Effect of Hypoxia on Endothelial Cell Surface Glycoprotein Expression: Modulation of Glycoprotein IIIa and Other Specific Surface Glycoproteins

Exposure to hypoxia alters many aspects of endothelial cell metabolism and function; however, changes in surface glycoconjugates under these conditions have not been extensively evaluated. In the current studies, we examined surface glycoproteins of cultured bovine aortic (BAEC) and pulmonary arterial (BPAEC) endothelial cells under standard culture conditions (21% oxygen) and following exposure to hypoxia (0% oxygen) for varying time periods (30 min to 18 h) using a system of biotinylation, lectin binding (concanavalin A, Con A; Griffonia simplicifolia , GSA; Arachis hypogaea, PNA; Ricinus communis, RCA; or Triticum vulgaris, WGA), subsequent strep-avidin binding, and staining. Using these methods, we identified differences in lectin binding between the two cell types cultured in 21% oxygen with all lectins except PNA. With exposure to 0% oxygen, there was no change in lectin binding to most surface glycoproteins. Several surface glycoproteins, including glycoprotein IIIa on both cell types, demonstrated a time-dependent decrease in lectin binding; in addition, there was an increase in lectin binding to a few specific surface glycoproteins on each cell type within 30-60 min of exposure to 0% oxygen. These changes in specific surface glycoproteins were confirmed in both cell types by ¹²⁵I labeling. Increased lectin binding was observed for Con A binding BAEC glycoproteins at molecular weight (MW) 116, 130, and 205 kDa, GSA binding BAEC glycoproteins at MW 120 and 205 kDa, and RCA binding BPAEC glycoproteins at MW 140 and 205 kDa. Increased binding of WGA or PNA was not observed during exposure to hypoxia. The specificity of lectin binding was further confirmed by competitive inhibition with the appropriate sugar. These studies demonstrate that there are baseline differences between BAEC and BPAEC cell surface glycoproteins and that exposure to hypoxia is associated with little change in lectin binding to most surface glycoproteins. There is, however, increased surface expression of a few glycoproteins that differ depending of the origin of the endothelial cell. Although the mechanism of this increase in lectin binding is not yet clear, subsequent studies suggested that it is due to increased availability of select carbohydrate moieties. The time course of these alterations suggests a possible role in the endothelial cell response to decreases in ambient oxygen tension.     

Trypanosoma brucei gambiense and T. b. rhodesiense: Concanavalin A Binding to the Membrane and Flagellar Pocket of Bloodstream and Procyclic Forms1

ABSTRACT We have measured binding of fluorescein-conjugated succinyl-concanavalin A (Fl-s-Con A) to bloodstream and procyclic forms of Trypanosoma brucei gambiense and to bloodstream forms of T. b. rhodesiense by flow cytofluorimetry. Bloodstream forms bound an order of magnitude less lectin than procyclic forms. Trypsin-treating cells enhanced binding of Fl-s-Con A to bloodstream forms 3–16-fold depending on the strain and the length of trypsinization but had little effect on Fl-s-Con A binding by procyclics. The trypsinization protocol used did not remove major common glycoproteins detected on lectin blots of either life cycle form but removed >95% of the variant specific glycoprotein and fragments derived from this protein of bloodstream forms. Microscopically detectable Fl-s-Con A binding to bloodstream forms was confined to the flagellar pocket. Trypsinized bloodstream forms and procyclics bound Fl-s-Con A in the flagellar pocket, on the flagellum, and on the cell surface. Lectin remained cell associated but appeared to redistribute towards the flagellum and pocket when cells that had bound lectin on ice were subsequently incubated at physiological temperatures. The Fl-s-Con A binding had specificity characteristic of the interaction between the lectin and oligosaccharides. These results are consistent with the hypothesis that the variant specific surface glycoprotein blocks binding of the lectin to surface glycoproteins of bloodstream forms and suggest that concanavalin A-binding glycoproteins are abundant in the flagellar pocket of both life cycle forms.     

