Identification and partial characterization of plasma membrane polypeptides of Trypanosoma brucei

A plasma membrane-enriched vesicle fraction has been prepared from Trypanosoma brucei by sonication and differential centrifugation on sucrose gradients. This fraction is enriched 5-fold in the plasma membrane marker enzymes adenyl cyclase (EC 4.6.1.1) and ouabain-inhibitable, ($\text{Na}{}^{\mathrm{+}}\text{+}\text{K}{}^{\mathrm{+}}$)-dependent adenosine triphosphatase (EC 3.6.1.3). It is also enriched up to 14-fold in iodinated surface proteins, and up to 4-fold in [³H]mannose-labeled glycoproteins, of which the major variable surface coat glycoprotein is the main constituent. Proteins of the plasma membrane fraction and other subcellular fractions have been identified by electrophoretic analysis in sodium dodecyl sulfate-polyacrylamide gradient slab gels. Several high molecular weight surface glycopeptides have been selectively investigated and partially characterized by a combination of metabolic labeling with [³H]mannose, lactoperoxidase-catalyzed surface iodination, and affinity chromatography on Con A-Sepharose. In addition to the major variable surface coat glycoprotein (estimated $\text{M}{}_{\mathrm{<mtext>r</mtext>}}\text{= 58 000}$), there are several minor surface glycopeptides ($\text{M}{}_{\mathrm{<mtext>r</mtext>}}\text{= 76 000, 86 000}\text{and}\text{92 000–100 000}$) which are apparent extrinsic membrane components, and two surface glycopeptides ($\text{M}{}_{\mathrm{<mtext>r</mtext>}}\text{= 42 000}\text{and}\text{130 000}$) which are intrinsic membrane components.     

Biochemical and morphological characterization of a plasma membrane-enriched fraction from bovine parathyroid cells

A fraction of enriched plasma membranes from bovine parathyroid cells has been prepared by differential centrifugation. Biochemical characterization shows that this fraction has a specific activity enrichment of 7.2-fold in ouabain-sensitive Na+-K+ ATPase, and 3.5-fold in 5'-nucleotidase. Less than 4% of the total mitochondria and lysosomes are present within the plasma membranes, while microsomal contamination accounts for 14% of total specific activity. Parathyroid hormone radioimmunoassay also reveals the presence of some secretory granules within the plasma membrane fraction. The characteristic morphological aspect of the unusual surface membrane is shown by freeze-fracture electron microscopy. In the enriched pellets, vesicles identified as having a plasma membrane origin have variable sizes, and 50% show an inside-out conformation. Even though the plasma membrane fraction described herein is not absolutely free from contamination by other subcellular components, this protocol represents the first attempt to purify surface membrane from parathyroid tissue and provide the starting material for understanding, at a molecular level, the properties of extracellular Ca2+ regulation and its coupling with secretion of parathyroid hormone.     

Analysis of the surface labelling characteristics of human spermatozoa and the interaction with anti-sperm antibodies

Washed ejaculated human spermatozoa were surface labelled with 125I, using solid phase (iodogen) or enzymic (lactoperoxidase) methods, while membrane components possessing terminal galactose or galactosamine residues were labelled with the galactose oxidase-sodium [3H]borohydride technique. All three procedures revealed the presence of 2 major labelled surface components. The first comprised a broad band of radioactivity migrating just behind the ion front on SDS-PAGE, which could be extracted with chloroform and methanol, suggesting a lipid-like composition. The second fraction exhibited properties consistent with a major glycoprotein component of the human sperm plasma membrane, giving a peak of radioactivity with Mr = 20,000, within which a discrete doublet of bands (Mr = 17,000 and 19,000) could be resolved by autoradiography. A more detailed analysis of the labelled protein fraction after TCA precipitation revealed a number of other surface components, the major ones of which exhibited Mr values of 30,000, 45,000, 66,000, 115,000 and 160,000. Western blot analysis was then used to determine whether any of the surface components described above interacted with the gamma-globulin fraction of antisera obtained from patients exhibiting idiopathic autoimmunity against sperm antigens. Using a purified membrane preparation as the target, antibodies were detected against numerous high molecular weight bands with Mr values similar to the major components of the human sperm surface (35,000, 45,000, 66,000, 90,000 and 150,000). The nature of the antigens targeted by these antisera did not correlate with the ability of the latter to stimulate or suppress sperm-oocyte fusion.     

