Detergent-solubilized Membrane Proteins Research Articles

Detection of a Thy-1 precursor in human T lymphoma cell lines

In an attempt to gain insight into the biosynthesis and processing of human Thy-1, rabbit antisera (RαTP) were raised against a synthetic peptide (TP) of 13 amino acid residues of identical amino acid sequence to that of residues 110–122 of the putative Thy-1 precursor deduced from the cDNA sequencing. Immunoblotting assay showed that the IgG fraction of RαTP (RαTP-IgG) recognized the synthetic peptide without demonstrable cross-reactivity with isolated human brain Thy-1 and detergent-solubilized membrane proteins of human brain cells. Immunohistochemical studies showed that both RαTP-IgG and HB-2S-1 (a monoclonal antibody which reacts with both membrane-bound Thy-1 on viable cells and detergent-solubilized Thy-1) stained cells of two human T lymphoma cell lines (HUT-78 and HUT-102) but they did not stain cells of a human B lymphoma cell line (Raji). In contrast, in cell surface indirect immunofluorescence assay, HB-2S-1 reacted with HUT-78 and HUT-102 cells while RαTP-IgG did not. Taken together, these data indicate the existence of a precursor form of human Thy-1 which is processed prior to anchoring to the cell surface.

Phagocytosis in Dictyostelium discoideum is inhibited by antibodies directed primarily against common carbohydrate epitopes of a major cell-surface plasma membrane glycoprotein

Using a water-soluble, reversible biotinylating reagent, we retrieved three surface-exposed proteins from a complex mixture of crude membrane proteins. The compound, sulfosuccinimidyl 2-(biotinamido)ethyl-1-3′-dithiopropionate (sulfo-NHS-SS-biotin), which has a cleavable disulfide bond, was used to label Dictyostelium discoideum amebae. Cells were lysed and a crude membrane preparation was isolated and solubilized with Triton X-100. Biotinylated molecules were bound to immobilized streptavidin and then eluted from the affinity matrix with dithiothreitol. Analysis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed that out of the original complex mixture of detergent-solubilized membrane proteins, three major species at 130, 100, and 77 kDa were specifically bound and eluted with thiol reagents. These three proteins were glycoproteins (gp) since they bound concanavalin A. As demonstrated by one-dimensional peptide mapping, the retrieved gp130 and gp100 also were present in specialized plasma membrane subdomains called contact regions which are regions of cell-cell cohesion isolated from aggregated, developed amebae. This finding provides preliminary evidence that the two proteins may be involved in cell-cell interactions during both the vegetative and aggregation stages of the D. discoideum life cycle. The retrieved gp130 species has a relative mobility on SDS-gels similar to that of gp126, a surface-exposed glycoprotein. gp126 has been suggested to play roles both as a phagocytosis receptor and as a cohesion molecule (C. M. Chadwick, J. E. Ellison, and D. R. Garrod, (1984) Nature (London) 307, 646). To test if the retrieved gp130 was the same as gp126, a polyclonal antiserum was raised against gelpurified, endoglycosidase F-treated gp130. The immune serum recognized epitopes, apparently carbohydrates, present on many D. discoideum membrane proteins. Univalent IgG fragments from this antiserum inhibited phagocytosis, suggesting that anti-carbohydrate activity was responsible for the functional inhibition of phagocytosis.

Neutron small angle scattering of micelles from detergents suitable for the solubilization of native membrane proteins

The dependence of the neutron small angle scattering of aqueous detergent solutions on the detergent concentration has been investigated. It has been shown that preferably the bean-shaped bile acids form micelles, which are so small that they do not disturb the scattering profile of detergent solubilized membrane proteins

Purification and characterization of the human interferon-gamma receptor from placenta.

