Concanavalin-mediated attachment to membranes of a cellular slime mould cyclic AMP phosphodiesterase.

In the cellular slime mould Dictyostelium, a membrane-bound cyclic AMP phosphodiesterase undergoes a tenfold increase in activity when amoebae reach the aggregation stage of development. Our previous studies had shown that when non-aggregating cells, which produce extracellular and intracellular forms of the enzyme, are treated with the lectin Concanavalin A (Con A), they exhibit prematurely high levels of the membrane bound enzyme. The present results indicate that this effect may be largely due not to the induction of the enzyme by Con A but rather to the binding of the intracellular form of the enzyme to membranes by Con A. This conclusion is based on the findings that: a) the enzyme activity associated with membranes from Con A treated cells can be decreased by treatment with the haptenic sugar alpha-methyl mannoside: b) mambranes from untreated cells having only low membrane-bound phosphodiesterase activity can acquire increased activity after incubation with Con A and intracellular phosphodiesterase; c) the intracellular phosphodiesterase binds to Sepharose-Con A and is eluted with alpha-methyl mannoside. These results raise the possibility that some of the effects attributed to Con A in the literature may not be due directly to Con A but to glycoproteins attached to membranes by Con A.     

A scanning electron microscope study of concanavalin a receptors on retinal rod cells labeled with latex microspheres

Con A-methacrylate microsphere conjugates prepared by a two-step glutaraldehyde reaction were used to label Con A-binding sites on bovine rod photoreceptor cells for visualization by scanning electron microscopy. A dense distribution of markers was observed on the surface of the rod outer segment, the inner segment, and the synaptic region. Disk membranes also appear to be heavily labeled with the Con A-microsphere conjugates. The Con A inhibitor, α-methyl mannoside, inhibited the binding of the conjugate to the surface of these visual cells.     

The regulation of lymphotoxin release from stimulated human lymphocyte cultures: the requirement for continual mitogen stimulation

Concanavalin A (Con A) activates nonimmune human lymphocytes in vitro to undergo transformation, DNA synthesis, and lymphotoxin (LT) secretion. LT secretion is inhibited (within minutes) when free and membrane-bound Con A are removed by washing or incubation with the competitive inhibitor, α-methyl mannoside. LT secretion can be reinitiated by addition of fresh Con A. While LT can be rapidly regulated, blast transformation and cellular DNA synthesis are under less restrictive control. Although they appear to be related, LT secretion and lymphocyte transformation seem to be regulated by independent control mechanisms. These studies indicate that recognition and contact of lymphocyte membrane sites initiate as well as regulate the efferent or destructive phase of cell-mediated immune (CMI) reactions. A model of how lymphocytes could employ LT in specific and nonspecific cytodestructive CMI reactions is presented.     

Role of haemagglutinins in adhesion of Erwinia carotovora to potato tissue

The adhesion of two strains each of Erwinia carotovora subsp. carotovora and Erwinia carotovora subsp. atroseptica to potato tuber discs, leaflets and tuber cell cultures was examined and found to occur independently of the presence of either mannose-sensitive (MSHA) or mannose-resistant (MRHA) haemagglutinins. Adhesion was generally greater when bacteria were grown in nutrient broth than on phosphate-buffered agar. The specific MSHA inhibitor α-methyl mannoside reduced the adhesion of two strains to tuber discs and leaflets and the specific MRHA inhibitor, asialofetuin inhibited strains only on leaflets. A reduction in adhesion of a MSHA-producing strain by α-methyl mannoside was observed by scanning electron microscopy which found that adhesion was localized at intercellular junctions.     