The isolation and characterization of the plasma membrane of cultured chinese hamster ovary cells.

Plasma membranes were prepared from cultured Chinese hamster ovary cells utilizing a two-phase polymer system and were characterized by enzymatic and chemical assay, and by electron microscopy. The usual degree of purification of presumptive membrane markers such as Na+-K+ ATPase (ATP phosphohydrolase, EC 3.6.1.3) ranged from three-to eightfold. Gel electrophoresis in SDS revealed several polypeptides and two glycopeptides which were enriched in the plasma membrane fraction.     

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.     

The isolation and analysis of the luminal plasma membrane of calf urinary bladder epithelium.

The luminal plasma membrane of calf urinary bladder epithelium (urothelium) has been isolated by a method designed to preserve enzymic activity as well as structural integrity. The yield was about 80 micrograms per calf bladder. Low levels of 5' nucleotidase, Mg2+-ATPase and (Na+ + K+)-ATPase activities were found in the luminal membrane fraction. Cerebroside was the major lipid present and dodecyl sulphate gel electrophoresis revealed a complex protein and glycoprotein composition in the whole membrane. A membrane fraction consisting of only the plaque areas was shown to have a simpler protein composition with major polypeptides of apparent Mr 12 000 and 22 000. These may associate to form a 30 000 apparent Mr complex which could represent the individual 'particles' of the dodecameric subunits seen by electron microscopy in the plaque regions.     

Isolation and characterization of a Mr = 110,000 glycoprotein localized to the hepatocyte bile canaliculus

A Mr = 110,000 glycoprotein, GP 110, was partially purified using wheat germ agglutinin-Sepharose affinity chromatography from a bile canalicular-enriched membrane fraction denoted N2u of rat liver. This fraction was subjected to preparative sodium dodecyl sulfate-polyacrylamide gel electrophoresis and the Mr = 110,000 polypeptide was excised and used as an immunogen in rabbits. The antisera were found to specifically recognize a Mr = 110,000 polypeptide, named GP 110, in the N2u membrane fraction. In isolated hepatocytes, GP 110 was readily accessible to cell surface iodination catalyzed by lactoperoxidase at 4 degrees C and was judged by immunoprecipitation studies to contain about 2% of total radioactivity incorporated into externally oriented proteins of the cell. Immunoprecipitated GP 110 was shown by two-dimensional polyacrylamide gel electrophoresis to migrate with an approximate pI of 4.9. Indirect immunofluorescence on frozen liver sections demonstrated that GP 110 was primarily localized in the bile canaliculus. In corroborative studies employing subcellular fractionation, it was found that GP 110 was enriched nearly 19-fold in P2, a plasma membrane fraction primarily derived from the sinusoidal domain, and 44-fold in N2u. In contrast, only low levels of GP 110 were present in endoplasmic reticulum, mitochondrial, cytosolic, and nuclear-enriched fractions of liver. The physiological function of GP 110 is as yet unknown; antisera to it did not immunoprecipitate other known bile canalicular proteins of similar molecular weights. GP 110 was found to be extensively glycosylated relative to other known membrane proteins; approximately 33% of the apparent molecular weight appear to be carbohydrate. In agreement, limited removal of N-linked carbohydrate chains indicated that there are approximately eight chains/GP 110 polypeptide. Neuraminidase treatment of GP 110 resulted in a desialylated Mr = 85,000 polypeptide suggesting that the majority of carbohydrate chains on GP 110 are of the complex type.     

Isolation of a domain of the plasma membrane in Chinese hamster ovary cells which contains iodinatable, acidic glycoproteins of high molecular weight

A light vesicle fraction, apparently derived from the plasma membrane, was obtained following breakage of Chinese hamster ovary (CHO) cells by means of a fluid pump disrupting device. The final preparation was enriched approx. 40-fold over the homogenate in K+,Na+-stimulated ATPase and phosphodiesterase I, but only approx. 10-fold in 125I specific radioactivity after lactoperoxidase-catalyzed iodination. This preparation was compared with another plasma membrane fraction purified as large sheets via a two-phase centrifugation procedure. Two-dimensional polyacrylamide gel electrophoresis followed by Coomassie blue staining indicated that both fractions were fairly similar in polypeptide composition, although a few consistent differences were evident. However, staining of glycoproteins by the periodic acid-Schiff technique or by overlaying with 125I-labeled concanavalin A showed that the vesicle fraction was highly enriched in groups of high molecular weight, acidic glycoproteins which stain only weakly with Coomassie blue. These glycoproteins also bound 125I-labeled ricin I agglutinin and wheat germ agglutinin. They appear to be the major receptors for wheat germ agglutinin on the CHO cell surface. After surface labeling of cells by the 125I-lactoperoxidase technique, the membrane sheet fraction contained a large number of iodinated polypeptides, whereas labeling in the vesicle fraction was restricted almost entirely to the high molecular weight, acidic glycoproteins. It is proposed that the vesicle fraction constitutes a specific domain of the cell surface which is coated on its exterior by this group of glycoproteins. These components probably mask underlying proteins of the plasma membrane from external labeling.     

