Enhanced glycosylation of a melanoma cell surface glycoprotein by retinoic acid: carbohydrate chain analysis by lectin binding

Retinoic acid (RA) inhibits the growth of mouse S91-C2 melanoma cells and enhances the glycosylation of a cell surface sialoglycoprotein (gp160). The present study analyzed the binding of 125I-labeled lectins to gp160 within polyacrylamide slab gels after electrophoretic separation of cellular macromolecules. Wheat germ agglutinin (WGA) and concanavalin A (Con A) bound to gp160 of RA-treated cells (RA-gp160) more extensively than to gp160 of control cells (C-gp160). Lens culinaris hemagglutinin (LCH), pokeweed mitogen (PWM), Ricinus communis agglutinin I (RCAI), and peanut agglutinin (PNA) failed to bind to either C-gp160 or to RA-gp160. The binding of WGA was greatly diminished after sialic acid removal. In contrast, desialylation made possible the binding of RCAI to RA-gp160. LCH, PWM and PNA did not bind to gp160 even after desialylation. Smith degradation exposed WGA-binding sites on RA-gp 160. These results suggest that gp 160 contains one or more highly branched, sialylated, N-linked complex-type side chains and lacks O-linked oligosaccharides and poly N-acetyllactosamine side chains.     

Identification and characterization of podocalyxin--the major sialoprotein of the renal glomerular epithelial cell

The glomerular epithelial polyanion is a specialized cell surface component found on renal glomerular epithelial cells (podocytes) that is rich in sialoprotein(s), as detected by staining with cationic dyes (colloidal iron, alcian blue) and wheat germ agglutinin (WGA). We have isolated rat glomeruli and analyzed their protein composition by SDS PAGE in 5-10% gradient gels. When the gels were stained with alcian blue or "Stains All," a single band with an apparent Mr of 140,000 was detected that also stained very prominently with silver, but not with Coomassie Blue. This band predominated in fluorograms of gels of isolated glomeruli that had been labeled in their sialic acid residues by periodate-[3H]borohydride. In lectin overlays, the 140-kilodalton (kd) band was virtually the only one that bound [125I]wheat germ agglutinin, and this binding could be prevented by predigestion with neuraminidase. [125I]Peanut lectin bound exclusively to the 140-kd band after neuraminidase treatment. An antibody was prepared that specifically recognizes only the 140-kd band by immunoprecipitation and immuneoverlay. By immunoperoxidase and immunogold techniques, it was localized to the surface coat of the glomerular epithelium and, less extensively, to that of endothelial cells. When analyzed (after electroelution from preparative SDS gels), the 140-kd band was found to contain approximately 20% hexose and approximately 4.5% sialic acid. These findings indicate that the 140-kd protein is the major sialoprotein of the glomerulus, and it is the only component of glomerular lysates with an affinity for cationic dyes and lectins identical to that defined histochemically for the epithelial polyanion in situ. Since this molecule is a major component of the cell coat or glycocalyx of the podocytes, we have called it "podocalyxin."     

Cell surface glycoproteins of 13762NF mammary adenocarcinoma clones of differing metastatic potentials

