Characterization of glycopeptides isolated from membranes of F9 embryonal carcinoma cells

From cells of a nullipotential line of embryonal carcinoma was isolated a membrane fraction enriched in the cell surface F9 antigen. More than 40% of the radioactive fucose and galactose incorporated by cells into nondialyzable material was recovered in this membrane preparation, corresponding to an approximately 10-fold purification of the labeled material. Extreme heterogeneity of membrane glycoproteins labeled with these sugars was revealed by sodium dodecyl sulfate gel electrophoresis. Glycopeptides prepared by extensive pronase digestion of membranes labeled with fucose or galactose showed properties similar to those already described for fucose-labeled glycopeptides from whole cells. Namely, large glycopeptides eluted near the excluded volume of Sephadex G-50 column were the predominant glycopeptide species, while complex glycopeptides of molecular weight around 2500 were minor components. Therefore, these large glycopeptides, characteristic of embryonal carcinoma cells, are derived mainly from a variety of glycoproteins closely associated with the membrane system, most probably cell-surface membrane of the cells. The large glycopeptides were also significantly labeled with glucosamine, but only slightly with mannose; major components of mannose-labeled glycopeptides from the membranes were high-mannose glycopeptides of low molecular weight. Several experiments excluded the possibility that the larg glycopeptides are mucopolysaccharides, glycolipids or mucin-type glycoproteins with short oligosaccharide chains.     

Identification of the milk fat globule membrane proteins: I. Isolation and partial characterization of glycoprotein B

The salt soluble proteins from the fat globule membrane of cow's milk were resolved into three fractions by Sephadex column chromatography in sodium dodecyl sulfate. One of the fractions, termed glycoprotein B, was purified by rechromatography to essentially one band on sodium dodecyl sulfate gel electrophoresis. It was found to contain 14% carbohydrate including sialic acid, mannose, galactose, glucose, glucosamine and galactosamine. The amino acid composition of glycoprotein B was determined; it has amino terminal serine and carboxyl terminal leucine. The molecular weight of this glycoprotein as estimated by sodium dodecyl sulfate gel electrophoresis is 49 500.     

A lipid-linked oligosaccharide intermediate in glycoprotein synthesis. Characterization of [Man-14C]glycoproteins labeled from [Man-14C]oligosaccharide-lipid and GDP-[14C]Man.

Endogenous proteins of cell-free preparations of hen oviduct labeled from GDP-[14C]Man or from [Man-14C]oligosaccharide-lipid have been compared by sodium dodecyl sulfate polyacrylamide gel electrophoresis. Under the conditions tested, a polypeptide chain of molecular weight about 25,000 was the principle acceptor for the oligosaccharide moiety of exogenous [Man-14C]oligosaccharide-lipid. The product labeled by [Man-14C]oligosaccharide-lipid appeared identical with one of three glycoproteins formed when GDP-[14C]Man was incubated with a crude membrane fraction. These three proteins (apparent molecular weight of 75,000, 55,000, and 25,000) accounted for nearly two-thirds of the [14C]mannose-labeled glycoprotein products using GDP-[14C]Man and either the crude membrane fraction or a total oviduct homogenate. Thus, all of the mannose acceptor proteins present in the oviduct homogenate appear to be membrane-bound. Analyses of the [Man-14C]glycoproteins labeled from GDP-[14C]Man in membrane fractions from hen kidney, liver, brain, and oviduct indicated that a labeled polypeptide of apparent molecular weight 25,000 was the only major protein product common to the four preparations.     

The transmembrane proteins in the plasma membrane of normal human erythrocytes. Evaluation employing lactoperoxidase and proteases.

The molecular architecture of the human erythrocyte membrane has been probed using lactoperoxidase-catalyzed iodination in conjunction with Pronase hydrolysis. Resealed, hemoglobin-free ghosts were labeled at the cytoplasmic surface and the external membrane surface was subsequently digested with Pronase. Changes in size of the components labeled at the cytoplasmic surface were readily detected by sodium dodecyl sulfate gel electrophoresis. The protein 3 molecular weight class labeled at the cytoplasmic surface was extensively hydrolyzed at the external surface to produce a major 65000 molecular weight fragment and a minor 45000 molecular weight fragment. When resealed membranes were labeled on the external surface the same 65000 molecular weight labeled component is produced. These results unequivocally demonstrate that the same polypeptides in the protein 3 molecular weight class that can be labeled by lactoperoxidase at the cytoplasmic membrane surface are digested by Pronase at the external surface and are, therefore, transmembrane components. Where it is possible to label one surface of a membrane with lactoperoxidase and reseal the membrane this procedure represents an alternate method for establishing transmembrane configuration of membrane proteins.     

