Isolation of glycophorin with deoxycholate

In a previous communication we reported that human erythrocyte glycophorin prepared by the lithium diiodosalicylate phenol procedure contains approximately 10 mol of lithium diiodosalicylate per mol of glycophorin, and further we showed that this bound lithium diiodosalicylate is difficult to remove by detergents or organic solvents (Romans, A.Y. and Segrest, J.P. (1978) Biochim. Biophys. Acta 511, 297–301). In the present communication we report an alternative purification procedure for glycophorin in which sodium deoxycholate is substituted for lithium diiodosalicylate; the sodium deoxycholate is subsequently removed by gel filtration. Utilizing this procedure, 25–30 mg glycophorin are obtained per gram of lyophilized erythrocyte ghosts. The glycophorin prepared by the sodium deoxycholate procedure, after a single gel filtration step, contains less than 1 mol of sodium deoxycholate per mol glycophorin and is colorless compared with glycophorin prepared by the lithium diiodosalicylate procedure, which has a distinct reddish-brown cast.     

Reassociation of disk components solubilized with deoxycholate.

Cattle disk membrane was solubilized in 2% deoxycholate in 0.05 m Tris-HCl buffer at pH 8.0. The sedimentation coefficient of rhodopsin in this solvent was 4.4 S. A sedimentation equilibrium study in H₂O-D₂O solvent showed that rhodopsin was mainly associated with deoxycholate and probably free of phospholipid when the deoxycholate concentration was higher than 0.8%. Bleaching of rhodopsin resulted in aggregation of the protein. Reduction of the deoxycholate concentration by dialysis promoted protein-phospholipid interaction in such a way that when the deoxycholate concentration was 0.2%, most of the rhodopsin present in the initial solution formed a soluble protein-phospholipid-deoxycholate complex with a molecular weight of 86,000. The complex, we believe, contains one molecule of rhodopsin, 50 to 55 molecules of phospholipid, and 20 to 25 molecules of deoxycholate.After dialysis against 0.05 m phosphate buffer at pH 6.5 that contained 20 mm MgCl₂ all the material was incorporated into vesicular membranes of diameters from 20 to 500 nm. Such results signify the dominance of rhodopsin-phospholipid interaction over either inter-rhodopsin or inter-phospholipid interactions in the initial decrease of deoxycholate concentration. The protein-phospholipid membrane was then formed by the two-dimensional aggregation of a rhodopsin-phospholipid complex. The presence of at least a part of the sugar moiety of rhodopsin on the outer surface of the vesicles was shown by using ferritin-labeled concanavalin A.     

Isolation of the cDNA clone for mouse glycophorin, erythroid-specific membrane protein

The cDNA clone for a major mouse glycophorin, transmembrane glycoprotein of erythrocytes has been isolated from a mouse spleen erythroblast cDNA library. The primary structure of a major glycophorin indicates that the protein is a single polypeptide chain of 168 amino acids (aa) clearly organized in three domains distinct in the glycophorin of other species. A strong homology of the mouse major glycophorin with human glycophorin A or B, but not with human glycophorin C is observed only in the hydrophobic stretch of 23 nonpolar aa, indicating that the major mouse glycophorin species cloned is similar to human glycophorin A. The glycophorin mRNA is absent in all non-erythroid organs or cell lines examined. The glycophorin mRNA is induced during the differentiation of murine erythroleukemia cells with dimethyl sulfoxide.     

Protein-lipid interactions glycophorin and dipalmitolyphosphatidylcholine.

The choline methyl groups of dipalmitoylphosphatidylcholine were enriched with ¹³C, and glycophorin extracted from human erythrocytes was included in bilayers of this phospholipid. At temperatures below the transition temperature, the ¹³C nuclear magnetic resonance spectra have two components, one sharp and one broad. The sharp signal is attributed to relatively “fluid” lipids in the immediate vicinity of the glycoprotein. In a defined ternary mixture consisting of ¹³C-labeled dipalmitoylphosphatidylcholine, dielaidoylphosphatidylcholine and glycophorin, the sharp ¹³C resonance signal disappears below the transition temperature of the mixture, indicating that the unsaturated lipid is preferentially associated with the glycoprotein under these conditions.     