Rat 13762NF mammary adenocarcinoma cell surface glycoproteins from s.c. tumor- or lung metastases-derived cell clones of differing spontaneous metastatic potentials were examined for their relationship to metastasis. After treatment with neuraminidase, lectin-binding assays showed that highly metastatic clone MTLn3 cells express approximately twice the quantity of peanut agglutinin (PNA) binding sites (approximately 2.3 X 10(8) sites/cell) than clones of lower metastatic potential. However, the number of wheat germ agglutinin (WGA)-binding sites on the various cell clones decreased slightly as the metastatic potential of the clones increased. The quantities of concanavalin A (conA)-binding sites were similar (approximately 1.7 X 10(8) sites/cell) in all cell clones and growth conditions. Glycoprotein analysis was performed by electrophoresis on polyacrylamide gels in the presence of sodium dodecyl sulfate (SDS-PAGE) and subsequent staining with 125I-labeled lectins. SDS-PAGE gels stained with 125I-labeled conA revealed mainly one glycoprotein (Mr approximately 150 kD), and the amounts of this glycoprotein did not correlate with metastasis. Differences in WGA-binding glycoproteins were detected between s.c. tumor- and lung metastases-derived cell clones. Several desialylated glycoproteins were detected with 125I-labeled PNA after SDS-PAGE, and the labeling intensity of one (Mr approximately 580 kD) correlated with the metastatic potentials of the various cell clones. This high Mr galactoprotein was further analyzed by [3H]glucosamine metabolic labeling, solubilization, sequential gel filtration, and chondroitinase ABC treatment prior to SDS-PAGE. The 580 kD galactoprotein was expressed in increased amounts on the more highly metastatic clones. Chemical labeling of cell surface sialic acid residues using periodate treatment followed by [3H]borohydride reduction showed an additional change in a major sialoglycoprotein (Mr approximately 80 kD), which decreased in labeling intensity on clones of increasing metastatic potential. The results suggest quantitative changes in cell surface glycoproteins rather than major qualitative alterations are associated with differences in the metastatic behavior of 13762NF tumor cell clones.     

Differential involvement of cell surface sialic acid residues in wheat germ agglutinin binding to parental and wheat germ agglutinin-resistant Chinese hamster ovary cells

Two Chinese hamster ovary (CHO) cell mutants selected for resistance to wheat germ agglutinin (WGA) have been shown to exhibit defective sialylation of membrane glycoproteins and a membrane glycolipid, GM3. The mutants (termed WgaRII and WgaRIII) have been previously shown to belong to different genetic complementation groups and to exhibit different WGA-binding abilities. These mutants and a WGA-resistant CHO cell mutant termed WgaRI (which also possesses a surface sialylation defect arising from a deficient N-acetylglucosaminyltransferase activity), have enabled us to investigate the role of sialic acid in WGA binding at the cell surface. Scatchard plots of the binding of 125I- WGA (1 ng/ml to 1 mg/ml) to parental and WgaR CHO cells before and after a brief treatment with neuraminidase provide evidence for several different groups of sialic acid residues at the CHO cell surface which may be distinquished by their differential involvement in WGA binding to CHO cells.     

The vasoactive intestinal peptide receptor on intact human colonic adenocarcinoma cells (HT29-D4). Evidence for its glycoprotein nature.

We have previously shown that the mono [125I]iodinated vasoactive intestinal peptide (125I-VIP) could be covalently cross-linked on intact colonic adenocarcinoma cells (HT29). A major Mr 67,000 and a minor Mr 120,000 cross-linked polypeptides have been characterized [Muller, Luis, Fantini, Abadie, Giannellini, Marvaldi & Pichon (1985) Eur. J. Biochem. 151, 411-417]. The glycoprotein nature of these species was investigated using endo-beta-acetylglucosaminidase F (Endo F) treatment, enzymic and chemical desialylation and wheat germ agglutinin (WGA)-Sepharose affinity chromatography. Affinity-labelled VIP-binding proteins solubilized by Nonidet P-40 bound to WGA-Sepharose and could be eluted specifically with N-acetyl-D-glucosamine. Treatment with Endo F resulted in an increased electrophoretic mobility of both polypeptides. The major and the minor VIP-binding proteins were converted respectively into Mr 47,000 and 100,000 species, indicating removal of 20 kDa of N-linked oligosaccharides. Deglycosylation with trifluoromethanesulphonic acid also led to a 20 kDa loss in mass of the Mr 67,000 component, indicating the absence of additional O-linked sugars on this polypeptide. The presence of sialic acid on the major VIP-binding protein was demonstrated after treatment of intact cells with neuraminidase or by chemical desialylation with hydrochloric acid. We conclude from this study that the VIP receptor from intact HT29-D4 cells is a glycoprotein with N-linked oligosaccharide side chains containing sialic acid.     

