Effect of concanavalin A on membrane-bound enzymes from mouse lymphocytes.

The ionic influence and ouabain sensitivity of lymphocyte mg-2+-atpase and Mg-2+-(Na+ +K+)-activated ATPase were studied in intact cells, microsomal fraction and isolated plasma membranes. The active site of 5'-nucleotidase and Mg2+-ATPase seemed to be localized on the external side of the plasma membrane whereas the ATP binding site of (Na+ +K+)-ATPase was located inside the membrane. Concanavalin A induced an early stimulation of Mg2+-APTase and (Na+ +K+)-ATPase both on intact cells and purified plasma membranes. In contrast, 5'-nucleotidase activity was not affected by the mitogen. Although the thymocyte Mg2+-ATPase activity was 3-5 times lower than in spleen lymphocytes, it was much more stimulated in the former cells (about 40 versus 20%). (Na+ +K+)-ATPase activity was undectectable in thymocytes. However, in spleen lymphocytes (Na+ +K+)-ATPase activity can be detected and was 30% increased by concanavalin A. Several aspects of this enzymic stimulation had also characteristic features of blast transformation induced by concanavalin A, suggesting a possible role of these enzymes, especially Mg2+-ATPase, in lymphocyte stimulation.     

Effect of phorbol myristate acetate and concanavalin A on the glycolytic enzymes of human peripheral lymphocytes

The effects of Concanavalin A and the tumor promoting agent, phorbol 12-myristate 13-acetate (PMA), on glycolytic enzymes in human peripheral lymphocytes have been studied. A combination of Concanavalin A plus PMA stimulates DNA and protein synthesis to a significantly greater extent than when each are added individually. PMA and concanavalin A together, but not individually, also increase the levels of the activity of the glycolytic enzymes in peripheral lymphocytes treated for 48 h. The increase in hexokinase activity induced by PMA plus concanavalin A appeared to be due to the expression of the isoenzyme form, hexokinase II. The results suggest that the expression of glycolytic enzymes in stimulated lymphocytes is a late event (perhaps associated with the S phase) which is regulated by a cellular signal system controlled by the combined action of PMA plus concanavalin A.     

Effect of concanavalin A on the activity of membrane-bound and detergent-solubilized Mg2+ -ATPase

Plasma membranes were isolated after binding liver and hepatoma cells to polylysine-coated polyacrylamide beads, and the effect of concanavalin A on the membrane-bound Mg2+ -ATPase and the Mg2+ -ATPase solubilized by octaethylene glycol monododecyl ether (C12E8) was studied. In the experiment of membrane-bound Mg2+ -ATPase, plasma membranes were pretreated with Concanavalin A and the activity was assayed. Concanavalin A stimulated the activity of both liver and hepatoma enzymes assayed above 20 degrees C. Concanavalin A abolished the negative temperature dependency characteristic of liver plasma membrane Mg2+ -ATPase. On the other hand, Concanavalin A prevented the rapid inactivation due to storage at -20 degrees C, which was characteristic of hepatoma plasma membrane Mg2+ -ATPase. With solubilized Mg2+ -ATPase from liver plasma membranes, the negative temperature dependency was not observed. Concanavalin A, which was added to the assay medium, stimulated the activity of the enzyme solubilized in C12E8 at a high ionic strength. However, Concanavalin A failed to show any effect on the enzyme solubilized in C12E8 at a low ionic strength. With solubilized Mg2+ -ATPase from hepatoma plasma membranes, Concanavalin A could not prevent the inactivation of the enzyme during incubation at -20 degrees C.     

Effect of concanavalin a on ganglioside metabolism of human lymphocytes

The effect of Con-A on the incorporation of radioactivity from [¹⁴C]-glucosamine into gangliosides of human lymphocytes was investigated. Compared with non-stimulated lymphocytes there was increased incorporation into gangliosides and total lipids within the first 24 hours of exposure to Con-A. Ganglioside synthesis also occurred in later time intervals within the 96 hour incubation period. GM3 accounted for 80% of the labeled ganglioside in Con-A stimulated cells at all times studied. Thus ganglioside synthesis is not only associated with cellular division, but also occurs within a few hours of lymphocyte activation representing an extremely early prereplicative event.     

