Topography of glycoproteins in the chick synaptosomal plasma membrane

Chick brain synaptosomes or synaptic subfractions were treated with neuraminidase (EC 3.2.1.18) and/or galactose oxidase (EC 1.1.3.9) preparations in which proteolytic activity was inhibited with phenylmethanesulfonyl fluoride followed, after washing, by reductive incorporation of sodium boro[³H]hydride to identify galactose residues exposed on the synaptosomal external surface. Control experiments to demonstrate restriction of labeling to the external surface involved comparing the radioactivity in synaptoplasmic, soluble polypeptides isolated after labeling with labeled, isolated synaptoplasm and examining incorporation into fractions incubated without enzymes. Intactness of the synaptic plasma membrane after labeling was shown by trypsin digestion studies. Polypeptides were separated on sodium dodecyl sulfate polyacrylamide gels and were detected by a liquid scintillation counting procedure. Eleven major radioactive peaks were found after galactose oxidase treatment and reduction of isolated synaptic membranes. When intact synaptosomes were labeled, the same components were detected. When isolated synaptic membranes or intact synaptosomes were treated with neuraminidase before galactose oxidase treatment, three additional components were labeled. These results suggest that (a) chick synaptic membranes have a complex mixture of glycoproteins, (b) all major chick synaptic membrane glycoproteins labeled by galactose oxidase have most or all carbohydrate groups exposed at the exterior surface of the synaptosome, (c) all major, externally-disposed polypeptides of these synaptic membranes are glycoproteins.     

Accessibility of Galactosyl Ceramides to Probe Reagents in Central Nervous System Myelin

The suitability of isolated central nerve myelin preparations for probe labelling studies was assessed and the accessibility of galactosyl ceramides in myelin to galactose oxidase and sodium periodate was determined. Isolated myelin preparations present a uniform external membrane surface to added probes because lamellae in the myelin sheath separate at their external apposition surfaces exclusively during isolation. The cytoplasmic apposition remains intact in isolated myelin. Cationised ferritin can gain access along external apposition regions of inner lamellae in multilamellar fragments of isolated myelin, indicating that proteins and lipids on the external membrane surface will be accessible to probes. Over 50% of the total galactosyl ceramides of myelin are accessible to galactose oxidase attack; hydroxy fatty acid- and nonhydroxy fatty acid-containing cerebrosides are equally attacked. Sodium periodate attacks over 90% of the galactosyl ceramides in isolated myelin at 20°C and electron micrographs of the periodate-treated myelin reveal changes at the external apposition only. Galactosyl ceramides in vesicles of myelin lipid vesicles are not so readily attacked by periodate. The disposition of galactosyl ceramides in the myelin lamellae is discussed.     

Loss of covalently labeled glycoproteins and glycolipids from the surface of newly transformed schistosomula of Schistosoma mansoni

Schistosomula of Schistosoma mansoni were labeled by oxidation with galactose oxidase or with periodate followed by reduction with NaB3H4 to study the loss of the surface membrane of these parasites in vitro. Grain counts of light microscope autoradiographs (LMARG) of radiolabeled schistosomula show that both galactose oxidase and periodate specifically label the surface of the organisms. Galactose oxidase labels 11 glycoproteins on the surface of skin and mechanical schistosomula, ranging in apparent molecular weight from 17,000 to greater than 105,000. These glycoproteins are lost from the surface of schistosomula with a halftime of 10-15 h in culture in defined medium. Most of these glycoproteins appear to be shed intact from the surface of the schistosomula rather than endocytosed and degraded, because greater than 50% of each of the lost proteins can be recovered by trichloroacetic acid precipitation of the culture medium and because there is no internalization of the radiolabels into cultured schistosomula examined by LMARG. In addition to glycoproteins, periodate labels at least seven glycolipids on the surface of mechanical schistosomula. After culture for 15 h, more than half of each of these periodate-labeled proteins and lipids are lost from the schistosomula, and their abundance relative to each other remains similar to that of freshly labeled organisms. Since both proteins and lipids are lost from the surface of the schistosomula at the same rate, we believe that we are observing a general loss of the parasite surface membrane.     