Solubilization of the Semliki Forest virus membrane with sodium deoxycholate.

The effects of increasing concentrations of sodium deoxycholate on Semliki Forest have been studied. Sodium deoxycholate begins to bind to the virus at less than 0.1 mM free equilibrium concentration and causes lysis of the viral membrane at 0.9 +/- 0.1 mM free equilibrium concentration when 2.2 +/- 0.2 - 103 mol of sodium deoxycholate are bound per mol of virus. Liberation of proteins from the membrane begins at 1.5 +/- 0.1 mM sodium deoxycholate and the proteins released are virtually free from phospholipid above 2.0 mM sodium deoxycholate. The overall mechanism of sodium deoxycholate solubilization of the viral membrane resembles that of Triton X-100 and sodium dodecyl sulphate except that with sodium deoxycholate the various stages of membrane disruption occur at about 10-fold higher equilibrium free detergent concentrations. At sodium deoxycholate concentrations higher than 2.3 mM the viral spike glycoproteins can be separated by sucrose gradient centrifugation or gel filtration into constituent polypeptides E1, E2 and E3. E1 carries the haemagglutinating activity of the virus.     

Studies on human red-cell membrane glycophorin A and glycophorin B genes in glycophorin-deficient individuals.

1. Genomic DNA derived from individuals who lack glycophorin A (GPA), glycophorin B (GPB) or both of these proteins was subjected to Southern-blot analysis using GPA and GPB cDNA probes. 2. Bands on the Southern blots were assigned to the GPA gene, GPB gene or to a putative pseudogene. 3. Genomic DNA derived from an individual of the Mk phenotype was shown to have deletions in the GPA and GPB genes. The simplest model for the results obtained is that a single deletion spans the GPA and GPB genes in the individual studied.     

Isolation of cDNA clones and complete amino acid sequence of human erythrocyte glycophorin C

Two cDNA clones for glycophorin C, a transmembrane glycoprotein of the human erythrocyte which carries the blood group Gerbich antigens, have been isolated from a human reticulocyte cDNA library. The clones were identified with a mixture of 32 oligonucleotide probes (14-mer) which have been synthetized according to the amino acid sequence Asp-Pro-Gly-Met-Ala present in the N-terminal tryptic peptide of the molecule. The primary structure of glycophorin C deduced from the nucleotide sequence of the 460 base-pair insert of the pGCW5 clone indicates that the complete protein is a single polypeptide chain of 128 amino acids clearly organized in three distinct domains. The N-terminal part (residues 1-57, approximately) which is N- and O-glycosylated is connected to a hydrophilic C-terminal domain (residues 82-128, approximately) containing 4 tyrosine residues by a hydrophobic stretch of nonpolar amino acids (residues 58-81, approximately) probably interacting with the membrane lipids and permitting the whole molecule to span the lipid bilayer. Northern blot analysis using a 265-base-pair restriction fragment obtained by DdeI digestion of the inserted DNA shows that the glycophorin C mRNA from human erythroblasts is approximately 1.4 kilobases long and is present in the human fetal liver and the human K562 and HEL cell lines which exhibit erythroid features. The glycophorin C mRNA, however, is absent from adult liver and lymphocytes, indicating that this protein represents a new erythrocyte-specific probe which might be useful to study erythroid differentiation.     

Reaction of ozone with glycophorin in solution and in lipid vesicles.

Glycophorin from human red blood cells was exposed to ozone in aqueous solution. Amino acid analysis of glycophorin exposed to a 10-fold molar excess of ozone showed that the only residue affected was methionine. Both methionine residues of the protein were oxidized to methionine sulfoxide. Exposure of the oxidized protein to cyanogen bromide caused no cleavage of the polypeptide chain. Glycophorin was incorporated into unilamellar lipid vesicles made from phosphatidylcholine. The protein containing vesicles were exposed to ozone in a 10-fold molar excess to the glycophorin. Gas chromatography of the methyl esters showed negligible change in the fatty acid composition. Amino acid analysis of the ozone-treated protein showed the oxidation of only one methionine residue per polypeptide chain to methionine sulfoxide. Ghosts of human erythrocytes were exposed to ozone. Cyanogen bromide treatment of the oxidized glycophorin yielded fragments showing that the only methionine residue oxidized by ozone was residue 8. These results indicate that in this membrane model (a) amino acid is more susceptible to ozone than is the lipid, and (b) amino acids external to the membrane are more susceptible than those in the polypeptide chain spanning the membrane.     