A Further Separation and Some Properties of β-Lactamase I

A carbohydrate binding protein was found in mid-lactating rat mammary gland. This rat mammary gland lectin agglutinated trypsinized rabbit erythrocytes and the hemagglutination was inhibited by the addition of β-d-galactosides such as lactose, melibiose, UDP-galactose and thio-d-galactoside. The lectin was partially purified by affinity chromatography on a column of Sepharose 4B to which asialo-fetuin had been covalently linked. Rat mammary gland lectin is a glycoprotein with a molecular weight of 14,800, estimated from SDS-PAGE, or 16,800 from gel filtration.

The occurrence of two glycoproteins, C4-casein and α-lactalbumin, is known in rat milk. Bovine κ-casein is a well-characterized glycoprotein. These glycoproteins were found to be bound by the rat mammary gland lectin, when they were desialylated by the action of neuraminidase. Neuraminidase-untreated α-lactalbumin also bound to the lectin but to a lesser extent. The level of the lectin in rat mammary gland was greatly reduced during regression of the gland after weaning.     

Lectin Receptors in Central Nervous System Myelin

Abstract: Proteins from central nervous system myelin were separated by high-resolution, sodium dodecyl sulfate-pore gradient slab gel electrophoresis and the glycoproteins were detected by autoradiography after direct application of radioiodinated lectins. A surprising heterogeneity of lectin binding proteins was found associated with this highly purified membrane fraction. Iodinated wheat germ agglutinin, which has a monosaccharide specificity for N-acetyl-D-glucosamine and N-acetylneuraminic acid, revealed six major bands and two minor bands. By correlating the molecular weights (M_r) of radioiodinated protein standards with the gel concentration at the position reached by the protein (%T) using the relationship log(M_r) versus log(%T) for gradient gel systems, molecular weight estimates of 128, 300, 109, 800, 75, 300, 48, 800, 26, 100 and 23, 700 were obtained for the major glycoprotein bands and molecular weights of 98, 300 and 86, 600 for the minor bands. When the isolated myelin was extracted with chloroform-methanol-a procedure that removes the major myelin proteins, including the proteolipid protein and most of the basic proteins and hence concentrates the minor high molecular weight proteins-and analyzed after gradient gel electrophoresis, additional glycoproteins of molecular weights 607, 700, 196, 900, 175, 100, 61, 800, 52, 200 and 42, 600 were resolved with this lectin. Radioiodinated soybean agglutinin, which has a specificity for N-acetyl-D-galactosamine and D-galactose, revealed seven bands, three of which were unique to this lectin (19, 600, 19, 100 and 17,000). Iodinated concanavalin A (d -mannose, d -glucose) revealed bands similar to the wheat germ agglutinin as well as additional bands of 40, 300, 37, 300, 35, 700, 21, 800 and 20, 400. The glycoprotein specificity for these lectin binding components was demonstrated by hapten carbohydrate binding inhibition and by organic solvent extraction for removal of glycolipids. Based on these experiments using three lectins with different carbohydrate specificity, 22 lectin-reactive components were identified; however, six of these bands were removed by chloroform-methanol extraction. The variations observed in the lectin binding capacity for these different bands suggest possible carbohydrate heterogeneity for these individual glycoproteins. Although many of these bands may be dissociated subunits (monomeric polypeptides) of oligomeric complexes, the observed multiplicity of these quantitatively minor glycoproteins associated with the purified myelin membrane implies a more intricate molecular organization for the myelin sheath complex than previously believed.     