Concanavalin A-Sepharose Affinity Chromatography of Axon Plasma Membrane Proteins. Heterogeneity of Cholinergic Binding Sites

Abstract: Lysolecithin-solubilized proteins from axon plasma membranes of lobster walking leg nerve bundles were chromatographed on concanavalin A (Con A)-sepharose. Bound glycoproteins were eluted with α-methyl-D- mannoside. Near quantitative recovery of total protein was observed, 20–30% of the total protein being eluted in the Con A-binding glycoprotein fraction. A 5-fold enrichment of acetylcholinesterase (AChE) activity was achieved, demonstrating the glycoprotein nature of the axonal enzyme. The chromatographed fractions were characterized for binding of [³H]quinuclidinyl benzilate (QNB), [³nicotine (Nic), and [1251]α-bung arotoxin (BgTx) in an attempt to distinguish possible "muscarinic" and "nicotinic" binding sites in axonal membranes. All of the high-affinity "muscarinic" [³H]QNB binding activity appeared in the non-Con A-binding protein fractions, while binding of the two "nicotinic" ligands, [³Nic and ¹²⁵I-BgTx, was found in both the glycoprotein and non-Con A-binding protein fractions. BgTx interaction with the Con A-binding glycoproteins could be blocked with dtubocurarine, but BgTx binding in the non-Con A-binding proteins was not inhibited by curare. The significance of multiple cholinergic binding sites in axonal membranes is discussed. These data suggest a closer similarity between the cholinergic ligand binding proteins of peripheral nerve membrane and ganglia than between the axonal cholinergic binding sites and the ACh receptor of the neuromuscular junction.     

BINDING OF CONCANAVALIN A TO THE SURFACE OF STARFISH FOLLICLE CELLS AND PRODUCTION OF 1-METHYLADENINE

Starfish follicle cells, treated with concanavalin A (Con A), continued to produce 1-methyl-adenine (1-MeAde), an inducer of starfish oocyte maturation, after rinsing with artificial seawater (ASW). On the other hand, they ceased to produce the substance if treated with methyl α-Dmannoside (αMM). These cells produced again 1-MeAde when re-stimulated with Con A after removal of αMM. An optical study with fluorescein revealed that Con A bound to the cells was not dissociated by rinsing with ASW, but was removed if the cells were treated with αMM. These results suggest that continuous binding of Con A to the surface of the follicle cells is essential for the production of 1-MeAde.     

Early increase in lymphocyte cyclic nucleotide phosphodiesterase activity upon mitogenic activation of human peripheral blood mononuclear cells.

Cyclic nucleotide phosphodiesterase activity of human peripheral blood mononuclear cells was significantly increased following a short (30 min) incubation with the mitogenic lectin Concanavalin A. Con A stimulated phosphodiesterase activity to the same extent whatever the subcellular compartment (homogenate, cytosol or pellet). Further separation of the Con A-activated mononuclear cells into lymphocyte-enriched and monocyte-enriched populations showed that the Con A-induced increase of phosphodiesterase activity exclusively affected the lymphocyte-enriched population. In lymphocytes, cyclic GMP phosphodiesterase activity was more importantly enhanced by Con A (+275%) than cyclic AMP phosphodiesterase activity (+75%). The increase of both activities occurred as early as from 10 min of Con A incubation and proved to be maximal with Con A doses of 2.5 and 5 micrograms per 10(6) cells, lower and higher doses being less effective. Inhibition experiments with reference inhibitors suggested that, among the high affinity phosphodiesterase isoforms, the cyclic GMP-inhibited enzyme might be more selectively enhanced by Con A than the cyclic AMP-specific, Rolipram-sensitive one. The non-mitogenic lectin Helix pomatia hemagglutinin, was not able to enhance cyclic nucleotide phosphodiesterase activity of human mononuclear cells whereas anti-CD3 monoclonal antibody, although being less effective than Con A, exhibited a significant stimulatory effect. Putting together these results suggest that the early increase in phosphodiesterase activity might be a normal step involved in the mitogenic activation of human lymphocyte.     

Cell Pairing during Mating in Tetrahymena: I. Does Phagocytosis or a Cell Surface Receptor Participate in Con A Block?1

The lectin, Concanavalin A (Con A), inhibits cell pairing during mating in Tetrahymena and binds to the surface of pairing cells via receptors concentrated around the conjugation junction. Concanavalin A is also ingested in large amounts into food vacuoles. To dispel the possibility that Con A inhibits pairing via uptake into food vacuoles or through induction of food vacuole formation and to strengthen the idea that pairing is blocked through binding of Con A to cell surface receptors, we have conducted three types of experiments: 1) attempts to inhibit pairing by feeding with nutrients and with tantalum, a non-nutritive reagent; 2) a temporal analysis of the presence of food vacuoles in mating cells fed with tantalum; and 3) analysis of the restoration of pairing following the addition of α-methyl mannoside to cells previously treated with inhibitory concentrations of Con A. The results of these studies support the idea that Con A inhibits pairing by binding to receptors located on the cell surface and not by induction of or uptake into food vacuoles. We also present evidence that cells grown in an enriched proteose peptone medium are able to pair and undergo morphogenesis more readily than cells grown in 2% proteose peptone.     

