Alterations in the cell surface proteins of Drosophila during morphogenesis

Summary Unevaginated and evaginated Drosophila imaginal discs were surface-labeled with ¹²⁵I. Relative labeling was greater in eleven peptides and lower in three peptides of evaginated discs compared to unevaginated discs. These results are compared to the effects of 20-hydroxyecdysone (20-HOE) on metabolic labeling of membrane proteins fractionated from imaginal discs, and on cell surface labeling of a hormone-responsive Drosophila tissue culture line. A group of ³⁵S-methionine labeled membrane fraction peptides whose metabolic labeling is 20-HOE dependent have isoelectric points and apparent molecular weights very similar to those of a group of proteins only labeled in iodinated evaginated discs, supporting the conclusion that these are hormone-dependent, cell surface proteins (Rickoll and Fristrom 1983). Based upon two-dimensional gel electrophoretic and immunological criteria three of the proteins showing increased labeling in evaginated discs are related to three proteins induced by 20-HOE in tissue culture cells. Two different subsets of radiolabeled peptides were observed in the imaginal discs based upon detergent solubility. Some of the proteins which are soluble in NP-40 plus urea but insoluble in NP-40 alone may be localized in the basal lamina of the imaginal discs, a structure which labels heavily with ¹²⁵I and is lacking in tissue culture cells. In discs, the majority of hormone-dependent changes in radiolabeled peptides were seen in the fraction solubilized by NP-40 and urea with a sulfhydryl reducing agent, while in tissue culture cells, the majority of differences is seen in the fraction solubilized by NP-40 only. We speculate that these proteins may be involved in similar processes, e.g., cell rearrangement, that occur during both disc morphogenesis and 20-HOE induced aggregation in tissue culture cells.This work was supported by grants CD-205 from the American Cancer Society, RR08132 from NIH to C.A.P. and GM 19937 from NIH to J.W.F.     

Lectin affinity chromatography of cell surface proteins of novikoff tumor cells

Novikoff hepatocellular carcinoma cells were radioiodinated by a cell surface-specific method using lactoperoxid ase/¹²⁵I. The iodinated proteins were solubilized in 0.5% Nonidet P-40 and subjected to affinity chromatography on Sepharose-conjugated lectins (Ricinus communis agglutinins I or II, soybean agglutinin, concanavalin A, or wheat germ agglutinin) and analyzed by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. Almost all the iodinated proteins bound to one or more of the Sepharose-conjugated lectins, presumptive evidence that these peptides are glycosylated. Lectin affinity chromatography resolved defined subsets of iodinated glycoproteins and suggested that certain glycoproteins could be fractionated on the basis of heterogeneity of their heterosaccharide moieties. Incubation of the iodinated cells with neuraminidase resulted in increased binding of iodinated proteins to Sepharose-conjugated Ricinus communis agglutinins I and II and soybean agglutinin and decreased binding to Sepharose-conjugated wheat germ agglutinin. Binding of iodinated proteins to concanavalin A was unaffected by neuraminidase treatment of the cells. These studies demonstrate the utility of lectins for the multicomponent analysis of plasma membrane proteins.     

Evaluation of an effective sample prefractionation method for the proteome analysis of breast cancer tissue using narrow range two-dimensional gel electrophoresis

One method of improving the protein profiling of complex mammalian proteomes is the use of prefractionation followed by application of narrow pH range two dimensional (2-D) gels. The success of this strategy relies on sample solubilization; poor solubilization has been associated with missing protein fractions and diffuse, streaked, and/or trailing protein spots. In this study, I sought to optimize the solubilization of prefractionated human cancer cell samples using isoelectric focusing (IEF) rehydration buffers containing a variety of commercially available reducing agents, detergents, chaotropes, and carrier ampholytes. The solubilized proteins were resolved on 2-D gels and compared. Among five tested IEF rehydration buffers, those containing 3-[(3-cholamidopropyl)dimethylamino]-1-propane sulfonate (CHAPS) and dithiothreitol (DTT) provided superior resolution, while that containing Nonidet P-40 (NP-40) did not significantly affect protein resolution, and the tributyl phosphine (TBP)-containing buffer yielded consistently poor results. In addition, I found that buffers containing typically high urea and ampholyte levels generated sharper 2-D gels. Using these optimized conditions, I was able to apply 2-D gel analysis successfully to fractionated proteins from human breast cancer tissue MCF-7, across a pH range of 4-6.7.     