Trypanosoma brucei brucei and Trypanosoma brucei gambiense: stage specific differences in wheat germ agglutinin binding and in endoglycosidase H sensitivity of glycoprotein oligosaccharides

Gel electrophoresis, lectin affinity blotting, and endoglycosidase H digestion have been used to analyze the glycoprotein profiles of bloodstream and procyclic forms of Trypanosoma brucei brucei and T. b. gambiense. Proteins resolved by polyacrylamide gel electrophoresis were stained with silver nitrate or electrophoretically transferred to nitrocellulose and probed with a horseradish peroxidase conjugate of either concanavalin A or wheat germ agglutinin. Silver staining showed, as expected, that the expression of the variant specific glycoprotein was restricted to the bloodstream forms. Twenty-three concanavalin A binding proteins were resolved in blots of bloodstream forms. Concanavalin A binding molecules corresponding in electrophoretic mobility to 21 of these 23 bloodstream form glycoproteins were detected in blots of procyclic forms. The two concanavalin A binding glycoproteins present only in bloodstream form extracts were variant specific glycoprotein and an 81-kDa protein designated glycoprotein 81b. One concanavalin A binding molecule of 84 kDa, glycoprotein 84p, was detected only in procyclic forms. The 19 major wheat germ agglutinin binding glycoproteins expressed by bloodstream forms were not detected in procyclic forms; only small proteins or protein fragments in procyclic form extracts bound wheat germ agglutinin. Incubating transferred proteins in endoglycosidase H eliminated subsequent binding of concanavalin A to most of the 22 common glycoproteins of bloodstream forms. Three major concanavalin A binding glycoproteins of bloodstream forms, variant specific glycoprotein, glycoprotein 81b, and a 110-kDa molecule (glycoprotein 110b), and other minor glycoproteins carried sugar chains that resisted endoglycosidase H digestion. In contrast, concanavalin A did not bind to any procyclic form glycoproteins, including a 110-kDa concanavalin A binding molecule (glycoprotein 110p) after endoglycosidase H treatment.(ABSTRACT TRUNCATED AT 250 WORDS)     

Interaction of Concanavalin A and a Divalent Derivative with Lymphocytes and Reconstituted Lymphocyte Membrane Glycoproteins

Both concanavalin A (con A) and its divalent derivative, succinyl-concanavalin A (S-con A) are mitogenic for porcine lymph node lymphocytes. We have compared the binding of these two lectins to intact porcine lymphocytes and phospholipid vesicles containing reconstituted lymphocyte membrane glycoproteins. Both con A and S-con A showed high- and low-affinity binding to intact cells, as indicated by LIGAND analysis of Scatchard plots of binding data. Despite the apparently identical saccharide specificities of the two lectins, high-affinity binding sites for S-con A were only one-third as numerous as high-affinity sites for the parent lectin. Large numbers of low-affinity binding sites existed for con A, while many fewer were present for S-con A. It is suggested that these sites result from hydrophobic association. Con A bound to lymphocytes in a positively cooperative fashion, while S-con A showed non-cooperative behavior. Lectin binding to large unilamellar phospholipid vesicles containing reconstituted lymphocyte membrane glycoproteins was measured using a rapid filtration assay, and was linear with the glycoprotein content of the vesicles. Almost all of the outward-facing glycoprotein was functional in terms of lectin binding. Reconstituted glycoproteins showed only a single class of high-affinity binding sites for both con A and S-con A, with association constants similar to those measured for intact cells. Con A, but not S-con A, showed positively cooperative binding to reconstituted vesicles. Cooperativity was observed in both gel phase and liquid crystalline phase lipid, and was thus not dependent on long-range lateral rearrangement of glycoprotein receptors. Results suggested that con A induces a microre-distribution of receptors on the lymphocyte membrane surface, leading to the exposure of glycoproteins that were previously inaccessible to the lectin. S-Con A does not cause glycoprotein redistribution, and a large fraction of the receptors remain cryptic.     