External labeling of human erythrocyte glycoproteins. Studies with galactose oxidase and fluorography.

Glycoproteins of the human erythrocyte membrane were labeled with tritiated sodium borohydride after oxidation of terminal galactosyl and N-acetylgalactosaminyl residues with galactose oxidase. After separation of the polypeptides on polyacrylamide slab gels, a scintillator was introduced into the gel, and the radioactive proteins were visualed by autoradiography (fluorography). The following results were obtained. (a) The erythrocyte membrane contains at least 20 glycoproteins, many of which are minor components. (b) The carbohydrate of all the labeled glycoproteins is exposed only to the outside, since no additional glycoproteins can be labeled in isolated unsealed ghosts. (c) The membrane contains two major groups of glycoproteins. The first group of proteins contains sialic acids linked to the penultimate galactosyl/N-acetylgalactosaminyl residues, which are efficiently labeled only after pretreatment with neuraminidase. The second group has terminal galactosyl/N-acetylgalactosaminyl residues which can be easily labeled without neuraminidase treatment. The glycoproteins from fetal erythrocytes all belong to the first group, whereas only five glycoproteins of erythrocytes from adults belong. (d) Trypsin cleaves the proteins containing sialic acids, and fragments containing carbohydrate remain tightly bound and exposed in the membrane. (e) Pronase cleaves Band 3 in addition to the sialic acid containing glycoproteins, but most of the glycoproteins still remain unmodified in the membrane. (f) No difference is seen between membrane glycoproteins from cells of different ABH blood groups.     

Topographic studies of glycoproteins of intact synaptosomes from rat brain cortex

Glycoproteins in the external surface of intact synaptosomes from rat brain cortex have been studied by oxidation of exposed galactose and galactosamine groups by galactose oxidase followed by reduction with labeled sodium borohydride. Purified synaptosomes were labeled, disrupted by osmotic shock, and the particulate components were fractionated on diatrizoate to give four synaptosomal membrane fractions (A to D) and a mitochondrial pellet (E). Fractions A and B represent highly purified synaptosomal plasma membranes. After separation of their polypeptides by electrophoresis, $\text{4}\text{5}$ of the label was present in two bands: one about 72 000 and the other between 7800 and 3200 daltons. Seven other bands were labeled to various degrees: 160 000, 96 000, 53 000, 39 000, 34 000, 23 000 and 16 000 daltons. With isolated membranes (which incorporate 5–6 times more label) $\text{4}\text{5}$ of label was present in polypeptides in three ranges: 160 000–96 000, 70 000–40 000 and 7800-3200. The number of polypeptides that can be labeled by treatment of isolated membranes is very large. In comparison, glycoproteins whose topographical distribution permits interaction with large molecules at the synaptic surface are very limited. It is further suggested that the external synaptosome membrane involves a relatively tight network of interacting molecules that cannot be readily penetrated by large molecules.     