Characterization of a soluble follitropin receptor from porcine testis.

Porcine testis receptors for follitropin (FSH) were solubilized by treatment with the non-ionic detergent Nonidet P-40 and receptor-bound and free ¹²⁵I-porcine FSH were separated by ammonium sulfate precipitation. The soluble receptor retained both its high affinity and specificity for FSH. The soluble hormone-receptor complex exhibited an equilibrium association constant of 4.7 × 10¹⁰ M^?1 at 4°C. Its hydrodynamic properties were consistent with those obtained for other solubilized peptide hormone receptors, and its molecular weight estimated to 244,000.     

Alkaline urea solubilization, two-dimensional electrophoresis and lectin staining of mammalian cell plasma membrane and plant seed proteins

Plasma membranes, isolated from Chinese hamster ovary cells and seed proteins from Arachis hypogaea (L.) were analyzed by two-dimensional electrophoresis. Polypeptides were solubilized without employing sodium dodecyl sulfate (SDS), using in its place 5 mm K₂CO₃ and 9.5 m urea. After addition of dithiothreitol and the nonionic detergent Nonidet P-40, more than 95% of the total protein remained in the supernatant fraction after the preparation was centrifuged at 100,000 g. The solubilization was comparable to that achieved with boiling SDS solution. This soluble material could be used directly for either isoelectric focusing or nonequilibrium pH gradient electrophoresis in narrow bore, tubular, polyacrylamide gels crosslinked by means of N,N′-diallyltartardiamide. Up to 750 μg of protein could be analyzed in one such 3 mm gel. Electrophoresis in polyacrylamide slab gels containing SDS was used for separations in the second dimension. The method allows large amounts of both basic and acidic insoluble proteins to be solubilized and then analyzed without employing SDS as a solubilizing agent. Classes of glycoproteins on the gels were detected by incubating with small volumes of ¹²⁵I-lectins in heat-sealed plastic bags. CHO cells contain several high molecular weight acidic glycoproteins that bind wheat germ agglutinin, but which do not stain with Coomassie blue. Several of the storage polypeptides in peanut seeds were also shown to bind wheat germ agglutinin and are probably, therefore, glycoproteins containing N-acetyl d-glucosamine.     

Interaction of isolated components from mammalian sperm and egg

In order to identify those proteins from the plasma membrane of hamster spermatozoa that exhibit an affinity for components of the zona pellucida we have used the Western blot technique. Zonae pellucidae from postovulatory hamster oocytes were solubilized by exposure to an acidic pH and then radiolabeled using the Bolton-Hunter reagent. These ¹²⁵I-zona pellucida proteins retain their immunoreactivity and migrate in three heterogeneous bands when submitted to SDS-PAGE electrophoresis. Membrane proteins from epididymal spermatozoa of mature hamsters were extracted by treatment with Nonidet P-40 and then submitted to electrophoresis (SDS-PAGE). The proteins in the gel were electrophoretically transferred to a nitrocellulose membrane and then probed with the radiolabelled zone pellucida proteins. ¹²⁵I-zona pellucida proteins bind preferentially to a sperm protein with a molecular weight of 26,400 ± 1,400 daltons (n = 9). Using a similar procedure it was shown that this protein also binds ¹²⁵I-Concanavalin A. The interaction between the sperm protein and the ¹²⁵I-zona pellucida proteins shows species specificity as demonstrated by the fact that the hamster ¹²⁵I-zona pellucida proteins do not bind to proteins extracted from ram, bull, and stallion spermatozoa. Whether this sperm protein could be implicated be implicated in the process of sperm-egg interaction is under investigation.     

