Lentil Lectin Affinity Research Articles

Purification and characterization of the extracellular acid phosphatase of Leishmania donovani

A tartrate sensitive acid phosphatase activity was purified from culture supernatants of Leishmania donovani promastigotes grown in a macromolecule-free defined medium. Purification was accomplished by ultrafiltration, lentil lectin affinity chromatography, and sucrose density gradient ultracentrifugation. This enzyme was determined to be an acid glycoprotein containing 0.37 mg hexose per mg protein. A molecular weight of 134 000 by sucrose density gradient centrifugation was observed, although on molecular sieve chromatography the enzyme eluted with an apparent molecular weight of >700 000. The specific activity of the purified enzyme was >200 μmol min −1(mg protein) −1 when assayed with 4-methylumbelliferylphosphate as the substrate. In addition to various hexose phosphates, the enzyme hydrolyzed phosphorylated amino acids, in particular phosphotyrosine. The purified enzyme was heterodisperse with respect to both protein and activity staining patterns upon polyacrylamide gel electrophoresis.

Affinity to lectin, biological and immunological characteristics of human chorionic gonadotropins from pregnant women and trophoblastic tumour patients

To compare the affinity to lectin of human chorionic gonadotropins (hCG) from pregnant women and trophoblastic tumour patients, a small amount of urine was fractionated by a lentil lectin (LcH) affinity chromatography. The LcH-bound fractions were eluted with 2% mannoside solution, and each fraction was assayed for hCG activities by radioimmunoassay. In pregnant women, more than 90% of hCG immunoactivity in urine was bound to the LcH column and eluted from it, whereas 8 to 26% and 37 to 51% of the activity were not adsorbed to the affinity column and were recovered in the LcH-unbound fraction in the patients with hydatidiform mole and choriocarcinoma, respectively. These results suggest that LcH affinity chromatography of urinary hCG contributes to differential diagnosis between pregnancy and trophoblastic tumours. To characterize the properties of hCG from the urine of choriocarcinoma patients, with or without the LcH affinity, hCG activities in both LcH-unbound and LcH-bound fractions were measured by in vivo and in vitro bioassays and each of the activities was compared with the activities measured by radioimmunoassay. The results showed that the LcH-unbound fraction contained hCG molecules defecting to induce the biological activity of hCG in vivo.

Characterization of human T cell-derived IgE-potentiating factor.

We have previously shown that Fc epsilon receptor-positive (Fc epsilon R+) T cell lines from patients with the hyper IgE syndrome secrete IgE-binding factors which selectively enhance IgE but not IgG synthesis in cultures of B cells obtained from patients with allergic rhinitis but not from nonatopic subject. In the present study we have tested the effect of supernatants from Fc epsilon R+ T cell lines on a large panel of B cells from atopic patients (n = 20). We found that IgE synthesis was selectively enhanced only in B cell cultures in which there was ongoing spontaneous synthesis of IgE. The target of IgE-potentiating factor(s) was a large low-density B cell present in the circulation of responding atopic donors. In addition, we further characterized IgE-potentiating factors derived from Fc epsilon R+ T cell lines. The factor(s) fractionated into 2 peaks on Sephadex G-75 with approximate molecular masses of 15,000 and 60,000 kDa, and had affinity for lentil lectin but not for peanut agglutinin. Release of IgE-potentiating factor(s) was enhanced by the addition of exogenous human IgE to Fc epsilon R+ T cell cultures and was inhibited by tunicamycin, an inhibitor of N-glycosylation. These studies suggest a close homology between the physicochemical characteristics of human and rodent IgE-potentiating factors and the immune signals which modulate production of these IgE regulatory factors.

Effects of horse and fetal calf serum on the expression of tumor-associated antigen and tumorigenicity of L5178Y leukemia/lymphoma cells

