Effect Of Wheat Germ Agglutinin Research Articles

The effects of wheat germ agglutinin on the adipocyte insulin receptor

Treatment of rat adipocytes with wheat germ agglutinin markedly enhances insulin binding by increasing the affinity of the insulin receptor. The structure of the adipocyte receptor following wheat germ agglutinin treatment was studied by column chromatography and polyacrylamide gel electrophoresis to determine if aggregates of the insulin receptor are present. Solubilization of the receptor by Triton X-100 and passage of this material over Sepharose 6B revealed two insulin binding activities: a major peak which has a Stokes radius of 87 Å and a minor peak with a Stokes radius of 47 Å. Wheat germ agglutinin treatment produced an increase in the binding activity of both peaks, but their molecular weights did not change. In addition, 125I-labeled insulin was covalently attached to the adipocyte insulin binding sites by a cross-linking reagent. Column chromatography of the insulin-receptor complex again indicated the presence of two species with Stokes radii of 87 Å and 47 Å. Heterogeneity in these complexes was also demonstrated by polyacrylamide gel electrophoresis in the presence of Triton X-100. Ferguson plots indicated that the peak of radioactivity had a molecular radius of 60 Å, a size found both in the presence and absence of wheat germ treatment of adipocytes. These findings suggest that the increase in receptor affinity produced by wheat germ agglutinin treatment is not caused by the formation of receptor clusters. Instead, it appears that a simple interaction between the plant lectin and the receptor is sufficient to induce the changes in the insulin binding properties of adipocytes.

Tissue Differences in Insulin Receptors: Acute Changes in Insulin Binding Characteristics Induced by Wheat Germ Agglutinin

The plant lectin, wheat germ agglutinin (WGA), produced differential effects on insulin binding by insulin receptors from rat adipocytes, rat liver, human placenta, and human monocytes. Treatment of adipocyte membranes with WGA (1 μg/ml) markedly enhanced insulin binding (500% of control, 100%) conducted with 5 × 10-10 M 125I-insulin. The lectin's effect on insulin binding by liver membranes was significantly less (260% of control) and required a larger WGA concentration (5 μg/ml) to produce the stimulation. Insulin binding by placental membranes was increased only slightly (140%) by WGA, and monocytes failed to respond. The rate of insulin dissociation from the membrane preparations was decreased by lectin treatment. This effect was most prominent with adipocyte membranes, followed next by liver membranes, whereas only a slight inhibition was found with placental membranes. Scatchard analysis of the adipocyte receptor binding data indicated that WGA treatment completely linearizes the normally curvilinear plot to one high-affinity component (Kd of 0.22 nM) and decreases total insulin binding capacity by 60%. Similar effects were found with liver membranes, except that the extent of the changes was not as dramatic. WGA treatment did not linearize the Scatchard plot determined for placental membranes. Adipocyte and liver receptors solubilized by Triton X-100 responded to WGA, although the response was less in the former (350% above control, 100%) and greater in the latter (500%). Solubilization of placental receptors did not improve their response to the lectin. These results suggest differences in the structure of the insulin receptors from different tissues. Furthermore, the effects of WGA on adipocyte and liver receptors indicate the potential usefulness of this lectin in studies of acute changes in the the insulin binding properties of these insulin target tissues.

Effects of wheat germ agglutinin on insulin binding and insulin sensitivity of fat cells.

The plant lectin (wheat germ agglutinin, WGA) produces several alterations in the ability of fat cells to bind and respond to insulin. Although WGA markedly stimulated glucose oxidation, it caused only a modest stimulation of glucose transport. WGA (0.25-20 micrograms/ml) increased the binding of insulin by adipocytes, apparently by increasing the binding affinity of the insulin receptor. With low WGA concentrations (0.25-2.5 micrograms/ml), the elevation in binding was accompanied by an increase in the sensitivity of the adipocytes to insulin stimulation of glucose transport. However, the sensitivity of these cells to vitamin K5 and H2O2 was not altered. With higher WGA concentrations (5-20 micrograms/ml), stimulation of the glucose transport system by insulin, vitamin K5, or H2O2 was markedly inhibited, an effect that is reversed by the addition of ovomucoid. These findings suggest that low WGA concentrations increase the affinity of the insulin receptor and the insulin sensitivity of the cells. At higher concentrations, the lectin appears to act at another site(s) to inhibit the activation of the transport system by insulin or other agents.

Effects of wheat germ agglutinin on the coupled transports of sodium and potassium in reconstituted (Na,K)-ATPase liposomes.

Effects of wheat germ agglutinin on the coupled transports of sodium and potassium in reconstituted (Na,K)-ATPase liposomes.

The effect of wheat germ agglutinin on sialyl and galactosyltransferases of rat liver Golgi membranes.