Solubilization of plant membrane proteins for analysis by two-dimensional gel electrophoresis

A plasma membrane-enriched fraction prepared from barley roots was analyzed by two-dimensional gel electrophoresis. Four methods of sample solubilization were assessed on silver stained gels. When membranes were solubilized with 2% sodium dodecyl sulfate followed by addition of Nonidet P-40, gels had high background staining and few proteins because of incomplete solubilization. Gels of membranes solubilized in urea and Nonidet P-40 had a greater number of proteins but proteins with molecular weights greater than 85,000 were absent and proteins with low molecular weights were diffuse. High molecular weight proteins were present in gels of membranes solubilized in 4% sodium dodecyl sulfate followed by acetone precipitation but background staining and streaking remained a problem. Gels of the best quality were obtained when membrane proteins were extracted with phenol and precipitated with ammonium acetate in methanol; background staining and streaking were diminished and proteins were clearly resolved. This method makes possible the resolution required for meaningful qualitative and quantitative comparisons of protein patterns on two-dimensional gels of plant membrane proteins.     

Cellular expression of bone-related proteins during in vitro osteogenesis in rat bone marrow stromal cell cultures

The regulation of cell surface fibroblast growth factor (FGF) receptors during the differentiation of F9 teratocarcinoma cells was investigated. The capacity of F9 cells to bind ¹²⁵I-basic FGF (FGF-2) increased upon induction of differentiation with dibutyryl cAMP and retinoic acid. No change in binding capacity was observed in the first 24 h after addition of differentiating agents, but a sixfold increase in binding capacity was observed after 48 h and a fivefold increase after 72 h. Scatchard analysis of the binding data indicated that the increased binding of ¹²⁵I-FGF-2 was due to an increase in the number of receptors with no change in their affinity. When ¹²⁵I-FGF-2 was cross-linked to cell surface receptors, an increase in FGF-2-receptor complexes with molecular weights of 140,000–160,000 was also observed in the differentiated F9 cells. Undifferentiated F9 cells are known to secrete FGF-4 and cease expression of this molecule upon differentiation. To determine whether the low level of receptors in undifferentiated cells might be related to their production of FGF ligands, the ability of suramin, a drug that can disrupt FGF-receptor interactions, to modulate receptor number on F9 cells was investigated. Suramin treatment increased ¹²⁵I-FGF-2 binding capacity of undifferentiated F9 cells threefold but had little effect on the binding capacity of differentiated cells. In addition, antibodies to FGF-4 increased the ¹²⁵I-FGF-2 binding capacity of undifferentiated F9 cells by 58%. These results suggest that undifferentiated F9 cells might be responding in an autocrine manner to their own FGF ligands resulting in downregulation of cell surface FGF receptors. The increased number of receptors observed in differentiated cells may partly result from the decreased production of FGF ligands by these cells. © 1994 Wiley-Liss, Inc.     

Assay of plant proteins with bicinchoninic acid for high resolution two-dimensional polyacrylamide gel electrophoresis

A method is described for estimating proteins in the same plant tissue sample that is solubilized for separation by two-dimensional polyacrylamide gel electrophoresis. The method uses a modified bicinchoninic acid (BCA) protein assay procedure and a modified standard urea solubilization buffer to estimate microgram values of unknown protein concentration, in the presence of 9 M urea and 4% Nonidet P-40, from a linear standard curve. A method for a quantitative determination of protein concentration by BCA in a sample containing 9 M urea and 4% Nonidet P-40 is also described. This method is effective for the determination of proteins in minute non-green and green plant tissue, and is especially designed for vegetative and floral shoot apices, and the primordia of inflorescences.     

Differentiation-associated changes in membrane proteins of mouse myeloid leukemia cells

The mouse myeloid leukemia cell line (M1) is known to differentiate in vitro into macrophages and granulocytes upon treatment with various inducer including mouse ascitic fluid. Changes of cell surface proteins during differentiation of M1 cells were analyzed by the lactoperoxidase-catalyzed radioiodination method and SDS-polycrylamide slab gel electrphoresis. Treatment of the cells with ascitic fluid changed the electrophoretic pattern of the iodinated proteins, the prominent change being the appearance of a new protein with a molecular weight of 180 000 (P180). Iodinated P180 was also detected in normal macrophages in granulocytes, which are similar to differentiated M1 cells. This protein was metabolically labeled with l-[¹⁴C]fucose, increasing with the period of the treatment. P180 was not expressed on ascitic fluid-treatment of a resistant clone of M1 cells that could not be induced to differentiate. These results indicate that P180 is a glycoprotein that is exposed on the outer surface of differentiated M1 cells, and that its expression is associated with differentiation of the cells.P180 was solubilized from ¹²⁵I-labeled macrophages with detergents bound to concanavalin A-Sepharose. This suggests that P180 is one of the receptors for concanavalin A. Therefore, P180 may contribute partly to the increases in agglutinability by concanavalin A and in the number of concanavalin A binding sites on the surface of M1 cells, which are known to be associated with differentiation of M1 cells.     