The interaction of human glycophorin with 8-anilino-1-naphthalene sulfonate.

Both the sialoglycoprotein of human erythrocyte membranes, glycophorin, and the sialic acid free protein, obtained by treatment of glycophorin with neuraminidase (EC 3.2.1.18), increase the fluorescence of 8-anilino-1-naphthalene sulfonate (ANS). Binding of ANS to glycophorin is weak compared with the binding to bovine serum albumin (BSA). equilibrium dialysis gives an apparent binding constant of about 4 X 10(3) M(-1) at neutral pH, but Ka increases 1.75 times when NaCl or CaCl2 are added and 10-fold when the pH is lowered to 3.0. Sialic acid groups do not significantly affect ANS binding, although they have some effect at low ionic strength and neutral pH. Fluorescence studies indicate only one to two binding sites for ANS, with apparent pK = 3.8 +/- 0.2, and located close to aromatic residues in glycophorin. Polarization and quantum efficiency of the fluorescence of ANS associated with glycophorin fail to indicate changes in the vicinity of the binding site when the pH is lowered.     

Effect of sodium deoxycholate on 5'-nucleotidase.

The 5'-nucleotidase localized in rat liver plasma membranes was purified to a single protein, which contained phospholipid. The molecular weight and the sedimentation constant were about 150 000 and 7 S in the presence of sodium deoxycholate, while the enzyme protein was aggregated when the preparation was dialyzed thoroughly. The purified 5'-nucleotidase exhibited the same properties as the 5'-nucleotidase in plasma membranes. The 5'-nucleotidase activity was increased by the addition of various bile salts or by the solubilization of membranes with trypsin, papain or phospholipase C. The solubilized and aggregated forms of the enzyme showed different substrate specificity for nucleotides, pH optimum, heat stability and Km. The purified enzyme catalyzed an exchange reaction between AMP and adenosine, which was diminished by the addition of sodium deoxycholate.     

Subunit structure of human erythrocyte glycophorin A.

Glycophorin A is a sialoglycoprotein isolated from human erythrocyte membranes which seems to exist as stable dimeric complexes in the presence of sodium dodecyl sulfate. When analyzed by dodecyl sulfate acrylamide electrophoresis this molecule forms two PAS-stainable bands (PAS-U and PAS-2) which are reversibly interconvertible. This change in electrophoretic mobility is dependent on the concentration of dodecyl sulfate, the use of Trisbuffer systems, the protein concentration in the incubation mixture, and the duration and temperature of incubation before electrophoresis. Reducing agents do no influence the results. Chromatography of the sialoglycopeptides on Sepharose columns in dodecyl sulfate before and after heat treatment gave similar results. A small hydrophobic peptide (T-6) derived from glycophorin A was able to prevent reassociation of the monomeric subunits back to the higher molecular weight form. This peptide was able to bind to the subunit of glycophorin A, but not to the high molecular weight complex. These results are consistent with a model of glycophorin A composed of two subunits which can dissociate and reassociate in the presence of detergents. These subunits may interact via the hydrophobic portions of the polypeptide chains.     

Interaction of tubulin with octyl glucoside and deoxycholate. 1. Binding and hydrodynamic studies

Tubulin purified from calf brain cytoplasm, normally a compact water-soluble dimer, is able to interact with the mild detergents octyl glucoside (a minimum of 60 detergent molecules) and deoxycholate (95 +/- 8 molecules). Binding is cooperative and approaches saturation below the critical micelle concentration of the amphiphiles. Binding is accompanied by a quenching of the intrinsic protein fluorescence, but no spectral shape changes indicating denaturation such as in the case of sodium dodecyl sulfate are observed. Glycerol, which is known to be preferentially excluded from the tubulin domain and to favor the folded and associated forms of this protein, inhibits the binding of the mild detergents. Octyl glucoside induces a rapidly equilibrating tubulin self-association reaction characterized by a bimodal sedimentation velocity profile with boundaries at approximately 5 and 12 S. Full dissociation of this detergent restores the normal sedimentation behavior to 90% of the protein. Binding of deoxycholate slows the sedimentation velocity of tubulin from s(0)20,w = 5.6 +/- 0.2 S to s(0)20,w = 4.8 +/- 0.3 S. Measurements of the molecular weight of the tubulin-deoxycholate complex indicate an increase from 100,000 to 143,000 +/- 5,000. The diffusion rate consistently decreases from (5.3 +/- 0.5) X 10(-7) to (3.8 +/- 0.2) X 10(-7) cm2 S-1. This is most simply interpreted as an expansion of the undissociated tubulin dimer upon detergent binding (a change in the frictional ratio, f/f min, from 1.35 to 1.86). It is concluded that tubulin shows a reversible transition between the water-soluble state and amphipathic detergent-bound forms which constitute a model system of tubulin-membrane interactions.     