The canine renal parathyroid hormone receptor is a glycoprotein: characterization and partial purification

Covalent labeling of the canine renal parathyroid hormone receptor with [125I]bPTH(1-34) reveals several major binding components that display characteristics consistent with a physiologically relevant adenylate cyclase linked receptor. Through the use of the specific glycosidases neuraminidase and endoglycosidase F and affinity chromatography on lectin-agarose gels, we show here that the receptor is a glycoprotein that contains several complex N-linked carbohydrate chains consisting of terminal sialic acid and penultimate galactose in a beta 1,4 linkage to N-acetyl-D-glucosamine. No high mannose chains or O-linked glycans appear to be present. The peptide molecular weight of the deglycosylated labeled receptor is 62,000 [or 58,000 if the mass of bPTH(1-34) is excluded]. The binding of [125I]bPTH(1-34) to the receptor is inhibited in a dose-dependent fashion by wheat-germ agglutinin, but not by either succinylated wheat-germ agglutinin or Ricinus communis lectin, suggesting that terminal sialic acid may be involved in agonist binding. A combination of lectin affinity chromatography and immunoaffinity chromatography affords a 200-fold purification of the covalently labeled receptor.     

Wheat germ agglutinin inhibits thrombin-induced rises in cytosolic free calcium and prostacyclin synthesis by human umbilical vein endothelial cells

To characterize the endothelial cell surface membrane glycoproteins that mediate thrombin stimulation of PGI2 synthesis by human umbilical vein endothelial cells (HUVEC), HUVEC were stimulated with thrombin in the presence or absence of different lectins. Of the lectins tested, only wheat germ agglutinin (WGA) inhibited thrombin-induced rises in cytosolic free calcium [( Ca2+]i), measured using Quin 2-loaded HUVEC and PGI2 production measured by radioimmunoassay. However, WGA by itself had no influence on baseline [Ca2+]i or PGI2 production and did not inhibit histamine-induced rises in [Ca2+]i. The inhibition of thrombin-induced rises in [Ca2+]i and PGI2 production by WGA was dose dependent, with half-maximal inhibition occurring at 2 micrograms/ml. WGA also inhibited thrombin-induced release of 3H-arachidonic acid. These effects of WGA were reversed by N-acetyl-glucosamine (GlcNAc) and N-acetyl-neuraminic acid, which bind specifically to WGA, but not by unrelated sugars. Succinylated WGA (succ-WGA), a chemically modified derivative of WGA that binds to GlcNAc but, unlike native WGA, not to sialoglycoproteins, did not inhibit thrombin-induced rises in [Ca2+]i and PGI2 production. These results suggest that thrombin induces rises in [Ca2+]i and PGI2 production by interacting with an endothelial surface membrane sialoglycoprotein.     

Modification and introduction of various radioactive labels into the sialic acid moiety of sialoglycoconjugates

A method for modifying and isotopic labeling the sialyl moiety of sialoglycoproteins is described. The basis of the procedure is the reductive amination of the exocyclic aldehyde group, generated on sialic acid by mild periodate oxidation, with a variety of amino compounds and sodium cyanoborohydride. Optimal conditions were selected to obtain maximum modification of sialic acid and minimal non-specific incorporation of the amino compound (glycine). The glycine modified model glycoproteins (α₁-acid glycoprotein, fetuin) yielded single homogenous peaks upon gel filtration and on ion exchange chromatography. On gel electrophoresis a major band accounting for 92–98% of the modified glycoprotein and two minor bands consisting of dimers and trimers of the glycoprotein were observed. The modification did not alter the ability of the sialoglycoproteins to bind to wheat germ agglutinin-Sepharose or to interact with antibodies. The modified sialic acid was only partially released by mild acid hydrolysis suggesting that the introduction of an amino compound into the polyol chain of sialic acid has a stabilizing effect on the ketosidic linkage of the sugar. Interestingly, the modification rendered the sialic acid resistant to a variety of sialidases. The potential uses of this modification procedure include 1) the introduction of different isotopic labels (³H,¹⁴C,³⁵S,¹²⁵I) into the sialic acid moiety of glycoproteins; 2) the preparations of biologically active sialoglycoprotein (hormones, enzymes, co-factors) with increased circulating half-lives in animals; 3) preparation of substrates to search for endoglycosidases; 4) the direct comparison of sialoglycoprotein patterns obtained in small amounts from normal and pathological cells or tissues, and 5) the isolation and purification of cell surface sialoglycoproteins.     