Isolation and identification of the major concanavalin A binding glycoproteins from murine-lymphocytes.

The major glycoproteins which bind concanavalin A have been isolated and identified from murine spleen cells, thymocytes,and purified thymus-derived (T) lymphocytes, and from the spleen cells of congenitally athymic (nude) mice. The cells were radiolabeled by lactoperoxidase catalyzed 125I iodination or by culturing the cells in media containing [3H]leucine or [3H]fucose. The cell membrane was solubilized with Nonidet P-40 and the concanavalin A binding proteins were isolated by affinity chromatography and analyzed according to their mobility on polyacrylamide gel electrophoresis in sodium dodecyl sulfate. The major proteins from various lymphocyte preparations were identified by immunoprecipitation with specific antisera. The molecules coded by the histocompatibility-2 complex acted as concanavalin A binding proteins H-2K and H-2D were isolated from T lymphocytes, thymocytes, and bone marrow derived (B) lymphocytes. The Ia antigens were identified from B lymphocytes and tentatively identified from T lymphocytes. In addition to these H-2 complex proteins, immunoglobulin M and D on B lymphocytes also bound concanavalin A binding. All these glycoproteins have previously been identified as cell surface molecules. The presence of certain minor unidentified concanavalin A binding proteins on lymphoid cells is indicated.     

Identification of Low Molecular Mass GTP-Binding Proteins in Membranes of the Halotolerant Alga Dunaliella salina

A family of specific guanine nucleotide-binding proteins in Dunaliella salina was studied. Polypeptides of different subcellular fractions were separated by electrophoresis and transferred to nitrocellulose or Immobilon membranes. Incubation of the transfer blots with [³⁵S]GTPγS or [α-³²P]GTP showed no evidence for GTP-binding proteins in the chloroplast and cytosol fractions. However, two GTP-binding proteins with molecular masses of 28 and 30 kilodaltons were present in the plasma membrane and microsomal fractions. An additional 29 kilodalton GTP-binding protein was detected in the plasma membrane. The mitochondrial fraction contained significant amounts of only the 28 kilodalton GTP-binding protein. Binding of [³²P]GTP to the protein blots was completely prevented by 10 micromolar GTP or guanosine 5′-O-(2-thiodiphosphate) (added in 3 × 10⁴-fold excess), whereas ATP or CTP had no effect on the binding. The 28 kilodalton GTP-binding protein was recognized by polyclonal antibodies to the ras-related YPT1 protein of yeast but not by the anti-ras Y13-259 monoclonal antibody. GTP-binding proteins present in the microsomal fraction could not be solubilized by incubation of microsomes with 1 molar NaCl or 0.2 molar Na₂CO₃, but some GTP-binding activity was solubilized when microsomes were treated with 6 molar urea. These results indicate that D. salina GTP-binding proteins are tightly associated with the membranes. The covalent attachment of fatty acids to these proteins was also investigated. Electrophoresis followed by fluorography of delipidated microsomal proteins extracted from [³H]myristic acid-labeled cells showed an intense labeling of a 28 kilodalton protein. We conclude that D. salina contains proteins resembling the ras-related proteins found in animal cells and higher plants.     

The Endothelin/Sarafotoxin‐Induced Increase of the Proliferation of Undifferentiated and DMSO‐Differentiated GEM‐81 Goldfish Erythrophoroma Cells is Mediated by ETB Receptors

