The nature of the stable noncovalent dimers of band 3 protein from erythrocyte membranes in solutions of Triton X-100

Stable noncovalent dimers of band 3 protein from human erythrocyte membranes, in which state the protein is thought to exist after solubilization by the nonionic detergent Triton X-100, do not occur when purified batches of the detergent are used. Instead, the protein is in a monomer/dimer/tetramer association equilibrium. The stable dimers do appear, however, when the detergent has been 'aged'. They thus seem to be artifacts.     

The state of association of band 3 protein of the human erythrocyte membrane in solutions of nonionic detergents

Band 3 protein, the anion transport protein of the human erythrocyte membrane, was solubilized and purified in aqueous solutions of two nonionic detergents: Ammonyx-LO (dimethyl laurylamine oxide) and C12E9 (nonaethylene glycol lauryl ether). The state of association of the purified protein was studied by analytical ultracentrifugation. Band 3 protein solubilized and studied in solutions of Ammonyx-LO was found to be in a monomer/dimer/tetramer association equilibrium. Band 3 protein freshly prepared in C12 E9 showed the same behaviour; however, during aging the protein was converted into stable noncovalent dimers. The conversion was retarded by the presence of beta-mercaptoethanol or by treatment of the samples with iodoacetamide; it seems to be due to oxidation of the protein by degradation products of the detergent. It is concluded that a monomer/dimer/tetramer association equilibrium is the native state of association of band 3 protein solubilized by nonionic detergents. Since nonionic detergents are assumed not to interfere with protein-protein interactions among membrane proteins, the results strongly support the claim that, in the erythrocyte membrane, band 3 is in a monomer/dimer/tetramer association equilibrium (Dorst, H.-J. and Schubert, D. (1979) Hoppe-Seyler's Z. Physiol. Chem. 360, 1605-1618).     

Purification of the Ca2+-stimulated ATPase activator from human erythrocytes. Its membership in the class of Ca2+-binding modulator proteins.

The activator of the Ca2+-stimulated ATPase of erythrocyte membranes was purified 13,000-fold to homogeneity from human erythrocytes. The protein gave a single band upon electrophoresis both with and without detergent, and upon isoelectric focusing. This protein was compared with Ca2+-binding modulator proteins from bovine brain and rat testis. All three proteins were homogeneous and co-migrated on electrophoresis both in the presence of detergent and without detergent at pH values on both sides of the isoelectric point of the protein. The amino acid compositions of the three proteins were nearly indistinguishable, and all three proteins contained 1 residue of the unusual amino acid, trimethyllysine. All three were also indistinguishable as measured by their ability to further stimulate the Ca2+-stimulated ATPase of human erythrocyte membranes. Thus, we conclude that they represent functionally the same protein. Upon storage of all three proteins, a second band was detectable by detergent gel electrophoresis; the biochemical activity and the behavior on nondetergent gels were not changed. The presence of this second band is probably responsible for previous reports of differences between the rat testis and bovine brain modulator protein. The possibility is discussed that this protein is a general intracellular Ca2+ receptor, which mediates the activities of Ca2+ as an intracellular messenger.     

Purification of proctolin-binding proteins from the foregut of the insect Blaberus craniifer.

A membrane protein that specifically binds the insect neuropeptide proctolin was purified using standard chromatography from cockroach foregut membranes. Proctolin-binding sites were efficiently solubilized with either the nonionic detergent digitonin or the zwitterionic detergent Chaps, as indicated by the specific binding of 3H-proctolin to solubilized samples. A solubilized sample obtained from 1600 foregut membranes was subjected to a five-step chromatographic purification including chromatofocusing, anion-exchange and size-exclusion chromatographies. The final size-exclusion separation resulted in the isolation of approximately 100 pmol of purified proctolin-binding proteins, eluting as a single peak at approximately 74 kDa. Analysis of the purified sample using SDS/PAGE and silver staining showed two bands at 80 kDa and 76 kDa. Densitometric analysis of the gel indicated that each band contained approximately 7-8 microg of protein, suggesting that one band corresponds to the proctolin-binding activity. Proctolin-binding proteins were thus purified 1800-fold using standard chromatography.     