Identification of differences between the surface proteins and glycoproteins of normal mouse (Balb/c) and human erythrocytes

Summary The topography of the external surface of the Balb/c mouse erythrocyte has been investigated and compared to the human erythrocyte by using a series of protein radiolabeling probes. After sodium dodecyl sulfate-polyacrylamide gel electrophoresis, the pattern of Coomassie Blue stained proteins was very similar for mouse and human erythrocyte ghosts, as was the distribution of radioactivity in protein bands after lactoperoxidase catalyzed radioiodination. The mouse erythrocyte glycoproteins identified by periodic-acid-Schiff and Stains-All reagents, sialic acid analysis of gel slices, binding of¹²⁵I-wheat germ agglutinin and¹²⁵I-concanavalin A to the gels, and glycoprotein radiolabeling techniques, differed markedly from the sets of proteins labeled by radioiodination, and also differed from the human erythrocyte glycoproteins. Instead of the PAS I to PAS IV series of sialoglycoproteins characteristic of human erythrocytes, the mouse erythrocyte possesses a broad band of sialoglycoproteins with several peaks ranging in mol wt from 65,000 to 32,000. The same group of sialoglycoproteins were labeled by the periodate/B³H₄ ^– technique specific for terminal sialic acid, and the galactose oxidase/B³H₄ ^– method (plus neuraminidase) specific for galactosyl/N-acetylgalactosaminyl residues penultimate to sialic acid. These results emphasize the necessity to employ a variety of protein radiolabeling probes based on different labeling specificities, to study the membrane topography of cells which are poorly understood compared to the human erythrocyte membrane.     

Developmental regulation of neuraminidase-sensitive lectin-binding glycoproteins during myogenesis of rat L6 myoblasts.

Intact monolayers of L6 myoblasts were treated with neuraminidase, with the aim of selectively removing sialic acid residues of cell-surface glycoproteins. Neuraminidase treatment unmasked binding sites for Ricinus communis agglutinin I and peanut agglutinin, thus allowing the identification of the major binding proteins for these lectins. For Ricinus communis agglutinin I these neuraminidase-sensitive glycoproteins had apparent Mr values of 136000, 115000, 87000, 83000 and 49000. For peanut agglutinin the major neuraminidase-sensitive glycoproteins had apparent Mr values of 200000, 136000, 87000 and 83000. We found highly reproducible, developmentally regulated, changes in the lectin-binding capacity of certain of these glycoproteins as L6 myoblasts differentiated into myotubes. Coincident with myoblast fusion there was a co-ordinate decrease in Ricinus communis agglutinin I binding by glycoproteins of apparent Mr of 136000 and 49000. There was also a co-ordinate shift in mobility of the broad band of glycoprotein, centred at an apparent Mr of 115000 in myoblasts, to a new average apparent Mr of 107000 in mid-fusion cultures and myotube cultures. Peanut agglutinin binding by the major protein of apparent Mr 136000 also decreased at the mid-fusion stage of myogenesis, and was barely detectable in 7-day-old fused cultures. These developmentally regulated changes in neuraminidase-sensitive glycoproteins were all inhibited by growth of myoblasts in 6.4 microM-5-bromo-2'-deoxyuridine, indicating that they are associated with myoblast differentiation. In contrast, an increase in fibronectin was seen in mid-fusion cultures, which was not inhibited by growth of myoblasts in 5-bromo-2'-deoxyuridine. This initial increase in fibronectin is, therefore, unlikely to be directly related to myoblast fusion or differentiation.     