Alterations of red cell membranes from phenylhydrazine-treated rabbits

Reticulocytosis was induced in rabbits by two methods: phlebotomy and injection of phenylhydrazine. Normal erythrocytes, reticulocytes from bed rabbits, reticulocytes from phenylhydrazine-treated rabbits, and erythrocytes treated in vitro with phenylhydrazine were compared with respect to their plasma membrane labeling by galactose oxidase and NaB³H₄, and lactoperoxidase-catalyzed incorporation of ¹²⁵I. Normal erythrocyte membranes and membranes from reticulocytes of bled rabbits showed almost identical labeling patterns, the presence of 2–3 glycoproteins with moderate to low mobilities on dodecyl sulfate acrylamide gel electrophoresis. Labeling in the absence of enzyme was negligible. In contrast, the reticulocytes from phenylhydrazine-treated rabbits exhibited a large incorporation of tritium without prior treatment with galactose oxidase. Even after prereduction with unlabeled NaBH₄ to remove this nonspecific labeling, the labeled glycoprotein components found in normal erythrocytes were not detectable. Normal erythrocytes treated in vitro with phenylhydrazine, washed, and labeled with galactose oxidase had labeling patterns, including high nonspecific incorporation of ³H, similar to those observed with in vivo phenylhydrazine treatment.Solubilization of membranes with lithium diiososalicylate followed by partitioning with phenol showed that the same glycoproteins were presented in normal or phenylhydrazine membranes, although only the former extract was labeled by galactose oxidase. Individual carbohydrates from the membranes were analyzed by gas-liquid chromatography and, in the case of hexosamines, on the amino acid analyzer. The results of these analyses indicated a slight decline in galactose content with phenylhydrazine treatment. Reticulocyte membranes from bled rabbits also showed a decrease in galactose content, although it was less pronounced.Most of the label incorporated by nonspecific borohydride labeling of membranes from phenylhydrazine-treated animals was found associated with protein. The modified amino acids from labeled proteins are similar to those formed in reactions of oxidized lipids and proteins in model systems.     

Analysis of the envelope of Rauscher murine oncornavirus: in vitro labeling of glycopeptides.

The identity and localization of the oligosaccharides of Rauscher murine type C viral glycoproteins have been examined by techniques of in vitro labeling. Terminal sialic acid was labeled with tritium by borohydride reduction after selective periodate oxidation, and galactose was labeled by borohydride reduction after specific enzymatic oxidation of the nonreducing terminal of the sugar. The results were compared with those of protein surface labeling with pyridoxal phosphate or lactoperoxidase catalyzed radioiodination. Examination of the labeled reaction products by polyacrylamide gel electrophoresis in sodium dodecyl sulfate showed that in every case the major component labeled was a glycoprotein of about 70,000 daltons. The identity of this glycoprotein as the virion envelope component was confirmed by immunoprecipitation with mono-specific antiserum prepared against purified Rauscher virus glycopeptides of 69,000 and 71,000 daltons. No other protein or glycoprotein on the surface of the virion was detected, and disruption of virions-before labeling did not reveal additional distinctive glycoproteins. There was minor labeling of sugar residues of other components, but these remain to be characterized and are not now identified as other viral proteins. Studies of the structural organization of virion proteins using the cross-linking reagent methyl-4-mercaptobutyrimidate showed only linkage of the virion envelope or core proteins to themselves. These results indicate that most, if not all, of the oligosaccharides at the surface of Rauscher virus are entities of the 69,000- and 71,000-dalton glycopeptides and that they contain a terminal sialic acid and galactose and a subterminal galactose.     

Platelet plasma membrane glycoproteins Identification of a proteolytic substrate for thrombin

The surface glycoproteins of the platelet plasma membrane were labeled by oxidation with galactose oxidase followed by reduction with (³H)-sodium borohydride. Of the glycoproteins labeled, only glycoprotein V (apparent molecular weight of 89,000) was decreased as a result of thrombin action. The affected glycoprotein appeared to be completely removed at a concentration of 1 U thrombin per 10⁹ platelets. A soluble glycopeptide hydrolytic product with an apparent molecular weight of 70,000 was released into solution.     

The isolation and characterization of a concanavalin A receptor from boar spermatozoa surface

Boar spermatozoa were radioactively labeled by either lactoperoxidase-catalysed iodination or galactose oxidase oxidation followed by reduction with tritiated sodium borohydride. Plasma membrane glycoproteins were solubilized with the non-ionic detergent Nonidet P40 and separated by affinity chromatography on concanavalin A-Sepharose. A major water-soluble concanavalin A receptor of molecular weight greater than 160 000 was isolated by gel filtration and ion-exchange chromatography. Its amino acid and carbohydrate composition were determined. This glycoprotein is susceptible to digestion by trypsin or chymotrypsin.     

