Immunoabsorption of membrane-specific antibodies for determination of exposed and hidden proteins in human erythrocyte membranes.

Human erythrocyte membrane proteins solubilized with the non-ionic detergent Berol EMU-043 have been characterized by crossed immunoelectrophoresis with rabbit antibodies raised against the membrane material. Three out of sixteen membrane-specific immunoprecipitates disappeared when the antisera were first absorbed with intact erythrocytes. This finding indicates that three antigens are exposed on the outside of the erythrocyte membrane. One of these antigens showed acetylcholinesterase activity, and another was the major glycoprotein (glycophorin) as shown by crossed-line immunoelectrophoresis. No antigenic determinants of the latter protein were detected within the membrane or on its inner surface. In crossed immunoelectrophoresis with antisera after absorption with washed, non-sealed membranes only one precipitate remained. This precipitate corresponded to albumin. Accordingly, several proteins seem to have antigenic determinants exposed on the inside of the membrane.     

Immunoabsorption of membrane-specific antibodies for determination of exposed and hidden proteins in human erythrocyte membranes

Human erythrocyte membrane proteins solubilized with the non-ionic detergent Berol EMU-043 have been characterized by crossed immunoelectrophoresis with rabbit antibodies raised against the membrane material. Three out of sixteen membrane-specific immunoprecipitates disappeared when the antisera were first absorbed with intact erythrocytes. This finding indicates that three antigens are exposed on the outside of the erythrocyte membrane. One of these antigens showed acetylcholinesterase activity, and another was the major glycoprotein (glycophorin) as shown by crossed-line immunoelectrophoresis. No antigenic determinants of the latter protein were detected within the membrane or on its inner surface.In crossed immunoelectrophoresis with antisera after absorption with washed, non-sealed membranes only one precipitate remained. This precipitate corresponded to albumin. Accordingly, several proteins seem to have antigenic determinants exposed on the inside of the membrane.     

Separation of the proteins of bovine milk-fat-globule membrane by electrofocusing with retention of enzymatic and immunological activity

1. Proteins of fat-globule membrane from bovine milk were solubilized with the non-ionic detergent Triton X-100 in the presence of protease inhibitors. Approximately 25% of the total membrane protein was solubilized and the extracts were shown to contain a sample of most of the major membrane proteins and glycoproteins. 2. The solubilized proteins were separated in flat-beds of Ultrodex by electrofocusing and the pI values for the major proteins, glycoproteins and certain enzymes determined. Several of the proteins displayed marked heterogeneity indicating the existence of protein variants and isoenzymes. Principal pI values for the enzymes assayed were as follows: xanthine oxidase, 7.35--7.55; NADH2: iodonitrotetrazolium reductase, less than 4.5; 5'-nucleotidase, 7.15--7.4; alkaline phosphatase, 5.4--5.7; phosphodiesterase, 4.6--4.8; gamma-glutamyl transpeptidase, 4.4--4.55. 3. Fractions after electrofocusing were analyzed by 'fused rocket' immunoelectrophoresis and crossed immunoelectrophoresis after separation in polyacrylamide gels containing sodium dodecyl sulphate. Major antigens of the membrane include xanthine oxidase and glycoproteins of apparent molecular weights 67 000, 49 500 and 46 000. The latter two components share common antigenic determinants and could not be separated by gel filtration, ion-exchange chromatography, lectin-affinity chromatography or preparative electrofocusing.     

The major sialoglycoprotein of the human erythrocyte membrane. Release with a non-ionic detergent and purification.

The major sialoglycoprotein of the human erythrocyte membrane has been selectively released by the non-ionic detergent Tween 20 and further purified in detergent-free buffers by hydroxyapatite chromatography and, finally, by hydrophobic interaction chromatography on pentyl-Sepharose. The purified glycoprotein shows one main zone, PAS-1, and up to three minor zones after staining both for protein and carbohydrate in polyacrylamide gel electrophoresis in the presence of dodecyl sulfate. The relative staining intensities are concentration dependent. When the purified glycoprotein has been heated to 100 degrees C in dodecyl sulfate, more stain appears in the most rapid zone, PAS-2, and less in the slower zones, indicating a disaggregation of oligomeric forms of this glycoprotein, including a dimer, PAS-1.     