Changes in the content and distribution of sialic acid on the basal surface of isoproterenol-stimulated mouse parotid acinar cells

The presence, distribution and content of sialic acid on the cell surface in collagenase-dispersed acini obtained both from unstimulated as well as from in vivo isoproterenol-stimulated mouse parotid have been studied. To this end, sialic acid residues have been qualitatively and quantitatively analyzed by 1) cytochemical labeling by wheat germ agglutinin (WGA), 2) biochemical procedures and 3) isotopic labeling by [3H]WGA (WGA-N-[acetyl-3H]-acetylated). Electron microscopy revealed striking differences in the binding of ferritin-conjugated WGA at the basal, lateral and apical cell surface. Unstimulated acinar cells showed a heavy patch-distributed binding of ferritin-conjugate on the basal cell surface while it was homogeneous and very scarce on the lateral one and absent on the apical cell surface. During the first few hours after isoproterenol, the WGA binding sites at the basal cell surface became homogeneously distributed. This fact was coincident with a loss of about 60 to 70% both in the content of neuraminidase-releasable sialic acid and in the binding of [3H]WGA to the acinar surface. These findings suggest that the release of sialic acid as free residues, which has been involved in the isoproterenol-triggered cell proliferation-inducing mechanism in the mouse parotid, would occur at the glycocalyx corresponding to the basal plasma membrane of the acinar cells.     

Cell surface properties of ascites sublines of the 13762 rat mammary adenocarcinoma : Relationship of the major sialoglycoprotein to xenotransplantability

The relationship between cell surface sialoglycoprotein and xenotransplantation has been investigated in ascites sublines of the 13762 rat mammary adenocarcinoma. Two of the five sublines (MAT-C and MAT-C1) can be transplanted into mice. These two sublines also have the greatest amounts of total, trypsin-releasable and neuraminidase-releasable sialic acid. Chemical labeling using periodate treatment followed by [³H]borohydride reduction indicates that most of the protein-bound sialic acid is associated with a single major sialoglycoprotein (or family of glycoproteins) with a low mobility on polyacrylamide gels in dodecyl sulfate (SDS). This glycoprotein, denoted ASGP-1, is also labeled by lactoperoxidase and ¹²⁵I, indicating its presence at the cell surface. Metabolic labeling with [³H]glucosamine shows that ASGP-1 is the major glycosylated protein in both xenotransplantable (MAT-C1) and non-xenotransplantable (MAT-B1) sublines, representing >70% of the protein-bound label in each. The labeling studies indicate that the non-xenotransplantable subline does not have a substantially greater amount of ASGP-1 on its cell surface. Likewise cationized ferritin labeling and transmission electron microscopy (TEM) do not show substantially greater amounts of negatively charged groups distributed along the cell surfaces of MAT-C1 than of MAT-B1 cells. The results indicate that the transplantation differences between these sublines cannot be explained solely by the presence of a major sialoglycoprotein at the cell surface.     

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.     