Ultracytochemistry as a tool for the study of the cellular and subcellular localization of membrane-bound guanylate cyclase (GC) activity. Applicability to both receptor-activated and receptor-independent GC activity

Membrane-bound guanylate cyclase activity was detected by ultracytochemistry at the electron microscope level in several mammalian tissues. The technique used in these studies allows the detection of active enzyme at the membrane site where it is located. In a few cases, such as normal and regenerating peripheral nerves and placenta, membrane-bound guanylate cyclase could be detected in the absence of stimulators of enzyme activity. However, in the majority of these studies membrane-bound guanylate cyclase was investigated following stimulation with natriuretic peptides, guanylin, or the Ca²⁺ sensor proteins, S100B and S100A1. In general, membrane-bound guanylate cyclase was localized to plasma membranes, in accordance with the functional role of this enzyme. Yet, in secretory cells the enzyme activity was localized on intracellular membranes, suggesting a role of membrane-bound guanylate cyclase in secretory processes. Finally, S100B and S100A1 were found to colocalize with membrane-bound guanylate cyclase on photoreceptor disc membranes and to stimulate enzyme activity at these sites in dark-adapted retinas in a Ca²⁺-dependent manner. The results of these analyses are discussed in relation to the proposed functional role(s) of this enzyme.     

Characterization and quantitation of concanavalin a binding by plasma membrane enriched fractions from soybean root

The binding of concanavalin A (Con A) to soybean root membranes in plasma membrane enriched fractions (recovered from the 34/45% interface of simplified discontinuous sucrose density gradients) was studied using a radiochemical assay employing tritiated (³H)-Con A. The effect of lectin concentration, time, and membrane protein concentration on the specific binding of ³H-Con A by the membranes was evaluated. Kinetic analyses showed that Con A will react with membranes in that fraction in a characteristic and predictable manner. The parameters for an optimal and standard binding assay were established. Maximal binding occurred with Con A concentrations in the range of 8 to 16% of the total membrane protein with incubation times greater than 40 min at 22 C. Approximately 10¹⁵ molecules of ³H-Con A were bound per microgram of membrane protein at saturation. Binding was reversible. Greater than 92% of the total Con A bound at saturation was released by addition of α-methyl mannoside.     

Ultrastructural and biochemical identification of con A receptors in the desmosome

Correlated ultrastructural and biochemical methods were used to identify and localize Concanavalin A (Con A) receptors in the desmosomes of bovine epidermis. Specific carbohydrate residues were labeled with ferritin-Con A in thin sections of tissue embedded in a hydrophilic resin. Quantitative mapping of ferritin distribution in labeled desmosomes revealed that Con A receptors are localized in the intercellular zone and concentrated along the desmosomal midline or central dense stratum. Labeling was almost entirely absent when sections were treated with ferritin-Con A in the presence of 0.1 M α-methyl mannoside, a hapten-inhibitor of Con A. “Whole” desmosomes and desmosomal intercellular regions (desmosomal “cores”) were purified from bovine muzzle epidermis. Sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis reveals a limited number of major desmosomal protein constituents. Certain of these are glycoproteins and are greatly enriched in the core fraction. Almost all the desmosomal glycoproteins are intensely labeled when electrophoretic gels of whole desmosome or core fractions are exposed to fluorescent Concanavalin A.     

Stimulation of hamster adipocyte cyclic 3':5'-nucleotide phosphodiesterase activity by ionophore A23187 and calcium.

