concanavalin-a and 2-aminoisobutyric-acid

In this study, we employed thiolated peptides of the conformationally constrained, strongly helicogenic α-aminoisobutyric acid (Aib) residue to prepare self-assembled monolayers (SAMs) on gold surfaces. Electrochemistry and infrared reflection absorption spectroscopy support the formation of very well packed Aib-peptide SAMs. The immobilized peptides retain their helical structure, and the resulting SAMs are stabilized by a network of intermolecular H bonds involving the NH groups adjacent to the Au surface. Binary SAMs containing a synthetically defined glycosylated mannose-functionalized Aib-peptide as the second component display similar features, thereby providing reproducible substrates suitable for the controlled display of bioactive carbohydrate ligands. The efficiency of such Aib-based SAMs as a biomolecular recognition platform was evidenced by examining the mannose-concanavalin A interaction via surface plasmon resonance biosensing.

Topics: Aminoisobutyric Acids; Concanavalin A; Electrochemical Techniques; Gold; Hydrogen Bonding; Immobilized Proteins; Mannose; Peptides; Protein Stability; Protein Structure, Secondary; Sulfhydryl Compounds; Surface Plasmon Resonance

In Madin-Darby canine kidney (MDCK) cells, specific plasma membrane binding of [125I]insulin was undetectable. Correspondingly, neither insulin-stimulated incorporation of [14C]glucose into glycogen nor insulin-induced uptake of radiolabeled alpha-aminoisobutyrate ([ 3H]AIB) could be demonstrated. These results suggested that MDCK cells lack specific cell surface insulin receptors. To further examine this question intact MDCK cells were preincubated with antireceptor serum and subsequently labeled with [125I]protein A; however, insulin receptors were not detected. Control H4 hepatoma cells bound insulin, responded with increased glycogen synthesis and amino acid uptake, and possessed immunologically recognizable insulin receptor components. The insulin-associated response of stimulated [3H]AIB uptake was induced in MDCK cells by the insulinomimetic lectins concanavalin A (130-140% of basal value at concentrations of 10-40 micrograms/ml) and wheat germ agglutinin (140-160% of basal value at concentrations of 10-30 micrograms/ml). This stimulation was abolished by the respective lectin-specific monosaccharides D-mannose and N-acetyl-D-glucosamine. Together, these data indicate that the insulin-like activity of concanavalin A and wheat germ agglutinin can be elicited in MDCK cells even in the apparent absence of specific plasma membrane insulin-binding sites.

Topics: Aminoisobutyric Acids; Animals; Cells, Cultured; Concanavalin A; Dogs; Glucose; Glycogen; Insulin; Kidney; Liver Neoplasms, Experimental; Protein Binding; Receptor, Insulin; Tumor Cells, Cultured; Wheat Germ Agglutinins

It was previously reported that the lectins wheat germ agglutinin (WGA) and concanavalin A (Con A) inhibit the mitogenic actions of multiple peptide growth factors in human fibroblasts without having a significant effect on mitogen binding. The current studies were designed to further examine the mechanisms of this antimitogenic action of lectins. Addition of WGA at progressively later times after stimulation of fibroblasts with peptide mitogens revealed significant inhibition of DNA synthesis even when the lectin was added 16-20 h after growth factors. This suggests an inhibitory effect on a pathway occurring late in G1, or close to the G1/S boundary. WGA also inhibited stimulation of DNA synthesis by non-peptide agents such as colchicine and vanadate ion, indicating that the lectin inhibits a common distal step in the mitogenic response, rather than acting primarily on events occurring at the level of the growth factor-receptor interaction. WGA had a rapid (within 30 min) inhibitory effect on insulin-stimulated amino acid uptake, but Con A, which like WGA blocked mitogen-stimulated 3H-dT incorporation, had little effect on stimulation of amino acid uptake. Thus the inhibition of DNA synthesis and amino acid uptake by lectins appear to be mediated by distinct mechanisms. WGA binding to fibroblasts persisted even when the lectin was removed from the incubation medium, but unlike 125I-EGF, which was rapidly internalized at 24 degrees C, little 125I-WGA was internalized. Incubation of fibroblasts for 20 h with WGA or Con A was not toxic to cells, since reversal of lectin binding by the appropriate saccharide allowed normal subsequent stimulation of DNA synthesis by EGF and insulin. However, the observation that cells exposed to antimitogenic lectins undergo a marked decrease in cell spreading suggests that changes in cell shape may be relevant to the mechanism by which lectin-treated fibroblasts become unresponsive to mitogenic stimulation.

Topics: Aminoisobutyric Acids; Cell Division; Cell Survival; Cells, Cultured; Concanavalin A; Epidermal Growth Factor; Fibroblasts; Humans; Lectins; Microscopy, Electron; Thymidine; Wheat Germ Agglutinins

The effect of plant lectins on amino acid uptake and DNA synthesis in cultured human skin fibroblasts stimulated by various peptide mitogens was studied. Wheat germ agglutinin (WGA), at a concentration of 5 micrograms/ml, which by itself had little effect on 3H-aminoisobutyric acid (AIB) uptake, markedly inhibited stimulation of 3H-AIB uptake by somatomedin-C, insulin, epidermal growth factor (EGF) and platelet-derived growth factor. This inhibition could not be overcome by increasing the concentration of peptide added. Neither WGA nor concanavalin A (Con A) significantly affected basal 3H-thymidine incorporation. However both lectins, at concentrations of 5-20 micrograms/ml, decreased EGF- and insulin-stimulated DNA synthesis while succinyl Con A, a divalent lectin derivative, did not. The inhibitory effects of lectins on mitogenic stimulation were reversed by alpha-methyl mannose (Con A) or N-acetylglucosamine (WGA), and were not due to a reduction in the binding of growth factors to their receptors. It is concluded that certain lectins noncompetitively inhibit the response of human fibroblasts to multiple peptide mitogens at the post-receptor level, possibly by interfering with lateral mobility and aggregation of mitogen-receptor complexes.

Topics: Acetylglucosamine; Adult; Aminoisobutyric Acids; Binding Sites; Concanavalin A; DNA Replication; Epidermal Growth Factor; Fibroblasts; Growth Substances; Humans; Insulin; Insulin-Like Growth Factor I; Lectins; Male; Platelet-Derived Growth Factor; Receptors, Cell Surface; Skin; Time Factors; Wheat Germ Agglutinins