Plasmodium berghei: modification of sialic acid on red cells from infected mouse blood

The surface membrane glycoproteins of normal mouse erythrocytes can be labeled by oxidation with either periodate or galactose oxidase in the presence of neuraminidase, followed by reduction with NaB³H₄. Without neuraminidase there is little galactose oxidase-catalyzed labeling of protein. Analysis of labeled proteins by SDS-polyacrylamide gel electrophoresis showed that both methods labeled the same set of glycoproteins. Plasmodium berghei infection dramatically reduced the sialoglycoprotein labeling of red blood cells from infected blood using the periodate/NaB³H₄ method. Provided neuraminidase was present, labeling by the galactose oxidase method gave identical results to normal erythrocytes. We conclude that the glycoprotein sialic acid of uninfected as well as infected red cells is modified during infection such that it is refractory to periodate oxidation. Acylation of the exocyclic hydroxyls of sialic acid is suggested to account for this. Lectin binding and cell agglutination experiments using Limulin, soybean and wheatgerm lectins, and concanavalin A confirmed and extended these observations. The possible implications of these results with regard to anemia induced by malaria are briefly discussed.     

Metabolic fate of cell surface glycoproteins during immunoglobulin-induced internalization

Goat antibodies directed against a subset of the externally oriented plasma membrane glycoproteins of hepatoma tissue culture (HTC) cells were used to follow the metabolic fate of the membrane antigens and the specifically bound immunoglobulin molecules in this cell type in cultures. Analyses of the immunoprecipitates from cells labeled in situ with neuraminidase and galactose oxidase, followed by reduction with tritiated sodium borohydride, indicate that about 40% of the galactose-labeled plasma membrane glycoproteins are recognized by the antiserum. Fluorescent microscopic analyses of cells treated with fluorescein-conjugated immunoglobulins and analyses of trypsin accessibility indicate that probably all of the antibodies bound to the cell surface are patched and internalized within about 4 hr when the cells are subsequently cultured at 37 degrees C in the presence of rabbit anti-goat immunoglobulins. At the same time, the antigens are also interiorized. Analyses of the cellular localization of the interiorized antigens and antibodies by cell fractionation on Percoll gradients show that the immunoglobulins to the cell surface antigens and the antigens themselves migrate to the same region of the Percoll gradient as lysosomal hydrolases. Although the antibodies bind to the cell surface glycoproteins and bring about patching and interiorization, there is no effect on the degradation of the plasma membrane antigens labeled via the galactose oxidase/borohydride reduction method. Furthermore, the iodinated antibodies directed against these membrane glycoproteins behave in their turnover properties like membrane antigens; the cell-bound specific immunoglobulins have the same half-life as the membrane glycoproteins. When the cells that had been reacted with the goat antibodies to membrane glycoprotein were cultured in the presence of rabbit anti-goat immunoglobulins, degradation of the former antibodies was effectively decreased. Similar results were obtained with concanavalin A and antibodies directed against this plant lectin.     

Glycoprotein synthesis in the adult rat pancreas. IV. Subcellular distribution of membrane glycoproteins