A tumor antigen (TA) associated with murine leukemia-lymphoma L5178Y cells has been identified by the enzyme-linked immunosorbent assay (ELISA) and indirect immunofluorescence (IIF) techniques. The antigen was present in both non-solubilized and 0.5% NP-40 solubilized membrane extracts. Rabbit anti-L5178Y lymphoma serum (RALS), extensively absorbed with normal mouse tissues, identified TA in extracts of L5178Y lymphoma and L5178Y leukemia cells grown in horse serum (L5178Y/HS), but not in extracts of L5178Y cells grown in fetal calf serum (L5178Y/FCS). Similarly, absorbed rabbit anti-L5178Y/HS serum specifically reacted with extracts of lymphoma and L5178Y/HS but not with L5178Y/FCS cells. Membrane IIF showed positive reactivity in 88% of lymphoma and 73% of L5178Y/HS cells, whereas splenic lymphocytes and L5178Y/FCS cells were negative. Goat anti-AKR virus serum reacted with soluble extracts of lymphoma, L5178Y/HS, and L5178Y/FCS as well as with normal DBA/2 tissues in the ELISA. However, goat anti-AKR virus serum did not block the reactivity of RALS to lymphoma in the blocking ELISA (BELISA). Expression of TA, but not murine leukemia viral antigen(s), was correlated with the in-vivo tumorigenicity of the L5178Y cells. The antigenic activity of lymphoma extract was reduced by incubation for 1 h at 56 and 65°C, by trypsin digestion, and by exposure to pH 2.8 or 11.0 for 1 h. The antigen, sequentially purified by gel filtration and Lentil-lectin affinity chromatography, was a glycoprotein, with a molecular weight of approx. 64,000 daltons, as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis.

Relationship among IgE-binding factors with various molecular weights.

The rat-mouse T cell hybridoma 23B6 forms IgE-binding factors on incubation with IgE. The hybridoma cells incubated with IgE contained intracellular IgE-binding factors of 60K, 30K, 14K, and 10K daltons, and secreted the 60K, 30K, and 14K IgE-binding factors. Both intracellular and extracellular IgE-binding factors of 60K and 14K daltons selectively suppressed the IgE response, whereas the 30K and 10K factors failed to do so. Reduction and alkylation of the extracellular 60K IgE-binding factors yielded fragments of 30K, 14K, and 10K daltons with IgE-binding activity, and the same treatment of the 30K "inactive" IgE-binding factor yielded the 14K and 10K fragments. Among the fragments obtained by reduction and alkylation, only the 14K IgE-binding factors selectively suppressed the IgE response. Monoclonal antibody OX 3, which recognizes an Ia determinant, bound the 60K and 30K IgE-binding factor, and the 30K fragment obtained by reduction and alkylation. None of the 14K and 10K IgE-binding factors and fragments of comparable size obtained by reduction and alkylation contained the antigenic determinant. The results indicate that 30K, 14K, and 10K IgE-binding factors are derived from the 60K precursor molecules. Transfection of COS 7 monkey kidney cells with a single cloned cDNA, 8.3, which encodes rodent IgE-binding factor, resulted in the formation of IgE-binding factors of 60K and 11K daltons. Both species of the factors had affinity for lentil lectin and selectively potentiated the IgE response, but only the 60K IgE-binding factor bound to the monoclonal anti-Ia antibody. Reduction and alkylation of the 60K IgE-binding factors from the cDNA clone yielded an 11K fragment having the same properties as the 11K IgE-binding factor, with respect to the affinity for lentil lectin, and the biologic activities. Because the cDNA clone was constructed from mRNA of 23B6 cells, it appears that the 60K IgE-binding factor molecule from 23B6 cells is composed of a single peptide chain, and that the naturally formed 30K, 14K, and 10K IgE-binding factors are formed by post-translational modification of the 60K peptide.

Humoral factors in very young NZB mice that enhance the maturation of normal B cell precursors. Partial purification and characterization.

Soluble factors that enhance maturation of murine B lineage precursor cells in vitro were partially purified from the serum of very young NZB mice and characterized biochemically and biologically. Activity was initially detected by induction of colony-forming activity and surface immunoglobulin (sIg) on normal sIg- marrow cells as well as responsiveness of a pre-B cell line. Pooled sera from 4- to 5-wk-old NZB mice were initially fractionated on Sephacryl S-300 and Sephadex G-100 superfine columns. Fractions with activity (corresponding to m.w. of 15,000 to 45,000) were pooled and further separated. The activity was eluted as a single peak by hydrophobic (phenyl-Sepharose, with 0.8 M (NH4)2SO4) and lentil lectin affinity chromatography but resolved into three distinct peaks in preparative isoelectric focusing (IEF), with pI values of 3.5, 7.8, and 8.4. The latter two merged into a single peak with a pI value of 8.8 when the sample was further treated with neuraminidase before IEF. These three IEF fractions, each of which were enriched at least 1000-fold in specific activity relative to starting serum, were then characterized. Each was stable at pH 2 but sensitive to trypsin, 10 M urea, and heat treatment (56 degrees C for 1 hr). In nonreduced SDS-poly-acrylamide gel electrophoresis, their mobilities corresponded to m.w. of 17,000 for peak I (pI 3.5), 15,000 for peak II (pI 7.8), and 15,000 for peak III (pI 8.4). Interleukin 1, interleukin 2, interleukin 3, colony-stimulating factor for granulocyte and macrophage progenitors, antiviral, or B cell growth factor type I-like activities were not demonstrable. Peaks II and III, but not peak I, induced Ig secretion of anti-stimulated B cells. Peak I was also less effective than peaks II and III in induction of sIg on an established pre-B cell line. However, all fractions were equally effective in enhancing maturation of normal sIg- B lineage cells. Thus, serum from 4- to 5-wk-old NZB mice contains at least two distinct soluble factors that can enhance the maturation of sIg- B lineage cells in vitro. The biologic and biochemical characteristics of these factors appear to differ from those of previously well-defined cytokines.

