Physical-chemical studies on the role of the metal ions in concanavalin A.

Substitution of Co²⁺ for Mn²⁺ in concanavalin A generates characteristic circular dichroism and magnetic circular dichroism spectra which are strongly affected by the concentration of Ca²⁺. With three equivalents of Ca²⁺ per protomer of [(Co²⁺)Con A], no spectral effects of addition of α-methyl-d-glucopyranoside can be demonstrated. With one equivalent of Ca²⁺, however, α-methyl-d-glucopyranoside alters the circular dichroism and magnetic circular dichroism spectra in a manner identical to that produced by adding further equivalents of Ca²⁺. Under these same conditions the higher molecular weight carbohydrates, trehalose and melezitose, cause no spectral alterations in the regions investigated.The magnetic circular dichroism spectrum of [(Co²⁺)Con A] is characterized by a negative peak centered at 510 nm (θ/gauss = ?0.28 °) and a pronounced shoulder at 462 nm (θ/gauss = ? 0.16 °). Comparison of this spectrum to that of Co(H₂O)₆²⁺ indicates that the transition metal ion exhibits octahedral geometry in solution and maintains this geometry in its interaction with carbohydrate moieties.Circular dichroism experiments in the far ultraviolet region indicate a change in secondary (presumed β) structure upon interaction of Apo Con A with Mn²⁺ consistent with a more ordered arrangement. Unlike Mn²⁺, cobalt alone will not induce these secondary changes until Ca²⁺ is added. Kinetic analysis, using a mannan light scattering assay, indicates that [(Mn²⁺)Con A] and [(Co²⁺)Con A] will slowly recover cross-linking function in the absence of Ca²⁺, suggesting that the role of the metal in S₂ is to accelerate a conformational change leading to binding or effector function.Overall, the data are consistent with a suggestion by Cuatrecasas (1973) that α-methyl-d-glucopyranoside binds to a locus different from the membrane binding (or agglutination) site. Nevertheless, there are strong conformational interactions between these two sites, since α-methyl-d-glucopyranoside will elute Con A from membrane surfaces.     

Effect of divalent metal ions on the digestibility of concanavalin A by endopeptidases.

Demetallized concanavalin A is degraded rapidly at pH 7.0 and 8.2 by alpha-chymotrypsin, thermolysin or trypsin, yielding peptide fragments devoid of ability to bind to Sephadex G-75. Addition of Ni2+ and of Ca2+ confers on concanavalin A high resistance towards proteolytic attack so that even after long periods of exposure to the enzymes, almost all of the saccharide-binding capacity is preserved. Ni2+ alone protects strongly at pH 7.0 but not at pH 8.2. Apparently, both the transition metal ion and Ca2+ play an important role in stabilizing the native conformation of the protein molecule. Digestion of demetallized concanavalin A with alpha-chymotrypsin or thermolysin readily yields small peptide fragments (Mr less than 10 000), while trypsin yields as the major product(s) larger peptide(s) (Mr approximately 20 000) of appreciable resistance to further fragmentation.     

Stabilization of concanavalin A by metal ligands.

Metal-free concanavalin A is readily and irreversibly inactivated by temperatures above 60 degrees. Manganese ion completely prevents the thermal aggregation of the protein at 60 and 70 degrees, and partially protects at 80 degrees, but shows no protective properties at 90 degrees. Managanese protection against thrermal aggregation was found to be maximal at pH 4-8. The precipitation between glycogen and Mn2+-stabilized conanavian A is partially inhibited at temperatures greater than 30 degrees, but can be reversed by cooling to room temperature...     

The activation of concanavalin A by lanthanide ions.