A nuclear specific glycoprotein representative of a unique pattern of glycosylation

Whole rat liver nuclei were reacted with UDP-[14C]galactose in the presence of bovine beta(1----4) galactosyltransferase. The reaction mixture was electrophoresed on a reducing sodium dodecyl sulfate-polyacrylamide gel. Autoradiograms of the gel demonstrated a major labeled broad band migrating with an apparent molecular weight of 65,000-66,000. A number of other less prominently labeled bands were also present. The labeled 65,000-66,000 band when cut from the gel and subjected to alkaline reduction while in the gel matrix exclusively yielded a 14C-labeled disaccharide that co-migrated with a [14C]Gal-GlcNAcol standard in descending paper chromatography. Treatment of this disaccharide with beta-galactosidase (beta-D-galactoside galactohydrolase; EC 3.2.1.23) from Aspergillus niger removed all the [14C]galactose label. Treatment of the labeled 65,000-66,000 polypeptide with Endoglycosidase F, however, did not remove the [14C]galactose label. Western transfer blots of sodium dodecyl sulfate-polyacrylamide gel electrophoresis gels performed with horseradish peroxidase-labeled succinyl wheat germ agglutinin, a lectin specific for GlcNAc, on unlabeled nuclei revealed a dominant band at 63,000-64,000. Subjecting 14C-labeled nuclei to this procedure resulted in a shift of the major horseradish peroxidase-labeled succinyl wheat germ agglutinin band to 65,000-66,000. The shifted band was coincident with the [14C]galactose band as visualized on an autoradiogram. A survey of other rat tissue nuclei revealed the same spectrum of [14C]galactose acceptor proteins with a dominant 65,000-66,000 galactose-labeled band.     

Glycoproteins from human colonic adenocarcinoma. Isolation and characterization of cell surface carcinoembryonic antigen from a cultured tumor cell line.

Alterations in cell surface glycoproteins have been implicated in malignancy. We examined surface membrane proteins of a cultured cell line, SKCO-1, which had been derived from a human colonic adenocarcinoma. Cell surface labeling of SKCO-1 cells with galactose oxidase, followed by reduction with sodium borotritide, revealed five major labeled glycoproteins upon sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis. At least three additional labeled glycoproteins could be detected if galactose oxidase treatment was preceded by neuraminidase treatment. Some, but not all, of the glycoproteins could be iodinated by lactoperoxidase. The predominantly labeled glycoprotein (GPI) had a molecular weight of 200,000 and co-migrated in SDS gel with carcinoembryonic antigen (CEA). GPI was not removed from the cell surface by EDTA, hypertonic saline, or sonication but was released from the membrane by detergents. This glycoprotein was subsequently purified using lectin-agarose columns and gel filtration. GPI was judged homogenous by protein- and carbohydrate-stained SDS-polyacrylamide gels and had an amino acid composition similar to that of CEA. The carbohydrate composition of GPI was qualitatively similar to CEA but quantitatively distinct. GPI had a greater proportion of sialic acid and galactosamine and less fucose and glucosamine than CEA. Immunological studies, however, demonstrated identity between GPI and CEA. A study of the turnover rate of GPI showed it to have a half-life of 5 days.     

SDS GEL ELECTROPHORESIS OF RAPIDLY TRANSPORTED PROTEINS IN GARFISH OLFACTORY NERVE

Abstract— Proteins undergoing rapid axonal transport in the garfish olfactory nerve were examined by sodium dodecyl sulphate gel electrophoresis. The distribution of polypeptides and the extent of their labeling by transported molecules was determined in several nerve subfractions including: total particulate, total membrane, mitochondrial and two membrane subfractions rich in axolemma. The polypeptide composition of the various fractions was found to be relatively similar, with each showing a major protein with an estimated MW of 58,000. Specific differences in the concentrations of certain proteins were noted between fractions, including differences between the lower and higher density axolemma rich subfractions. Axonally transported radioactivity was predominantly localized among high molecular weight proteins, with all fractions, except mitochondrial pellet, displaying a major peak of radioactivity centered at 126,000-MW. Several major proteins including the 58,000-MW band were labeled by rapid transport to a much smaller extent. Certain labeled peaks were found to be concentrated in individual fractions, particularly a polypeptide (MW 35,000) more predominantly found in the lower density axolemma rich fraction.
Systemic labeling of the nerve is found to give a general distribution of radioactivity on gels, which is clearly different from the pattern obtained after axonal transport labeling.     