Isolation of human platelet glycoproteins

Human platelet glycoproteins were isolated from whole platelets by two methods. The first method, that of affinity chromatography on wheat germ agglutinin, is based on the known affinity of lectins for cell surface glycoproteins. When solubilized whole platelets are used as starting material for this procedure, elution with N-acetylglucosamine yields primarily a glycoprotein of M_r ≈ 150 000 as estimated by sodium dodecyl sulfate-acrylamide gel electrophoresis. The second method is based on the ability of the chaotropic salt lithium diiodosalicylate to extract glycoprotein from particulate cell fractions in water-soluble form. This method yields three major glycopeptides with apparent molecular weights after sulfhydryl reduction of 145 000, 125 000, and 95 000 as estimated on 5.6% sodium dodecyl sulfate-acrylamide gels. Carboxymethylation of these preparations in the presence of sulfhydryl-reducing agent further resolves a glycoprotein of M_r ≈ 165 000.Treatment of whole platelets by periodate oxidation and sodium[³H]borohydride reduction labels the three major glycoproteins extracted by lithium diiodosalicylate and the glycoprotein of M_r ≈ 150 000 isolated on wheat germ agglutinin confirming their surface orientation. However, glycoprotein with M_r ≈ 165 000 resolved by carboxymethylation of the lithium diiodosalicylate extracted glycoprotein mixture was not labelled by this method, suggesting that it represents the granule protein with similar electrophoretic characteristics described by others.Phosphorylation of intact platelets with ³²P_i also results in labelling of glycoproteins isolated by both methods, suggesting that these molecules traverse the     

Influence of lectins on the binding of 125I-labeled EGF to human fibroblasts.

Lectins that interact with mannose (concanavalin A), galactose (ricin, abrin), or N-acetylglucosamine (wheat germ agglutinin) block ¹²⁵I-labeled EGF binding to the surface of cultured human fibroblasts at 37° or 5°. Lectins specific for fucose or N-acetylgalactosamine, soybean agglutinin or gorse lectin, respectively, do not interfere with growth factor binding. The inhibition of ¹²⁵I-labeled EGF binding by concanavalin A at 37° or 5° could be reversed rapidly by the addition of α-methyl mannoside. The results suggest that the fibroblast membrane receptor for EGF is, or is closely associated with, a glycoprotein or glycolipid that contains mannose, galactose and N-acetylglucosamine residues.     

SOLUBLE AND MEMBRANE LECTIN-BINDING GLYCOPROTEINS OF THE CHROMAFFIN GRANULE

Abstract— Fluorescein isothiocyanate-labelled lectins were used to identify lectin-binding glycoproteins of the chromaffin granule after electrophoresis of the membrane and soluble granule proteins on sodium dodecyl sulphate polyacrylamide slab gels. The glycoprotein nature of all lectin-binding bands was confirmed by staining the gels for carbohydrates, and the specificity of the lectin-binding was demonstrated by hapten sugar inhibition of binding. In samples of granule membrane proteins reduced with dithiothreitol 10 concanavalin A (Con A), 5 wheat germ agglutinin, 8 Ricinus communis agglutinin-60, and 7 Ricinus communis agglutinin-120 (RCA-120) binding glycoproteins were identified. Molecular weights of these glycoproteins varied from 20,000 to 200,000 daltons. All but two of the Con A-binding bands and one of the RCA-120 binding bands appeared to react with more than one lectin, suggesting possible carbohydrate heterogeneity in these membrane glycoproteins. The band identified as dopamine β-hydroxylase reacted most intensely with all four lectin tested, and in the soluble core material this enzyme was the sole significant lectin binding glycoprotein.     

Global Identification of Prokaryotic Glycoproteins Based on an Escherichia coli Proteome Microarray

Glycosylation is one of the most abundant protein posttranslational modifications. Protein glycosylation plays important roles not only in eukaryotes but also in prokaryotes. To further understand the roles of protein glycosylation in prokaryotes, we developed a lectin binding assay to screen glycoproteins on an Escherichia coli proteome microarray containing 4,256 affinity-purified E.coli proteins. Twenty-three E.coli proteins that bound Wheat-Germ Agglutinin (WGA) were identified. PANTHER protein classification analysis showed that these glycoprotein candidates were highly enriched in metabolic process and catalytic activity classes. One sub-network centered on deoxyribonuclease I (sbcB) was identified. Bioinformatics analysis suggests that prokaryotic protein glycosylation may play roles in nucleotide and nucleic acid metabolism. Fifteen of the 23 glycoprotein candidates were validated by lectin (WGA) staining, thereby increasing the number of validated E. coli glycoproteins from 3 to 18. By cataloguing glycoproteins in E.coli, our study greatly extends our understanding of protein glycosylation in prokaryotes.     

