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.     

Isolation and characterization of a plasma membrane fraction from sea urchin sperm exhibiting species specific recognition of the egg surface

A method is described for isolating preparative quantities of plasma membranes from sea urchin sperm. The final membrane fraction is homogeneous by sucrose density sedimentation and is enriched in adenylate cyclase as well as in the four glycoproteins accessible to radioiodination of intact sperm. The electrophoretic profiles of sperm membranes from three sea urchin species are very similar. The membrane preparation consists primarily of sealed vesicles which release carboxyfluorescein when exposed to detergents or distilled water. Ninety-two percent of the 125I-labeled vesicle material binds to wheat germ lectin columns, suggesting a right-side-out orientation. The isolated sperm membrane vesicles exhibit species specific adhesion to the surfaces of sea urchin eggs; this adhesion is blocked by pretreatment of the vesicles with trypsin or egg jelly. This method will be useful for isolating biologically active sperm membrane components involved in sperm-egg recognition during fertilization.     

Glycoproteins in the whorls of membrane produced by oligodendroglia in culture

Two glycoproteins of 99 kDa and 77 kDa which exhibit intense binding to wheat germ agglutinin have been purified from the whorls of membrane produced by oligodendroglia in culture. The whorls of membrane were isolated by gradient centrifugation from purified bovine oligodendroglia maintained in culture. The two glycoproteins were solubilized from the membranes using a non-ionic detergent and purified by Sephadex LH-60 chromatography, wheat germ agglutinin affinity chromatography, and SDS-polyacrylamide pore gradient gel electrophoresis. HPLC peptide mapping of the 99-kDa and 77-kDa glycoproteins revealed structural differences between the two proteins. Peptide mapping suggested that the 99-kDa glycoprotein from the whorls of membrane may be homologous to that from the plasma membranes. The 77-kDa glycoproteins from both sets of membrane may also be structurally related. Lectin binding studies showed that both glycoproteins from the whorls of membrane bound to wheat germ agglutinin, succinylated wheat germ agglutinin, concanavalin A, and lentil lectin, indicating the presence of high mannose and hybrid type oligosaccharide side-chains.     

Isolation of the dorsal, ventral and intracellular domains of HeLa cell plasma membranes following adhesion to a gelatin substrate

The plasma membrane is a complex organelle responsible for many cellular functions. In addition to mediating the exchange of components with the extracellular fluid, the plasma membrane is involved in cell adhesion to matrix proteins in vivo and in vitro. In vitro, adherent cells have three distinct plasma membrane domains to carry out these functions: one attached to the substrate (ventral); another exposed to the media (dorsal); and an intracellular domain involved in endocytosis and secretion. A technique has been developed for the rapid isolation of these specific domains from HeLa cells immediately following adhesion to a gelatin substrate. The isolation procedure utilizes the tight binding of cationic colloidal silica to the dorsal plasma membrane domain of attached cells. Following silica binding and cell lysis, the silica-coated dorsal plasma membrane domain is readily separated from intracellular plasma membrane components by virtue of the high density of the silica pellicle, and the intact ventral plasma membrane domain remains attached to the gelatin substrate. Fluorescence and electron microscopy and biochemical studies using 125I-lactoperoxidase labeling, 125I-labeled wheat germ agglutinin binding, and [3H]-fucose incorporation into plasma membrane glycoproteins confirmed the separation of these three topologically distinct plasma membrane domains. The fractions isolated by the technique contained essentially all of the plasma membrane components present in intact cells. This unique membrane-isolation procedure is now being used to analyze membrane flow during plasma membrane domain formation accompanying cell adhesion to an extracellular matrix.     

