concanavalin-a and Pheochromocytoma

It has been shown that in PC12 and its subclone PC12h treatment of the cells with nerve growth factor (NGF) induces a selective decrease in the incorporation of radioactive phosphate into a 100,000-dalton protein, designated in an earlier study as Nsp100, in the subsequent phosphorylation of soluble extracts from cells with (gamma-32P)ATP. In the present study, we show that plant lectins, wheat germ agglutinin (WGA), concanavalin A (Con A), and lens culinaris agglutinin (LCA), inhibit the action of NGF on Nsp100 phosphorylation in PC12h cells. Treatment of the cells with WGA, which binds to N-acetylglucosamine and sialic acid residues on glycoproteins, strongly blocked the inhibitory action of NGF on the protein phosphorylation. Con A and LCA, both of which recognize the same specific sugars (mannose, glucose), displayed only a moderate blocking effect. Unlike the native lectin, succinylated WGA, which has the ability to bind to N-acetylglucosamine but not to sialic acid residues, and other lectins examined in this study did not inhibit the action of NGF on Nsp100. WGA-mediated inhibition of NGF action was reversed by the addition of N-acetylglucosamine and by the addition of a much lower concentration of a sialoglycoprotein, mucin, into the culture. Since the binding of succinylated WGA to N-acetylglucosamine residues of cell-surface glycoconjugates is not sufficient to prevent the action of NGF, WGA might act on sialic acid residues of the NGF receptor molecule to effect the inhibition of biological actions of NGF.

Topics: Adrenal Gland Neoplasms; Animals; Concanavalin A; Lectins; Nerve Growth Factors; Pheochromocytoma; Phosphorylation; Plant Lectins; Protein Kinases; Rats; Tumor Cells, Cultured; Wheat Germ Agglutinins

A large-sized glucose polymer was isolated by pronase digestion from line PC12 pheochromocytoma cells metabolically labeled with [1-3H]galactose. The polymer was included on a column of concanavalin A-Sepharose and could be eluted with 10 mM methyl-alpha-mannoside. Its slight retention in a column of Bio-Gel A-5m suggested that its molecular weight was in the several millions. Glucose was the component monosaccharide and there were two minor lipophilic components present. The polymer was digested with alpha-amylase into a series of oligosaccharides and was cleaved by glucoamylase into glucose residues. The disaccharide obtained by digestion with alpha-amylase was identified as maltose in several HPLC systems and by NMR spectroscopy. NMR measurement revealed the trisaccharide to be maltotriose. Susceptibility of the polymer molecule to alpha-amylase, and the digestion products obtained, indicated a resemblance to glycogen. An analysis for saccharide compositions before and after reduction of the polymer suggested the presence of an aglycon part. Contrary to expectations based on the presence of this moiety, the polymer displayed good solubility in neutral organic solvents. Two-thirds of the glucose polymer was also soluble in 10% TCA. A similar glucose polymer was isolated from neuronal cells of rat embryos metabolically labeled with [1-3H]galactose. Mouse neuroblastoma cells did not synthesize the polymer.

Topics: alpha-Amylases; Animals; Cells, Cultured; Chromatography, Affinity; Chromatography, Gel; Concanavalin A; Galactose; Glucan 1,4-alpha-Glucosidase; Glucans; Maltose; Neurons; Pheochromocytoma; Rats; Tritium

Rat pheochromocytoma cells (clone PC12) display cell surface receptors for both nerve growth factor (NGF) and epidermal growth factor (EGF) and therefore provide a useful model system with which to study the role of these receptors in the regulation of proliferation and differentiation. In this paper PC12 cells are demonstrated to possess two classes of EGF receptors, a high-affinity class with 7,600 sites per cell and an apparent dissociation constant (Kd) of 0.05 nM, and a low-affinity class with 62,000 sites per cell and a Kd of 14.1 nM. These findings are contrary to literature data (Huff et al., 1981; Vale and Shooter, 1983) but can be explained in part by differences in experimental conditions. Binding studies at 37 degrees C compared with room temperature demonstrated similar affinities of both classes, but during prolonged incubation at 37 degrees C, the binding capacities of both classes decreased. Furthermore the high-affinity class was sensitive to lectins, such as concanavalin A (Con A), and to the tumor-promoting phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA). Both compounds caused a decrease of the affinity of the high-affinity class without affecting the low-affinity class. At high concentrations of Con A or TPA, a decrease of the apparent number of binding sites of the low-affinity class was also observed. The similarities between the characteristics of EGF binding and NGF binding in PC12 cells are striking and will be discussed.

Topics: Animals; Cell Membrane; Cells, Cultured; Concanavalin A; ErbB Receptors; Pheochromocytoma; Phorbols; Rats; Receptors, Cell Surface; Temperature; Tetradecanoylphorbol Acetate

1. Bovine adrenal medulla dopamine beta-hydroxylase, a glycoprotein with terminal mannose residues in carbohydrate moiety, did not precipitate with any lectins tested except concanavalin A. After digestion with neuraminidase, the enzyme was shown to interact with ricin, and a good correlation was found between the amount of liberated sialic acids and the extent of agglutination. This finding show either that more than one type of carbohydrate unit occurs on the protein or that three are multibranched chains in the carbohydrate moiety. 2. Dopamine beta-hydroxylase from human serum, pheochromocytoma and normal adrenal were incubated with concanavalin A, ricin, wheat germ agglutinin and lectins from Dilochos biflorus and Robinia pseudoacacia. 83% of dopamine beta-hydroxylase from pheochromocytoma was precipitated by ricin, whereas the enzyme from human serum precipitated to a lesser extent (5-15%). Neuraminidase digestion of human serum dopamine beta-hydroxylase led to an increase of precipitation with ricin. The low extent of native human serum dopamine beta-hydroxylase precipitation with ricin can be explained by the attack of plasma membrane sialidases of liver cells, whereas the greater ricin precipitation of pheochromocytoma and normal adrenal dopamine beta-hydroxylases could be due to post-mortem effects. The clinical implications of possibility of difference concerning the carbohydrate moiety structures of pheochromocytoma and normal enzymes is discussed.

Topics: Adrenal Medulla; Animals; Cattle; Concanavalin A; Dopamine beta-Hydroxylase; Humans; Lectins; Neuraminidase; Pheochromocytoma; Plant Lectins; Plants, Toxic; Protein Binding; Ricinus