Developmental changes in membrane-bound enzymes of Dictyostelium discoideum detected by concanavalin A-Sepharose affinity chromatography.

Plasma membrane-enriched fractions isolated from $\text{Dictyostelium discoideum}$ at early stages of development were detergent extracted and subjected to affinity chromatography on a concanavalin A-Sepharose column. Alkaline phosphatase, 5′-nucleotidase, and cAMP phosphodiesterase activities were totally bound to the column when logarithmically growing cells were examined. As the cells entered development, however, a progressive decrease in the ability of these activities to bind to the affinity column was evident.     

Effect of concanavalin A on cellular slime mold development: Premature appearance of membrane-bound cyclic AMP phosphodiesterase

Concanavalin A delays aggregation of slime mold amoebae, apparently by interfering with the cells' response to the chemotactic agent, cyclic AMP. Concanavalin A also induces the premature appearance in non-aggregating cells of a membrane-bound cyclic AMP phosphodiesterase normally found only at the time of aggregation. The appearance of this enzyme is not due to activation of an inactive form of the enzyme.     

Change in acid phosphatase and N-acetyl-beta-glucosaminidase activity in mouse lymphocytes induced by concanavalin A

The treatment with concanavalin A (5 micrograms/ml) of mouse lymphocytes containing 70-72% of T cells entails an increase in the activity of acid phosphatase and a decrease in the activity of N-acetyl-beta-glucosaminidase. These changes were detectable 15 h after lymphocyte incubation with Con A. After 24 h of incubation acid phosphatase activity rose 2-fold whereas that of N-acetyl-beta-glucosaminidase dropped 45-50%. Possible mechanisms of these changes are discussed.     

Activity of deoxyribonucleoside-activated nucleotidase in cells from various lymphoid mouse tissues and in concanavalin A-stimulated lymphocytes

The specific activity in cells from lymph nodes, spleen and thymus was 32, 28 and 25 nmol/min per mg of cytosol protein, respectively, whereas that in bone marrow cells was significantly lower (10 units/mg of protein). No difference in specific DAN activity between isolated B- and T-lymphocytes was observed. Two types of lymphoid mouse cell lines (MOPC-31C plasmacytoma cells, S49 Cyc- lymphoma cells) showed specific activities similar to the normal lymphoid cells. In concanavalin A- stimulated spleen lymphocytes in culture there was a rapid increase in DAN activity shortly after maximum DNA synthesis, reaching a plateau 2-3 times the original level. The enzyme (DAN) of mouse tissues possessed the characteristic properties previously detected for the rat enzyme.     

Stimulation of the membrane-bound, magnesium-dependent adenosine triphosphatase of mouse neuroblastoma by concanavalin A and wheat germ agglutinin.

The effect of the plant lectins concanavalin A and wheat germ agglutinin on the membrane-bound Mg⁺²-dependent ATPase of an adrenergic clone of mouse neuroblastoma was examines. When cell membranes were treated with concanavalin A or wheat germ agglutinin, a dose-related increase in ATPase-specific activity was observed. Maximal stimulation was greater with wheat germ agglutinin than with concanavalin A; half-maximal and maximal stimulation occurred at similar lectin concentrations. Concanavalin A-dependent stimulation was blocked by α-methylmannoside but not by N-acetylglucosammine. Conversely, stimulation with wheat germ agglutinin was prevented by N-acetylglucosamine but not by α-methylmannoside. The combined effects of concanavalin A and wheat germ agglutinin were greater than the individual effects of either, but were not additive. The results suggest that these lectins interact specifically with membrane glycoproteins or glycolipids, resulting in enhancement of Mg⁺²-dependent ATPase activity.     

Isolation of a concanavalin A receptor from mouse L cells.