Reactivity with lectins of the saccharide components of rhodopsin in reconstituted membranes. Orientation of the carbohydrates

Rhodopsin-containing liposomes may provide a model for investigating the interaction of intrinsic membrane glycoproteins in biological systems. As part of the characterization of this preparation, the surface orientation of the carbohydrates of rhodopsin, assembled from purified bovine rhodopsin and egg phosphatidylcholine was examined, and is the topic of this report. The major tool used in these studies was the interaction with the carbohydrate-specific reagents, plant lectins. Two techniques were used: lectin-mediated aggregation of the liposomes, as measured by light scattering; the binding of 125I-labeled succinylated concanavalin A, and Scatchard analysis as a measure of affinity. The preparation most extensively examined had a mole ratio of rhodopsin:phospholipid of 1:100. Among a variety of lectins which were examined, only concanavalin A, succinylated concanavalin A, and wheat germ agglutinin were able to mediate the aggregation of rhodopsin-containing liposomes, as expected. The aggregation with concanavalin A was prevented by the presence of sugars having the alpha-D-glucopyranosyl configuration, and that brought about with wheat germ agglutinin, by N-acetylglucosamine (GlcNAc). In addition, the aggregation with concanavalin A was reversed with methyl alpha-D-mannoside, and with wheat germ agglutinin, by GlcNAc, suggesting that membrane fusion did not take place. On a molar basis, wheat germ agglutinin brought about a greatly reduced extent of aggregation as compared to concanavalin A, suggesting the relative inaccessibility of GlcNAc residues in the liposomes as compared to mannose. The initial rate of the aggregation, however, were similar with either lectin. The first-order rate constants were unaffected by wide variation in the concentrations of concanavalin A and wheat germ agglutinin, and by variation in the mole ratios of rhodopsin in the liposomes from 0.2 to 19 moles per 100 moles of egg lecithin. Rhodopsin-liposomes were also prepared from a total lipid extract of rod outer segments instead of egg lecithin. Similar kinetic properties were exhibited by this preparation as were obtained with the liposome prepared with the purified phospholipid. Scatchard analysis of the binding of 125I-labeled succinylated concanavalin A by rhodopsin liposomes indicated the presence of a single class of binding site as the preferred fit, with an apparent Kd of 2.8 X 10(-7) M. The binding was destroyed or extensively interfered with by trypsinization and by periodate treatment.     

Postnatal changes in biosynthesis of microvillus membrane glycans of rat small intestine: I. Evidence of a developmental shift from terminal sialylation to fucosylation

We present evidence of a change from sialylation to fucosylation of intestinal microvillus membrane oligosaccharides during postnatal development in the rat. The initial high sialic acid to fucose molar ratio in native and delipidated membranes was completely reversed after weaning. The specific binding of 125I-labeled wheat germ agglutinin to neuraminidase-sensitive sites in the native and delipidated membranes decreased markedly from early suckling to weaning ages. The binding of 125I-labeled Ulex europeus agglutinin I showed an age-related pattern opposite to that of wheat germ agglutinin. The changes in membrane reactivities to these lectins were entirely consistent with the existence of a developmentally-controlled shift from terminal sialyl to fucosyl substitutions among various glycoconjugate classes. This could play a key role on the functional transformation experienced by the intestinal epithelium of suckling rats.     

Interactions of lectins with plasma membrane glycoproteins of the Ehrlich ascites carcinoma cell.

Several aspects of the interaction of various lectins with the surface of Ehrlich ascites carcinoma cells are described. The order of agglutinating activity for various lectins is Ricinus communis greater than wheat germ greater than or equal to concanavalin A greater than or equal to soybean greater than Limulus polyphemus. No agglutination was noted for Ulex europaeus. Using 125I-labeled lectins it was determined that there are 1.6 and 7 times as many Ricinus communis lectin binding sites for concanavalin A and soybean lectins. Sodium deoxycholate-solubilized plasma membrane material was subjected to lectin affinity chromatography and sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The lectin receptors of the plasma membrane appeared to be heterogeneous and some qualitative differences could be discerned among the electrophoretically analyzed material, which bound to and was specifically eluted from the various lectin affinity columns. The characteristics of elution of bound material from individual lectin columns indicated secondary hydrophobic interactions between concanavalin A or wheat germ agglutinin and their respective lectin receptor molecules.     