Endothelins (ETs) and sarafotoxins (SRTXs) have been reported to exert ET_B‐mediated effects on vertebrate pigment cells. GEM‐81 cell line, a red pigment cell‐derived cutaneous tumor of the teleost Carassius auratus, expresses ET_B receptors and can be differentiated with 1.5% DMSO treatment, thus constituting an useful model to investigate ET and SRTX effects on cultured fish pigment cells. Our aim was to characterize the pharmacology and biological effects mediated by ET receptors in DMSO‐differentiated and undifferentiated cells. ET subtype receptors and their respective Ki values in both cell types were determined by competitive binding assays using ¹²⁵I ET‐1 and BQ‐485 (an ET_A antagonist) or BQ‐788 (an ET_B antagonist). BQ‐788, but not BQ‐485, significantly reduced ¹²⁵I‐ET‐1 binding in both cell types, with similar low (Ki > nM) affinities. To determine the proliferation effects of ETs/SRTXs, cells were treated for 72 h with the hormones, and counted in a hemocytometer. The proliferation assays were repeated for SRTX S6c in the presence or absence of BQ‐788. The results demonstrated that, with the exception of ET‐1 (biphasic effect) and ET‐3 (no significant effect) in undifferentiated GEM‐81 cells, all the tested hormones induced increases in the proliferation of both types of cells. The hormones were equipotent in DMSO‐differentiated cells, which exhibited increased sensitivity to ETs, but not to SRTXs, as compared with undifferentiated cells. The BQ‐788 antagonistic effect was also exerted on the proliferation responses to SRTX S6c. These results corroborate the long and important evolutionary history of the ET/SRTX receptor system in vertebrate pigment cells.     

A matrix approach to human class II histocompatibility antigens: Reactions of four monoclonal antibodies with the products of nine haplotypes

Three putative HLA-DC-specific monoclonal antibodies, Genox 3.53, BT3/4 and anti-Leu-10, and the HLA-DR-specific antibody, L243, were compared. Their interactions with molecules from homozygous cell lines expressing DR types 1 through 9 were studied. Indirect radioimmunoassays on 29 cell lines demonstrated that Genox 3.53 reactivity correlated with DR1, 2, 6; BT3/4 reactivity correlated with DR 1, 2, 4, 6, 8; and anti-Leu-10 reactivity correlated with DR1, 2, 4, 5, 6, 8, and 9. In addition, one of six DR3-positive cells and three DR7, DRw10-positive cells reacted with anti-Leu-10 and one of two DR9-positive cells reacted with BT3/4. Binding studies with soluble antigen and competitive radioimmunoassays demonstrated that all three antibodies reacted with the DC1 molecule. Preincubation with BT3/4 blocked anti-Leu-10 binding; Genox 3.53 and L243 did not. Genox 3.53 and L243 were only blocked by themselves. Serial immuno-precipitation showed anti-Leu-10 reacted with non-HLA-DR molecules from cells expressing DR types 1–6, 8 and 9. However, the molecules precipitated by anti-Leu-10 were characteristic class II major histocompatibility complex (MHC) molecules. Their and chains were of lower apparent molecular weight than the DR chains in all haplotypes. They also comigrated with the DC1 molecule precipitated by Genox 3.53. Serial immuno-precipitation also showed that anti-Leu-10 removed all Genox 3.53 reactive molecules from cell lysates, but Genox 3.53 removed only a subset of anti-Leu-10 reactive molecules. These studies show Genox 3.53, BT3/4, and anti-Leu-10 react exclusively with class II MHC molecules that are not HLA-DR, and most likely define different polymorphisms of DC molecules, the human equivalent of mouse I-A products.Abbreviations used in this paper BSA bovine serum albumin - PBS phosphate-buffered saline, pH 7.4 - RIA radioimmunoassays - ¹²⁵I-RAM ¹²⁵I-labeled F(ab)₂ of rabbit anti-mouse IgG - NP40 Nonidet P40 OVA-LB, 0.1% ovalbumin/0.5% NP40, 10mM Tris pH 7.3, 1MM M9Cl₂ 0.5% phenyl methyl sulfonyl fluoride - SDS-PAGE sodium dodecyl sulfate-polyacrylamide gel electrophoresis - MHC major histocompatibility complex - KD kilodaltons     

Characterization of muscarinic acetylcholine receptors on intact neuroblastoma × glioma NG108-15 cell upon induced differentiation

