved oxygen at a rate as high as 3,400 mumol (mg Chl)-1 h-1 at 45 degrees C with ferricyanide as an electron acceptor. The core complex emitted thermoluminescence B2-, B1- and Q-bands arising from S2QB-, S3QB- and S2QA- charge recombinations at respective emission temperatures of 45, 38 and 20 degrees C, all of which were higher by about 15 degrees C as compared with those in mesophilic spinach BBY membranes. These results indicated that the isolated core complex well retained the intact properties of thermoluminescence of thermophilic cyanobacterial cells, the deeper stabilization of PSII charge pairs. The isolated complex was extremely stable in terms of both protein composition and function, exhibiting no release of extrinsic proteins, no proteolytic degradation in any of its subunits, accompanied by only a slight (less than 10%) loss in oxygen evolution, after dark-incubation at 20 degrees C for 8 d. These properties of the thermophilic PSII core complex are highly useful for various types of studies on PSII.

Maturation-dependent apoptotic cell death of oligodendrocytes in myelin-deficient rats.

Mutations in the proteolipid protein gene (PLP/plp), which encodes the major intrinsic membrane protein in central nervous system (CNS) myelin, cause inherited dysmyelination in mammals. One of these mutants, the myelin-deficient (md) rat, has severe dysmyelination that is associated with oligodendrocyte cell death. Using the terminal deoxynucleotidyl transferase (TdT)-mediated dUTP-biotin nick end-labeling (TUNEL) assay, which labels apoptotic cells, we find that cell death is increased in multiple white matter tracts of md rats. The tracts that myelinate the earliest show the earliest increase in cell death, and cell death persists for at least 22 days, the lifespan of these mutant animals. In all tracts, and at all developmental ages examined, apoptotic cells expressed the markers of mature oligodendrocytes, such as myelin basic protein, myelin-associated glycoprotein, and the Rip antigen, but not chondroitin sulfate proteoglycan, a marker of oligodendrocyte precursors. Mature oligodendrocytes fail to accumulate in md brain because they die before they fully mature.

A new look at the lipid composition of the plasma membrane of human blood platelets relative to the GPIIb/IIIa (integrin cxIIβ3) content

The total lipids of the human platelet plasma membrane (HPPM) from 50 ml of blood of healthy subjects were extracted, quantified and related to the mass content of the major intrinsic membrane protein, the glycoprotein IIb/IIIa (GPIIb/IIIa). The HPPM total lipid/GPIIb/IIIa weight ratio determined was 5.40 ± 0.20, independently of the membrane washing procedure used, with the cholesterol/GPIIb/IIIa and phospholipid/GPIIb/IIIa molar ratios of 800 ± 50 and 1200 ± 40, respectively. If the distribution of lipids around each intrinsic protein were proportional to its mass, the lipids around a molecule of GPIIb/IIIa will occupy about 120 nm(2) of the membrane plane, which is about one and a half times the cross-sectional area of the extracellular head of GPIIb/IIIa, as estimated by electron microscopy. The lipid extracts were further subjected to thin-layer chromatography to separate and quantify the different phospholipid fractions, the free fatty acids and the neutral lipid fraction and the distribution of fatty acids in each fraction was determined by gas chromatography after methanolysis. The phospholipid molar distribution was SPM(22.3 ± 0.9%), PC(36.2 ± 1.0%), PE(24.9 ± 0.9%), PS(12.1 ± 0.6%) and PI(4.5 ± 0.4%) and the free fatty acid fraction represented 2.9 ± 0.4% of the total fatty acids in HPPM. The fatty acid chain length ranged from 14 to 24 carbons, comprising unsaturated fatty acids (47.3% molar per cent of the total) of which 40.7 ± 2.0% were monosaturated and 40.7 ± 0.9% tetraunsaturated. Palmitic, stearic, oleic and arachidonic acids represent 66% of the total fatty acids of HPPM, being: 68.9 ± 5.3% of palmitic acid and 63.3 ± 6.9% of oleic acid in PC; 50.9 ± 3.8% of arachidonic acid in PE; and 30.5 ± 2.4% of stearic acid in PS. We discuss the methodological modifications and the new data in relation with the major differences in HPPM lipid composition found in the literature. The data obtained provides a comprehensive and accurate description of the lipid composition of HPPM on which to rely as a reference for basic and medical studies.

