Membrane mobility and the Concanavalin A binding system of the plasmalemma of higher plant protoplasts

The binding of a colloidal gold-Concanavalin A (ConA) complex to the plasmalemma of tobacco leaf protoplasts has been investigated using scanning electron microscopy. At 5° C the particles of gold-ConA appear to be randomly distributed over the surface of the protoplast. If the temperature is raised, the particles associate into clusters. Saturation of the membrane with particles can only occur when the weight of ConA in solution exceeds 1 g/10⁴ protoplasts in suspension, and when its concentration exceeds 15 g/ml. These results are discussed in terms of the properties of the ConA binding site and the mobility of such sites within the membrane surface.Abbreviations ConA Concanavalin A - AuConA Colloidal gold-Concanavalin A complex     

Ultrastructural evidence of maintenance of concanavalin A binding sites in enucleated mammalian cells

Mouse L cells were enucleated by centrifugation in cytochalasin B. Following enucleation, the enucleated cells were incubated in fresh medium for 30 min, 4, 20, or 24 h before being fixed for electron microscopy. After fixation the cells were incubated in concanavalin A and then horseradish peroxidase was bound to the ConA. Electron microscopy of these enucleates revealed that the concanavalin A-binding sites (CABS) are present on the cell surface of the enucleates even at 24 h after enucleation. Although the method does not detail the number of sites present, the inherent distribution of sites appears similar in normal and enucleated cells. Furthermore, the sites are still functional in that the live enucleated cells are agglutinated by ConA to the same extent as are normal L cells—about 80% agglutination in each instance. The results of this study indicate that surface CABS are maintained in the absence of a nucleus and they are still present even after the Golgi apparatus is morphologically disrupted. Turnover of these sites and their relationship to nuclear function are discussed.     

Metal ion binding to concanavalin A

Metal ion activation of saccharide binding has been studied for concana-valin A near pH 7.0. Although two metal ions, a transition metal ion and a Ca²⁺ ion, can bind, both are not required. Ca²⁺ alone, Mn²⁺ alone, or Ca²⁺ with other transition metal ions can activate this lectin. Only one Ca²⁺ ion per subunit or only one Mn²⁺ per subunit is sufficient. Metal ion binding was studied by magnetic resonance techniques and direct binding assays. Saccharide binding activity was monitored by following the fluorescence of 4-methylumbelliferyl a-D-mannopyranoside. When Ca²⁺ binds to demetalized concanavalin A, the transition metal ion site is hindered. When Mn²⁺ alone binds to demetalized concanavalin A, saccharide binding activity is induced. A subsequent conformational change, not necessary for carbohydrate binding activity, covers the Mn²⁺.     

Immobilization of plant protoplasts: Viability studies

Protoplasts of Daucus carota Ca68 and Catharanthus roseus have been immobilized by entrapment in gelforming polysaccharides (kappa-carrageenan, agarose and alginate). Uniform spherical beads of carrageenan and agarose containing the protoplasts have been prepared by utilizing an inert hydrophobic phase (vegetable oil). The entrapped protoplasts are viable and stabilized towards osmotic shock by the polymeric backbone. Standard methods have been used to study the viability and integrity of the entrapped protoplasts. Upon incubation in a relatively simple medium the immobilized protoplasts show a much higher viability after 14 days as compared to free protoplasts under the same conditions. The viability of D. carota protoplasts has also been monitored by an enzyme activity present in the cells (digitoxigenin 58-hydroxylase).     

Studies on haptoglobin binding to concanavalin A

Ascitic fluid haptoglobins 1-1, 2-1 and 2-2 and their tryptic glycopeptides were fractionated by affinity chromatography on Con A-Sepharose. Three peaks were obtained, corresponding to non-binding, weakly binding and strongly binding fractions. Concanavalin A-non-binding and concanavalin A-binding fractions of haptoglobin and of glycopeptide III 2-2 consisted of a series of polymers with increasing molecular mass, except for the non-binding fraction of glycopeptide III 1-1. After reduction there was no difference between the subunit composition of the glycopeptides and their concanavalin A fraction. Concanavalin A-non-binding fractions from haptoglobin 2-1 and glycopeptides III 1-1 and III 2-2 did not form an active complex with hemoglobin and, in crossed immunodiffusion, showed a reaction of partial identity with haptoglobin 2-1, glycopeptides III 1-1, III 2-2 and their concanavalin A-binding fractions. Concanavalin A-binding fractions of the above preparations exhibited with hemoglobin higher peroxidase activity than before their separation on Con A-Sepharose and immunodiffusion gave a reaction of identity among themselves and with unfractionated preparations. The concanavalin A-binding glycopeptide III is the biologically active part of the haptoglobin beta-chain.     