Summary Studies have established that major increases in muscarinic acetylcholine receptor (mAchR) binding in the brain appear to coincide with synaptogenesis. The neuroblastoma × glioma hybrid NG108-15 cell line has been demonstrated to possess numerous functional characteristics of intact neurons, including synapse formation with myotubes. The present study examines and characterizes the mAchR on the hybrid NG108-15 cells during differentiation, induced by 1 mM dBcAMP. Specific binding of [³H]-QNB for differentiated cells increases gradually to a final level of 130% (P < 0.05) over the control undifferentiated cells during the first 24 hr of incubation. Further, this increase of receptor sites appears to correlate proportionately to the degree of neurite extension of the differentiating cells. The dissociation rate constant at equilibrium (K_d) and maximum binding capacity (B_max) have been determined to be 5.6 nM and 920 fmol/10⁶ cells, respectively, for differentiated cells, and 4.4 nM and 400 fmol/10⁶ cells, respectively, for undifferentiated cells. Computer analyses of the data obtained from saturation experiments reveal a single class of binding sites for [³H]-QNB on both differentiated and undifferentiated cells. The Hill plot analysis of the QNB-binding indicates a Hill coefficient (nH) of 1.0 and 0.91 for differentiated and undifferentiated cells, respectively, suggesting the unity of receptor sites with no co-operativity. Our results depict that increases of mAchRs on intact cells correlate with the degree of cellular differentiation.     

Characterization of the cholera toxin receptor on Balb/c 3T3 cells as a ganglioside similar to, or identical with, ganglioside GM1. No evidence for galactoproteins with receptor activity.

Balb/c 3T3 cells contain a large number [(0.8-1.6) x 10(6)] of high-affinity (half-maximal binding at 0.2 nM) binding sites for cholera toxin that are resistant to proteolysis, but are quantitatively extracted with chloroform/methanol. The following evidence rigorously establishes that the receptor is a ganglioside similar to, or identical with, ganglioside GM1 by the galactose oxidase/NaB3H4 technique on intact cells was inhibited by cholera toxin. (2) Ganglioside GM1 was specifically adsorbed from Nonidet P40 extracts of both surface- (galactose oxidase/NaB3H4 technique) and metabolically ([1-14C]palmitate) labelled cells in the presence of cholera toxin, anti-toxin and Staphylococcus aureus. (3) Ganglioside GM1 was the only ganglioside labelled when total cellular gangliosides separated on silica-gel sheets were overlayed with 125I-labelled cholera toxin, although GM3 and GD1a were the major gangliosides present. In contrast no evidence for a galactoprotein with receptor activity was obtained. Cholera toxin did not protect the terminal galactose residues of cell-surface glycoproteins from labelling by the galactose oxidase/NaB3H4 technique. No toxin-binding proteins could be identified in Nonidet P40 extracts of [35S]-methionine-labelled cells by immunochemical means. After sodium dodecyl sulphate/polyacrylamide-gel electrophoresis none of the major cellular galactoproteins identified by overlaying gels with 125I-labelled ricin were able to bind 125I-labelled cholera toxin. It is concluded that the cholera toxin receptor on Balb/c 3T3 cells is exclusively ganglioside GM1 (or a related species), and that cholera toxin can therefore be used to probe the function and organisation of gangliosides in these cells as previously outlined [Critchley, Ansell, Perkins, Dilks & Ingram (1979) J. Supramol. Struct. 12, 273-291].     

The Glycoproteins of Plant Seeds : ANALYSIS BY TWO-DIMENSIONAL POLYACRYLAMIDE GEL ELECTROPHORESIS AND BY THEIR LECTIN-BINDING PROPERTIES

Protein from the jack bean, peanut, soybean and kidney bean seeds were extracted with a solution containing 9.3 molar urea, 5 millimolar K₂CO₃, 0.5% dithiothreitol and 2% Nonidet P-40 and then subjected to two-dimensional gel electrophoresis. After electrophoresis, the slab gels were stained with a variety of ¹²⁵I-labeled lectins and the lectin-binding proteins were identified after autoradiography. Incubation of slab gels of jack bean with concanavalin A, peanut with peanut agglutinin, soybean with soybean agglutinin, and kidney bean with phytohemagglutinin showed that the majority of the polypeptides in each seed type were able to bind to their homologous lectins. Control slab gels in which incubations were carried out with identical amounts of proteins, ¹²⁵I-lectin and an appropriate sugar inhibitor showed little or no lectin binding to the polypeptides. Additionally, incubation of slab gels of peanut proteins with ¹²⁵I-ricin, ¹²⁵I-wheat germ agglutinin, ¹²⁵I-concanavalin A, and ¹²⁵I-soybean agglutinin each revealed a clearly distinct binding pattern compared to the one observed with the peanut agglutinin. The results demonstrate that a large number of legume seed polypeptides are glycoproteins and that the carbohydrate groups within a seed species are heterogeneous in structure, thus indicating the existence of complex glycosylating enzyme systems in legume seeds. It is suggested that the high degree of binding between seed proteins and their homologous lectins might have some functional significance in maintaining large aggregates of protein in compact, insoluble form.     