Characterization of gamma-glutamyltranspeptidase from human exocrine pancreatic carcinomas

Two different types of gamma-glutamyltranspeptidase (γ-GTP) have been found in normal human pancreas following bromelain treatment. On the other hand, three human pancreatic ductal cell carcinomas have only a single type of γ-GTP upon analysis with polyacrylamide gel electrophoresis, anion-exchange column chromatography and isoelectric focusing. Carcinoma γ-GTPs were almost identical to one of the two types of normal pancreatic γ-GTPs. The γ-GTP from pancreatic carcinomas bound to anion-exchange column and was eluted at the same NaCl fractions as normal pancreatic γ-GTP. The properties of pancreatic carcinoma γ-GTP, as assessed by binding to concanavalin A and lentil lectin affinity columns, were also similar to one of the two enzymes of normal pancreas. No apparent difference in isoelectric points was found between the carcinoma γ-GTPs and one of the two normal pancreatic γ-GTPs.

Modification of the N-linked oligosaccharides in cell surface glycoproteins during chick embryo development. A using lectin affinity and a high resolution chromatography study.

Important differences in asparagine-linked glycopeptides were observed in vitro cultured fibroblasts derived from chick embryo at different stages of development. Cells from 8-day and 16-day embryos were labeled metabolically with [3H]mannose. Cell surface glycopeptides obtained after mild trypsin treatment were extensively digested with pronase and then chromatographed on concanavalin-A-Sepharose and other immobilized lectins. The most important changes concerned the complex type chains. The ratio between triantennary plus tetraantennary and biantennary chains increased about 2.5-fold from the 8th to the 16th day of development. In the same way, complex chains with bisecting N-acetylglucosamine increased from 8-day to 16-day cells as shown by Phaseolus-vulgaris-erythroagglutinin--agarose chromatography. In 16-day cells, the majority of triantennary chains (60%) with alpha-linked mannose substituted at C2 and C6 positions and biantennary chains (50%) were shown to contain fucosyl (alpha 1----6)N-acetylglucosaminyl structure in the core region by their ability to bind to a lentil lectin affinity column. Similarly, in 8-day cells, triantennary chains (50%) were more fucosylated than biantennary chains (35%). Thus, complex structures exhibited an increased fucosylation of their invariable core from the 8th to the 16th day of development, except for fucosylated triantennary chains which were retained on Phaseolus vulgaris Leucoagglutin and on lentil lectin. These latter structures were present at the surface of 8-day cells and absent at the surface of 16-day cells. After chromatography on Bio-Gel P6 and treatment with endo-beta-N-acetylglucosaminidase H, the [3H]-mannose-labeled glycopeptides were separated by high resolution chromatography into glycopeptides with complex chains and glycopeptides with high-mannose chains. Analysis of the high-mannose oligosaccharides released after endo-beta-N-acetylglucosaminidase H treatment by chromatography on Bio-Gel P4 indicated that the same type of high-mannose chains were present at the surface of 8-day and 16-day cells. Quantification of mannose, galactose and sialic acid residues using gas liquid chromatography was consistent with a decrease of the relative amount of oligomannose chains and an increase of the relative amount of complex type chains in 16-day cells compared to 8-day cells. Thus N-linked oligosaccharides derived from cell surface glycoproteins undergo changes during embryo development resulting in greater complexity of carbohydrate chains.