Purification of the human interferon-gamma (IFN-gamma) receptor was facilitated by identification of human placenta as a large-scale receptor source. When analyzed in radioligand binding experiments, intact placental membranes and detergent-solubilized membrane proteins expressed 1.3 and 5.9 X 10(12) receptors per mg of protein, respectively, values that were 13-163 times greater than that observed for U937 membranes. Two protocols were followed to purify the IFN-gamma receptor from octyl glucoside-solubilized membranes: (i) sequential affinity chromatography over wheat germ agglutinin- and IFN-gamma-Sepharose and (ii) affinity chromatography over columns containing receptor-specific monoclonal antibody and wheat germ agglutinin. Both procedures resulted in fully active preparations that were 70-90% pure. Purified receptor migrated as a single molecular species of 90 kDa either when analyzed on silver-stained NaDodSO4/polyacrylamide gels or when subjected to electrophoretic transfer blot analysis using a labeled IFN-gamma receptor-specific monoclonal antibody. The identity of the 90-kDa component as the receptor was confirmed by demonstrating its ability to specifically bind 125I-labeled IFN-gamma following NaDodSO4/PAGE and transfer to nitrocellulose. Certain receptor preparations converted into a 55-kDa fragment either during purification or upon storage at 4 degrees C. On the basis of N-Glycanase digestion studies, the IFN-gamma receptor appeared to contain 17 kDa of N-linked carbohydrate. The ligand binding site, the epitope for the receptor-specific monoclonal antibody, and all of the N-linked carbohydrate could be localized to the 55-kDa domain of the molecule.

Structure and dynamics of a detergent-solubilized membrane protein: measurement of amide hydrogen exchange rates in M13 coat protein by 1H NMR spectroscopy.

The coat protein of bacteriophage M13 is inserted into the inner membrane of Escherichia coli where it exists as an integral membrane protein during the reproductive cycle of the phage. The protein sequence consists of a highly hydrophobic 19-residue central segment flanked by an acidic 20-residue N-terminus and a basic 11-residue C-terminus. We have measured backbone amide hydrogen exchange of the protein solubilized in perdeuteriated sodium dodecyl sulfate using 1H nuclear magnetic resonance (NMR) spectroscopy. Direct proton exchange-out measurements in D2O at 24 degrees C were used to follow the exchange of the slowest amides in the protein. Multiple exponential fitting of the exchange data showed that these amides (29 +/- 3 at pH 4.5) exchanged in two kinetic sets with exchange rates [(1.2 +/- 0.4) x 10(-4) s-1 and (4.1 +/- 1.2) x 10(-7) s-1] that differed by more than 100-fold, the slower kinetic set being retarded 10(5)-fold relative to poly(DL-alanine). The exchange rate constant for the slowest set of amides exhibited an unusual pD dependence, being proportional to [OD-]1/2. It is shown that this is an artifact of the multiple exponential fitting of the data, and a new method of presentation of exchange data as a function of pD is introduced. Steady-state saturation-transfer techniques were also used to measure exchange. These methods showed that 15-20 amides in the protein are very stable at 55 degrees C and that about 30 amides have exchange rates retarded by at least 10(5)-fold at 24 degrees C. Saturation-transfer studies also showed that the pH dependence of exchange in the hydrophilic termini was unusual. This is explained as being due to long-range electrostatic effects arising both from the protein itself and also from the anionic detergent molecules. Hydrogen exchange studies on the products of proteinase K digestion of the protein localized the slowly exchanging amides to the hydrophobic core of the protein. Relaxation [Henry, G.D., Weiner, J.H., & Sykes, B.D. (1986) Biochemistry 25, 590-598] and solid-state NMR experiments [Leo, G.C., Colnago, L.A., Valentine, K.G., & Opella, S.J. (1987) Biochemistry 26, 854-862] have previously shown that the majority of the protein backbone is rigid on the picosecond to microsecond time scale, except for the extreme ends of the molecule which are mobile.(ABSTRACT TRUNCATED AT 400 WORDS)

The use of high-performance liquid chromatography for the determination of size and molecular weight of proteins: A caution and a list of membrane proteins suitable as standards

We propose a list of 15 water-soluble globular proteins and 13 forms of detergent-soluble membrane proteins of known Stokes radii for the calibration of high-performance liquid chromatography columns. It is shown that it is advisable to use different sets of standards for these two types of proteins as the detergent-solubilized membrane proteins may behave differently, being excluded or retarded, depending upon the gel support. A smooth, although nonlinear, relationship between Stokes radii and erf −1(1 − K D ) is observed while a large scatter of points exists if the calibration is expressed as the molecular weight as a function of K D .