Parathyroid hormone receptors are plasma membrane glycoproteins with asparagine-linked oligosaccharides

In the preceding article, we described physicochemical and kinetic properties of parathyroid hormone (PTH) receptors in clonal rat osteosarcoma cells (ROS 17/2.8) using photoaffinity ligand labeling and showed that the physiologically relevant receptor-ligand complex has an apparent Mr = 80,000. In this study, the photoaffinity labeled Mr = 80,000 receptor was localized exclusively on the cell surface plasma membrane and its glycoprotein nature was demonstrated through the use of lectin affinity-chromatography and specific exo- and endoglycosidases. Rinsing ROS cells, preincubated in the dark with 125I-labeled [Nle8, N-epsilon-(4-azido-2-nitrophenyl)Lys13,Nle18,Tyr34]bovine PTH-(1-34)-NH2 (NAP-NlePTH) (4 h, 15 degrees C, equilibrium conditions) with acidic phosphate-buffered saline (pH 2.5, 30 s, 4 degrees C) before photolysis resulted in selective and nearly total disappearance of the labeled Mr = 80,000 receptor. PTH receptor integrity to acid rinsing and photolysis was shown by relabeling the Mr = 80,000 receptor after a second incubation of these cells with 125I-labeled NAP-NlePTH, followed by photolysis. Adsorption of Triton X-100-solubilized, 125I-labeled NAP-NlePTH receptors to wheat germ agglutinin-agarose is nearly complete and highly selective, and elution with N-acetylglucosamine resulted in virtually total recovery of the labeled receptors from the column. The wheat germ agglutinin-retarded PTH receptors show increased electrophoretic mobility upon treatment with neuraminidase which was inhibited by simultaneous addition of 2,3-dehydro-3-desoxy-N-acetylneuraminic acid, a specific neuraminidase inhibitor. Endoglycosidase F treatment of the Mr = 80,000 receptors generated a single, labeled polypeptide with a Mr = 59,000 which migrated as a narrow band. PTH receptors on ROS 17/2.8 cells appear to be monomeric plasma membrane glycoproteins with an apparent Mr of 80,000 which contain a Mr = 59,000 polypeptide backbone and a polymeric arrangement of N-acetylglucosamine with N-acetylneuraminic acid as major terminal sugar residues.     

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 Mr approximately 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 Mr approximately 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 Mr approximately 150 000 isolated on wheat germ agglutinin confirming their surface orientation. However, glycoprotein with Mr approximately 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 32Pi also results in labelling of glycoproteins isolated by both methods, suggesting that these molecules traverse the bilipid layer of the platelet membrane, bearing reactive groups on both outer and cytoplasmic surfaces.     

Lectin receptors in Trypanosoma cruzi. An N-acetyl-D-glucosamine-containing surface glycoprotein specific for the trypomastigote stage

We have investigated the interaction of three lectins, differing in their sugar specificities, with the surface of the three differentiation stages of Trypanosoma cruzi. The Scatchard constants for each lectin and parasite stage imply that differentiation of T. cruzi is accompanied by changes in the cell surface saccharides. Trypomastigotes obtained from two different sources do not differ appreciably as to the number and affinity of binding sites for the three lectins employed, suggesting a similar cell-surface saccharide composition. These conclusions are reinforced by sodium dodecyl sulfate-polyacrylamide gel electrophoretic analysis of the 131I-labeled surface glycoproteins, following isolation by affinity chromatography. The surface membrane of trypomastigotes, the infective stage to T. cruzi for mammalian cells, possesses a specific glycoprotein of apparent Mr 85000 (Tc-85) which is absent from the other two stages and can be isolated by affinity chromatography on wheat germ agglutinin-Sepharose columns. This glycoprotein also binds to concanavalin A, but not to Lens culinaris lectin. The binding of Tc-85 to wheat germ agglutinin is unaffected by treatment of either the isolated glycoprotein or intact living trypomastigotes with neuraminidase. Since N-acetyl-D-glucosamine inhibits internalization of trypomastigotes by cultured mammalian cells, it is suggested that Tc-85 might be involved in adhesion and/or interiorization of T. cruzi into mammalian cells, possibly via recognition of an ubiquitous host-cell surface N-acetyl-D-glucosamine-specific receptor activity.     