The major sialoglycoprotein of the human erythrocyte membrane. Release with a non-ionic detergent and purification

The major sialoglycoprotein of the human erythrocyte membrane has been selectively released by the non-ionic detergent Tween 20 and further purified in detergent-free buffers by hydroxyapatite chromatography and, finally, by hydrophobic interaction chromatography on pentyl-Sepharose. The purified glycoprotein shows one main zone, PAS-1, and up to three minor zones after staining both for protein and carbohydrate in polyacrylamide gel electrophoresis in the presence of dodecyl sulfate. The relative staining intensities are concentration dependent. When the purified glycoprotein has been heated to 100 °C in dodecyl sulfate, more stain appears in the most rapid zone, PAS-2, and less in the slower zones, indicating a disaggregation of oligomeric forms of this glycoprotein, including a dinier, PAS-1.     

Identification of immunologically distinct antigens in pseudorabies virus

Antigenic proteins of pseudorabies viruses (PrV) are poorly understood. Proteins from purified PrV and membrane proteins from these viral infected cells, therefore, have been studied by antigenic analysis, using virus neutralization and agargel immunoelectrophoresis tests and by polyacrylamide gel electrophoresis, respectively. The study of crossed immunoelectrophoresis against specific antiviral serum antibodies revealed four immunologically distinct antigens involved in PrV. According to their electromobilities, these four immunologically distinct antigens were designated as Ag 1, Ag 2, Ag 3 and Ag 4. The study of dodecyl sulfate-polyacrylamine gel electrophoresis of a membrane-bound but detergent solubilized viral antigenic complex from PrV infected cells also demonstrated the involvement of four glycoprotein antigens. By interpolations of relative mobilities between known protein markers, the molecular weights of these four glycoproteins were estimated to be 61,500, 68,000, 75,000, and 88,000. Results from two dimentional immunoelectrophoresis seemed to be concordant with those obtained by dodecyl sulfate-polyacrylamide gel electrophoresis. This report, therefore presents results, which strongly suggest antigenic similarities in the virion of PrV and cellular membrane glycoproteins of cells infected by this agent. The molecular weight of these four immunologically distinct antigens, Ag 1, Ag 2, Ag 3 and Ag 4, are presumed to have the following molecular weights of 88,000, 75,000, 68,000 and 61,500, respectively.     

Cell membrane IgD: demonstration of IgD on human lymphocytes by enzyme-catalyzed iodination and comparison with cell surface Ig of mouse, guinea pig, and rabbit.

A substantial fraction of human cord blood and peripheral blood lymphocytes have recently been shown to bear IgD. Although IgD has not been identified in mice, it has been suggested that it is also a major surface immunoglobulin of murine lymphocytes. Thus, lactoperoxidase-catalyzed iodination of surface immunoglobulin of mouse spleen and lymph node cells reveals the existence of an IgH chain differing from mu, gamma, and alpha-chain both antigenically and by mobility on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. This new H chain class has been previously proposed to be the mouse homologue of delta-chain. In this paper, we analyzed human, mouse, guinea pig, and rabbit lymphoid cell membrane Ig by lactoperoxidase-catalyzed iodination, extraction with non-ionic detergent precipitation with a variety of specific anti-Ig sera, and electrophoresis of dissolved reduced precipitates on sodium dodecyl sulfate-polyacrylamide gels. Our studies confirm the previous reports of a new mouse cell membrane H chain with a mobility more rapid than that of mu-chain. However, we fail to detect a molecule with this electrophoretic mobility on the surface of guinea pig or rabbit lymph node and spleen cells. Moreover, neither anti-kappa nor anti-delta antibody precipitates a molecule with an H chain of this mobility from labeled extracts of human cord blood or peripheral blood lymphocytes. Cell surface delta was identified on both human cord blood and peripheral blood lymphocytes, but it proved to have mobility similar to human and mouse mu-chain. This result indicates either that mouse delta-chain has an electrophoretic mobility on sodium dodecyl sulfate-polyacrylamide gels which differs appreciably from that of human membrane delta-chain or that the newly described mouse H chain is not the homologue of human delta-chain.     