Purification and characterization of a human T-lymphocyte-derived glial growth-promoting factor.

Antigen- or lectin-stimulated T lymphocytes and human T-cell leukemia virus (HTLV)-infected cell lines secrete lymphokines that can influence the growth and function of a variety of cell types. We recently demonstrated that supernatants from the HTLV-II-infected Mo-T-cell line stimulate the proliferation of rat brain oligodendrocytes and astrocytes. We have now purified a glial growth-promoting factor (GGPF) from these supernatants. Purification from serum-free conditioned medium was accomplished by sequential concentration, ammonium sulfate precipitation, lentillectin affinity chromatography, gel filtration, and reversed-phase high-performance liquid chromatography. GGPF is assayed by its ability to stimulate DNA synthesis in oligodendrocytes, as measured by [3H]thymidine uptake. The purified GGPF has an apparent Mr of 30,000 when analyzed by sodium dodecyl sulfate/polyacrylamide gel electrophoresis under nonreducing conditions. Under reducing conditions, however, GGPF appears as a single band of Mr 18,000. Both reduced and unreduced forms have biological activity, suggesting that GGPF exists in both a functional monomeric and dimeric form. Purified GGPF appears to be a biochemically and functionally distinct lymphokine.

Recognition by a mouse monoclonal antibody of a glycoprotein antigen of rat brain which is expressed intracellularly by neurons

We describe a previously uncharacterised glycoprotein antigen of rat brain. The antigen was localised by immunofluorescence on 10 μm cryostat tissue sections, and was found to be present intracellularly in neurons. No other cell types or structures within the brain were stained. The antigen is recognised by a mouse monoclonal antibody called NGP41. The antibody was produced after immunising a mouse with glycoproteins purified by lentil lectin affinity chromatography of solubilised rat brain membranes. Spleen cells from the immunised mouse were fused with the myeloma P3X63Ag8. The antigen is expressed by neurons in all brain regions, and also in the dorsal root ganglion neurons of the peripheral nervous system. In all brain regions, the large projection neurons are the most intensely stained by immunofluorescence, but some small neurons also express the antigen. Although dendrites were not stained, sections of sciatic nerve were stained by NGP41, suggesting that the antigen is expressed by axonal processes. The cell bodies of neurons in the inferior olive were stained by NGP41, but their terminals on Purkinje cell dendrites in the cerebellar cortex were not stained, suggesting that the antigen is absent or expressed below the limit of detection in terminals. Both crude brain membranes and a lentil lectin affinity purified brain glycoprotein fraction absorbed the antibody, suggesting that the antigen is a membrane bound glycoprotein. In immunoblotting experiments, the antigen was detected in homogenates of brain and spinal cord membranes, where it appeared as a triplet of bands with molecular weights of 41K, 38K and 36K. Antigen was not detected by immunoblotting in homogenates of six different tissues of non-nervous origin. The antigen was enriched in glycoprotein fractions from adult and juvenile cerebellum as assessed by immunoblotting. Adult brain glycoprotein preparations had a triplet structure similar to that in the homogenates, although most of the antigenic activity of the juvenile preparation was found in a position corresponding to the upper two bands of the triplet.

Purification and characterization of human T-lymphocyte-derived erythroid-potentiating activity.

The human T-lymphoblast cell line, Mo, secretes a number of lymphokines, including erythroid-potentiating activity (EPA), an important early regulator of erythropoiesis. We report purification of EPA to homogeneity, from serum-free Mo-conditioned medium. Purification was accomplished by sequential concentration, ammonium sulfate precipitation, lentil lectin affinity chromatography, gel filtration, and reverse-phase high-performance liquid chromatography. EPA was assayed by its ability to stimulate the growth of large erythroid colonies (bursts) from normal human peripheral blood. The purified EPA has a molecular weight of 28,000 and appears as a single band when analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis under reducing or nonreducing conditions. Purified EPA stimulates the growth of both early and late erythroid precursors from human bone marrow, as well as colony formation by the K562 human erythroleukemia cell line. Purified EPA has no colony-stimulating factor activity nor does it appear to be a structural protein of the human T-cell leukemia virus subtype II which infects the Mo cells.

IgE-binding factors from mouse T lymphocytes. II. Strain differences in the nature of IgE-binding factor.