Biochemical characterization of domain-specific glycoproteins of the rat hepatocyte plasma membrane

Seven integral proteins (CE 9, HA 21, HA 116, HA 16, HA 4, HA 201, and HA 301) were isolated from rat hepatocyte plasma membranes by immunoaffinity chromatography on monoclonal antibody-Sepharose. Six of the proteins (all but HA 16) exhibit domain-specific localizations (either bile canalicular or sinusoidal/lateral) about the hepatocyte surface. We identified three of these protein antigens as leucine aminopeptidase (HA 201), dipeptidyl peptidase IV (HA 301), and the asialoglycoprotein receptor (HA 116). We also developed 125I-lectin blotting procedures that, when used in conjunction with chemical and glycosidase treatments, permitted a comparison of the types of oligosaccharides present on the seven proteins. All seven are sialoglycoproteins, based upon the effects of prior neuraminidase and periodate-aniline-cyanoborohydride treatments of blots on labeling by 125I-wheat germ agglutinin. 125I-labeled Ricinus communis agglutinin I and 125I-peanut agglutinin blotting of the desialylated proteins revealed few if any conventional O-linked oligosaccharides, suggesting that the sialyl residues represent termini of N-linked complex-type oligosaccharides. Depending upon the protein, we estimated the presence of 2-26 N-linked oligosaccharides/polypeptide chain from the Mr reductions accompanying chemical or enzymatic deglycosylation. Three of these mature plasma membrane proteins (HA 21, HA 116, and HA 4) have both high mannose-type and complex-type oligosaccharides on every copy of their polypeptide chains. The labeling of these three proteins by 125I-concanavalin A was sensitive to treatment with endoglycosidase H, and each exhibited a quantitative reduction in Mr after the treatment, as assessed independently by 125I-wheat germ agglutinin blotting. At this level of analysis, we were unable to discern differences in the types of oligosaccharides present on these seven glycoproteins that correlate with their patterns of expression within the plasma membrane domains of this polarized epithelial cell.     

Isolation and properties of gamma protein,the major transmembrane sialoglycoprotein of the HeLa cell

The surface of the HeLa cell is composed of a heterogeneous population of sialogly coproteins which undergo lectin-mediated endocytosis (Kramer and Canellakis, Biochim Biophys Acta 551:328, 1979). One such sialoglyco-protein, gamma protein, is the major periodate-Schiff-reactive and [³H]-glucosamine-labeled component of the plasma membrane; it has an apparent molecular weight of 165,000. Gamma protein is also the major [¹²⁵I]-wheat germ agglutinin-binding component in sodium dodecyl sulfate gels. Neuraminidase digestion of HeLa cells abolishes binding of [¹²⁵I]-wheat germ agglutinin to gamma protein, and pretreatment of cells with wheat germ agglutinin protects gamma protein from desialation by neuraminidase. suggesting that wheat germ agglutinin binds to the sialic acid residues of gamma protein at the cell surface. Gamma protein can be extracted with various detergents but not with high-salt, chelating, or chaotropic agents. Intact inside-out plasma membrane vesicles have been prepared from HeLa cells that had phagocytosed latex particles. Treatment of these isolated vesicles with trypsin reduces the molecular weight of gamma protein. These results suggest that gamma protein is an integral membrane protein that spans the plasma membrane. Gamma protein can be purified to homogeneity by sequential lithium diiodosalicylate-phenol extraction, wheat germ agglutinin-agarose affinity chromatography, and preparative sodium dodecyl sulfate-polyacrylamide gel electrophoresis.     

Glycoprotein changes in the rat sperm plasma membrane during maturation in the epididymis.