A cell membrane-associated lectin of the oyster hemocyte

The presence of a lectin in association with hemocytes of the American oyster, Crassostrea virginica, has been demonstrated by utilizing a microhemagglutination assay. The plasma membrane association of this lectin is shown by its copurification with the plasma membrane fraction of disrupted hemocytes, using sucrose density gradient centrifugation, and also by the binding of ¹²⁵I-labeled glycoproteins to intact hemocytes at 4°C. Based upon agglutinating spcificity for a range of vertebrate erythrocytes, both untreated and enzyme-treated, along with hemagglutination-inhibition assays and crossed-absorption tests, it is apparent that there are also two serum (soluble) lectins, each having a distinct serological agglutination specificity, and that the hemocyte membrane-associated lectin has a specificity that is identical with one of these two serum lectins. It is proposed that the hemocyte membrane-associated lectin may be a true integral membrane protein, and therefore may function as a membrane receptor in nonself recognition by molluscan hemocytes.     

Stimulation of the biosynthesis of membrane glycoproteins from zajdela ascites hepatoma cells by Robinia lectin

Membrane glycoprotein biosynthesis of ascites hepatoma cells is followed by [¹⁴C]glucosamine and [³H]leucine incorporation into cells in culture. The rate of incorporation is strongly increased by the addition of Robinia lectin in culture medium. Labeled glycoproteins are released from lectin stimulated and non-stimulated ceils by trypsin digestion. Studies of labeled trypsinates on sodium dodecyl sulfate gel electrophoresis and Sephadex G-200 filtration exhibit two fractions both labeled with [¹⁴C]glucosamine and [³H]leucine and having different molecular weights, one over 200 000 and the other about 2000. Identical results are obtained when external membrane glycoproteins are solubilized by sodium deoxycholate. Comparison of surface glycoproteins isolated by trypsinization from control cells labeled with [³H]glucosamine and from lectin stimulated cells labeled with [¹⁴C]glucosamine displays no significant qualitative differences between glycoprotein fractions released from both cell groups.     

Glycoprotein Mediated Cell Binding of Lectin Coated Liposomes

Abstract

The malignant transformation of cells may cause changes in the oligosaccharide composition of their surface glycoproteins. These could be utilized for specific targeting of exogenous lectin probes to tumor cells. However, because of the usually low affinity of monovalent carbohydrate/lectin reactions, the formation of multiple cluster bonds would be required for stable binding. Because of the lateral mobility of protein molecules incorporated into lipid bilayers, lectin coated liposomes were supposed to be promising multivalent probes. This hypothesis was tested by determining the binding affinities of lectin coated liposomes toward erythrocytes bearing multiantennary glycophorin molecules as a major glycoprotein on their surface, and lymphoblastoid Croco II cells exhibiting glycoligands of a different nature. Actually, the measured affinities provide no or only poor evidence for lectin cluster effects. However, with erythrocytes a complementary affinity enhancing effect was observed that is caused by the multiantennary nature of the glycophorin molecule. Apparently, the presentation of 16 rather equivalent oligosaccharide substituents along the polypeptide backbone of this glycoprotein is cluster-like too, and may cause high affinity cell binding of lectin probes. Lectin coated liposomes might therefore be considered for targeting cells that bear mucin-like glycoligands on their surface.     