Normal spleen cells from high IgE responder, BDF1 mice, formed IgE-potentiating factor upon incubation with mouse IgE, whereas those from low IgE responder, SJL mice, formed IgE-suppressive factor. The majority of the factors from BDF1 spleen cells had affinity for lentil lectin; those from SJL mice lacked affinity for the lectin. BDF1 spleen cells could be switched, however, to form IgE-suppressive factor if the cells were incubated with IgE in the presence of lipomodulin , a phospholipase inhibitory protein. In contrast, SJL spleen cells could form IgE-potentiating factor in the presence of lysolecithin, which enhances the glycosylation of IgE-binding factors. When the two strains were primed with alum-absorbed ovalbumin and their spleen cells were stimulated with homologous antigen, IgE-binding factors were detected in culture filtrates. The factors formed by BDF1 spleen cells selectively potentiated the IgE response, however, whereas those formed by SJL spleen cells selectively suppressed the response. Analysis of the cellular mechanisms for the selective formation of IgE-potentiating factor by antigen-primed BDF1 spleen cells revealed that antigenic stimulation of Lyt-1+ T cells resulted in the formation of two T cell factors, i.e., an "inducer" of IgE-binding factor and a glycosylation-enhancing factor, and that these factors induced the selective formation of IgE-potentiating factor. In SJL spleen cells, antigenic stimulation of Lyt-1+ T cells resulted in the formation of an "inducer" and a glycosylation-inhibiting factor, and those factors in turn stimulated the formation of IgE-suppressive factor. An additional difference between the two strains was found in Fc gamma R+ Ly-1+ T cells that were the cell source of IgE-binding factors. Upon stimulation with IgE or "inducer", BDF1 Lyt 1+ cells formed IgE-potentiating factor, whereas the same subset of T cells from SJL mice formed IgE-suppressive factor. The results indicate that the genetic differences between the two strains with respect to the nature of IgE-binding factors appear to be expressed in the process of glycosylation of IgE-binding factors.

Purification and characterization of Rana pipiens brain Thy-1 glycoprotein.

The occurrence of Thy-1 antigens in Rana brain has been studied by the use of heterologous anti-Rana brain antisera raised in rabbit and BALB/c mouse (Thy-1.2) and AKR/J mouse (Thy-1.1) strains and by monoclonal anti-mouse Thy-1.1 and anti-mouse Thy-1.2 antibodies with the use of quantitative absorption assays. Three antigenic determinants were defined on Rana brain and referred to as: 1) the Rana-specific xenoantigen, 2) the Rana-mouse cross-reacting xenoantigen, and 3) the Thy-1.1 antigen. Thy-1 antigenic activities were solubilized from crude brain membranes in deoxycholate and followed by measuring the Rana-specific and the Thy-1.1 antigenic determinants. After solubilization, Rana brain Thy-1 antigens were purified by lentil lectin affinity chromatography and gel filtration on Sephadex G-200. A 605-fold and 400-fold enrichment in the Rana-specific and the Thy-1.1 antigenic activities with a yield of 25% and 17%, respectively, were obtained. Both antigenic activities were associated with a single glycoprotein of molecular size 3.1 nm and m.w. estimated at 27,000 by SDS-polyacrylamide gel electrophoresis. The serologic and biochemical properties of our purified Rana brain Thy-1 glycoprotein were very similar to those of the mammalian Thy-1 molecule, suggesting the conservation of the gene coding for Thy-1 during vertebrate evolution.

Monoclonal antibodies to rat liver cell membrane glycoproteins

Monoclonal antibodies were raised against membrane glycoproteins prepared from rat liver using lentil-lectin affinity chromatography. Monoclonal antibodies to rat hepatocytes with three different characteristics were obtained and designated as HAM.1, HAM.2 and HAM.3. The binding characteristics of these three monoclonal antibodies were analysed by flow cytofluorography and indirect radio-immunoassay; HAM.3 recognized a membrane antigen restricted mainly to hepatocytes. However, most rat hepatoma cell lines tested failed to express this antigen. Thus, this antibody might be useful for analysing the molecular nature of a liver-specific antigen or the membrane molecules on the normal and malignantly transformed hepatocytes. All the rat hepatoma cell lines tested were labeled with HAM.l which recognized a membrane antigen on both hepatocytes and lymphocytes, although the amount of antigen expression was quite low on lymphocytes. HAM.2 was found to recognise the Class 1 (RT-1A) histocompatibility antigen. This antigen was purified from rat liver with a HAM.2-affinity column.