To investigate surface glycoprotein changes during post-testicular maturation, plasma membranes were isolated from proximal caput, distal caput, and cauda epididymal rat spermatozoa. Membrane glycoproteins were identified on Western blots of SDS-PAGE fractionated samples using biotinylated lectins and Vecta-stain reagents; these were compared to glycoproteins present in cauda epididymal luminal fluid. Lens culinaris agglutinin, Pisum sativum agglutinin, peanut agglutinin, wheat germ agglutinin, Ricinus communis agglutinin, Ulaex europaeus agglutinin, and Dolichol biflorus agglutinin each bound a specific subset of the polypeptides present. Several types of glycoprotein changes were noted including their appearance, loss, alteration of staining intensity, and alteration of electrophoretic mobility. Some maturation-dependent sperm surface glycoproteins co-migrated with glycoproteins present in epididymal fluid. This approach of direct analysis of the glycoproteins in purified plasma membranes identifies a broader spectrum of maturation-related surface changes occurring within the epididymis than are noted with surface labeling procedures.     

Isolation of lectins of different specificities on a single affinity adsorbent.

Affinity chromatography on Sepharose-fetuin columns was used in a single step procedure to isolate the lectins concanavalin A, Favin, phytohemagglutinin, wheat germ agglutinin, and Limulus hemagglutinin. New lectins with unknown binding specificities were also purified by the same procedure from extracts of small California white beans, Idaho red beans, and white pea beans. The purified lectins exhibited different cell surface mapping properties on erythrocytes, lymphocytes, and sperm cells. It was particularly striking that neither 131I-labeled concanavalin nor 125I-labeled wheat germ agglutinin had any effect on the binding of the other to mouse spleen cells. In accord with this observation, gel electrophoretic analysis of radiolabeled lymphocyte receptors for these two lecithins yielded different patterns. These results indicate that highly purified lectins prepared by affinity chromatography on the same adsorbent can possess strikingly different binding specificities for cell surface receptors.     

Preparation and characterization of plasma membrane-enriched fractions from rat pancreatic islets

Methods have been developed for the isolation on a semi-micro scale of a plasma membrane-enriched fraction from rat islets of Langerhans. An important feature of these experiments is the use of 125I-labeled wheat germ agglutinin as a specific probe for plasma membrane-containing fractions. The partly purified plasma membrane fraction had a density in sucrose of about 1.10 and was enriched in the activities of 5'- nucleotidase, alkaline phosphatase, sodium-potassium, and magnesium- dependent ATPase and adenylate cyclase. It contained only very low levels of acid phosphatase, cytochrome c oxidase, insulin, and RNA. Further purification was hampered by the relatively small amounts of fresh plasma membrane material that could be obtained from 16-24 rats in each experiment. When islets were prelabeled with radioactive fucose, the plasma membrane-enriched fraction contained radioactivity at a four- to fivefold higher specific acivity than the whole islet homogenate. Sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis of plasma membrane-enriched fractions pooled from several experiments revealed a distinctive pattern of protein bands as compared with other less pure fractions. With respect to rapidity, apparent specificity, and easy reversibility of the labeling of the plasma membrane fraction, 125I-wheat germ agglutinin provides a highly useful tool for the detection of microgram quantities of plasma membrane components which should be applicable to many other systems as well.     

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.     

Specific glycoprotein changes during development of the chick neural retina

After separation of whole proteins of chick neural retina by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate (SDS), a number of glycoproteins can be detected by staining the gels with 125I-labeled wheat germ agglutinin (WGA) and other lectins. The glycoprotein patterns show both quantitative and qualitative changes between days 7 and 13 of development. Some of these glycoproteins can be separated by chromatography on columns of insolubilized lectins. These observations suggest that purification of some of these glycoproteins identified by staining with radioactive lectins would yield retinal antigens which may be specific for developmental stage and cell type.     