Studies on the surface coat of Paramecium aurelia

Summary The plasma membrane of Paramecium aurelia is covered with a ruthenium red stainable surface coat. Results obtained after digestion with pronase, trypsin and neuraminidase suggest the glycoprotein nature of this structure. Lipid extraction also affects the surface coat forming material. The results are consistent with the model proposed by Ginsburg and Kobata dealing with spatial configuration of the surface coat components.Authors are grateful to Mrs. D. Kucharczyk for very efficient technical assistence, to Mrs. Z. Kaminska for sectioning the material and Mr. A. Renski for help with the electron microscope service.     

Purification of glycoproteins IIb and III from human platelet plasma membranes and characterization of a calcium-dependent glycoprotein IIb-III complex

Human platelet membrane glycoproteins IIb and III are two major integral membrane components that have been identified as sites mediating thrombin-induced aggregation. For purposes of our study, glycoproteins IIb and III were solubilized by extracting platelet plasma membranes with a buffer containing 0.1% Triton X-100 and were separated by gel filtration chromatography on Sephacryl S-300, employing Triton X-100-containing column buffers with or without urea or guanidine hydrochloride. The physical properties of the purified glycoproteins were: for glycoprotein IIb, Rs = 61 A, s20.w = 4.7, f/f0 = 1.7, Mr = 125,000 (hydrodynamic values), Mr = 136,000 (sodium dodecyl sulfate gels); for glycoprotein III, Rs = 67 A, s20,w = 3.2 f/f0 = 2.1, Mr = 93,000 (hydrodynamic values), Mr = 95,000 (sodium dodecyl sulfate gels). Although the amino acid compositions of the two glycoproteins were similar, antibodies raised against glycoprotein IIb did not crossreact with glycoprotein III. If divalent cations were not chelated in the Triton extract, glycoproteins IIb and III coeluted during gel filtration chromatography (apparent Stokes radius of 71 A) and co-sedimented on sucrose gradients (apparent s20.w of 8.6), from which Mr = 265,000 was calculated. Glycoproteins IIb and III were coprecipitated by an antibody monospecific for glycoprotein IIb. The two glycoproteins dissociated into monomers when EDTA was added to Triton lysates. Readdition of Ca2+ caused them to reassociate into a complex with physical properties similar to those of the complex in the original Triton lysate. The data show that glycoproteins IIb and III are a heterodimer complex, that complex formation depends upon the presence of Ca2+, and that chelation of Ca2+ causes dissociation into monomeric glycoproteins.     

Purification and characterization of two plasma membrane domains from ejaculated bull spermatozoa

Plasma membranes were detached from ejaculated bull spermatozoa by a brief sonication in a moderately hypotonic medium, and the released plasma membranes were partially purified by differential centrifugation. The resulting fraction was enriched 8- and 15-fold in alkaline phosphatase and 5' nucleotidase activities, respectively, compared with the starting sonicated spermatozoa. This total plasma membrane fraction was separated into two distinct fractions by equilibrium density centrifugation on a continuous linear sucrose gradient. Two peaks of light scattering material were formed at densities of 1.117 and 1.148 g/ml. The denser peak contained most of the protein of the plasma membrane fraction, whereas nearly all the concanavalin A binding activity was found in the lighter peak. The two bands had distinctly different polypeptide compositions when analyzed by SDS PAGE. Polyclonal antibodies were raised in rabbits against a major integral membrane glycoprotein of each fraction (Mr of 92,000 in the light peak and 98,000 in the dense peak). The two antigens were detected on the surface of intact spermatozoa by indirect immunofluorescence microscopy. The 92-kD protein (present in the lighter band) was detected only on the plasma membrane of the acrosomal and anterior postacrosomal regions of the head. The 98-kD antigen, present in the heavier band, was localized to the surface of the postacrosomal region of the head, to the principal piece of the tail, and to the connecting piece between the head and tail. The exclusive localization of the 92-kD polypeptide to the surface of the anterior portion of the head was confirmed by immunoelectron microscopy. These data show that the two fractions isolated on the sucrose gradient originate from different regions of the sperm cell plasma membrane.     