The sialyltransferase and galactosyltransferase activities of the Golgi-rich fraction from rat liver were enhanced by the binding of wheat germ agglutinin (WGA). The sialytransferase was more sensitive than the galactosyltransferase to the WGA. Maximal stimulation of the galactosyltransferase activity resulted from the binding of 60--80 micrograms WGA to the Golgi membrane, while only 40 micrograms of WGA produced a maximal enhancement in the sialyltransferase activity. Within 5 min of WGA binding, the Golgi sialytransferase activity was doubled. After the initial binding of WGA to the Golgi fraction, the galactosyltransferase activity was decreased by 30%. However, in 15 min the activity was doubled by the binding of WGA. The activities of both enzymes were further enhanced by incubation for up to 90 min. The stimulation of both sialyltransferase and galactosyltransferase activities by WGA was reversed by N-acetyl-D-glucosamine (GlcNAc), the specific inhibitor of agglutination by WGA. Complete reversal of the enhanced activity was observed after 20--30 min in the presence of 1 micromol GlcNAc. The association constant for the binding of WGA to the Golgi membranes was calculated to be 4.16 X 10(-6) M from a Steck-Wallach plot. The 'n' value or mean binding sites was calculated as 5.26 X 10(-5) M/mg of Golgi membrane protein.

An ultrastructural study of the effects of wheat germ agglutinin (WGA) on cell cortex organization during the first cleavage of Xenopus laevis eggs. II. Cortical wound healing.

Uncleaved fertilized eggs of Xenopus laevis treated with wheat germ agglutinin (WGA) have been pricked at the animal pole both inside and outside the regressed furrow region. The wounded cortex of both regions has been studied with the electron microscope and compared with the same region of wounded, untreated eggs. In all 3 cases, filaments are organized in an annular zone in the damaged cortex. When the surface is pricked outside the regressed furrow of WGA-treated embryos, bundles of microfilaments radiate from the ring and extend in deep folds which form a 'star' around the wound at the surface of the embryo. However, when the surface is pricked in the new membrane of the regressed furrow, filaments are intermingled with internalized portions of the plasma membrane. It is suggested that, when the surface is pricked outside the furrow region, more filaments are mobilized to counteract the tangential retraction of the membrane which has acquired more rigidity after WGA binding.

Transmembrane effects of lectins: I. Effects of wheat germ agglutinin and soybean agglutinin on furrow formation and cortical wound healing in Xenopus laevis eggs

Studies on the binding of FITC-WGA and FITC-SBA to normal and cytochalasin (CB)-treated Xenopus eggs have demonstrated the presence of lectin receptors all over the egg surface; there are aggregates of receptors in the region where microvilli are numerous and microfilaments well organized. Addition of WGA or SBA to the incubation medium provokes inhibition of furrow formation provided that the lectins are added before the streak stage, or arrest of contraction when added during furrow formation. This arrest of contraction is followed by furrow regression. The lectins also affect wound healing. All these effects may be explained by an inhibition of membrane proteins movement and an alteration of the relations between the surface and the cytoskeleton.

Studies on growth inhibition by lectins of Penicillia and Aspergilli.

It has previously been shown in our laboratory that wheat germ agglutinin (WGA) binds to Trichoderma viride and inhibits growth of this fungus. Here we report on the effect of WGA, soybean agglutinin (SBA) and peanut agglutinin (PNA) on Penicillia and Aspergilli. Binding of the lectins to the fungi was examined with the aid of their fluorescein isothiocyanate (FITC) conjugated derivatives. FITC-WGA bound to young hyphal walls of all species, in particular to the hyphal tips and septa, in agreement with the chitinous composition of the cell walls of the two genera. Hyphae of all species examined were labelled, though in different patterns, by FITC-SBA and FITC-PNA, suggesting the presence of galactose residues on their surfaces. Young conidiophores, metulae (of the Penicillia), vesicles (of the Aspergilli), sterigmata and young spores, were also labelled. The three lectins inhibited incorporation of [3H]acetate, N-acetyl-D-[3h]glucosamine and D-[14C]galactose into young hyphae of Aspergillus ochraceus, indicating interference with fungal growth. Inhibition of spore germination by the three lectins was also observed. Preincubation of the lectins with their specific saccharide inhibitors prevented binding and the inhibitory effects. We conclude that lectins are useful tools for the study of fungal cell surfaces, and may also serve as an important aid in fungal classification. The present findings also support the suggestion that one role of lectins in plants is protection against fungal pathogens.