Fatty acid-acylated proteins in secretory mutants of Saccharomyces cerevisiae.

Yeast secretory (sec) mutants that are blocked in the transport of secretory proteins and accumulate membrane organelles were used to study the biosynthesis of fatty acid-acylated proteins. Four proteins were labeled with [3H]palmitate in sec mutants accumulating endoplasmic reticulum membranes. Three of these (molecular weights approximately equal to 20,000, 50,000, and 120,000) were N-linked glycoproteins, based on their ability to be labeled with [3H]mannose and their sensitivity to endoglycosidase H. The fourth protein (molecular weight approximately equal to 30,000) also was labeled with [3H]mannose but was insensitive to endoglycosidase H; it appeared to contain O-linked sugars. In sec mutants accumulating Golgi membranes or post-Golgi vesicles, a 35-kilodalton protein was labeled with [3H]palmitate. Analysis of Staphylococcus aureus protease V8 digests and pulse-chase experiments indicated that the 30-kilodalton protein was a precursor of 35 kilodaltons. None of these proteins was labeled with [3H]palmitate in a sec mutant that blocked the penetration of nascent polypeptides into endoplasmic reticulum; thus, acylation occurred in endoplasmic reticulum. All four proteins could be recovered from fractions enriched for yeast membranes. Fatty acids were not released from proteins by boiling in sodium dodecyl sulfate or extraction with organic solvents but were recovered as methyl esters after proteins were treated with KOH-methanol, a reaction characteristic of an acyl ester linkage.     

Subcellular location and identification of a large molecular weight substrate for the liver plasma membrane transglutaminase

When the particulate fraction from a rat liver homogenate was incubated with [3H]putrescine and calcium, the radioactive amine was incorporated into the membranes via a transglutaminase-mediated reaction. Fractionation of the membranes by isopycnic density gradient centrifugation revealed that the radioactive label was coincident with the 5'-nucleotidase and transglutaminase activities which serve as markers for the plasma membrane (Slife, C. W., Dorsett, M. D., Bouquett, G. T., Register, A., Taylor, E., and Conroy, S. Arch. Biochem. Biophys. 241, 329-336). If the labeled membranes were treated with digitonin and fractionated, the radioactivity and the plasma membrane enzyme activities coincidentally shifted to a greater density. Examination of the [3H]putrescine-labeled membranes by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and autoradiography showed that the largest amount of radioactivity was associated with a large molecular weight material that did not enter the acrylamide gel. Pulse-chase experiments indicated that the large aggregate already was present in the native membrane, or that it was formed very rapidly during the putrescine incubation. The complex did not result from putrescine cross-linking between proteins since dansylcadaverine and [3H]histamine were also selectively incorporated into it. These data show that there are protein substrates in the plasma membrane which are accessible to the membrane-associated transglutaminase and that the substrates form a large molecular weight aggregate which is not dissociated by sodium dodecyl sulfate and disulfide reducing agents.     

Isolation and characterization of plasma membrane associated immunoglobulin from cultured human diploid lymphocytes

The lactoperoxidase iodination method was adapted to label surface proteins of cultured diploid human lymphocytes. Membrane associated immunoglobulin of the μ,K type was isolated from WIL₂-A3 cells as well as from their purified membrane preparations by detergent solubilization of labeled membrane proteins and subsequent precipitation with specific antisera. These data indicate that using our conditions all of the labeled immunoglobulin was membrane bound. The molecular weight of the bound molecule was estimated to be 265,000±15,800 by sodium dodecyl sulfate gel electrophoresis and on reduction was separated into proteins with molecular sizes identical to μ and light-chain markers. The combination of two μ and two light chains to give an “IgM monomer” configuration should give a molecular weight of 180,000 to 200,000. Possible reasons for this discrepancy are discussed.     