Incubation of hamster isolated fat cells with the ionophore A23187 and calcium for 20 minutes caused 30-40% increases in the cyclic 3':5'-nucleotide phosphodiesterase (EC 3.1.4.17) activity of adipocyte homogenates when either 0.6 micron cyclic AMP or 0.6 micron cyclic GMP was the enzyme substrate. The stimulation of adipocyte cyclic AMP phosphodiesterase activity by A23187 and calcium was not antagonized by the adrenergic receptor blocking agents phentolamine and propranolol. The changes in enzyme activity produced by the ionophore and calcium were not associated with elevated intracellular cyclic AMP levels. Furthermore, A23187 and calcium acted to enhance adipocyte phosphodiesterase activity before, but not after, homogenization of the fat cells. These data suggest that the phosphodiesterase activity of hamster isolated fat cells may, at least in part, be regulated by fluctuations in intracellular calcium concentrations.     

Purification of 5'-nucleotidase from human placenta after release from plasma membranes by phosphatidylinositol-specific phospholipase C

5'-Nucleotidase was purified greater than 1000-fold from human placenta by treatment of plasma membranes with S. aureus phosphatidylinositol-specific phospholipase C and affinity chromatography on Con A Sepharose and AMP-Sepharose. The resulting enzyme had a specific activity of greater than 5000 mumol/hr/mg protein and a subunit molecular weight of 73,000. Goat antibodies against 5'-nucleotidase inhibited enzyme activity and detected 5'-nucleotidase after Western blotting. These antibodies also recognized a soluble form of 5'-nucleotidase and residual membrane-bound 5'-nucleotidase which could not be released by phosphatidylinositol-specific phospholipase C treatment, suggesting that the three forms of the enzyme are structurally related. The soluble 5'-nucleotidase may be derived from the membrane-bound form by the action of an endogenous phospholipase C. The structural basis for the inability of some of the membrane-bound 5'-nucleotidase to be released by phosphatidylinositol-specific phospholipase C is unknown.     

Increase in plasma membrane phosphodiesterase activity in contact-inhibited 3T3 cells and in phenotypically reverted SV-3T3 cells

Summary Contact-inhibited 3T3 mouse fibroblast cells, in contrast to logarithmically growing 3T3 cells and SV-3T3 transformed cells, have increased levels of plasma membranebound phosphodiesterase (oligonucleotidase, E.C. 3.1.4.19; nucleotide pryrophosphatase, E.C. 3.6.1.9) activity. The increase in enzyme, recorded as increased specific activity, is reversible, as evidenced by the return to normal values following dilution of confluent 3T2 cells and re-initiation of growth. Increased enzyme activity is induced again when the cells regain the confluent state. Transformed SV-3T3 cells can be induced to mimic the contact inhibited state, including increased plasma membrane phosphodiesterase activity, by exposure to a combination of: (i) agents that are known to induce increased intracellular cAMP levels and (ii) additions of purified 3T3 or SV-3T3 plasma membranes. Additions of either alone fails to induce the increase in membrane phosphodiesterase activity, although each alone can significantly suppress cell growth, as measured by incorporation of³H amino acids.We suggest that the elevation of plasma membrane phosphodiesterase activity may serve as a measure of conversion to the contact-inhibited state in both normal cells and phenotypically reverted transformed cells.     

Biological and Biochemical Characterization of Macrophage Activating Factor (MAF) in Murine Lymphocytes: Role of Mannopyranosyl Residue of the MAF Molecule in Macrophage Activation

Activation of macrophages with macrophage activating factor (MAF) was evaluated by measuring the intracellular killing activity of murine macrophages against Salmonella typhimurium. Concanavalin A (Con A)-induced MAF-rich fraction was obtained by a Sephadex G-100 column, which contained molecules ranging from 25,000 to 67,000 daltons. The intracellular killing ability of mouse peritoneal macrophages against S. typhimurium was found to be increased by 0.1 m d-mannose as well as by Con A-induced MAF-rich fraction. Both 0.1 m d-mannose and MAF exhibited a similar timing pattern for macrophage activation. The same concentration of d-glucose or l-rhamnose did not change bacterial uptake and intracellular killing by macrophages. Moreover, when MAF-rich fraction was applied to a Con A-Sepharose column, a fraction that was adsorbed on Con A and eluted with 0.1 m α-methyl d-mannoside exhibited MAF activity. These results suggest the possibility that mannopyranosyl residues in the MAF molecules play an important role as a ligand in macrophage activation.     