Visualization of lactotransferrin brush-border receptors by ligand-blotting

The uptake of iron (III) mediated by lactotransferrin to human biopsies from upper intestine has suggested the presence of specific receptors for human lactotransferrin at the brush border (Cox, T., Mazurier, J., Spik, G., Montreuil, J. and Peters, T.J. (1979) Biochim. Biophys. Acta 588, 120–128). In the present data, using 125I-radiolabeled transferrins, we have demonstrated that a preparation of microvillous membrane vesicles, from rabbit jejunal brush-border specifically binds human lactotransferrin. This binding is specific, saturable and calcium dependent. Scatchard plots analysis of lactotransferrin binding indicates 1.5 · 10 13 sites per mg of membrane proteins with an equilibrium constant of 1.2 · 10 6 M −1. Sodium dodecyl sulfate solubilization of the brush-border proteins allows the lactotransferrin receptor to retain its binding activity. Moreover, the ligand blotting of the detergent solubilized membrane proteins on nitrocellulose sheet and after incubation with 125I-labeled lactotransferrin, has shown that the receptor is a protein of about 100 kDa. In the same experimental conditions, the rabbit microvillous membrane vesicles do not specifically bind rabbit serotransferrin indicating the absence of serotransferrin receptors at the brush border.

Pre- and postnatal development of differentiated functions in rat intestinal epithelial cells

A panel of monoclonal antibodies to intestinal cell surface components has been used to compare the expression of differentiation-specific antigens in the epithelial cells of fetal, suckling, and adult rat small intestine. Indirect immunofluorescence staining, and immunopurification of detergent-solubilized membrane proteins, followed by single- and two-dimensional slab gel electrophoretic analysis, have demonstrated that fetal intestinal cells (at day 21 of gestation) express most differentiation-specific markers typical of adult absorptive villus cells. A marked heterogeneity in antigen expression was observed among different villus cell populations in suckling rat intestine, and three cell surface components were identified which are exclusively present during this period of intestinal development. STriking changes in the patterns of antigen expression in crypt and villus cells, and variations in the apparent isoelectric points for most luminal membrane components, were associated with the maturation of the intestinal mucosa at weaning. These changes could not be prematurely induced by cortisone injection in newborn rats, suggesting that factors other than glucocorticoids are responsible for the postnatal development of the intestinal epithelium. These results suggest that basic differences in biological properties and regulatory mechanisms exist among intestinal epithelial cells at different stages of pre- and postnatal maturation.

Phenyl-Sepharose-mediated detergent-exchange chromatography: its application to exchange of detergents bound to membrane proteins.

Detergent-saturated phenyl-Sepharose was used to exchange detergents for one another in the presence of membrane proteins. The alkyl detergents lauryl maltoside, octyl glucoside, and dodecyl sulfate were each successfully exchanged for Triton X-100, Triton N-101, or Nonidet P-40 present in a solution of either cytochrome c oxidase, a mixture of inner mitochondrial membrane proteins, or a mixture of erythrocyte membrane proteins. The method involves (1) saturating a small column of phenyl-Sepharose (1-2 mL) with one of the alkyl detergents at a pH of 8 or 9 and an ionic strength of 0.01, (2) applying a detergent-solubilized membrane protein sample containing as much as 20 mg/mL of Triton X-100, Triton N-101, or Nonidet P-40, and (3) eluting the protein with buffer containing the detergent with which the resin had been saturated. With this approach, 90-99% of the detergent in the initial protein sample was exchanged for the second detergent with an 80-100% recovery of protein. The advantages of this method over previous approaches for exchanging detergents include the rapidity of the technique and the apparent general applicability of the method to a wide variety of detergents and membrane proteins.

Characterization of detergent-solubilized membrane proteins. Hydrodynamic and sedimentation equilibrium properties of the insulin receptor of the cultured human lymphoblastoid cell.

Characterization of detergent-solubilized membrane proteins. Hydrodynamic and sedimentation equilibrium properties of the insulin receptor of the cultured human lymphoblastoid cell.

Use of gel chromatography for determination of size and molecular weight of proteins: Further caution

The permeation properties of chromatographic gels with large pore sizes have been examined by the use of selected, water-soluble proteins. We observed that plots of Stokes radius vs erf −1 (1 − K d ) of Sepharose gels were nonlinear, probably reflecting the presence of two classes of pore sizes of the gel. This was also the case for Sephacryl S-300 columns, although to a lesser degree. An apparent anomaly in the elution of hemoglobin could be attributed to dissociation into dimer. The elution of detergent-solubilized membrane proteins on Sepharose columns was somewhat enhanced relative to the calibration curve. On the basis of these results the use and limitations of gel chromatography for estimation of Stokes radius and molecular weight of proteins is discussed.

Identification of the membrane glycoprotein that is the C3b receptor of the human erythrocyte, polymorphonuclear leukocyte, B lymphocyte, and monocyte.