Zymogen granule membranes from the rat exocrine pancreas displays distinctive, simple protein and glycoprotein compositions when compared to other intracellular membranes. The carbohydrate content of zymogen granule membrane protein was 5-10-fold greater than that of membrane fractions isolated from smooth and rough microsomes, mitochondria and a preparation containing plasma membranes, and 50-100-fold greater than the zymogen granule content and the postmicrosomal supernate. The granule membrane glycoprotein contained primarily sialic acid, fucose, mannose, galactose and N-acetylglucosamine. The levels of galactose, fucose and sialic acid increased in membranes in the following order: rough microsomes less than smooth microsomes less than zymogen granules. Membrane polypeptides were analyzed by polyacrylamide gel electrophoresis in sodium dodecyl sulfate. The profile of zymogen granule membrane polypeptides was characterized by GP-2, a species with an apparent molecular weight of 74 000. Radioactivity profiles of membranes labeled with [3H]glucosamine or [3H]leucine, as well as periodic acid-Schiff stain profiles, indicated that GP-2 accounted for approx. 40% of the firmly bound granule membrane protein. Low levels of a species similar to GP-2 were detected in membranes of smooth microsomes and the preparation enriched in plasma membranes but not in other subcellular fractions. These results suggest that GP-2 is a biochemical marker for zymogen granules. Membrane glycoproteins of intact zymogen granules were resistant to neuraminidase treatment, while those in isolated granule membranes were readily degraded by neuraminidase. GP-2 of intact granules was not labeled by exposure to galactose oxidase followed by reduction with NaB3H4. In contrast, GP-2 in purified granule membranes was readily labeled by this procedure. Therefore GP-2 appears to be located on the zymogen granule interior.     

Enzymatic and chemical oxidation of gangliosides in cultured cells: effects of choleragen.

Cell surface glycolipids of normal human fibroblasts and NCTC2071 cells (transformed mouse fibroblasts) were labeled by incubating the intact cells with either galactose oxidase or sodium periodate, followed by reduction of the oxidized sugar residues with NaB3H4. In intact human fibroblasts, incorporation of 3H was increased with increasing time of exposure to galactose oxidase prior to treatment with NaB3H4. Following limited exposure to galactose oxidase, more label was incorporated into the larger glycolipids. Although labeling of the monosialoganglioside GM1 was maximal by 16 h, not all of the GM1 in the intact cells appeared to be accessible to galactose oxidase, since 10 to 12 times more GM1 was labeled when cells were disrupted before incubation with the enzyme. The human fibroblasts contained approximately 8 X 10(6) molecules of GM1 per cell. Maximal binding of choleragen (5 X 10(5) molecules of [125I]choleragen per cell) completely prevented cholevented oxidation of GM1 in intact fibroblasts by galactose oxidase but only partially protected the sialic acid moiety of GM1 from oxidation by periodate. Choleragen had little effect on the enzymatic or chemical oxidation of other glycolipids. NCTC 2071 cells do not contain endogenous GM1 but incorporate exogenous GM1 from the culture medium. When bound to NCTC 2071 cells, exogenous GM1 was protected by choleragen from oxidation by galactose oxidase or whether endogenous or taken up from the incubation medium, are, after interaction with choleragen, less accessible to oxidation by periodate or galactose oxidase.     

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.     

Glycoproteins associated with myelin in the central nervous system

Purified myelin fractions from the central nervous system contain one major myelin-associated glycoprotein and approximately 16 minor glycoproteins. While the genuine association of the major myelin-associated glycoprotein with the oligodendroglial myelin unit is demonstrated, the possibility exists that several of the minor glycoproteins have their origin in contaminating membranes not related to myelin. The major myelin-associated glycoprotein is probably not present in compacted myelin, but immunocytochemical and subfractionation studies indicate that it is confined to the periaxonal and paranodal region of the myelin sheath. In experimental demyelination and multiple sclerosis, the major glycoprotein is the first myelin constituent to be affected. Its localization on the membrane surface where myelin and axolemma are in close contact, and other indirect evidence indicate that the major glycoprotein, and possibly other myelin-associated glycoproteins, could play a role in the process of myelination and myelin maintenance.     