Effects of wheat germ agglutinin on membrane transport

1. (1) Low concentrations of wheat germ agglutinin are cytotoxic toward several tissue culture lines, including Chinese hamster ovary cells, Swiss 3T3 cells, mouse L cells and baby hamster kidney cells. The LD 50 ranged from 1 to 5 μg wheat germ agglutinin per ml. Similar concentrations of the lectin inhibited the transport of the non-utilizable amino acids α-aminoisobutyric acid and cyclo-leucine and inhibited the uptake of thymidine. In contrast, 2-deoxy- d-glucose uptake was not altered and colchicine uptake was enhanced. 2. (2) The inhibition of α-aminoisobutyric acid uptake occurred within minutes after lectin addition and was maximal by 1 h. Maximal inhibition ranged from 50 to 70% of control values. Studies of the kinetics of the uptake demonstrated that wheat germ agglutinin decreased the V of the uptake by 70% without affecting the apparent K m . Ovomucoid, a haptene inhibitor of wheat germ agglutinin-binding to cell surface receptors, prevented the wheat germ agglutinin-induced inhibition of α-aminoisobutyric acid transport. Three other lectins (Concanavalin A, Phaseols vulgaris E-phytohemagglutinin and l-phytohemagglutinin) inhibited the uptake by 20% or less at doses up to 50 μg/ml. 3. (3) We propose that the cytotoxicity of wheat germ agglutinin probably results in part, if not totally, from membrane alterations which impair multiple membrane transport systems

Isolation of wheat germ agglutinin-resistant clones of Chinese hamster ovary cells deficient in membrane sialic acid and galactose.

Several clones of Chinese hamster ovary cells have been selected for their resistance to the toxic effects of wheat germ agglutinin. The clones do not bind wheat germ agglutinin as well as parent cells and are 5- to 250-fold more resistant to the toxic effects of the lectin. Of three clones studied in detail, all exhibit a decrease in wheat germ agglutinin binding affinity. Two have normal numbers of wheat germ agglutinin binding sites, while one (Clone 13) has a 65% decrease in binding sites. Crude membrane preparations of the clones have a decrease in sialic acid content relative to parent cells, and Clone 13 membranes are also deficient in galactose, while the mannose and hexosamine contents of all three clones are normal. The membrane sugar deficiencies affect both glycoproteins and glycolipids. Sialyl-lactosylceramide is the major glycolipid in parent cells, while Clones 1 and 1021 have lactosylceramide and Clone 13 has glucosylceramide as the predominant glycolipid. Labeling experiments with N-[G-3H]acetylmannosamine suggest that Clone 1021 cells have a block in the transfer of sialic acid from CMP-sialic acid to glycoprotein and glycolipid acceptors. Yet CMP-sialic acid:glycoprotein sialyl-transferase activity in cell lysates of Clone 1021 cells is 80% of normal. While CMP-sialic acid:lactosylceramide sialyl-transferase activity is only 25% of normal, it can be restored to normal or elevated levels by sodium butyrate induction without an associated increase in cellular sialyl-lactosylceramide content. Similarly, the galactose-deficient Clone 13 can synthesize UDP-galactose and has normal levels of UDP-galactose:glycoprotein galactosyltransferase and UDP-galactose:glucosylceramide galactosyltransferase when assayed in vitro. The glycosyltransferases of both these clones can utilize their own glycoproteins as sugar acceptors in in vitro assays. These data suggest that the variant cells fail to carry out specific glycosyltransferase reactions in vivo despite the fact that they possess the appropriate nucleotide sugars, glycoprotein and glycolipid acceptors, and glycosyltransferases.

Effects of wheat germ agglutinin on fertilization of mouse ova in vivo and in vitro.

Mouse ova with or without follicular cells (denuded) were treated with wheat germ agglutinin (WGA) in concentrations ranging from 5-500 mug/ml and fertilized in vitro. Ova subjected to concentrations greater than 10 mug/ml WGA were incapable of fertilization in vitro, although sperm were observed adhering to the zona pellucida after treatment in concentrations as high as 500 mug/ml WGA. At concentrations of 50 mug/ml or higher, WGA adversely affected the shape of the vitellus. Of various saccharides employed to block the effects of WGA D-glucose was the most effective. Injections of 0.03 ml of 100 mug/ml WGA into the ovarian bursa two to three hours before the expected time of ovulation had little or no effect on fertilization in vivo.

Drosophila cell fusion induced by wheat germ agglutinin.

The effects of wheat germ agglutinin on Drosophila embryonic cell lines growing on cover-glasses was examined by scanning electron microscopy. At low concentrations of the lectin (5-10 mug/ml), cells spread against the glass surface and fused to form syncytia. At high concentration, damage to the cell surface was evidenced as extensive membrane shrivelling and loss of surface microfilaments. Fusion also occurred under these conditions. There was some indication that the morphology of cells in division remains undisturbed by wheat germ agglutinin. The coalescence of cells and morphologic disotrtion induced by wheat germ agglutinin were not inhibited by N-acetylglucosamine, the hapten inhibitor of the lectin, under the conditions utilized in this study.