A study of the interaction of immobilized delipidized rhodopsin with G-proteins

It was shown that delipidated rhodopsin immobilized on concanavalin A-Sepharose (Rimm) binds with high selectivity transducin from total extracts of rod outer segment protein as well as the regulatory GTP-binding Gi- and Go-like proteins from solubilized membranes of bovine brain. The Rimm-bound proteins are eluted in the presence of the nonionic detergent, octyl glucoside, and GTP. This suggests that Rimm can be used as an affinity adsorbent for the isolation and purification of G-proteins.     

Phase separation of integral membrane proteins in Triton X-114 solution

A solution of the nonionic detergent Triton X-114 is homogeneous at 0 degrees C but separates in an aqueous phase and a detergent phase above 20 degrees C. The extent of this detergent phase separation increases with the temperature and is sensitive to the presence of other surfactants. The partition of proteins during phase separation in solutions of Triton X-114 is investigated. Hydrophilic proteins are found exclusively in the aqueous phase, and integral membrane proteins with an amphiphilic nature are recovered in the detergent phase. Triton X-114 is used to solubilize membranes and whole cells, and the soluble material is submitted to phase separation. Integral membrane proteins can thus be separated from hydrophilic proteins and identified as such in crude membrane or cellular detergent extracts.     

Destabilization of collagen structure by amides and detergents in solution

The effects of amides and detergents on collagen to gelatin transition have been studied at neutral pH. Simple amides denature the protein. The substitution of H-atoms by the alkyl groups at the nonpolar end of amide increases the effectiveness of the compounds in destabilizing the collagen structure whereas substitution of the H-atom at the polar amide end shows marginal effects on the collagen transition. The capabilities of these reagents to denature collagen are much less pronounced than their effects on denaturing globular proteins. Anionic detergents are found to destabilize collagen at very low concentrations (below their cmc values). In this respect, the effects of the detergents on collagen are comparable to the denaturing effects of the detergents on globular proteins. The effect of detergents increases with the increase in the length of the alkyl chain. The structure of the anion in the detergent is also important as seen from the lower potency of the sulfonate containing detergent compared to the sulfate containing detergent in denaturing collagen. Cationic and nonionic detergents do not denature collagen.     

Structural stability of the erythrocyte anion transporter, band 3, in native membranes and in detergent micelles.

The exothermic thermal denaturation transition of band 3, the anion transporter of the human erythrocyte membranes, has been studied by differential scanning calorimetry, in ghost membranes and in nonionic detergent micelles. In detergent micelles the transmembrane domain of band 3 gave an irreversible denaturation transition (C transition). However, no thermal transition was observed for the N-terminal cytoplasmic domain when band 3 was solubilised in detergent micelles. A reduction in enthalpy (190-300 kcal mol-1) with an accompanying decrease in thermal denaturation temperatures (48-60 degrees C) for the C transition was observed in detergent solubilised band 3 when compared with ghost membranes. Unlike ghost membranes, two thermal transitions for band 3 in detergent micelles were observed for the C transition when in the presence of excess covalent inhibitor, 4,4'-diisothiocyanostilbene-2,2'-disulphonate (DIDS), which derive from the thermal unfolding of a single protein with two different thermal stabilities; DIDS-stabilised (75 degrees C) and DIDS-insensitive (62 degrees C). A reduction in the denaturation temperature for the transmembrane domain of band 3 was observed when compared with intact band 3 although no significant differences was observed in the corresponding enthalpy values. This indicates some cooperativity of the two domains of band 3 in maintaining the transmembrane conformation. The results presented in this study show that detergents of intermediate micelle size (e.g. Triton X-100 and C12E8) are required for optimal thermal stability of band 3.     

Effect of stilbenedisulfonate binding on the state of association of the membrane-spanning domain of band 3 from bovine erythrocyte membrane

The membrane-spanning domain of bovine band 3, the anion transport protein of erythrocyte membrane, was purified in the presence of nonaethyleneglycol lauryl ether (C12E9) and the effect of a covalent attachment of 4,4'-diisothiocyanostilbene-2,2'-disulfonate (DIDS), a potent transport inhibitor, on the state of association of the domain isolated (the 58 kDa fragment) was studied via gel filtration, gel electrophoresis and sedimentation velocity experiments. It was indicated that the DIDS-unlabeled fragment in C12E9 solution forms heterogeneous aggregates which are larger in size than the dimer. This contrasted with the behavior that bovine band 3 is present as dimers or tetramers in the same medium (Nakashima and Makino (1980) J. Biochem. 88, 933-947). When DIDS was covalently attached, the fragment was present as a single molecular species which was indicated to be a dimer by molecular weight determination. The secondary structure of the fragment was not affected by DIDS. The change in the state of association caused by the DIDS-binding was also found in the presence of sucrose monolaurate (SE12), which was a more potent detergent for extraction of the 58 kDa fragment from membranes than C12E9. However, the complex with SE12 was extremely unstable.     