A rapid procedure for the enrichment of undenaturated, antigenically active herpes simplex virus glycoproteins

The usefulness of lentil lectin affinity chromatography for the rapid enrichment of HSV glycoproteins in an undenatured state for both research and clinical purposes was investigated. In order to compare the lentil lectin-binding characteristics and immunologic specificities of undenatured HSV-1 and HSV-2 glycoproteins, [ 35S]methionine-labelled extracts of virus-infected HEp-2 cells were subjected to lentil lectin affinity chromatography. Individual HSV-1 and HSV-2 glycoproteins in bound and unbound fractions were identified using monoclonal antibodies. With the exception of a portion of pgD and gD, all major viral glycoprotein species (gA, gB, gC, gD, gE and gF) and their glycosyiated processive intermediates bound to lentil lectil indicating that all possess predominantly mannosyl and/or glucosyl carbohydrate moieties. Although the unbound pgD and gD species were glycosyiated, no gD and only a portion of pgD bound to lentil lectil when reapplied to the column indicating that these subspecies possess alterations in factors required for efficient lectin binding. Immunoprecipitation of undenatured lectin-bound glycoproteins from infected cells using HSV-1- and HSV-2-specific rabbit and human antisera confirmed previous findings that the predominant type-specific glycoproteins of HSV-1 and HSV-2 are gC and gE/gF, respectively.

Presence of an antigenic determinant common to rat IgE-potentiating factor, IgE-suppressive factor, and Fc epsilon receptors on T and B lymphocytes.

Polyclonal antibodies against purified IgE-potentiating factor were prepared in guinea pigs, and mouse monoclonal antibodies were prepared against inactive IgE-binding factor from a T cell hybridoma. The polyclonal antibodies and two of the monoclonal antibodies bound the IgE-potentiating factor, the IgE suppressive factor, and the inactive IgE-binding factor. The results indicate that the three IgE-binding factor molecules share a common antigenic determinant. The polyclonal antibodies and the monoclonal antibody bound intracellular IgE-binding factors that did not have affinity for either lentil lectin or peanut agglutinin, which suggests that the antibodies were directed against the peptide portion of IgE-binding factors. Pretreatment of Fc epsilon R+ T hybridoma cells and Fc epsilon R+ B cells with either the monoclonal or the polyclonal anti-IgE-binding factor (anti-IgE-bF) antibody prevented rosette formation of the cells with IgE-coated erythrocytes, whereas the same treatment failed to affect rosette formation of Fc gamma R+ cells with IgG-coated erythrocytes. Specific binding of the guinea pig anti-IgE-bF antibodies to Fc epsilon R on both B and T cells was confirmed by immunofluorescence. The results indicate that Fc epsilon R on B and T cells share a common antigenic determinant with IgE-binding factors. The F(ab')2 fragments of the guinea pig anti-IgE-bF induced the expression of Fc epsilon R on Fc gamma R+ lymphocytes and the formation of IgE-binding factors. The capacities of not only IgE, but also anti-IgE-bF, to induce both Fc epsilon R expression and IgE-binding factor formation indicate that Fc epsilon R on lymphocytes, rather than IgE, transmit the signal for the formation of IgE-binding molecules.

Purification to electrophoretic homogeneity of human alpha lymphotoxin from a cloned continuous lymphoblastoid cell line IR 3.4

The 70–90,000 molecular weight (MW) alpha (α) component of the human lymphotoxin (LT) system has been purified to electrophoretic homogeneity. The α LT containing supernatants were obtained from a phorbol myristate (PMA) stimulated cloned continuous human B lymphoblastoid cell line IR 3.4. Supernatants were subjected to a biochemical separation scheme that consisted of chromatography on control pore glass beads, DEAE ion-exchange chromatography, lentil-lectin affinity chromatography, and electrophoresis on 7% native preparative polyacrylamide gels. The specific activity of the α LT in the final fractions was from 10 7 to 5 × 10 7 units of LT activity/mg protein. Approximately 3 to 5% of the initial α LT was recovered in the final fractions and a purification factor of 25,000 to 30,000 fold was required to achieve homogeneity. The α LT preparation from preparative PAGE exhibited concident migration of bioactivity and radioactivity on 5 and 7% native PAGE tube gels. Only a single protein peak was observed when the radiolabeled α LT was subjected to a two-dimensional SDS-reducing slab gel.