A cell surface glycoprotein receptor for concanavalin A (Con A) has been isolated from mouse L cells. The isolation procedure involved dissolving whole L cells in 0.3 M lithium diiodosalicylate and extracting with aqueous phenol. The Con A receptor, which was found in the aqueous phase of this extract, was further purified by affinity chromatography on a column of Con A-Sepharose; the receptor was adsorbed to Con A-Sepharose and eluted with 0.1 M methyl alpha-D-glucopyranoside or with 0.1 M methyl alpha-D-mannopyranoside, but not with other monosaccharides. The cell surface location of the Con A receptor purified in this way was confirmed by showing that it can be isolated from purified L cell plasma membranes and by demonstrating that it can be labeled from the exterior surface of intact L cells by the nonpenetrating galactose oxidase-KB3H4 system. Biochemical studies of the Con A receptor have shown that it migrates on sodium dodecyl sulfate-polyacrylamide gels as a single component having an apparent molecular weight of approximately 100,000. Its N-terminal amino acid is valine and it has carbohydrate attached at several (at least five) different sites along the polypeptide chain.     

Effect of concanavalin A and succinyl-concanavalin A on nucleoside and sugar uptake in mouse embryo fibroblasts

We have examined the effect of the tetrameric and dimeric form of Con A at a dose of 50μg ml^?1 on nucleoside and glucose uptake using synchronized mouse embryo fibroblasts undergoing S phase. We have found that thymidine and uridine uptake were depressed by Con A but not significantly by succinyl-Con A. The inhibition was gradual, as it required a suitable time of incubation to become fully manifest and it was of non-competitive type. By contrast the uptake of 2-deoxy glucose was inhibited promptly and to a similar extent by Con A regardless of molecular structure. Kinetic analysis of the modalities of the sugar uptake process indicated an inhibition of competitive type.     

Mitogenic stimulation and the redistribution of concanavalin A receptors on lymphocytes

When mouse spleen (Ig⁻) cells undergo maximal mitogenic stimulation by optimal concentrations of concanavalin A (conA), the Ig⁻ cells form caps of conA very slowly, with 50% of maximum cap formation occurring after about 10 h and maximal capping after about 24 h. Anti-conA antibody added after optimal conA accelerates the rate of cap formation and effectively blocks mitogenic stimulation (< 10%) by optimal conA concentrations when the rate of capping is increased more than about 2-fold. The effect of anti-conA antibody in accelerating cap formation by optimal conA is antagonized by cytochalasin D (CD), which substantially restores the mitogenic action of optimal conA. Thus there is an inverse relationship between rate of cap formation and extent of mitogenic stimulation. Further experiments showed that if anti-conA antibody, α-methyl mannoside or EGTA were added at increasing intervals after the addition of conA, these inhibitors block the stimulation of the cells with very similar time courses. Addition of appropriate concentrations of an inhibitor at the same time as optimal conA blocks mitogenic stimulation completely, but has negligible effects after 24 h. The extent of stimulation which occurs after the addition of inhibitor at intermediate times closely follows the extent of cap formation at the same time. The simplest interpretation of these results is that mitogenic action by optimal conA can be blocked by (i) accelerated capping of uncapped cells; or (ii) by the removal of either conA or calcium before, but not after, cap formation has occurred. These results suggest that the rate of cap formation by conA, and the presence of external calcium (>10⁻⁴ M) in the medium for some unspecified period before cap formation occurs are both significant factors in generating the primary mitogenic signals which commit the cells to DNA synthesis.     

Effect of concanavalin A on the sensitivity of mouse L cell surface glycoproteins to proteolysis

Binding of Concanavalin A to the outer membrane of mouse L cells is found to result in the protection of cell surface glycoproteins from proteolytic digestion by trypsin. Complete sensitivity to proteolysis, however, is restored after removal of bound Con A from the cells.     

Human monocyte activation by supernatants from concanavalin A (con A) stimulated lymphocytes

Human peripheral lymphocytes were stimulated with Concanavalin A (Con A) in the absence of serum. Supernatants were collected from control and mitogen stimulated lymphocyte cultures and fractions pooled according to the elution before, together with or after human serum albumin which was added as a marker. Only one fraction derived from Con A stimulated lymphocyte culture Supernatants which eluted immediately after human serum albumin had a significant effect on the metabolism and structure of human monocytes in vitro. Monocytes separated by human serum albumin and incubated with this fraction for 20 hr had an increase in nuclear RNA synthesis. Monocytes attached to cover slips in Leighton tubes showed an increase in the percentage of phagocytizing cells and phagocytic activity. Electron microscopy demonstrated highly phagocytic cells containing numerous Golgi associated granules and strands of nondilated rough surfaced endoplasmic reticulum in presence of the active fraction.