Biosynthesis of intestinal microvillar proteins. Evidence for an intracellular sorting taking place in, or shortly after, exit from the Golgi complex

Pig small intestinal mucosal explants, labelled with [35S]-methionine, were fractionated into Mg2+-precipitated (intracellular and basolateral) and microvillar membranes, and the orientation of newly synthesized aminopeptidase N (EC 3.4.11.2) in vesicles from the two fractions was studied by its accessibility to proteolytic cleavage. The mature polypeptide of Mr 166 000 from the latter fraction was cleaved by trypsin, proteinase K and papain, consistent with an extracellular location of the enzyme at its site of function. In contrast, both the mature form and the transient form of Mr 140 000 from the Mg2+-precipitated fraction were equally well protected from proteolytic cleavage (in the absence of Triton X-100). This indicates that the basolateral plasma membrane is unlikely to be involved in the post-Golgi transport of newly synthesized aminopeptidase N and suggests instead a direct delivery of the enzyme to the apical plasma membrane. A crude membrane preparation from labelled explants was used in immunoelectrophoretic purification of membranes to determine at what stage during intracellular transport newly synthesized microvillar enzymes are sorted, i.e., accumulated in areas of the membrane from where other proteins are excluded. The transient form of aminopeptidase N was only moderately enriched by immunopurification, using antibodies against different microvillar enzymes, but the mature form was enriched approximately 30-fold from explants, labelled for 30 min. This suggests that for microvillar enzymes, the aspects of sorting studied take place in, or shortly after exit from, the Golgi complex.     

Isolation of synaptosomal plasma membranes from cholinergic nerve terminals and a comparison of their proteins with those of synaptic vesicles

Plasma membranes were purified from purely cholinergic nerve endings (synaptosomes) isolated from the electric organ of Torpedo marmorata. Synaptosomes were lysed, membranes recovered and further separated by density gradient centrifugation. A fraction was obtained enriched in 5'-nucleotidase, Na+, K+-activated ATPase and acetylcholine esterase. Morphological examination showed abundant membrane fragments of the size range of synaptosomes and few of vesicle size. The fraction has a characteristic protein composition upon gel electrophoresis. Five reproducible major bands with apparent Mr of 100000, 75000, 52000, 42000 and 35000--33000 are found. A gel-electrophoretic comparison with proteins from synaptic vesicles from the same source (major bands Mr 160000, 147000, 34000 and 25000) was made. Comigration of major bands was detected in one-dimensional gel electrophoresis with the 42000-Mr, 35000--33000-Mr and 34000-Mr components. Upon two-dimensional gel electrophoresis the 42000-Mr component comigrates with a similar component in vesicles, recently characterized as actin; the other components are different. The presence of tubulin-like polypeptides is unlikely. Beside actin, all major vesicle proteins are often detected in small amounts in the plasma membrane preparation. It cannot be decided if they result from fused or contaminating vesicle membranes, but since they are essentially absent in some preparations, it seems that the plasma membrane does not contain vesicle proteins.     

Studies on thyroid cell surface glycoproteins: Isolation of plasma membranes and characterization of carbohydrate units

Plasma membranes were isolated from calf thyroid microsomes and further resolved into two subfractions by sucrose density gradient centrifugations. The lighter and major membrane fraction was obtained in a yield of 10 mg/100 g of thyroid and was enriched 38-fold with respect to 5′-nucleotidase activity compared to the homogenate. It differed from the denser plasma membrane fraction in containing greater amounts of phospholipid and cholesterol but had a similar total carbohydrate content (16 mg/100 mg protein) and monosaccharide composition. The membranes were found to retain most (80%) of their carbohydrate after delipidation. The major protein-bound sugars present in the lighter membrane fraction expressed as micromoles per 100 mg of peptide were: galactose 24, mannose 17, fucose 3, glucosamine 23, galactosamine 4, and sialic acid 9. Polyacrylamide gel electrophoresis in sodium dodecyl sulfate of the lipid-free membranes revealed at least 18 protein bands and 3 periodic acid-Schiffreactive glycoprotein components. Incubation of the delipidated membranes with Pronase resulted in the solubilization of 95% of the saccharide portion which upon filtration through Bio-Gel P-6 and P-10 columns yielded several glycopeptide fractions. While some of the carbohydrate was found in glycopeptides which appeared to contain the well-known complex and polymannose asparagine-bound oligosaccharides, as well as small O-glycosidically linked units, approximately half was recovered in high molecular weight components which contained galactose and glucosamine as their principal sugar constituents, and which were similar in composition to glycopeptides recently isolated (T. Krusius, J. Finne, and H. Rauvala, 1978, Eur. J. Biochem.92, 289–300) from human erythrocyte membranes.     