Unusual heterogeneity in the glycosylation of the G protein of the Hazelhurst strain of vesicular stomatitis virus

The asparagine-linked oligosaccharides of the G protein of the Hazelhurst subtype of the New Jersey serotype of vesicular stomatitis virus (VSV) have been compared with the oligosaccharides from the G protein of the well-characterized Indiana serotype of VSV, with baby hamster kidney cells in monolayer culture as the host for both viruses. [ 3H]Glucosamine- and [ 3H]mannose-labeled glycopeptides from the G protein of purified virus were analyzed by the combined techniques of endo-β- N-acetylglucosaminidase H (ENDO-H) digestion, concanavalin A and lentil lectin affinity chromatography, and Bio-Gel P-4 chromatography. Although almost all of the Indiana G protein oligosaccharides were acidic-type structures, as expected from previous studies; the Hazelhurst G protein contained a mixture of acidic-type, hybrid-type containing sialic acid, and neutral-type (predominantly Man 5–6GlcNAc 2-Asn) structures. The vast majority of acidic-type oligosaccharides from both the Hazelhurst and Indiana G proteins were diantennary structures, with less than half containing fucose linked to the innermost N-acetylglucosamine. Additional analysis of the Hazelhurst G protein by ENDO-H digestion and gel electrophoresis suggested that some of the mature G polypeptides contained acidic-type structures at both glycosylation sites, whereas the remainder contained an ENDO-H-resistant, acidic-type structure at one site and an ENDO-H-sensitive, hybrid- or neutral-type structure at the other site.

Modulation of the biologic activities of IgE-binding factors. III. Switching of a T cell hybrid clone from the formation of IgE-suppressive factor to the formation of IgE-potentiating factor.

Incubation of rat-mouse T cell hybridoma cells, 23B6, with rat immunoglobulin E (IgE) results in the formation of the 15,000-dalton IgE-suppressive factor and the 30,000-dalton IgE-binding factor, which has neither potentiating activity nor suppressive activity on the IgE response. Another T cell hybridoma, 23A4 cells, produces the 30,000-dalton "inactive" IgE-binding factor upon incubation with IgE. Both the 15,000-dalton IgE-suppressive factor and the 30,000-dalton IgE-binding factor lacked affinity for lentil lectin but bound to peanut agglutinin. When the 23B6 cells were incubated with IgE in the presence of lysolecithin, the majority of the 15,000-dalton IgE-binding factor formed by the cells gained affinity for lentil lectin, and this factor selectively potentiated the IgE response. The glycosylation-enhancing factor, which was formed by stimulation of normal spleen cells with lymphocytosis-promoting factor (LPF or pertussigen), also switched 23B6 cells from the formation of IgE-suppressive factor to the formation of IgE-potentiating factor. It was also found that the 30,000-dalton "inactive" IgE-binding factor, formed by both 23B6 and 23A4 cells, gained the ability to potentiate the IgE response, when the cells were cultured with IgE in the presence of glycosylation-enhancing factor. The results indicate that IgE-potentiating factor and IgE-suppressive factor share common precursors, and that biologic activities of IgE-binding factors are decided by their carbohydrate moieties. Incubation of the two hybridoma cells with lysolecithin or glycosylation-enhancing factor results in an increase in the proportion of FC epsilon R+ cells, suggesting that the assembly of N-linked oligosaccharide to precursor molecules is intrinsic for the expression of FC epsilon R.

Clathrin-coated vesicles purified from a human B lymphocyte cell line contain newly synthesized glycoproteins.

Clathrin-coated vesicles were purified from the cultured human B lymphocyte cell line RPMI 1788. These preparations were shown to be substantially free of contaminating membranes by the following criteria: (a) polypeptide composition, as assessed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, (b) electron microscopy, (c) lack of binding of intact vesicles to concanavalin A-Sepharose, (d) migration of coated vesicles as a single major peak when applied to a Sephacryl S-1000 gel filtration column. An association between HLA-A, -B, and -DR antigens and immunoglobulins with coated vesicle fractions was demonstrated by lentil lectin affinity chromatography and by immunoprecipitation using antisera directed against these glycoproteins. [35S]Methionine pulse-chase experiments revealed that there was a kinetic correlation between the expression of newly synthesized glycoproteins and their presence in coated vesicles. These results indicate that coated vesicles may be involved in the transport of cell surface and secreted eukaryotic glycoproteins through the cell to their final destinations at the plasma membrane or in the extra-cellular medium.