Posttranslational Processing of Carboxypeptidase E, a Neuropeptide-Processing Enzyme, in AtT-20 Cells and Bovine Pituitary Secretory Granules

Abstract: Carboxypeptidase E (CPE) functions in the posttranslational processing of peptide hormones and neurotransmitters. Like other peptide processing enzymes, CPE is present in secretory granules in soluble and membrane-associated forms that arise from posttranslational processing of a single precursor, “proCPE.” To identify the intracellular site of proCPE processing, the biosynthesis and posttranslational processing were investigated in the mouse anterior pituitary-derived cell line, AtT-20. Following a 15-min pulse with [³⁵S]Met, both soluble and membrane-bound forms of CPE were identified, indicating that the posttranslational processing event that generates these forms of CPE occurs in the endoplasmic reticulum or early Golgi apparatus. The relative proportion of soluble and membrane-bound forms of CPE changed when cells were chased for 2 h at 37°C but was unaffected when cells were chased at either 20 or 15°C, suggesting that further processing of membrane forms to the soluble form occurs in a post-Golgi compartment. Treatment of the cells with chloroquine did not alter the relative distribution of soluble and membrane forms, suggesting that an acidic compartment is not required for this processing event. Overexpression of CPE did not influence the distribution of soluble and membrane forms of CPE, indicating that the CPE-processing enzymes are not rate-limiting. To examine directly CPE-processing enzymes, bovine anterior pituitary secretory vesicles were isolated. An enzyme activity that releases the membrane-bound form of CPE was detected in the purified secretory vesicle membranes. This enzyme, which removes the C-terminal region of CPE, is partially inhibited by EDTA and phenylmethylsulfonyl fluoride and is activated by CaCI₂. Together, the data indicate that posttranslational processing of CPE occurs in secretory granules and that this activity may be mediated by a prohormone convertase-like enzyme.     

Studies on adenosine 3′,5′-monophosphate phosphodiesterase of human erythrocyte membranes

In this work we show the existence of cyclic AMP phosphodiesterase (EC 3.1.4.17) in human erythrocyte membranes and have clarified some properties of the enzyme. In human erythrocytes, about 23% of the total cyclic AMP phosphodiesterase activity is in a membrane-bound form. Although it could be solubilized with Triton X-100 in 5 mM Tris-HCl buffer (pH 8.0), it was not solubilized by a low or high concentration of salt. The enzyme seems to be localized in the cytoplasmic surface, since it is detected in sealed inside-out vesicles of human erythrocyte membranes, but not in intact human erythrocytes. The optimum pH was found to lie between 7.4 and 8.0, and Mg²⁺ was found to be necessary for its activity. Ca²⁺ and calmodulin could not stimulate the activity of this enzyme. Theophylline was a strong inhibitor, but cyclic GMP could not inhibit the enzymic hydrolysis of cyclic [³²P]AMP and this membrane-bound enzyme therefore seems to be specific to cyclic AMP.     

Calcium alginate entrapped preparation of α-galactosidase: its stability and application in hydrolysis of soymilk galactooligosaccharides

Thermostable α-galactosidase from Aspergillus terreus _GR was insolubilized using concanavalin A obtained from jack bean extract and in order to maintain the integrity of complex in the presence of its substrate or products, this complex was crosslinked with glutaraldehyde. Soluble α-galactosidase entrapped in calcium alginate retained 82% of enzyme activity whereas, Con A-α-galactosidase complex entrapped in calcium alginate and crosslinked Con A-α-galactosidase complex entrapped calcium alginate retained 74 and 61% activity, respectively. A fluidized bed reactor was constructed for continuous hydrolysis of galactooligosaccharides in soymilk using crosslinked Con A-α-galactosidase complex entrapped calcium alginate. Optimum conditions such as pH (5.0) and temperature (65°C) were the same for all immobilized enzyme preparations and soluble enzyme. Crosslinked Con A-α-galactosidase entrapped complex exhibited enhanced thermostability and showed 62% of activity (38%) after 360 min at 65°C. Entrapped crosslinked Con A-α-galactosidase complex preparation was superior in the continuous hydrolysis of oligosaccharides in soymilk by batch processes compared to the other entrapped preparations. The entrapped crosslinked concanavalin A-α-galactosidase complex retained 95% activity after eight cycles of use.     