Specific Removal of Proteins from the Envelope of Escherichia coli by Protease Treatments

When the envelope fraction of Escherichia coli was treated by trypsin, about 40% of total envelope proteins were removed from the fraction without changing its phospholipid content. Analysis of envelope proteins by acrylamide gel electrophoresis in 0.5% sodium dodecyl sulfate revealed that trypsin treatment was very specific; one of the major proteins (molecular weight, 38,000) and all proteins of molecular weight greater than 70,000 were completely removed by the treatment. On the other hand, three other major proteins were found to be resistant to the treatment, including protein Y, which was previously shown to be related to deoxyribonucleic acid replication. The trypsin treatment of the envelope fractions composed of a five electron-dense layered structure formed vesicles with a triple-layered membrane (two electron-dense layers). Pronase treatment of the envelope fraction removed about 60% of the envelope proteins without changing its phospholipid content. A major protein of molecular weight of 58,000 was found to be the only protein resistant to the Pronase treatment. Application of these treatments is useful for purification and structural studies of envelope proteins.     

Isolation and characterization of the putative canalicular bile salt transport system of rat liver

Through labeling with the sodium salt of the photolabile bile salt derivative (7,7-azo-3 alpha,12 alpha-dihydroxy-5 beta-[3 beta-3H]cholan-24-oyl)- 2-aminoethanesulfonic acid, a bile salt-binding polypeptide with an apparent molecular weight of 100,000 was identified in isolated canalicular but not basolateral (sinusoidal) rat liver plasma membranes. This labeled polypeptide was isolated from octyl glucoside-solubilized canalicular membranes by DEAE-cellulose and subsequent wheat germ lectin Sepharose chromatography. The purified protein still contained covalently incorporated radioactive bile salt derivative and exhibited a single band with an apparent molecular weight of 100,000 on sodium dodecyl sulfate-gels. Antibodies were raised in rabbits and their monospecificity toward this canalicular polypeptide demonstrated by immunoblot analysis. No cross-reactivity was found with basolateral membrane proteins. The antibodies inhibited taurocholate uptake into isolated canalicular but not basolateral membrane vesicles. In addition, the antibodies also decreased efflux of taurocholate from canalicular vesicles. If the canalicular bile salt-binding polypeptide was immunoprecipitated from Triton X-100-solubilized canalicular membranes and subsequently deglycosylated with trifluoromethanesulfonic acid, the apparent molecular weight was decreased from 100,000 to 48,000 (sodium dodecyl sulfate-polyacrylamide gel electrophoresis). These studies confirm previous results in intact liver tissue and strongly indicate that a canalicular specific glycoprotein with an apparent molecular weight of 100,000 is directly involved in canalicular excretion of bile salts.     

Purification of a human plasma membrane glycoprotein from human red blood cells by affinity chromatography using a monoclonal antibody

Using the monoclonal antibody LICR-LON-Fib75.1 coupled to Sepharose as an affinity chromatography column, a membrane glycoprotein with an apparent molecular weight of 18,000 on sodium dodecyl sulfate-polyacrylamide gels has been purified from human red blood cells. The purified protein contained 25% carbohydrate by weight, the predominant sugars being galactose, mannose, and glucosamine. Amino acid analysis indicated that the protein was relatively rich in aspartate, glutamate, valine, and leucine and had a low proline and methionine content. The molecule could be removed from intact red blood cells by trypsin and could be labeled with iodine by lactoperoxidase-catalyzed cell surface iodination of red blood cells. The protein could also be labeled using the lipidsoluble photoactivatable reagent 3-(trifluoromethyl)-3-(m-[125I]iodophenyl) diazirine) and partitioned into the lower phase of the phase-separable detergent Triton X-114. During size-exclusion chromatography in different detergents alterations were observed in the apparent molecular weight of the protein. These results suggest that this Fib75.1-binding protein is an external red blood cell membrane glycoprotein which is capable of binding detergent. Proteins with a similar molecular weight have also been isolated from two human tumor cell lines by immunoprecipitation with this monoclonal antibody.     

Limited proteolysis of some egg surface components is an early event following fertilization of the sea urchin, Strongylocentrotus purpuratus.