Solubilization of Myelin Membranes by Detergents

The major proteins of myelin have classically been extracted in organic solvents. Here we investigated some of the characteristics of brain myelin solubilization in aqueous detergent solutions. At comparable molar concentrations, two nonionic detergents, i.e., octyl glucoside and Lubrol PX, proved relatively better myelin solubilizers than the detergents related to the bile salts, i.e., cholate and CHAPS. The two former detergents solubilized more protein than lipid and the two latter ones more lipid than protein from myelin membranes. All four detergents solubilized the phospholipid more efficiently than the cholesterol component of myelin. The detergent concentrations required for myelin solubilization were reduced substantially if the temperature and the salt concentration of the media were increased. As much as 3 mg of lyophilized myelin (about 1 mg of protein) were solubilized readily per milliliter of a solution containing 30 mM octyl glucoside and 0.1 M sodium sulfate in 0.1 M sodium phosphate buffer, pH 6.7. Each of the detergents studied, including the above four, sodium dodecyl sulfate (SDS). Triton X-100, and Zwittergent 3-14, had its own advantages and drawbacks as myelin protein extractors. The nonionic amphiphiles and CHAPS left a small residue mainly composed of proteins of the Wolfgram fraction, as revealed by SDS-polyacrylamide gel electrophoresis. Octyl glucoside was preferred, given its versatility as solubilizer, ultraviolet transparency, and high critical micellar concentration. Observations on possible difficulties that may be encountered are also included.     

Effect of detergent on protein structure. Action of detergents on secondary and oligomeric structures of band 3 from bovine erythrocyte membranes

With special interest in the mode of action of zwitterionic detergents on proteins, a variety of detergents were examined for their ability to disrupt the secondary and quaternary structures of an anion transport protein, band 3, and its cytoplasmic 38 kDa fragment from bovine erythrocyte membranes and for their effect on the binding of an anion transport inhibitor to band 3. Nonionic detergents and Chaps also acted as a nondenaturant in these instances, as well accepted for other proteins. Though deoxycholate and cholate inhibited the binding of an anion transport inhibitor to band 3, these detergents did not show any effect on the native structure of band 3. Zwitterionic detergents (Zwittergent 3-10, Zwittergent 3-12 and N, N-dimethyl-N-dodecyl glycine) were suggested to denature the water-soluble 38 kDa fragment at concentrations above the critical micelle concentration, but to be weak in disrupting interacting forces between hydrophobic membrane-bound domains of band 3. The results indicated that these zwitterionic detergents are similar in the mode of denaturing action to dodecyltrimethylammonium bromide rather than sodium dodecyl sulfate.     

A new major transmembrane glycoprotein, gp155, in goat erythrocytes. Isolation and characterization of its association to cytoskeleton through binding with band 3-ankyrin complex

Erythrocyte membranes from goat contain a considerable amount, more than 10% of the total amount, of a glycoprotein with Mr = 155,000 (gp155) on sodium dodecyl sulfate-polyacrylamide electrophoresis gel. This report describes the first isolation and characterization of gp155. This gp155 has major trypsin-sensitive sites at each side of the plasma membrane to generate membrane-bound fragments, indicating that the gp155 spans the lipid bilayer several times. This protein consists of a single polypeptide containing about 1,200 amino acid residues corresponding to Mr = 134,000 and some complex type N-linked oligosaccharide chains. A fraction (15-20%) of the gp155 is recovered in nonionic detergent-extracted ghosts along with 25-30% of band 3 and other cytoskeletal proteins and is completely released into solution by extraction with 1 M KCl. Immunoprecipitation with anti-gp155 and anti-ankyrin antibodies of detergent-solubilized membranes separated on a gel permeation chromatography column showed that a part of the gp155 is tightly linked to band 3 with a molar ratio of 1:2 to 1:3. This gp155-band 3 complex in turn is associated to ankyrin through the binding of band 3 to ankyrin. These data indicate that, in native erythrocyte membranes, as well as in detergent solution, gp155 could play a physiological role in controlling cellular integrity and elasticity by forming the gp155-band 3-ankyrin complex. Partial amino acid sequences of the tryptic peptides are also determined.     