Characterization of the ovine-lens plasma-membrane protein-kinase substrates.

The cAMP-dependent protein-kinase-catalyzed phosphorylation of the two major intrinsic lens fiber cell plasma membrane proteins, MP20 and MP26, is likely restricted to the inner cortical and nuclear regions of the lens in vivo. The ovine-lens-specific connexin, MP70, that has been identified as Cx50 in mice and Cx45.6 in the chick, is also a protein kinase substrate although it does not appear to be phosphorylated by a number of protein kinases including cAMP-dependent protein kinase, calmodulin-dependent protein kinase or protein kinase C. Rather, an extrinsic lens membrane fraction was isolated which contained protein kinase activity that catalyzed the phosphorylation of MP70; this protein kinase activity was cAMP-independent, Ca(2+)-independent, Mg(2+)-dependent, phosphorylated MP70 on a serine residue(s) and migrated with a molecular mass of 35 kDa on a gel filtration column. Both MP70 phosphorylation and the endogenous protein kinase activity were restricted to the lens outer cortical region. This membrane-associated protein kinase activity represents the first reported partial characterization of an endogenous lens fiber cell protein kinase activity that catalyzes the phosphorylation of a lens connexin protein. The phosphatase-induced shift in the electrophoretic mobility of MP70 is not reversed by this protein kinase, indicating that MP70 is likely phosphorylated on different residues by two or more protein kinases.

Transcriptional regulation of the rat PLP promoter in primary cultures of oligodendrocytes.

Proteolipid protein (PLP) is the major intrinsic membrane protein of CNS myelin and is expressed in oligodendrocytes as part of a coordinate program of myelin-specific gene activation. In order to identify the DNA sequences and proteins involved in the regulation of PLP transcription, we have analyzed the 5' flanking sequences of the rat PLP gene by transient transfections into primary cultures of developing oligodendrocytes, the glial tumor line, C6, and L cells. High levels of expression of the CAT reporter gene in oligodendrocytes and C6 cells were obtained with constructs containing both 4270 and 225 bp of PLP promoter. A fusion construct containing 1061 bp of the PLP promoter, however, showed two-fold lower CAT expression. In addition, the activity of these promoter fusion constructs in oligodendrocytes was 2.5-4.6 higher than that observed in C6 cells, while very little expression was found in L cells. These data suggest that 225 bp of PLP promoter is sufficient for oligodendrocyte-specific regulation of PLP expression. Furthermore, both positive and negative elements within the PLP promoter are involved in this process. Finally, primary cultures of developing oligodendrocytes are a useful model system for the analysis of myelin-specific gene activation.

Interaction of synaptotagmin with the cytoplasmic domains of neurexins

Synaptotagmin, a major intrinsic membrane protein of synaptic vesicles that binds Ca 2+, was purified from bovine brain and immobilized onto Sepharose 4B. Affinity chromatography of brain membrane proteins on immobilized synaptotagmin revealed binding of α- and β-neurexins to synaptotagmin in a Ca 2+-independent manner. Using a series of recombinant proteins in which glutathione S-transferase was fused to the cytoplasmic domains of three different neurexins or of control proteins, it was found that synaptotagmin specifically interacts with the cytoplasmic domains of neurexins but not of control proteins. This interaction is dependent on a highly conserved, 40 amino acid sequence that makes up most of the cytoplasmic tails of the neurexins. Our data suggest a direct interaction between the cytoplasmic domains of a plasma membrane protein (the neurexins) and a protein specific for a subcellular organelle (synaptotagmin). Such an interaction could have an important role in the docking and targeting of synaptic vesicles in the nerve terminal.

Regional differences in the polypeptide composition of pig lens intracellular matrix and plasma membrane.

Analysis of pig lens urea-soluble fractions (USF) by gel filtration and SDS-PAGE revealed that the cortical USF is characterized by a high content of noncrystallin bands of 42, 44, 52, 55, 100 and 200 kD. These bands are markedly decreased in the nuclear USF though the 44-kD band to a lesser extent. These results suggested that pig lens nucleus contains much less cytoskeletal proteins than the cortex. Analysis of pig lens membrane showed that MP26 and MP17 are the major intrinsic membrane proteins and the relative amount of alpha A and beta B1b chains attached to the membrane are more abundant in the nucleus than in the cortex.