Erythrocyte spectrin. Purification in deoxycholate and preliminary characterization.

Erythrocyte spectrin, isolated by aqueous extraction of erythrocyte ghosts, may be freed from contaminating membrane lipids and small amounts of other proteins by gel chromatography in 5 or 10 mM deoxycholate. The purified protein, in deoxycholate, is a mixture of monomers and dimers, both highly asymmetric molecules. The hydrodynamic properties of the dimer closely resemble those of muscle myosin, and spectrin and myosin also have similar circular dichroism spectra. The proportion of dimer to monomer in the purified protein varies from one preparation to another, an observation for which there is no simple explanation. In the absence of deoxycholate, spectrin associated beyond the dimer stage, possibly by loose end-to-end aggregation involving hydrophobic forces.     

Interaction of human serum apolipoprotein B with sodium deoxycholate

Preparation of apolipoprotein B (Apo B)-deoxycholate (DOC) complexes by gel filtration chromatography in the presence of 20 mM DOC, pH 8.5, gave two populations of particles with 5% (peak I) and 13% (peak II) lipid remaining bound. These complexes were initially shown to be very large and elongated, with partition radii of approx. 131 +/- 0.5 A, weight average molecular weights of approx. 164 000 +/- 1 000, and an intrinsic viscosity of 80.19 +/- 2.21 ml/g. Additionally, they appeared very similar to native low-density lipoprotein on sodium dodecyl sulfate-polyacrylamide gels, giving one major band. Incubation of these samples for 10 days under nitrogen at 4 degrees C in the presence of antibiotics and protease inhibitor resulted in dissociation to many smaller subunits. Results of scanning molecular sieve chromatography and analytical ultracentrifugation showed that dissociation of these complexes was relatively slow and indicated the presence of at least two classes of components in fresh samples: one a very elongated complex with a radius directly correlated to the DOC/Apo B ratio and inversely correlated to sample aging; and another of much smaller radius which was independent of DOC/Apo B ratio but directly correlated to sample aging; indicating that these dissociated subunits interact with each other to an appreciable extent. Furthermore, these complexes were found to undergo a preferential hydration upon interaction with DOC, which may contribute to large changes in their effective specific volumes, as well as to dissociation of subunits.     

Protein chromatography on adsorbents with hydrophobic and ionic groups. Purification of human erythrocyte glycophorin.

Human erythrocyte glycophorin was purified rapidly by (a) chromatography of a Triton X-100 extract of erythrocyte 'ghosts' on N-(3-carboxypropionyl)aminodecyl-Sepharose in buffers containing Triton X-100 or sodium dodecyl sulphate, or (b) chromatography of whole 'ghosts', solubilized in sodium dodecyl sulphate, on dodecyl-Sepharose, in buffers containing sodium dodecyl sulphate. The products contained 85-95% glycophorin (electrophoretic band PAS-1) and the major contaminants were glycoproteins PAS-2 (possibly a subunit of glycophorin) and PAS-3.     

Interaction of fatty acid sodium salts with sodium deoxycholate

Interaction of homologous fatty acids (C3-C18) with sodium deoxycholate was investigated. From NMR and ultrasonic results it was found that short chain homologues (up to C9) do not participate in the formation of mixed micelles with sodium deoxycholate. Fatty acid homologues with longer chains (starting with C9) form mixed micelles by "burying" hydrophobic chains in hydrophobic environment of a sodium deoxycholate micelle.