Photosynthesis of leaf cell protoplasts and permeability of the plasmalemma to some solutes

Photosynthesis was measured in mesophyll protoplasts isolated from spinach leaves. Under high intensity illumination and in the presence of 21% O₂, half-saturation of photosynthesis by CO₂ required CO₂ concentrations between 8 and 12 μm at different pH values of the suspending medium. Concentrations of HCO ₃ ^- needed for half-saturation increased correspondingly with the pH of the media. The pH profile of protoplast photosynthesis was much broader than that of CO₂ assimilation by isolated chloroplasts. The data indicate that leaf cells possess mechanisms to maintain considerable differences between external and internal pH over prolonged periods of time. Protoplast photosynthesis was inhibited by nitrite, acetate and bicarbonate; inhibition was more pronounced at low than at high pH and was attributed to stroma acidification. Nitrite was reduced in the light by protoplasts and chloroplasts. At pH 7.6, the apparent K_m NO ₂ ^- was about 0.6 mM for chloroplasts and 25 mM for protoplasts. Approximate permeability coefficients for NO ₂ ^- and HNO₂ were calculated from nitrite-dependent oxygen evolution at low nitrite concentrations, known nitrite or HNO₂ gradients, data on the surface area of protoplasts and chloroplasts and the pH profile of nitrite inhibition of photosynthesis. The membrane potential was assumed to be-100 mV. For the chloroplast envelope, permeability coefficients were 1.5·10^-3 ms^-1 (HNO₂) and 2·10^-8 ms^-1 (NO ₂ ^- ) and for the plasmalemma 4·10^-5 ms^-1 (HNO₂) and 5·10^-10 ms^-1 (NO ₂ ^- ). The values calculated for anion penetration probably represent upper limits of permeability. The protoplasts appeared to be largely impermeable to phosphate and phosphate esters. A rapid metabolic response of cells or cellular strands to added anionic substrates such as phosphate esters as reported in the literature appears to be possible only in damaged cells. It requires the presence of open channels between the cytosol and external medium.     

Relationship of prolactin receptors to concanavalin A binding

Concanavalin A, which binds to specific carbohydrate determinants on the cell surface, was used to investigate the binding of prolactin to its receptors in liver membranes from female rats. The binding of ¹²⁵I-labeled ovine prolactin to receptors was sharply inhibited by concanavalin A. This effect was reversed by the competitive sugar α-methyl-D-mannopyranoside and thus required the presence of specifically bound lectin. Concentrations of concanavalin A of up to 50 μg/ml caused a progressive decrease in the apparent affinity of the prolactin receptor for hormone. When higher concentrations were used, the number of available binding sites decreased. Concanavalin A-resistant receptors, about 30% of the total, had the same dissociation constant (K_d) as the controls. The binding of ¹²⁵I-labeled concanavalin A in the same membrane preparations showed the presence of two distinct types of concanavalin A binding. At low concentrations, the lectin bound with high affinity (K_d ≈ 6.6 · 10^?8 M). At high lectin concentrations, low affinity (K_d ≈ 6.7 · 10^?5 M) binding predominated. Since high affinity concanavalin A binding was saturated at 50 μg/ml, this class of binding most likely alters the affinity of the prolactin receptor for hormone; low affinity concanavalin A binding may mask prolactin receptors, making them inaccessible to the hormone.Binding sites for concanavalin A and prolactin appear to be independent but closely related since (i) concanavalin A did not displace bound prolactin from its receptor, and (ii) detergent-solubilized ¹²⁵I-labeled prolactin-receptor complexes bound to concanavalin A-Sepharose and were eluted by α-methyl-D-mannopyranoside.     

Calorimetric study of concanavalin A binding to saccharides

The binding of concanavalin A in the dimer form to various saccharides has been studied by calorimetry, and estimates of the binding enthalpy and binding constants have been calculated. Methyl α-d-mannoside and methyl α-d-glucoside have a — ΔH⁰ of 21.5 and 11.5 kJ/mol, respectively, at both pH 4 and 4.5. The p-nitrophenyl derivatives react with enthalpic values of 15.6 and 14.6 kJ/mol. The galactosepyranosides show no heat effects during mixing with the protein solutions.The apparent binding enthalpies calculated from the variations of the equilibrium constants with temperature are in good agreement with the values measured experimentally. The two binding sites of the dimer form of concanavalin A are equal and independent, and the low enthalpies obtained do not justify a large conformational change during the reaction.The binding reaction has also been estimated for other sugars normally contained in glycoproteins.     