Electron microscopic observations of reconstituted proteoliposomes with the purified major intrinsic membrane protein of eye lens fibers.

The purified major intrinsic protein of the lens fiber plasma membrane (MP26) reconstituted into liposomes favored membrane-to-membrane close contacts as visualized by freeze fracture and immunoelectron microscopy. Reconstituted apposed unilamellar vesicles formed pentalaminar profiles, and multilamellar liposomes showed regions of stacked bilayers. Immunogold labeling, using antibody directed against MP26, demonstrated that this polypeptide is present in regions of membrane-to-membrane close interaction. Fracture faces displayed both randomly distributed clusters of 8-nm polygonal intramembrane particles and membrane domains where a bidimensional lattice of repeating subunits was present. The structural pleomorphism which characterized the MP26-reconstituted proteoliposomes seems quite comparable to that visualized in natural fiber plasma membrane domains.

Experimental allergic encephalomyelitis mediated by murine encephalitogenic T cell lines specific for myelin proteolipid apoprotein.

T cell lines specific for bovine myelin proteolipid apoprotein (PLP) were established from SJL/J mice. The line cells bore surface phenotypes of T helper/inducer cells (Lyt-1+, Lyt-2-, L3T4+) and responded well to bovine, rat, and guinea pig PLP but not to myelin basic protein. One line responded to major PLP, and another responded to both major PLP and DM-20, which are the two major intrinsic membrane proteins of the central nervous system (CNS) myelin. Intraperitoneal inoculation of 4 to 30 X 10(6) PLP-activated line cells followed by injection of pertussis vaccine induced acute inflammatory disease of the CNS, with typical clinical signs of EAE mostly in a week in recipient mice that had been treated with low-dose irradiation. Almost all animals recovered completely, and two of the 12 animals relapsed 42 or 75 days after inoculation. The lesions were restricted to the CNS and were characterized by perivascular and parenchymal infiltration of inflammatory cells, fibrin deposit, and demyelination. In the severe lesions, axons were also damaged. These observations suggest that PLP is a definite encephalitogen, and PLP-sensitized effector T cells induce inflammatory demyelination in the CNS.

A specific immunological probe for the major myelin proteolipid: Confirmation of a deletion in DM-20

Major myelin proteolipid (MMPL, also called PLP) and DM-20 are the two major intrinsic membrane proteins of CNS myelin. A specific immunological probe was obtained for MMPL by raising antibodies against the synthetic tridecapeptide 117–129 of MMPL. Antibodies against this peptide reacted with the MMPL but dit not cross react with DM-20, while both proteolipids had been shown previously to be recognized by antibodies directed against the C-terminal hexapeptide of MMPL. This is in accordance with previous findings showing that DM-20 diners only from MMPL by a deletion of residues 100–140 (±few units). Furthermore, this site-specific immunological probe also recognizes MMPL in its native form in oligodendrocytes in primary glial cell cultures.

Study of the expression of myelin proteolipid protein (lipophilin) using a cloned complementary DNA.

We have prepared a lambda gt10 cDNA library with the mRNA isolated from fetal calf brains which were actively myelinating. Using two oligonucleotides made according to the known amino acid sequence of myelin proteolipid protein (PLP or lipophilin), we have isolated several cDNA clones for this major intrinsic membrane protein of myelin. One of these clones, designated as pLP1, is found to contain 444 bp of coding sequence for the C-terminal half of PLP and 486 bp of 3' untranslated sequence. Using pLP1 as a hybridization probe, we have studied the regulation of PLP at the mRNA level during rat brain development. Our results show that the relative amounts of mRNA for PLP and that for the major extrinsic protein of the myelin membrane, myelin basic protein, increase coordinately during the course of myelination in the brain.