Isolation of a membrane protein by chromatofocusing: Cytochrome b-561 of the adrenal chromaffin granule

Chromatofocusing, a form of ion-exchange chromatography in which proteins are separated on the basis of their differing isoelectric points, has been adapted for use with membrane proteins, solubilized by the non-ionic detergent Nonidet P-40. Using a two-step detergent extraction followed by chromatofocusing under high pressure, the highly hydrophobic protein cytochrome b-561 was isolated from chromaffin granule membranes and purified to near homogeneity in a functionally active form, in less than 5 h. Chromatofocusing conditions were optimized empirically since the behaviour of the chromaffin granule membrane proteins conformed less to the theory than that of soluble proteins, and the various factors affecting yield and resolution are discussed. The speed, high resolution and focusing effect could make this method particularly suitable for rapid isolation in a functionally active form of the many membrane proteins that are unstable in dilute solution and when removed from their lipid environment.     

Molecular characterization of the human erythrocyte anion transport protein in octyl glucoside

Band 3 protein, the anion transport protein of the human erythrocyte membrane, was purified in the presence of the nonionic detergent octyl glucoside. A molecular characterization was carried out to investigate whether the native structure of the protein was retained in the presence of this detergent. Band 3 bound octyl glucoside below the critical micelle concentration (cmc) of the detergent, approaching saturation above the cmc. At 40 mM octyl glucoside, close to saturating concentrations, 0.64 g of octyl glucoside is bound per gram of band 3 protein, corresponding to 208 molecules of detergent bound per monomer of band 3. Sedimentation velocity and gel filtration studies, performed at 40 mM octyl glucoside, indicated that the band 3-octyl glucoside complex had an average molecular weight of 1.98 X 10(6), which corresponds to a dodecamer. Sedimentation equilibrium experiments confirmed that band 3 in octyl glucoside exists in a heterogeneous and high oligomeric state. This high oligomeric state did not change dramatically over octyl glucoside concentrations ranging from 6 to 60 mM. The circular dichroism spectrum of band 3 changed only slightly over this range of octyl glucoside concentrations. The alpha-helical and beta-sheet contents of band 3 in 2 mM octyl glucoside were calculated to be 40% and 27%, respectively, indicating that no gross alteration in the secondary structure of the protein had occurred in octyl glucoside. The ability of band 3 to bind 4-benzamido-4'-aminostilbene-2,2'-disulfonate (BADS), a potent inhibitor (Ki = 1 microM) of anion transport, was measured to assess the integrity of the inhibitor binding site of the protein in octyl glucoside.(ABSTRACT TRUNCATED AT 250 WORDS)     

Heterogeneity in the conformation of different protein fractions from the human erythrocyte membrane

We have isolated 5 families of proteins from human red blood cell membranes and characterized their secondary structure by ultraviolet circular dichroism measurements. The protein families were prepared by selective solubilization from ghosts under nondenaturing conditions. We find that the intact ghost has a mean α-helix fraction of 0.37, whereas a low-ionic-strength extract (bands 1, 2, 5, “spectrin”) has a substantially higher helix fraction, 0.55. Further extraction of the ghosts with para-chloromercuribenzoate yields bands 2.1, 4.1, 4.2, and 6; their helix content is only 0.17. Finally, the major intrinsic protein, band 3, was solubilized by a nonionic detergent. Its helix fraction is 0.38.     

Microtubule-membrane interactions in cilia. I. Isolation and characterization of ciliary membranes from Tetrahymena pyriformis