Characterization of sodium dodecyl sulfate-stable Staphylococcus aureus bacteriolytic enzymes by polyacrylamide gel electrophoresis.

Profiles of the bacteriolytic activities of Staphylococcus aureus culture supernatants, sodium dodecyl sulfate cell extracts, LiCl cell extracts, cell wall extracts, and cell membranes were analyzed in sodium dodecyl sulfate-polyacrylamide gels containing Micrococcus luteus or S. aureus. A total of 20 distinct bands of bacteriolytic activity could be detected in gels containing M. luteus, 8 of these bands were found in culture supernatants. The sodium dodecyl sulfate cell extracts, the LiCl cell extracts, and the cell membranes each contained 20 bands (P1 to P20), but no activity was found in cell wall extracts. Less bacteriolytic activity could be detected in gels containing S. aureus, although three bands were found in culture supernatants and LiCl extracts and cell membranes contained one major band, P13. Crude cell extracts showed five bacteriolytic bands of which the major bacteriolytic bands were distributed in an identical manner in all 10 strains of S. aureus studied. The effects of chemical and physical factors were determined, and it was shown that iodoacetic acid, Hg2+, and Cibacron Blue 3G-A reduced activity, and an optimum pH for enzyme detection was between 7 and 8. Preincubation at 100 degrees C for 30 min reduced the activity of P1 and P2 bands.     

Membrane composition and virus susceptibility of Acholeplasma laidlawii.

The membrane composition of 11 strains of Acholeplasma laidlawii, including three strains persistently infected with mycoplasmaviruses MVL51, MVL2, and MVL3, was studied and correlated with mycoplasmavirus sensitivity. Membranes of the strains had similiar sodium dodecyl sulfate-polyacrylamide gel electrophoresis patterns, and all strains were inhibited by an antiserum produced against membranes from one of the strains. The amounts of integral membrane proteins solubilized by the nonionic detergent Tween 20 differed considerably. Therefore, characteristic crossed immunoelectrophoresis patterns were obtained for each strain. Strains persistently infected with MVL2 and MVL3 were notably different from the noninfected host. The ability to propagate any of the viruses was not correlated with sodium dodecyl sulfate-polyacrylamide gel electrophoresis or crossed immunoelectrophoresis patterns. The persistently infected strains had a characteristic lipid composition. MVL51-resistant strains, including a resistant clone selected from a sensitive strain, were characterized by a large monoglucosyldiglyceride/diglucosyldiglyceride ratio and trace amounts of diphosphatidylglyceol (as opposed to the sensitive strains). Differences in lipid composition in A. laidlawii seem to affect the relationship between cells and viruses.     

Crossed immunoelectrophoresis from sodium dodecyl sulfate-polyacrylamide gels into antibody-containing agarose: an improved method for evaluation of the number of immunochemical determinants in polypeptides, with reference to spectrin.

A simple method is described for performing crossed immunoelectrophoresis into antibody-containing agarose when the first-dimension gel contains peptides separated by electrophoresis in sodium dodecyl sulfate. Artifacts produced by sodium dodecyl sulfate are avoided by incorporation of Triton X-100 in the agarose layer. Peptides are located by prestaining (before SDS-acrylamide electrophoresis) with the cycloheptylamylose complex of fluorescamine. Injection of ink into prestained peptide bands produces a line extending from the peptide band location to its precipitin arc, thereby allowing unambiguous assignment of arcs to peptides in situations where peptide bands are not widely separated. The utility of this procedure is illustrated for the erythrocyte membrane protein spectrin.     

Complexes of chromogranin A and dopamine beta-hydroxylase among the chromogranins of the bovine adrenal medulla