The porcine LH/hCG receptor. Characterization and purification

Porcine luteal LH/hCG receptor (LH/hCG R) was solubilized with 70-80% recovery from the crude plasma membrane fraction by Triton X-100 in the presence of 25% glycerol and protease inhibitors. The solubilized receptor maintained 90% of original activity at -60 degrees C for 90 days. Equilibrium association constant (Ka) values of 1.92, 2.22, and 2.03 X 10(10) M-1 were observed for the whole homogenate, plasma membrane fraction, and solubilized LH/hCG R preparations, respectively. The specific binding capacity for the same fractions were 49, 70, 55 fmol/mg protein, respectively. Complexes of LH/hCG R and Triton X-100 were resolved into two components with approximate Mr = 2.7 X 10(5) and 5.4 X 10(5) by gel filtration on Sepharose 6B and two glycoprotein components by chromatography on concanavalin A-Sepharose. Solubilized porcine LH/hCG R was purified by two cycles of affinity chromatography on highly purified hCG-Sepharose with an overall recovery of 30-35% of the initial activity in the Triton extract. Purified porcine LH/hCG R had a specific binding capacity of 2300 pmol/mg protein and a Ka = 1.5 X 10(10) M-1. Silver staining of sodium dodecyl sulfate-polyacrylamide gel electrophoresis gels demonstrated that the major protein in porcine LH/hCG R preparations has Mr = 68,000. A weakly staining band at Mr = 45,000 was also observed in the purified receptor preparation. Analysis of iodinated purified LH/hCG R by autoradiography has confirmed these results. Porcine LH/hCG R was purified 40,000-fold by this method.     

Ca2+-dependent ATPase activity of alveolar macrophage plasma membrane.

A plasma membrane fraction was isolated from lysates of Bacillus Calmette-Guérin-induced alveolar macrophages of rabbit. On the basis of morphological and biochemical criteria this fraction appeared to be minimally contaminated by other subcellular organelles. Concentrations of Ca2+, but not of Mg2+, from 6.10(-8) to 1.10(-5) M markedly stimulated the basal ATPase (EC 3.6.1.3) activity of the plasma membrane, with an apparent Km (Ca2+) of 1.10(-6) M. The specific activity of the Ca2+-ATPase assayed at pCa = 5.5 was enriched about 8-fold in the plasma membrane fraction over the macrophage lysate. In contrast, the specific activity of the K+, EDTA-activated ATPase, associated to macrophage myosin, increased only 1.3-fold. Oligomycin and -SH group reagents exerted no influence on the Ca2+-ATPase activity, which was on the contrary inhibited by detergents such as Triton X-100 and deoxycholate. The activity of the Ca2+-ATPase was maximal at pH 7, and was decreased by 50 mM Na+ and 5 mM K+. On the contrary, the activity of Mg2+-ATPase, also present in the plasma membrane fraction, had a peak at about pH 7.8, and was stimulated by Na+ plus K+. On account of its properties, it is suggested that the Ca2+-ATPase is a component of the plasma membrane of the alveolar macrophage, and that its function may be that of participating in the maintenance of low free Ca2+ concentrations in the macrophage cytosol.     

Plasma Membrane Glycoproteins of Ciona intestinalis Spermatozoa that Interact with the Egg1

In the ascidian Ciona intestinalis the species-specific interaction between the spermatozoon and the egg occurs between the vitelline coat (VC) of the egg and the plasma membrane of the apical part of the head of the spermatozoa. Concanavalin A (Con A)-binding sites are present on this area of the sperm surface. We used Con A to identify and isolate the spermatozoon plasma membrane components that may be involved in the interaction with the VC. These glycoproteins have been identified on SDS-PAGE of a sperm membrane fraction (SMF) enriched with the extermal proteins, after incubation of the gel with ³H-Con A. Affinity chromatography on Con A-agarose has been used for the purification of sperm plasma membrane proteins with and affinity for the lectin. The biological activity of the Con A-retained fraction was determined with binding and fertilization assays.     