Tetrahymena ciliary membranes were prepared by four different techniques, and their protein composition was analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), electron microscopy, and two-dimensional thin-layer peptide mapping. Extraction of the isolated cilia by nonionic detergent solubilized the ciliary membranes but left the axonemal microtubules and dyneine arms intact, as determined by quantitative electron microscopy. The proteins solubilized by detergent included a major 55,000-dalton protein, 1-3 high molecular weight proteins that comigrated, on SDS-PAGE, with the axonemal dynein, as well as several other proteins of 45,000-50,000 daltons. Each of the major proteins contained a small amount of carbohydrate, as determined by PAS-staining; no PAS-positive material was detected in the detergent-extracted axonemes. The major 55,000- dalton protein has proteins quite similar to those of tubulin, based on SDS-PAGE using three different buffer systems as well as two- dimensional maps of tryptic peptides from the isolated 55,000-dalton protein. To determine whether this tubulin-like protein was associated with the membrane or whether it was an axonemal or matrix protein released by detergent treatment, three different methods to isolate ciliary membrane vesicles were developed. The protein composition of each of these differetn vesicle preparations was the same as that of the detergent-solubilized material. These results suggest that a major ciliary membrane protein has properties similar to those of tubulin.     

A new procedure for the isolation of the brain myelin basic protein in a lipid-bound form

Myelin basic protein has been isolated from bovine brain using the nonionic detergent n-octyl-polydisperse oligooxyethylene. The purified basic protein contains large amounts of heterogeneous lipids.     

Integral membrane protein interaction with Triton cytoskeletons of erythrocytes

The organization of erythrocyte membrane lipids and proteins has been studied following the release of cytoplasmic components with the non-ionic detergent Triton X-100. After detergent extraction, a detergent-resistant complex called the erythrocyte cytoskeleton is separated from detergent, solubilized lipid and protein by sucrose buoyant density sedimentation. In cytoskeletons prepared under isotonic conditions all of the major erythrocyte membrane proteins are retained except for the integral protein, glycophorin, which is quantitatively solubilized and another integral glycoprotein, band 3, which is only 60% removed. When cytoskeletons are prepared in hypertonic KCl solutions, band 3 is fully solubilized along with bands 2.1 and 4.2 and several minor components. The resulting cytoskeletons have the same morphology as those prepared in isotonic buffer but they are composed of only three major peripheral proteins, spectrin, actin and band 4.1. We have designated this peripheral protein complex the 'shell' of the erythrocyte membrane, and have shown that the attachment of band 3 to the shell satisfies the criteria for a specific interaction. Although Triton did affect erythrocyte shape, cytoskeleton lipid content and the activity of membrane proteases, there was no indication that Triton altered the attachment of band 3 to the shell. We suggest that band 3 attaches to the shell as part of a ternary complex of bands 2.1, 3 and 4.2.     

A new protein of the brain myelin: isolation and chemical characterization

An unknown protein has been isolated from bovine brain myelin. This protein, purified in the nonionic detergent n-octylpolydisperse oligooxyethylene, reveals on SDS gel electrophoresis a large number of bands in the higher MW region. However, chemical analysis and gel chromatography indicate the presence of a single, small protein containing large amounts of bound phosphatidylserine. N-terminal and C-terminal sequences, aminoacid composition, and the anomalous electrophoretic behaviour led us to exclude the protein as a fragment of other already known myelin proteins.     

Solute excluded-volume effects on the stability of globular proteins: A statistical thermodynamic theory

A statistical thermodynamic theory is developed to investigate the effects of solute excluded volume on the stability of globular proteins. Proteins are modeled as two states in chemical equilibrium: the denatured state is modeled as a flexible chain of tangent hard spheres (pearl-necklace chain) while the native state is modeled as a single hard sphere. Study of model proteins bovine pancreatic trypsin inhibitor and lysozyme in a McMillan-Mayer model solution of hard spheres indicates that the excluded volume of solutes has three distinct types of effects on protein stability: (1) small-size solutes strongly denature proteins, (2) medium-size solutes stabilize proteins at low solute concentrations and destabilize them at high concentrations, and (3) large-size solutes stabilize native-state proteins across the whole liquid region. The study also finds that increasing the chain length of hard-chain polymer solutes has an effect on protein stability that is similar to increasing the diameter of spherical solutes. This work qualitatively explains why stabilizers tend to be large size molecules such as sugars, polymers, polynols, nonionic, and anionic surfactants while denaturants tend to be small size molecules such as alcohols, glycols, amides, formamides, ureas, and guanidium salts. Quantitative comparison between theoretical predictions and experimental results for folding free energy changes shows that the excluded-volume effect is at least as important as the binding and/or electrostatic effects on solute-assisted protein-denaturation processes. Our theory may also be able to explain the effect of excluded volume on the Φ condensation of DNA. © 1996 John Wiley & Sons, Inc.