1. The core proteins of chromaffin granules have been examined by polyacrylamide gel electrophoresis and crossed immunoelectrophoresis against monospecific antisera. 2. Dopamine beta-hydroxylase (dopamine beta-monooxygenase, EC 1.14.17.1) appeared as the major immunogen of the core proteins and accounted for 4 and 8% by weight of the crude lysate and membrane-containing fractions, respectively. 3. The non-ionic detergent, Berol, solubilized dopamine beta-hydroxylase from the membranes in a form which was immunologically identical but of lower relative mobility by crossed immunoelectrophoresis. In the absence of detergent a difference in relative mobility was also noted between the purified enzyme and that contaminated by chromogranin A. These observations suggest that several molecular forms of dopamine beta-hydroxylase may occur which differ in size and/or charge due to interactions with the contaminants under the experimental conditions. 4. The main chromogranin in the crude lysate was absent from electropherograms of the acidic chromogranins (95--96% of total protein in lysate). These were obtained free of dopamine beta-hydroxylase by concanavalin A adsorption at high ionic strength or by acidification in 2 M acetic acid. The main band reappeared upon recombination with dopamine beta-hydroxylase, indicating the presence of some dopamine beta-hydroxylase, possibly as dimers, in this main, chromogranin A band. A protein concentration-dependent aggregate of dopamine beta-hydroxylase-free chromogranin A was detected, with a relative mobility slightly faster than the main band of the crude lysate.     

Subunits of the calcium ion-pump system of sarcoplasmic reticulum.

Sarcoplasmic reticulum membranes with high content of Ca2+ -ATPase (80% of total protein) were dissolved in a non ionic medium and were submitted to isoelectric focusing in polyacrylamide gels. The membrane protein was resolved into six main bands whose isoelectric points range from 6 to 5. The mol. wt. of these peptides is about 100 000 as estimated by second dimension electrophoresis in sodium dodecyl sulfate-polyacrylamide system. The electrophoretic behaviour of the purified ATPase enzyme is similar to that of crude membranes.     

Characterization of an orthorhombic crystal form of iron-containing superoxide dismutase from Escherichia coli B.

Human erythrocytes were treated with the diazonium salt of oligodeoxythymidylic acid 5′-p-aminophenylphosphate, a reagent that does not penetrate the plasma membrane. Ghosts were isolated, and the oligomers, covalently linked at their 5′ ends to the outer surface of the membrane, were extended by treatment with terminal deoxynucleotidyl transferase in the presence of deoxythymidine triphosphate. The membranes were dissolved in sodium dodecyl sulfate, and complexes containing cell surface components were isolated by hybridization to polyriboadenylic acid-agarose. The cell surface components were regenerated by treatment with nuclease P₁ in the presence of Triton X100. Sodium dodecyl sulfate/polyacrylamide gels of the regenerated material showed bands III, PAS-1, PAS-2, and PAS-3, i.e. the major proteins known to be accessible at the outer surface of the human erythrocyte. The method should be useful for the isolation of surface components in other cell types.     

Studies on the brush border membrane of the mouse duodenum. I. Membrane isolation and analysis of protein components.

Brush border membranes have been isolated from villus epithelial cells of the adult Swiss mouse duodenum. Preparations of these membranes are not contaminated by other organelles as judged from electron-micrographs of sectioned pellets of brush borders. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of proteins from brush borders solubilized in Tris-sodium dodecyl sulfate buffer reveals a reproducible Coomassie Brilliant Blue pattern of 17 bands. By comparing the brush border protein band positions with those of standard proteins run concurrently on sodium dodecyl sulfate-polyacrylamide gel slabs it is estimated that the 17 brush border proteins and subunits have molecular weights ranging from over 250,000 to around 16,000. Periodate-fuchsin sulfite staining shows that the five more slowly migrating, high molecular weight proteins are glycoproteins. The two proteins of smallest molecular size react positively with Oil Red O but have very small amounts of lipophilic amino acid residues, which indicates that the lipid extractable from the gels in these areas is a contaminant and is not bound to the proteins.     

Alcian blue as a protein stain for sodium alkyl sulfate-polyacrylamide gels: Application to analysis of polypeptide components of the human platelet

The cationic dye, Alcian blue, previously used as a glycoprotein-specific stain on cellulose acetate and polyacrylamide gels, was found to be capable of staining a variety of purified proteins and each of the components of the human platelet presently identifiable with Coomassie blue R or periodic acid-Schiff (PAS) reagent in sodium alkyl sulfate-polyacrylamide gel electrophoretic preparations. Evidence was obtained to indicate that staining of detergent-protein complexes by Alcian blue occurs by virtue of the affinity of the stain for accessible sulfate groups of detergent molecules, especially sodium tetradecyl sulfate, hydrophobically associated with polypeptide chains. Thus, Alcian blue fails to stain nonglycosylated proteins when pure sodium dodecyl sulfate (C₁₂) is used as the detergent, but does so readily when small quantities of sodium tetradecyl sulfate are also present. The advantages of using Alcian blue to determine platelet protein composition and to make quantitative comparisons between bands in sodium alkyl sulfate gels are discussed.     