Galactose-rich glycoproteins are on the cell surface of herpes virus-infected cells. 1. Surface labeling and serial lectin binding studies of Asn-linked oligosaccharides of glycoprotein gC

Cell-surface glycoproteins of mock-infected and herpes simplex virus type 1 (HSV-1)-infected BHK-21 and HEp-2 cells were radiolabeled by incubation with galactose oxidase followed by reduction with NaB3H4. The incorporation of radiolabel into glycoconjugates in both BHK-21 and HEp-2 cells was increased several fold following infection with HSV, showing an increase in surface-exposed Gal residues in the infected cells. This was further confirmed by an increase in binding of cell-surface-labeled glycoproteins gC and gB from HSV-infected BHK-21 cells to Ricinus communis agglutinin I, which is specific for beta-D-Gal residues. Prior treatment of cells with Clostridium perfringens neuraminidase enhanced the surface radiolabeling by the galactose oxidase/NaB3H4 method: HEp-2 cells exhibited over sixfold enhancement in labeling, while BHK-21 cells showed only a slight increase. HSV glycoprotein gC was the predominant cell-surface glycoprotein radiolabeled by the galactose oxidase/NaB3H4 method in virus-infected BHK-21 cells. The glycoprotein gC was purified by immunoaffinity column chromatography on monoclonal anti-gC-antibody-Sepharose. The radiolabel in the glycopeptides of gC was resistant to beta elimination, showing that it was associated only with Asn-linked oligosaccharides. A serial lectin affinity chromatography of glycopeptides on columns of concanavalin A-Sepharose, lentil (Lens culinaris) lectin-Sepharose, and Ricin I-agarose allowed the assignment of minimal oligosaccharide structures bearing terminal Gal residues in gC.     

Glycoprotein synthesis in the adult rat pancreas: IV. Subcellular distribution of membrane glycoproteins

Zymogen granule membranes from the rat exocrine pancreas displays distinctive, simple protein and glycoprotein compositions when compared to other intracellular membranes. The carbohydrate content of zymogen granule membrane protein was 5–10-fold greater than that of membrane fractions isolated from smooth and rough microsomes, mitochondria and a preparation containing plasma membranes, and 50–100-fold greater than the zymogen granule content and the postmicrosomal supernate. The granule membrane glycoprotein contained primarily sialic acid, fucose, mannose, galactose and $\text{N-}\text{acetylglucosamine}$. The levels of galactose, fucose and sialic acid increased in membranes in the following order: rough microsomes < smooth microsomes < zymogen granules.Membrane polypeptides were analyzed by polyacrylamide gel electrophoresis in sodium dodecyl sulfate. The profile of zymogen granule membrane polypeptide was characterized by GP-2, a species with an apparent molecular weight of 74 000. Radioactivity profiles of membranes labeled with [³H]glucosamine or [³H]leucine, as well as periodic acid-Schiff stain profiles, indicated that GP-2 accounted for approx. 40% of the firmly bound granule membrane protein. Low levels of a species similar to GP-2 were detected in membranes of smooth microsomes and the preparation enriched in plasma membranes but not in other subcellular fractions. These results suggest that GP-2 is a biochemical marker for zymogen granules.Membrane glycoproteins of intact zymogen granules were resistant to neuraminidase treatment, while those in isolated granule membranes were readily degraded by neuraminidase. GP-2 of intact granules was not labeled by exposure to galactose oxidase followed by reduction with NaB³H₄. In contrast, GP-2 in purified granule membranes was readily labeled by this procedure. Therefore GP-2 appears to be located on the zymogen granule interior.     

Combining results from lectin affinity chromatography and glycocapture approaches substantially improves the coverage of the glycoproteome