The surface proteins of eggs from Stronglocentrotus purpuratus were labeled with ¹²⁵I by lactoperoxidase-catalyzed iodination. The eggs were examined after solubilization and disaggregation in sodium dodecyl sulphate (SDS) by electrophoresis on SDS-polyacrylamide slab-gels. Seventy-five percent of the label was found in material with a molecular weight greater than 130,000. About 5% of the radioactivity was excluded from the gels. Upon fertilization, embryos show a redistribution of the radioactively labeled species. There is a decrease in the amount of very high molecular weight material but an increase (35–40%) in material excluded from the gel. In addition, new radioactive bands of lower molecular weight are found. This change of distribution in the radioactive bands is blocked by inclusion of soybean trypsin inhibitor either before or immediately after fertilization, which completely inhibits the cortical granule protease. The disappearance of high molecular weight components is prevented by treatment of the eggs with procaine during fertilization, although the appearance of low molecular weight bands (approximately 20,000 and 30,000) is not completely blocked by procaine treatment. Parthenogenic activation of eggs by butyric acid or partial metabolic activation by ammonia each leads to changes in the egg surface proteins which are similar but not identical to those seen after fertilization. The data suggest that the labeling occurs on the vitelline membrane, plasma membrane and jelly layer. The possible significance of limited proteolysis in fertilization is discussed.     

Application of a new surface labeling reagent, EDTA derivative, on erythrocytes and platelets.

The modes of binding of a new class of impermeant metal-chelating probe, the complex of 111In3+ to 1-(p-benzenediazonium) ethylenediamine tetraacetic acid (azo-phenyl-EDTA), to human and rabbit erythrocyte membranes and the effect of binding on the function of rabbit platelets have been studied. The metal chelate, azo-phenyl-EDTA.[111In3+] bound covalently to membrane proteins following reaction with intact erythrocytes. The amount and the pattern of labeling was assessed by sodium dodecyl sulfate (SDS)-polyacrylamide disc and slab gels for radioactivity. The pattern of labeling of intact human erythrocytes by azo-phenyl-EDTA.[111In3+], by pyridoxal phosphate-NaB3H7 and by galactose oxidase-NaB3H4 was also compared. The following results were obtained: (a) The pattern of labeling of intact human erythrocyte by azo-phenyl-EDTA.[111In3+] differed from other commonly used probes for labeling external membrane surfaces. Five polypeptides were labeled by the metal chelates. In addition to the known major proteins (protein band III, PAS-1, PAS-2 and PAS-3 of Fairbanks et al. (1972) Biochemistry 10, 2606--2617) a protein (radioactive band 4) which migrated slightly slower than PAS-3 in SDS gel was labeled heavily by the metal chelate. This protein has an apparent molecular weight of 37,500 in 8.4% acrylamide-SDS gel. About 40% of bound radioactivity was found in this protein. The diazo linkage of the metal chelate to this protein was found to be especially unstable to heat. (b) In rabbit erythrocyte membranes, the metal chelate bound to three polypeptides with apparent molecular weights of 96,000, 43,000 and 33,000 in 8.4% acrylamide gel. They are probably glycoproteins in nature. (c) The binding of the probe to platelets did not affect the platelet aggregability induced by adenosine diphoshpate. In vivo studies indicated that the labeled platelets accumulated at the plague of atherosclerotic rabbits. (d) The bifunctional analog of EDTA may permit new applications of metals with useful physical properties for studies of cell membranes.     

Characterization of surface glycoproteins of mouse lymphoid cells

We have labeled exposed surface glycoproteins of mouse lymphoid cells by the galactose oxidase-tritated sodium borohydride technique. The labeled glyco-proteins were separated by polyacrylamide slab gel electrophoresis and visualized by autoradiography (fluorography). The major thymocyte surface proteins have molecular weights of 170,000 and 125,000. Thymocytes from TL antigen-positive mouse strains showed an additional band with a molecular weight of 27,000. Highly purified T lymphocytes contain two major surface glycoproteins with molecular weights of 180,000 and 125,000. Purified B lymphocytes have one major surface glycoprotein with a molecular weight of 210,000. When T lymphocytes are stimulated in vitro by concanavalin A or phytohemag-glutinin, the major proteins characteristic of T cells are relatively weakly labeled, but new components of lower molecular weights appear on the cell surface. A similar change is seen in B lymphocytes stimulated by Escherichia coli lipopolysaccharide. T lymphoblasts isolated from mixed lymphocyte cultures show a slightly different surface glycoprotein pattern. A polypeptide with a molecular weight of 57,000, which was labeled without enzymatic treatment by tritiated sodium borohydride alone, is strongly labeled in proliferating cells.     