Biochemical and immunological characterization and the synthesis of rat intestinal glycoproteins following the induction of rapid synchronous vitamin A deficiency

Glycoproteins and proteins were extracted from segments or scrapings of the intestine in tube-fed, vitamin-A-deficient and control rats on the eight day after withdrawal of retinoic acid from the diet by using either 1% sodium dodecyl sulfate (SDS) or aqueous 5 mM EDTA (pH 7.4). They were then fractionated on columns of Sepharose 4B. Water-soluble peak I material contained large (M_r > 10⁶; S₂₀ = 11.7) glycoprotein aggregates which were rich in hexose, fucose and sialic acid. These aggregates dissociated into several non-identical glycoprotein and protein subunits upon treatment with dithiothreitol. The protein matrix was rich in threonine, valine, proline, serine, glutamate and aspartate. Peak II consisted of smaller proteins and glycoproteins, the latter with much lower carbohydrate content. Some peak II glycoproteins also dissociated into subunits in the presence of dithiothreitol. Peak III consisted mainly of a heterogenous assortment of proteins, including some glycoproteins of low carbohydrate content. Antibodies either to peak II or to peak III reacted both with peaks II and III but not with peak I.The total weight, carbohydrate composition of glycoproteins and the ratio of carbohydrate to protein in the total extract or in each of the three fractions were not significantly affected in vitamin A deficiency despite decreased incorporation of all labeled precursors. Rather, the relatively lower incorporation (approx. 0.8) of radioactive sulfate, D-glucosamine and L-fucose into total SDS-soluble duodenal glycoproteins of vitamin-A-deficient rats could be explained on the basis of a reduced prevalence of goblet cells alone. In contrast, the relative incorporation rate of L-fucose into peak I, but not into peaks II and III, ranged from 0.25 to 0.45, less than expected on the basis of fewer goblet cells alone. The incorporation of radioactive threonine into all protein fractions was reduced to 60% of normal in vitamin A deficiency. Thus, the well established observation that intestinal tissue of vitamin-A-deficient rats synthesizes high molecular weight glycoproteins poorly might be due to several interacting factors: (1) a reduced prevalence of goblet cells, (2) a lower rate of protein synthesis, (3) a lack of retinyl phosphate for the formation of mannosyl or other carbohydrate derivatives, and (4) secondary, and as yet undefined, cellular changes which preferentially reduce the rate of synthesis of high molecular weight fucose- and sialic-acid-enriched glycoproteins.     

Carbohydrate-binding profile of a pregnancy-associated rat uterine glycoprotein

Sugar-binding proteins obtained from the peri-implantation uterine tissue have been thought in recent years to have significant roles in embryo implantation, where carbohydrate moieties of the protein are actively involved. Based on this rationale a mannose-containing glycoprotein/lectin (named uterine agglutinin or UA) was purified by Concanavalin A (Con A) affinity chromatography in a previous study. A modification of the original purification procedure to include a 33% ammonium sulfate fractionation improves the yield of the protein significantly. An alternative purification procedure by Mannan affinity matrix, indicates that apart from containing mannose, UA possesses mannose-binding properties as well.In this paper, we report some of the biochemical and more specifically, the carbohydrate-binding characteristics of UA. The protein is seen to contain mannose-6-phosphate (M-6-P)-binding sites, which is of importance since M-6-P receptors have a large number of biologically significant roles, including that of binding to growth factors.SDS-PAGE, gel filtration chromatography and alkaline PAGE indicate the homogenous nature of the protein with subunit molecular weights of 36 kDa and 19 kDa, and a native size of 64kDa. Amino acid analysis shows glycine, glutamic acid and aspartic acid to be the major constituents.UA is a glycoprotein and shows presence of N-acetyl glucosamine and galactose, apart from mannose.De nove synthesis studies in the presence of tunicamycin show that the carbohydrate moiety of the glycoprotein is attached by N-linkage to the protein. Binding characteristics of the protein is studied quantitatively in which (¹²⁵I)-labelled lectin is bound to Mannan-Sepharose affinity matrix. The sugar inhibition pattern of this binding shows -methyl mannopyranoside and M-6-P to be equally effective as inhibitors. Scatchard analysis of the binding of UA to (¹⁴C)-mannose shows a Ka of 6.43×10⁵ (M^–1) and that 1 mole of UA can bind to 8 moles of mannose. The possible role of the protein in implantation has also been discussed.Abbreviations b.w. body weight - BSA Bovine Serum Albumin - Con A Concanavalin A - cpm counts per minute - Endo H endoglycosidase H - GlcNAc N-acetyl glucosamine - Man mannose - M-6-P mannose-6-phosphate - MEM-deficient Minimum Essential Medium Eagle-deficient modification - NaBH₄ sodium borohydride - NaN₃ sodium azide - (NH₄)₂SO₄ ammonium sulphate - p.c. post coitum - PMSF phenyl methyl sulphonyl fluoride - PTA phosphotungstic acid - RCA Ricinus communis Agglutinin - SDS-PAGE sodium dodecyl sulphate-polyacrylamide gel electrophoresis - TCA trichloroacetic acid - UA Uterine Agglutinin - WGA Wheat-germ Agglutinin     