Targeting of large liposomes with lectins increases their binding to plant protoplasts

Soybean agglutinin, peanut agglutinin, and concanavalin A were covalently bound by condensation reaction to gangliosides and ceramides incorporated within the bilayer of multilamellar and unilamellar liposomes. These modified liposomes had a much higher affinity for carrot and tobacco protoplasts except when concanavalin A was used.

In addition, soybean agglutinin and concanavalin A were attached by ligand-specific binding to liposomes containing cholesterol molecules derivatized with each lectin-specific sugar. This procedure allowed efficient crosslinking of liposomes to protoplasts. The same effect was achieved with soybean agglutinin and peanut agglutinin when derivatized cholesterol was replaced by gangliosides. The implications of these findings for the liposome-mediated nucleic acid transfer into protoplasts are discussed.

     

Ultrastructural visualization of sites binding concanavalin a on the cell membrane ofDaucus carota

Summary Protoplasts fromDaucus carota showed differences in binding to Concanavalin A according to which of three enzyme preparations tested were used for their isolation. Protoplast bound Concanavalin A was visualized ultrastructurally using a peroxidase-diaminobenzidine staining. The variable amount of staining was supposed to be due to differences in the composition of contaminating membrane active enzymes in the crude enzyme preparations. Enzymatically removed binding sites for Concanavalin A in the plasmalemma were rapidly resynthesized when the isolated protoplasts were incubated in a sorbitol solution. At room temperature, Concanavalin A bound to the plasmalemma was found in clusters, while at 4 °C and on prefixed protoplasts the binding sites were homogeneously distributed. These results and the effects of the crude enzyme preparations on the cell membrane of the protoplasts will be discussed in relation to the fluid membrane model.     

Kinetic analysis of calcium binding to concanavalin A

The kinetics of calcium binding to concanavalin A was studied utilizing ultraviolet difference spectral measurements. The results show that calcium binds to the lectin in a biphasic process: a rapid and reversible phase, followed by a relaxation phase with a k_obs of 0.012 sec^?1. Kinetic measurements were used to calculate the association constant, K_a, for calcium binding to concanavalin A of 2.7 x 10⁴ M^?1, in reasonable agreement with values obtained by equilibrium methods.     

Proton relaxation rate and fluorometric studies of manganese and rare earth binding to concanavalin A

The binding of Mn²⁺, Ca²⁺, and rare earth ions to apoconcanavalin A has been studied by water proton relaxation enhancement, electron paramagnetic resonance spectroscopy, and fluorescence spectroscopy. An electron paramagnetic resonance and water proton relaxation rate study of the titration of apoconcanavalin A with Mn²⁺ gives evidence of two equivalent binding sites per monomer with K_D = 50 μm ± 4 μm. When a similar Mn²⁺ titration of apoconcanavalin A is performed in the presence of Ca²⁺ ion, very little free Mn²⁺ is detected by electron paramagnetic resonance until the two Mn²⁺ binding sites per monomer are filled. The substitution of a rare earth ion for Ca²⁺ ion in the above experiment often resulted in a slight displacement of Mn²⁺ from the transition metal site as detected by electron paramagnetic resonance. A water proton relaxation rate study of the titration of apoconcanavalin A with Gd³⁺ reflects two binding sites with a K_D = 40 μm ± 4 μm and two with a K_D = 200 μm ± 50 μm. The fluorescence emission spectrum of concanavalin A (λ_em = 340 nm) is slightly quenched by the addition of Tb³⁺ while Tb³⁺ fluorescence is greatly enhanced. A fluorometric titration of apoconcanavalin A with Tb³⁺ also reflects two sites with a K_D = 40 μm ± 15 μm and two with a K_D = 270 μm ± 50 μm.     

ABA-induced 'lipid melting' and its reversal by umbelliferone in the plasmalemma of guard cell protoplasts: a breakthrough in plant hormone-receptor binding and hormone action.

The dynamics of stomatal opening and closure had to date been ascribed largely to the K(+)-fluxes and cell wall elasticity. Using protoplasts of guard cells of Vicia faba as model system, we document convincing first hand evidence a that lipid phase alterations could regulate ABA-induced closure of stomates and its reversal by umbelliferone. Backed up by the presence of plasmalemma-located ABA-receptor in guard cells, a novel theory could be put forth explaining guard cell opening and closure mediated by hormone induced reconfiguration via a probable lipid-protein lattice modification. The phase reversal of the plasmalemma by umbelliferone is postulated to be through modified hormone receptor complex structure, which is yet to be substantiated.