A human erythrocyte membrane glycoprotein of 205,000 mol wt (gp205) has been identified as the C3b receptor of the erythrocyte, polymorphonuclear leukocyte (PMN), B lymphocyte, and monocyte. Initially, gp205 was sought and characterized as a constituent of the human erythrocyte membrane that can impair activation of the alternative complement pathway by inducing loss of function of the properdin-stabilized amplification C3 convertase (C3b,Bb,P) through displacement of Bb from C3b and by promoting cleavage-inactivation of C3b by C3b inactivator. These inhibitory activities of gp205 suggested that this membrane glyeoprotein had an affinity for C3b and prompted an analysis of its possible identity as the C3b receptor of human peripheral blood cells. The F(ab')2 fragment of rabbit IgG anti-gp205 inhibited the formation of rosettes with sheep EC3b of human erythroeytes, B lymphocytes, monocytes and PMN in a dose-response manner; the 50 percent inhibitory doses were 0.13/mug/ml, 0.90 mug/ml, 1.25 mug/ml, and 1.20 mug/ml of F(ab')2, respectively. Anti-gp205 did not impair the formation of rosettes by monocytes and B lymphocytes with sheep EC3bi or with EC3d. Scatchard analysis of the number of specific (125)I-F(ab')(2) anti-gp205 binding sites/cell revealed 950 sites/erythrocyte, 21,000 sites/cell of B lymphocyte preparation, 57,000 sites/PMN, and 48,000 sites/monocyte, indicating that the higher concentrations of antibody that had been required for inhibition of rosette formation by the nucleated cells reflected larger numbers of receptors on these cells. Direct evidence for the identity of gp205 as the C3b receptor of the four cell types was obtained when detergent-solubilized membrane proteins of the surface-radioiodinated cells were reacted with anti- gp205 and the immunoprecipitate was analyzed by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. In each instance, the antigenic material reacting with anti-gp205 represented a single protein with an apparent 205,000 mol wt. Thus, gp205 is the C3b receptor of human erythrocytes, PMN, B lymphocytes, and monocytes.

The induction of virus-specific cytotoxic T lymphocytes with solubilized viral and membrane proteins.

Reconstituted membranes were prepared from detergent solubilized P815 (H-2d) tumor cell membranes and solubilized Sendai virus protein. These reconstituted membranes stimulated a virus-specific H-2-restricted secondary CTL response. Stimulating activity was dependent upon the presence of both viral and P815 protein in the same membrane and was restricted to the H-2 specificity present in the reconstituted membrane. Liposomes prepared from solubilized Sendai virus proteins and partially purified H-2 alloantigen also had activity for CTL induction. The results demonstrate the feasibility of using detergent solubilized membrane proteins to study antigen recognition by virus-specific, H-2 restricted cytolytic T lymphocytes.

Membrane biosynthesis in the frog retina: opsin transport in the photoreceptor cell.

Rhodopsin biosynthesis and transport in the photoreceptor cell have been analyzed by subcellular fractionation of frog retinas after short periods of radioactive amino acid incorporation in vivo. Labelled membrane proteins were identified by autoradiography or sodium dodecyl sulfate-polyacrylamide gels. One of the most intensly labeled proteins in retina had a molecular weight comparable to opsin isolated from purified rod outer segments (ROS). Incorporation of label into this protein was rapid; the relative specific activity then diminished after the first 2 hr as radioactivity was transferred from retinal subcellular fractions to ROS. The kinetics of this transfer resembled rates previously observed by Hall et al. (Hall, M. O., Bok, D., and Bacharach, A.C.E. (1969), J. Mol. Biol. 45, 397). To identify the rapidly labeled protein as opsin we devised a new technique of two-dimensional immunoelectrophoresis of detergent solubilized membrane proteins. Antibodies were prepared against both whole ROS and opsin. After initial separation of retinal proteins on sodium dodecyl sulfate-polyacrylamide gels, a second dimension of electrophoresis in agarose, containing antisera, resulted in the formation of specific immunoprecipitates. Immunochemical analysis of all membranous and soluble retinal subcellular fractions indicated that newly synthesized opsin was membrane bound upon completion of synthesis. At no period of incorporation was a soluble form of newly synthesized opsin detectable. On this basis, we suggest that this protein is apparently transported as a water-insoluble membrane-bound molecule through the cytoplasm or along membranes of the inner segment to its assembly site near the base of the outer segment.