Isolation of the membrane glycoproteins of human blood platelets by lectin affinity chromatography

The major platelet membrane glycoproteins have been solubilized in 1.0% sodium deoxycholate and subjected to affinity chromatography on the lectins from Lens culinaris, wheat germ and Abrus precatorius. Polyacrylamide gel electrophoresis in the presence and absence of a reducing agent together with the differential binding of the lectins to the glycoproteins permitted the distinction of at least seven separate glycoprotein entities. A new nomenclature for the glycoproteins is proposed to accomodate the additional data.Using combinations of lectin columns, glycoproteins Ia and Ib could be prepared in a pure state and IIb and IIIa could be greatly purified. The binding of lectins to glycoprotein Ib has been strongly implicated as a necessary step in the aggregation response of platelets to lectins.     

Impairment of glycoprotein secretion by phenobarbital in rat liver slices

The effects of phenobarbital on protein and glycoprotein synthesis and secretion were studied in rat liver slices. Phenobarbital (2 mM) decreased [¹⁴C]-glucosamine and [¹⁴C]leucine incorporation into liver proteins and markedly inhibited their incorporation into medium (secretory) proteins. This inhibitory effect of phenobarbital was dose dependent and not reversible under the conditions of this study. In the presence of cycloheximide, an inhibitor of peptide synthesis, phenobarbital still inhibited the release of glycoproteins into the medium; however, the specific activity of liver glycoproteins was increased. The effects of phenobarbital on hepatic macromolecular secretion, independent of its effects on synthesis, were determined by prelabeling proteins in a liver slice system with either [¹⁴C]leucine of [¹⁴C]glucosamine. When phenobarbital was present, the secretion of these prelabeled proteins into the medium was impaired. 12 h after intraperitoneal injections of phenobarbital, glycoprotein secretion was inhibited from liver slices prepared from the pretreated rats. This inhibition of secretion occurred even though protein synthesis was stimulated and intracellular glycosylations unaffected. The results of this study indicate that phenobarbital impairs the secretion of glycoproteins by the liver.     

Cell surface proteins during early Xenopus development: analysis of cell surface proteins and total glycoproteins provides evidence for a maternal glycoprotein pool

Summary The populations of cell surface proteins and total glycoproteins were investigated in early Xenopus embryos through lectin staining, affinity binding of glycoproteins to lectins, and use of a succinimide ester to biotinylate cell surface molecules. Lectin staining shows that the egg is endowed with a thick layer of surface glycoprotein, and that glycoprotein is immediately detected on the newly formed membranes of nascent blastomeres. The amount of glycoprotein found in eggs and early embryos remains constant, and electrophoretic analysis reveals no changes in abundant lectin-binding glycoproteins through the neurula stage. In contrast, the amount of cell surface protein increases dramatically from the 2-cell to the gastrula stages. Despite this quantiative increase, only a small number of differences in cell surface proteins were detected during this period. A series of bands was detected which appears to be specific to the outer surface of the embryo. Because the populations of surface proteins and of total glycoproteins overlap to a great extent, the increase in cell surface protein, in the absence of a change in total glycoprotein, indicates the presence of a maternal glycoprotein pool in the Xenopus egg, from which the cell surface proteins of embryonic blastomeres are recruited.     