Characterization of mucin isolated from rat tracheal transplants

Subcutaneous rat tracheal grafts yield several milligrams of secretions from which a homogeneous mucin fraction was isolated and purified. Histological evidence demonstrated that a normal mucociliary epithelium and mucous secretion were maintained for the 4-6 weeks of the experiment. The collected secretions were initially characterized by column chromatography on Sepharose CL-6B which separated the excluded high molecular weight mucins (unpurified mucin fraction) from most of the serum-type glycoproteins and proteins, including albumin. A reductive alkylation treatment of the unpurified mucin fraction followed by Sepharose CL-4B chromatography removed contaminating protein and most of the mannose-containing material from the mucin fraction. The void volume material from this column produced a single high molecular weight band upon sodium dodecyl sulfate agarose/acrylamide gel electrophoresis. The purified mucin fraction contained 16.5% protein and primarily galactose, N-acetylglucosamine, N-acetylgalactosamine, and sialic acid. This fraction also underwent beta-elimination in the presence of alkaline borohydride, demonstrating the presence of O-glycosidic linkages.     

Isolation of a sodium dodecyl sulfate-insoluble transglutaminase substrate from liver plasma membranes

Rat liver plasma membranes contain transglutaminase activity and a large molecular weight protein complex which serves as a substrate for this enzyme. When plasma membranes were solubilized in sodium dodecyl sulfate and disulfide-reducing agents the transglutaminase substrate was recovered in the detergent-insoluble fraction. The insolubility of the complex suggested that it might be further studied by adsorbing membranes onto glass slides, then extracting with the detergent and reducing agent. After extraction, dark field light microscopy revealed numerous flattened sheets which varied in size from 4 to 12 micrometers. To confirm that these structures were the large molecular weight transglutaminase substrate, the plasma membranes were solubilized in sodium dodecyl sulfate and dithiothreitol and sedimented through a sucrose gradient containing the agent. The large molecular weight substrate was the only material found at the 1.11/1.23 g/cm3 interface. Microscopic examination showed the same structures previously observed on the glass slides. We conclude that the large molecular weight transglutaminase substrate is a sodium dodecyl sulfate-insoluble, morphologically distinct, protein complex. Due to its considerable size, nondissociable nature, and association with the lateral membrane, the sodium dodecyl sulfate-insoluble transglutaminase substrate may serve as a type of skeleton or scaffolding for this plasma membrane domain.     

Proteins of the human erythrocyte membrane as modified by pronase

Pronase degrades proteins on the outer surface of the human erythrocyte membrane which run in polyacrylamide-gel electrophoresis in the presence of sodium dodecyl sulfate at a molecular weight of approximately 125,000. Carbohydrate and sialic acid are removed, but fragments of molecular weight 50,000 to 100,000 remain attached to the membrane. The most prominent fragment, one of molecular weight about 73,000, can be labeled with a membrane-impermeable reagent (sulfanilic acid diazonium salt), so it is still accessible from the outside of the cell. Pronase rapidly inactivates membrane-bound acetylcholinesterase, but it has relatively little effect on the facilitated diffusion of glucose; both are inhibited by the diazonium salt. Extensive digestion leads to potassium loss and osmotic lysis. Ghosts prepared in 15 mosm-Tris (pH 7.6) are extensively degraded by pronase: essentially all the protein shifts to low molecular weight. Pronase is even more potent in 3% sodium dodecyl sulfate. Ghosts prepared from intact cells which have been treated with the enzyme hydrolyze when dissolved in the detergent unless steps are taken to inhibit proteolysis.     

Investigations on the outer membrane proteins of Salmonella typhimurium.

The number, nature and organization of the outer membrane proteins of Salmonella typhimurium have not yet been resolved. Therefore these proteins were isolated using a concentrated solution of guanidine hydrochloride and studied using different analytical techniques. Upon chromatography on Sephadex G-200 four fractions were obtained. Only the fraction containing a protein of molecular weight 13,000 produced immunoprecipitation reactions with the antisera raised against the whole bacteria. On polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulphate, 7 major proteins were found, with molecular weights between 13,000 and 43,000. Isoelectric focusing on 4.6% polyacrylamide gels resolved the outer membrane proteins into 10 bands with apparent isoelectric points between 5.0 and 8.4. Finally these proteins could be further resolved into as many as 50 spots where a two-dimensional electrophoresis was carried out with isoelectric focusing in the first dimension, and polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulphate in the second dimension. These results demonstrated that the outer membrane proteins of S. typhimurium are extremely heterogeneous. To investigate the mode of organization of lipopolysaccharides in the outer membrane, the membrane proteins were separated by the liquid isoelectric focusing technique. Lipopolysaccharides were primarily found to be associated with a protein of isoelectric point 7.8.