Interactions of lectins with plasma membrane glycoproteins of the Ehrlich ascites carcinoma cell

Several aspects of the interaction of various lectins with the surface of Ehrlich ascites carcinoma cells are described. The order of agglutinating activity for various various lectins is Ricinuscommunis > wheat germ concanavalin A soybean >Limuluspolyphemus. No agglutination was noted for Ulex europaeus. Using ¹²⁵I-labeled lectins it was determined that there are 1.6 and 7 times as many Ricinus communis lectin binding sites as sites for concanavalin A and soybean lectins. Sodium deoxy-cholate-solubilized plasma membrane material was subjected to lectin affinity chromatography and sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The lectin receptors of the plasma membrane appeared to be heterogeneous and some qualitative differences could be discerned among the electrophoretically analyzed material, which bound to and was specifically eluted from the various lectin affinity colums. The characteristics of elution of bound material from individual lectin columns indicated secondary hydrophobic interactions between concanavalin A or wheat germ agglutinin and their respective lectin receptor molecules.     

Lymphocyte plasma membranes. VI. Plasma membrane glycoproteins of thymic and splenic lymphocytes from inbred rats.

Plasma membranes of splenic and thymic lymphocytes from ACI rats were analyzed for their protein and glycoprotein components by surface radioiodination with 125I and SDS-polyacrylamide gel electrophoresis. The glycoproteins were extracted with lithium diiodosalicylate, characterized and assayed with antisera to thymic antigen. Plasma membranes of both cell types showed more than 25 proteins of which 10--15 were glycoproteins. Both cells showed five major glycoproteins but their apparent molecular weights or intensities differed. Surface radioiodination showed a 120 000 daltons component, common to both cell types, and a 27 000 daltons thymus-specific component as the most exposed surface glycoproteins. Lithium diiodosalicylate extracts of the plasma membranes contained almost all of the glycoprotein components and comprised 5-6 percent of the total membrane protein and 40-50 percent of the total membrane carbohydrate, with sialic acid content in thymus twice that of the spleen cells. About 1 percent of the total plasma membrane protein and 7 percent of the total isolated glycoproteins from thymocytes were reactive with rabbit anti-rat thymocyte antiserum and the immune precipitates showed two components with apparent molecular weights of 72 000 and 27 000.     

Cell surface glycoproteins of CHO cells. I. Internalization and rapid recycling

The major cell surface proteins of Chinese hamster ovary (CHO) cells have been investigated after reacting cells at 4 degrees C with the membrane-impermeant reagent, trinitrobenzenesulfonate (TNBS). Immunoprecipitation and subsequent two-dimensional, sodium-dodecyl sulfate, polyacrylamide gel electrophoresis (SDS-PAGE) of proteins from derivatized cells that had been labelled previously with [3H]D-glucosamine or [3H]L-leucine showed that TNBS reacted with most of the high molecular weight (HMW) acidic glycoproteins that became labelled with iodine by the lactoperoxidase technique and that bind the lectin, wheat germ agglutinin (WGA). After warming the cells to allow endocytosis to proceed, molecules haptenized with trinitrophenol (TNP) groups were followed radiochemically by means of [125I]anti-DNP antibodies. The half-life for internalization of proteins tagged with either [125I]anti-DNP IgG or Fab averaged about 5 min. A similar result was obtained when a monoclonal antibody directed against a single plasma membrane glycoprotein was used, or when the rate of surface loss of TNP groups unoccupied by antibodies was measured. Within 15 min at 37 degrees C, a steady-state between surface and cytoplasmic label was reached, with about 65% of the hapten located internally. Recycling of internalized TNP groups back to the cell surface also occurred rapidly (t 1/2 approximately 5 min). Most of the intracellular radioactivity was associated with a membrane fraction of density similar to that of the plasma membrane. Over a 4-h period, there was no significant entry of labeled molecules into lysosomes. By contrast, the fluid-phase marker, horseradish peroxidase, became associated with the lysosomes within 1 h. Our results are consistent with the view that the majority of plasma membrane glycoproteins are continuously being internalized and recycled at a high rate.