Lectin-gold cytochemistry of the Golgi apparatus in rabbit luteal cells, with special emphasis on the formation of a lysosomal-type membrane

In rabbit luteal cells embedded in glycolmethacrylate and stained with PTA at low pH highly glycosylated membrane patches can be observed after vesiculation of the trans-Golgi network. As these membranes could be prelysosomal, their sialic acid content was investigated by post-embedding labeling with Limax flavus agglutinin (LFA)/fetuin-Au. Additional labeling of the Golgi apparatus was performed with Wheat germ agglutinin (WGA)/ovomucoid Au, Ricinus communis agglutininI (RCAI)/Au and Helix pomatia agglutinin (HPA)/Au. The sections were then counterstained with PTA at low pH, which allows a clear distinction between the elements of the trans-Golgi network (G2-G1) and the saccules of the stack (g). With WGA, LFA and RCAI the trans-Golgi network was observed to be clearly more reactive than the stack. After vesiculation most intense labeling was found over the highly glycosylated vacuolar membranes derived from the G2-element. The limiting membrane of lysosomes, the MvB's and the plasma membrane also reacted strongly. Colloidal gold particles were also found over the membranes of the vacuoles derived from G1. The Golgi stack showed a lower reactivity and label for all three lectins could be found over three to four saccules of the stack (g3-g4). The matrix of the lysosomes was slightly labeled. Labeling with HPA was absent from the trans saccules and was consistently found in the cis and cis-most (g4-g5) saccules of the stack. Some cytoplasmic vesicles near the cell border were also labeled. With our procedure the Golgi apparatus can easily be detected and it is apparent that in rabbit luteal cells the highest lectin reactivity is found in the trans-Golgi network.(ABSTRACT TRUNCATED AT 250 WORDS)     

Membrane glycoproteins of the nerve growth cone: diversity and growth regulation of oligosaccharides

A subcellular fraction prepared from fetal rat brain and enriched in growth cone membranes is analyzed for its lectin-binding proteins. Growth-associated glycoproteins are identified by comparing the growth cone glycoproteins with those of synaptosomes. Protein was resolved in one- or two-dimensional gels, electroblotted, and blots probed with radioiodinated concanavalin A, wheat germ agglutinin, and Ricinus communis agglutinins I and II. In one-dimensional gels, each lectin recognizes approximately 20 polypeptides (with substantial overlap) most of which migrate diffusely and have relatively high molecular masses (range 30-200 kD). The seven major Coomassie-staining proteins of the membrane fraction (34-52 kD) are not the major lectin-binding proteins. In two-dimensional gels, the lectin-binding proteins are either streaked across the pH gradient or exist as multiple spots, indicating broad charge heterogeneity. Seven wheat germ agglutinin- and Ricinus communis agglutinin II-binding glycoproteins are present in greater abundance in growth cone fractions compared with synaptosomes. Most notably, an acidic, sialic acid-rich protein (27-30 kD, pI 4.0; termed gp27-30) is most abundant at postnatal day 4, but absent from adult brain. The protein's very acidic isoelectric point is due, at least in part, to its high sialic acid content. Growth regulation of specific protein-linked oligosaccharides suggests that they play a special role in growth cone function. In addition, the great diversity of growth cone glycoproteins from whole brain suggests glycoprotein heterogeneity among growth cones from different neuron types.     

Characterization of plasma-membrane glycoproteins from functional domains of the rat hepatocyte.

Plasma-membrane glycoproteins from the three different functional domains of the rat hepatocyte were radioactively labelled by oxidation with NaIO4, followed by reduction with NaB3H4. Analysis of the radioactively labelled glycoproteins by polyacrylamide-gel electrophoresis revealed the presence of at least 12 major sialoglycoproteins in each different region of the hepatocyte surface. The Mr-110 000 component was homogeneously distributed over the plasma membrane, whereas the Mr-90 000 polypeptide was only located at the sinusoidal face. These radiolabelled glycoproteins were solubilized in 1% Triton X-100, and the soluble fraction was subjected to affinity chromatography on Sepharose-conjugated wheat-germ agglutinin (WGA). The labelled glycoproteins were poorly bound to WGA. Membrane glycoproteins were also labelled by the galactose oxidase/NaB3H4 method. The results show that the polypeptides with apparent Mr 170 000 from the sinusoidal, 230 000 from the canalicular and 170 000 from the lateral membranes were specifically labelled. When the membranes were treated with neuraminidase and galactose oxidase/NaB3H4, the electrophoretic patterns showed changes in the apparent Mr values of the glycoproteins, owing to loss of sialic acid, and a clear increase in labelling in the sinusoidal and canalicular membranes compared with the lateral membranes. When these labelled membranes were solubilized in 1% Triton X-100 and subjected to affinity chromatography on Sepharose-conjugated Ricinus communis agglutinin and/or Lens culinaris agglutinin, the results showed that the former columns efficiently bound the radiolabelled glycoproteins, whereas the latter columns bound poorly. The results show that there is a differential distribution of glycoproteins along the hepatocyte's surface.     