Glycans of synaptosomal plasma membrane glycoproteins from adult rat forebrain: Characterization after fractionation by concanavalin A-Sepharose affinity chromatography

Glycopeptides obtained by exhaustive proteolytic digestion of synaptosomal plasma membranes from adult rat forebraini were separated by affinity chromatography on concanavalin A-Sepharoe. Concanavalin A-binding glycopeptides are essentially made up of mannose and N-acetylglucosamine in a molar ration of 3.45:1, whereas glycopeptides not bound to concanavalin A have a complex monosaccharide composition. By gel filtration on Bio-Gel P-30, concanavalin A-binding glycopeptides appear as low-molecular-weight glycopeptides (migrating like ovalbumin glycopeptides), whereas glycopeptides not bound to concanavalin A behave as high-molecular-weight glycopeptides (migrating like fetuin glycopeptides). Comparison of concanavalin A-binding glycopeptides from rat brain synaptosomal plasma membranes with concanavalin A-binding glycoproteins isolated from the same membrane fraction shows clear differences in monosccharide composition. We demonstrate here that this discrepancy is due to the presence on most concanavalin A-binding glycoprotein subunits of at least two different types of glycan: in addition to the concanavalin A-binding glycans, these glycoprotein subunits carry other glycans which do not interact with concanavalin A. Biological implications of the presence of two (or more) types of glycan on the same polypeptide are discussed.     

125I-wheat germ agglutinin blotting: increased sensitivity with polyvinylpyrrolidone quenching and periodate oxidation/reductive phenylamination

Two substantial improvements in sensitivity in the identification of 125I-wheat germ agglutinin-binding glycoproteins on nitrocellulose blots of sodium dodecyl sulfate-polyacrylamide gels are reported. The major improvement in sensitivity (about 30-fold) derives from the use of 2% (w/v) polyvinylpyrrolidone (average Mr 40,000) instead of bovine serum albumin or denatured hemoglobin as the quenching agent (or carrier) during incubation with 125I-wheat germ agglutinin in detergent-free, phosphate-buffered saline. Under these conditions, specific labeling with 125I-wheat germ agglutinin is observed for orosomucoid derivatives that display N-acetylglucosamine or sialic acid residues at the nonreducing termini of their oligosaccharides, as well as for a number of glycoprotein components of a rat hepatocyte plasma membrane fraction. An additional improvement in sensitivity (up to 10-fold) results from an increase in the binding of 125I-wheat germ agglutinin to sialic acid-containing glycoproteins after treatment of the blots with 5 mM sodium metaperiodate followed by 5 mM aniline in the presence of 30 mM sodium cyanoborohydride. This treatment appears to cause the sequential oxidation and reductive phenylamination of the side chain of glycoprotein sialic acid residues.     

Cell surface carbohydrates of embryonal carcinoma cells: polysaccharidic side chains of F9 antigens and of receptors to two lectins, FBP and PNA

Three cell surface components of mouse embryonal carcinoma (EC) cells, F9 antigens and the receptors to the lectins FBP and PNA, have been isolated from radiolabeled EC cells by indirect immunoprecipitation. All three were efficiently labeled with fucose, galactose and glucosamine, but scarcely at all with mannose. The high molecular weight glycopeptides characteristic of early embryonic cells were released as the major glycopeptides upon pronase digestion of the three markers. The binding sites to the two lectins are present in the high molecular weight glycopeptides. Furthermore, a close correlation exists between the disappearance of the high molecular weight glycopeptides from differentiating EC cells and the disappearance of the three markers from the surface of these cells. The large glycopeptides from the three markers have the following properties in common. First, they are not mucin-type glycopeptides with short oligosaccharides, glycolipids and acidic mucopolysaccharides, nor are they products of incomplete pronase digestion of conventional complex-type glycopeptides. Second, they do not contain appreciable amounts of Fucα1→2Gal or Fucα1→6GlcNAc linkages. Third, a significant fraction of the glycopeptides have the GlcNAcβ→Gal sequence in their core structure. We propose that the cell surface markers of EC cells have a class of large carbohydrate chains not found in typical surface markers of adult cells such as H-2, la and LETS.