76] Light-regulated binding of proteins to photoreceptor membranes and its use for the purification of several rod cell proteins

Publisher Summary This chapter discusses the light-regulated binding of proteins to photoreceptor membranes and its use for the purification of several rod cell proteins. Rhodopsin is the major intrinsic membrane protein of rod outer segments (ROS). A number of additional proteins are present in ROS whose plasma membrane has not been too damaged during isolation and purification. Several proteins that are extractable from dark-kept ROS become membrane-bound when the ROS are illuminated. These proteins sediment with the bleached membranes on centrifugation and therefore are absent, or present in reduced amounts, in the supernatants. They can be extracted from the bleached membranous pellets by various subsequent treatments; this may be used for their purification. The differential solubilities of some of the ROS proteins in light and darkness at different ionic strengths can be used to purify them by a few simple centrifugation steps carried out under the appropriate conditions of ionic strength and illumination, using the disk membrane as an “affinity chromatography support” material for specific adsorption and desorption of the proteins.

Peptide mapping by limited proteolysis in sodium dodecyl sulfate of the main intrinsic polypeptides isolated from human and bovine lens plasma membranes

The major intrinsic lens plasma membrane protein isolated from calves, young cows and young humans, is an approx. 26 000 dalton protein. In the adult human lens, a second major intrinsic protein ( M r 22 000 ) is found. The relationships between the bovine 26 000 dalton protein and the human 26 000 dalton protein, as well as between the human 26 000 dalton protein and the human 22 000 dalton protein, were examined using peptide mapping by limited proteolysis is sodium dodecyl sulfate. Purified membrane proteins were obtained by preparative sodium dodecyl sulfate gel electrophoresis. At concentrations of 1 mg/ml or more, the purified 26 000 dalton and 22 000 dalton proteins tend to aggregate in sodium dodecyl sulfate solution and form dimers, trimers and tetramers, as well as higher-order multimers. These aggregates cannot be dissociated by β-mercaptoethanol. Staphylococcus aureus protease exhibits differential proteolytic activity toward the intrinsic major plasma membrane proteins and the soluble lens proteins which are difficult to remove from membrane preparations. While the soluble lens proteins are very effectively cleaved by S. aureus into fragments of less than 12 000 daltons, both the human and bovine 26 000 dalton major intrinsic membrane proteins lost only an approx. 4000 dalton peptide. Under the same conditions, this protease did not proteolyse the human native approx. 22 000 dalton protein. The resistance to further proteolysis of the approx. 22 000 dalton proteins which were produced by the action of the protease on the human and bovine 26 000 dalton proteins, as well as the resistance to further proteolysis under the same conditions of the native 22 000 dalton human protein, suggest that all these proteins possess similar conformation and composition. Additional proof that the human 26 000 dalton and 22 000 dalton membrane proteins as well as the bovine 26 000 dalton protein are all closely related comes from the α-chymotrypsin peptide maps obtained from the purified proteins. These results suggest that the human 22 000 dalton membrane protein is a natural cleavage product of the 26 000 dalton main intrinsic plasma membrane protein.

Purification and characterization of Band 3, the major intrinsic membrane protein of the bovine erythrocyte membrane.

Band 3 from bovine erythrocyte membranes was isolated in a state of high purity by the following steps in the presence of a nonionic detergent, nonaethyleneglycol n-dodecyl ether (C12E9): (1) selective removal of Band 2.6 from ghosts by solubilization with 2% C12E9 (2) extraction of Band 3-rich fraction with 4% C12E9 from 2% C12E9-treated membrane residues, and (3) purification of Band 3 by aminoethyl-conjugated Sepharose 4B column chromatography. Human Band 3 was also purified in good yield by aminoethyl-conjugated Sepharose 4B column chromatography of erythrocyte membrane proteins solubilized with 1% C12E9 and treated with 2,3-dimethymaleic anhydride. There were no significant differences in CD spectra in C12E9, amino acid compositions, and migration mobilities in sodium dodecyl sulfate-gel electrophoresis between bovine and human Band 3. Calculations of average hydrophobicity and discriminant function demonstrated that bovine Band 3 could be categorized as a typical integral membrane protein. Bovine Band 3 showed a tendency to form a dimer and higher aggregates in 0.1% C12E9; these were resistant to dissociation into monomers in sodium dodecyl sulfate solution and, further, the protein retained residual secondary structure in highly concentrated guanidine hydrochloride solution, indicating the possible presence of an extended sequence of hydrophobic amino acid residues.