Identification of glycosylated proteins, especially those in the plasma membrane, has the potential of defining diagnostic biomarkers and therapeutic targets as well as increasing our understanding of changes occurring in the glycoproteome during normal differentiation and disease processes. Although many cellular proteins are glycosylated they are rarely identified by mass spectrometric analysis (e.g. shotgun proteomics) of total cell lysates. Therefore, methods that specifically target glycoproteins are necessary to facilitate their isolation from total cell lysates prior to their identification by mass spectrometry-based analysis. To enrich for plasma membrane glycoproteins the methods must selectively target characteristics associated with proteins within this compartment. We demonstrate that the application of two methods, one that uses periodate to label glycoproteins of intact cells and a hydrazide resin to capture the labeled glycoproteins and another that targets glycoproteins with sialic acid residues using lectin affinity chromatography, in conjunction with liquid chromatography-tandem mass spectrometry is effective for plasma membrane glycoprotein identification. We demonstrate that this combination of methods dramatically increases coverage of the plasma membrane proteome (more than one-half of the membrane glycoproteins were identified by the two methods uniquely) and also results in the identification of a large number of secreted glycoproteins. Our approach avoids the need for subcellular fractionation and utilizes a simple detergent lysis step that effectively solubilizes membrane glycoproteins. The plasma membrane localization of a subset of proteins identified was validated, and the dynamics of their expression in HeLa cells was evaluated during the cell cycle. Results obtained from the cell cycle studies demonstrate that plasma membrane protein expression can change up to 4-fold as cells transit the cell cycle and demonstrate the need to consider such changes when carrying out quantitative proteomics comparison of cell lines.     

FRACTIONATION OF GLYCOPROTEINS ASSOCIATED TO ADULT RAT BRAIN MYELIN FRACTIONS

Abstract— The chloroform-methanol insoluble residue of adult rat brain myelin fractions (My-CMI) contains only 20% of protein but all myelin-associated glycoproteins (Z anetta et al ., 1977a). After solubilization in sodium dodecyl sulphate, these glycoproteins were separated by sequential affinity chromatography on 4 immobilized lectins. Ten fractions (9 of which contained only glycoproteins) were obtained. Glycoproteins added up to at least 25% of My-CMI proteins. Many minor glycoproteins were detected in the different fractions. However most of them appeared not to be intrinsic to myelin. On the contrary a major glycoprotein electrophoretic band, component A, appeared to be intrinsic to myelin although its presence also on oligodendrocyte plasma membrane cannot be excluded. Component A was tentatively identified with the'major myelin associated glycoprotein'described by QUARLES (1972, 1973 a, b ). It accounted for less than 0.4% of proteins and 8% of glycoproteins of myelin fractions and consisted of at least two'glycopolypeptides'which, both, bind to concanavalin A and to the Ulex europeus lectin. The other major glycoprotein, component B, did not bind to any of the lectins used and, thus, must have N -acetyl neuraminic acid as only terminal sugar. The separation of myelin-associated glycoproteins according to their affinity for lectins allowed a tentative identification of the lectin binding sites of myelin sheath.     

Iodination and identification of surface membrane antigens in procyclic Trypanosoma rhodesiense

Surface membrane proteins and glycoproteins of procyclic Trypanosoma rhodesiense were labeled with 125I by the use of the insoluble catalyst Iodo-Gen. Autoradiography of whole solubilized procyclic trypanosomes after sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) showed a minimum of 25 surface components to have incorporated radioactivity. These components ranged in m.w. from approximately 10,000 to approximately 285,000. Immunoprecipitation with rabbit antisera of Triton X-100 extracts of radiolabeled trypanosomes revealed a subset of at least 14 surface antigens. Two of these antigens (m.w. of 63,000 and 96,000) showed heavy incorporation of label and may be major proteins of the procyclic membrane. Sera from trypanosome-infected mice recognized an overlapping but different subset of surface antigens, including a doublet of very high m.w. Lectin precipitation using antilectin conjugates or bead-bound lectins indicated that many of the labeled surface components are glycoproteins including the two major proteins precipitated by rabbit antisera. Radiolabeled glycoproteins identified by these methods bear alpha-methyl-mannopyranoside and/or galactose residues but not N-acetyl glucosamine or fucose residues in quantity. The use of these methods in identifying potentially pathogenic trypanosomal antigens is suggested.