Inhibition of prostaglandin E2-stimulated cAMP accumulation by lipopolysaccharide in murine peritoneal macrophages.

Treatment of murine peritoneal macrophages with 100 nM prostaglandin E2 (PGE2) produced a rapid biphasic increase in intracellular cAMP that was maximal at 1 min and sustained through 20 min. Pretreatment of macrophages with 100 ng/ml of lipopolysaccharide (LPS) for 60 min prior to PGE2 decreased the magnitude of cAMP elevation by 50%, accelerated the decrease of cAMP to basal levels, and abolished the sustained phase of cAMP elevation. The effect of LPS was concentration-dependent, with maximal effect at 10 ng/ml in cells incubated in the presence of 5% fetal calf serum and at 1 microgram/ml in the absence of fetal calf serum. LPS also inhibited cAMP accumulation in cells treated with 100 microM forskolin, but the decrease was about half that seen in cells treated with PGE2. LPS concentrations that inhibited cAMP accumulation produced a 30% increase in soluble low Km cAMP phosphodiesterase activity while having no effect on particulate phosphodiesterase activity. The nonspecific phosphodiesterase inhibitor, 3-isobutyl-1-methylxanthine, as well as the more specific inhibitors rolipram and Ro-20-1724 were effective in inhibiting soluble phosphodiesterase activity in vitro, producing synergistic elevation of cAMP in PGE2-treated cells, and blocking the ability of LPS to inhibit accumulation of cAMP. Separation of the phosphodiesterase isoforms in the soluble fraction by DEAE chromatography indicated that LPS activated a low Km cAMP phosphodiesterase. The enzyme(s) present in this peak could be activated 6-fold by cGMP and were potently inhibited by low micromolar concentrations of Ro-20-1724 and rolipram. Using both membranes from LPS-treated cells and membranes incubated with LPS, no decrease in adenylylcyclase activity could be attributed to LPS. Although effects of LPS on the rate of synthesis of cAMP cannot be excluded, the present evidence is most consistent with a role for phosphodiesterase activation in the inhibitory effects of LPS on cAMP accumulation in murine peritoneal macrophages.     

Investigation of the subcellular distribution of cyclic-AMP phosphodiesterase in rat hepatocytes, using a rapid immunological procedure for the isolation of plasma membrane.

The distribution of cyclic-AMP phosphodiesterase was investigated in subcellular fractions prepared from homogenates of rat liver or isolated hepatocytes. When measured at 1 mM or 1 microM substrate concentration, approx. 35% or 50%, respectively, of enzyme activity was particulate. The soluble activity appeared to be predominantly a 'high Km' form, whereas the particulate activity had both 'high Km' and 'low Km' components. The recovery of cyclic-AMP phosphodiesterase was measured using 1 microM substrate concentraiton, in plasma membrane-containing fractions prepared either by centrifugation or by the use of specific immunoadsorbents. The recovery of phosphodiesterase was lower than that of marker enzymes for plasma membrane, and comparable with the recovery of markers for intracellular membranes. It was concluded that regulation of both 'high Km' and 'low Km' phosphodiesterase could potentially make a significant contribution to the control of cyclic AMP concentration, even at microM levels, in the liver. the 'low Km' enzyme, for which activation by hormones has been previously described, appears to be located predominantly in intracellular membranes in hepatocytes. The immunological procedure for membrane isolation allowed the rapid preparation of plasma membranes in high yield. Liver cells were incubated with rabbit anti-(rat erythrocyte) serum and homogenized. The antibody-coated membrane fragments were then extracted onto an immunoadsorbent consisting of sheep anti-(rabbit IgG) immunoglobulin covalently bound to aminocellulose. Plasma membrane was obtained in approx. 40% yield within 50 min of homogenizing cells.