The concanavalin a receptor from human erythrocytes in lipid bilayer membranes. Interaction with concanavalin A and succinyl-concanavalin A

The concanavalin A receptor from human erythrocyte membranes has been isolated by affinity chromatography using the mild, readily-dialyzable detergent dodecyltrimethylammonium bromide. The purified protein has been reincorporated into large unilamellar phospholipid vesicles using a detergent dialysis technique. The mean diameter of these vesicles increases as the lipid: protein ratio decreases. Binding of succinyl-concanavalin A to these vesicles was quantitated using ¹²⁵I-labelled lectin in a filtration assay. The concanavalin A receptor in lipid bilayer vesicles provides specific high affinity binding sites for succinyl-concanavalin A with an association constant of 2.13·10⁶ M^?1. Scatchard plots indicate positive cooperativity of binding at very low lectin concentrations, a characteristic also seen in concanavalin A binding to intact human erythrocytes. The presence of bovine serum albumin has little effect on lectin binding and is not required for expression of cooperativity. Concanavalin A effectively competes with succinyl-concanavalin A for binding to the vesicles with an association constant of 4.83·10⁶ M^?1. Receptor-bearing vesicles are readily agglutinated by concanavalin A but not by its succinylated derivative. The kinetics of vesicle agglutination are biphasic, with an initial rapid phase followed by a pseudo-first order process. We suggest that studies on reassembled receptor proteins in lipid bilayers can provide valuable insight into receptor involvement in transmembrane signalling events and the factors involved in cell membrane behaviour and cell agglutination.     

Effects of trypsin on binding of insulin and concanavalin A and on glucose and proline transport in the R3230AC mammary adenocarcinoma

Effects of trypsin treatment on insulin and concanavalin A binding to, and glucose and proline transport in, dissociated R3230AC mammary adenocarcinoma cells were examined. Reduction of binding of ¹²⁵I-labelled insulin was dependent on the amount of trypsin used, the temperature and the time of the incubation period. Under conditions that reduced insulin binding by greater than 75%, transport of glucose and proline was reduced by less than 15%. Scatchard analysis of insulin binding after trypsin treatment yielded slopes similar to those from cells not exposed to trypsin, assuming either two classes of receptors or an average affinity, $\text{K}\text{?}{}_{\mathrm{<mtext>e</mtext>}}$. Dissociation of bound insulin from untreated or trypsin-treated cells was enhanced by addition of excess unlabelled ligand. Insulin added in vitro, which decreased glucose transport in untreated cells, produced a decrease in glucose transport in cells treated with trypsin for 5 min (insulin binding was decreased 35%), but not in cells treated for 45 min (insulin binding was decreased 90%). Binding of the plant lectin concanavalin A was also reduced by trypsin treatment, but to a lesser extent and with a different time-course than for insulin. Scatchard analysis of the binding of concanavalin A in untreated and trypsin-treated cells yielded comparable values for K_d. The insulinomimetic actions of concanavalin A on glucose transport were abolished after brief exposure to trypsin. Pre-treatment of cells with concanavalin A reduced insulin binding and partially protected insulin receptors from trypsin digestion, but the inability to remove all of the concanavalin A precluded its use as a method to protect insulin receptors. Thus, in this rat mammary tumor, the number, but not the affinity or functional activity, of insulin receptors can be reduced by trypsin treatment without significant effects on glucose or A system amino acid transport.