Direct determination of bound sialic acids in sialoglycoproteins by acidic ninhydrin reaction

A simple and rapid method for sialic acid determination in sialoglycoproteins by acidic ninhydrin reaction is described. The method is based on the reaction of sialic acids with an acidic ninhydrin reagent (K. Yao and T. Ubuka (1987) Acta Med. Okayama 41, 237-241). By heating a sample solution containing sialoglycoprotein with the reagent at 100 degrees C for 10 min, a stable color with an absorption maximum at 470 nm was produced. The standard curve was linear in the range of 20 micrograms to 3 mg of fetuin, a sialoglycoprotein, per 3.0 ml of the reaction mixture. The reaction is specific only for sialoglycoproteins among various proteins examined. The acidic ninhydrin method was applied to the determination of sialic acids in sialoglycoproteins in ascites fluids of Ehrlich ascites tumor-bearing mice.     

The effect of oral contraceptives on rat platelet membrane glycoproteins

Female rats were administered oral contraceptives and the levels of sialic acid on platelet membrane and granule glycoproteins were compared to controls using a sialic acid assay and a fluorescein-conjugated wheat germ agglutinin binding assay and also by measuring the binding of ¹²⁵I-labelled wheat germ agglutinin to glycoprotein bands from platelets separated by polyacrylamide electrophoresis. The contraceptive-treated rats showed increased levels of glycoprotein sialylation which may partly explain the altered physiological function of the platelets.     

Maturational changes in terminal glycosylation of small intestinal microvillar proteins in the rat

Studies were performed to identify rat intestinal microvillar proteins which undergo changes in terminal glycosylation during postnatal development. Pulse-labeling with [3H]fucose or N-[3H]acetylgalactosamine showed significantly higher incorporation into purified microvillar membranes of weanling than suckling rats. In contrast, the incorporation of [3H]sialic acid after pulse-labeling with N-[3H]acetylmanosamine was higher in suckling rats. SDS-polyacrylamide gel electrophoresis revealed these developmental differences in radioactive sugar incorporation to involve mainly proteins above Mr 90,000. 125I-labeled peanut lectin autoradiography revealed an Mr greater than 330,000 binding protein in suckling rats. Neuraminidase treatment of the membranes revealed the presence of sialyl-substituted sites in this protein in suckling, weaning and weanling animals, but the unmasking of sites decreased with advancing maturation. 125I-labeled Ulex europeus I autoradiography showed marked increases in binding of this lectin to Mr 66,000, 92,000, 130,000, 150,000 and greater than 330,000 proteins from weaning to weanling periods. Similar age-related increases in soybean lectin binding to Mr 130,000-150,000, and greater than 330,000 proteins were demonstrated by affinity chromatography. The Mr values of the major lectin-binding proteins were close to those reported for several hydrolases (trehalase, alkaline phosphatase, sucrase-isomaltase and glucoamylase). Comparison of the Coomassie blue-stained electrophoretograms from each age-group against the corresponding autoradiograms of lection-binding proteins led us to conclude that, while the content of these proteins in the membrane achieve their mature levels at or before weaning, their terminal glycosylation (desialylation, fucosylation, N-acetylgalactosamination) is not fully established until later development.