Isolation, purification, and partial characterization of platelet membrane glycoproteins IIb and IIIa

Platelet membrane glycoproteins IIb and IIIa were isolated and purified from human platelet membranes using lentil lectin affinity chromatography and electrophoretic elution from sodium dodecyl sulfate-polyacrylamide gels. Two-dimensional immunoelectrophoresis of a mixture of the purified proteins against monospecific antisera showed antigenic uniqueness of the separate polypeptides. Computerized analysis of autoradiographs of two-dimensional tryptic 125I peptide maps revealed that the two glycoproteins had completely different structures. Monospecific anti-glycoproteins IIb and IIIa Fab'2 fragments, either singly or in combination, induced platelet agglutination but did not inhibit or alter the platelet aggregation response to physiologic stimuli. The results demonstrate that human platelet membrane glycoproteins IIb and IIIa are separate molecular entities. In the native state, the membrane macromolecular IIb.IIIa complex may play an important role in mediating platelet-platelet interactions.     

Isolation and characterization of cytoplasmic membranes and chlorosomes from the green bacterium Chloroflexus aurantiacus.

A method was developed which allows the isolation and purification of cytoplasmic membranes and chlorosomes from cells of Chloroflexus aurantiacus grown under different light conditions. The dipolar ionic detergent Deriphat (0.08%) and a sodium iodide gradient centrifugation were used in isolating cytoplasmic membranes. Chlorosomes were prepared with 0.16% of the dipolar ionic detergent Miranol and purified by a sucrose gradient centrifugation. Cytoplasmic membrane fractions prepared from either high- (3,000 W m-2), medium-(200 W m-2) or low- (7 W m-2) light-grown cells had near infrared absorption bands at 866, 808, and 755 nm in a constant characteristic absorbance ratio of 6:3.8:1. In all cytoplasmic membrane preparations, the amount of bacteriochlorophyll a (Bchl a) per cytochrome, the amount of Bchl a per reaction center, and reaction center per milligram of cytoplasmic membrane protein was found to be constant. No Bchl c was present. Five respiratory enzyme activities have been measured in the cytoplasmic membrane fraction. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of denatured cytoplasmic membrane showed many bands, but a major polypeptide with an apparent molecular weight of 8,000. In contrast, sodium dodecyl sulfate-polyacrylamide gel electrophoresis of purified chlorosomes did not contain the 8,000-molecular-weight band but revealed only three distinct protein bands with molecular weights of 15,000, 12,000, and 6,000. Isolated chlorosomes contained Bchl c and a small, yet constant, amount of Bchl a (absorbing at 790 nm) in a molar ratio of 25:1. The data indicated that the components of the photosynthetic apparatus in the cytoplasmic membrane of Chloroflexus aurantiacus remained constant and only the amount of antenna Bchl c varied with light conditions.     

Spectrin band 1 and 2 are antigenically distinct and are not composed of subunits.

Human erythrocyte membranes and freshly isolated spectrin were separated into their constituent peptides by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The peptides were electrophoresed from slices of such gels into agarose gels containing anti-spectrin antibodies and Triton X-100. In fresh preparations, precipitin arcs were observed only against peptides migrating as bands 1 and 2. It was found that bands 1 and 2 did not cross-react. There were two major arcs from band 1 and one principal arc from band 2, plus minor splitting of these arcs. None of the band 1 arcs fused with band 2 arcs. In fresh erythrocyte ghosts only bands 1 and 2 reacted with anti-spectrin; bands 2.1, 3, and 5, in particular, showed no precipitin arcs. However, in aged ghosts, arcs appeared in the band 3 region; in aged isolated spectrin, arcs appeared in the band 2.1 region; and in trypsin-degraded spectrin, reactive species occurred in all molecular weight classes. It is concluded that spectrin has no subunits smaller than 220,000 molecular weight and that bands 1 and 2 are immunochemically distinct.