Two detergent-insoluble proteins of the human lymphocyte membrane are enriched in an isolated membrane fraction

Human lymphocytes isolated from peripheral blood on Ficoll/Paque density gradients were surface-labelled by ¹²⁵I/lactoperoxidase or ³H/reductive alkylation and lysed in buffer solutions containing non-ionic or amphoteric detergents (octylphenylpolyoxyethylenes, octylglucoside, cholylamidopropyldimethylammoniopropane sulfonate) under a variety of conditions. The cell lysate was fractionated by sedimentation or by density gradient centrifugation. The large majority of the labelled proteins is solubilized by the detergents. Two proteins of 45 000 and 30 000 molecular weight are the main detergent-insoluble, surface-labelled components. They can be fractionated from detergent lysates of cells in relatively pure form from the other membrane proteins and from nuclear material on density gradients. The same two proteins are specifically enriched in a membrane fraction isolated from a detergent-free cell homogenate by density gradient centrifugation. Cytoskeletal and other intracellular proteins remain associated with these two proteins when fractionated by either of these two independent methods.     

Changes in pattern and accessibility for 125I-labelling of cell-surface proteins after mesenchymal differentiation of embryonal carcinoma cells

Cell-surface proteins of the embryonal carcinoma line C17-S1 1003 (1003) and of some of its mesenchymal derivatives were studied. The surface proteins were labelled with ¹²⁵I using the lactoperoxidase-glucose-glucose oxidase system either on the cells attached to the culture dishes or after their dissociation. Iodinated proteins were analyzed by two-dimensional gel electrophoresis. The patterns obtained with embryonal carcinoma cells 1003 and with two mesenchymal cell types derived from them, namely embryonic mesenchymal cells (line 10035) and fibroblastic cells (line 10031), were different one from the other, especially when considering the group of proteins labelled on the attached cells. The pattern of cell-surface proteins of the myoblastic line 1168, also derived from C17-S1, was found to be similar to that of 10031 fibroblastic cells. This result is discussed in the light of the phenotypic transition toward myogenesis, which can be obtained with 10031 fibroblastic cells but not with 10035 embryonic mesenchymal cells. A direct method of detection of lectin-binding proteins permitted us to identify the major concanavalin A-binding proteins. Two of them are common to all cell lines studied. They were labeled with ¹²⁵I on the attached undifferentiated 1003 cells, while in all differentiated derivatives they became available for labelling after the cell detachment only.     

Detection of the Initial Step of Mesenchymal Differentiation of Teratocarcinoma Cells Using the Monoclonal Antibody Eccd-11

The monoclonal antibody ECCD-1 recognizes the Ca²⁺-dependent cell-cell adhesion molecule of teratocarcinoma stem cells (EC cells) and of a certain class of differentiated epithelial cells. It actively disrupts cell-cell adhesion when added to monolayer cultures of these cells, but does not affect adhesion of mesenchymal or neuronal cells. When ECCD-1 was added to clonal cultures of EC cells (PCC3/A/1 line), all the cells were initially sensitive to the antibody, but after 5 to 6 days of culture a fraction of the cells in certain colonies no longer reacted with the antibody although they expressed alkaline phosphatase activity, which is a marker of undifferentiated EC cells. We isolated these ECCD-1-resistant cells by recloning and examined their differentiation by clonal culture. Most of them differentiated into fibroblastic cells and a few into skeletal muscle-like cells, but none differentiated into any other cell types. From these observations, we suppose that the ECCD-1-resistant population of EC cells are committed to mesenchymal differentiation. The use of ECCD-1, thus, permitted us to detect EC cells at the initial stage of a particular differentiation pathway.