Divalent cadmium and lead and the trivalent lanthanides bind in the trasition metal site (S1) of concamavanlin A and induce saccharide binding to the protein in the presence of calcium. Partial activation of the protein in the presence of lanthanides alone indicates these ions bind into both transition metal (S1) and calcium sites (S2). The activity of a lanthanide-protein derivative may be increased by the addition of either calcium or a transition metal ion. The saccharide binding activity decreases in the order Zn2+ is greater than Ni2+ is greater than Co2+ is greater than Mn2+ is greater than Cd2+ reflecting the order of binding constants for these ions in the transition metal site. Like the lanthanides, divalent cadmium substitutes for both the transition metal ion and calcium ion to partially activate the protein. Divalent lead substitutes only for the transition metal ion and partially activates the protein upon addingcalcium. The data are consistent with a model in which saccharide binding activity is independent of the metal size in S1 but critically dependent upon metal size in S2.     

Effect of metal ions and EGTA on the optical properties of concanavalin A at alkaline pH

In our earlier communications, we reported the effect of salts and alcohols on alpha-chymotrypsinogen [1] and the existence of stable intermediates at low pH in bromelain [2] and glucose oxidase [3]. In the present study, the role of metal ions and EGTA on the conformation of concanavalin A at alkaline pH was studied by near- and far-UV circular dichroism, fluorescence emission spectroscopy and binding of a hydrophobic dye, 1-anilino-8-naphthalene sulfonate (ANS). Far-UV CD spectra showed the transition from an ordered secondary structure at pH 7 with a trough at 223 nm to a relatively unordered state at pH 12. Near-UV CD spectra showed the loss of signal at 290 nm, thereby indicating the disruption of native three dimensional structure. Maximum ANS binding occurred at pH 12 suggesting the presence of an intermediate or molten globule-like state at alkaline pH.     

Ocular changes accompanying eye loss in the spider family uloboridae

In uloborid spiders, eye loss is accompanied by increased visual angles, optical material investment, and potential visual acuity of the retained eyes. Relative to carapace volume, the six-eyed Hyptiotes cavatus and two four-eyed Miagrammopes species have greater retinal hemisphere areas and lens volumes than do the eight-eyed uloborids Waitkera waitkerensis, Uloborus glomosus, and Octonoba sinensis. In Waitkera, in which the eyes have little visual overlap, and in Miagrammopes, in which eye loss simplifies the spiders' patterns of visual overlap, increased retinal cell density enhances potential visual acuity. However, this occurs at the expense of potential retinal cell sensitivity.     

Activation of lymphocytes by concanavalin A requires calcium ions.

Mouse spleen cells were exposed to a short pulse of the mitogenic lectin concanavalin A (con A). After removal of con A mitogenesis was measured by the incorporation of tritiated thymidine into DNA. It was found: (a) the number of cells responding to con A was proportional to the time of exposure to con A; (b) exposure of cells to con A in the absence of extracellular calcium failed to initiate mitogenesis; (c) for a mitogenic effect an extracellular calcium concentration greater than 10(-5)M was required during the time that the cells were exposed to con A.     

Interaction of metal ions with polynucleotides and related compounds. XXII. Effect of divalent metal ions on the conformational changes of polyribonucleotides

Changes in the conformation of poly(G), poly(C), poly(U), and poly(I) in the presence of divalent metal ions Mg²⁺, Ca²⁺, Mn²⁺, Co²⁺, Ni²⁺, Cu²⁺, Cd²⁺, and Zn²⁺ have been measured by means of ORD and u.v. spectra. Mg²⁺ and Ca²⁺ ions stabilize helical structures of all the polynucleotides very effectively at concentrations several orders of magnitude lower than the effective concentration of Na⁺ion. Cu²⁺ and Cd²⁺ destabilize the helical structure of polynucleotides to form random coils. Zn²⁺, Ni²⁺, Co²⁺, and Mn²⁺ions do not behave in such a clear-cut manner: they selectively stabilize some ordered structures, while destabilizing others, depending on the ligand strength of the nucleotide base as well as the preferred conformation of that polynucleotide.     

The role of metal ions in substrate recognition and stability of concanavalin A: a molecular dynamics study

The binding of carbohydrate substrates to concanavalin A (Canavalia ensiformis agglutinin (ConA)) is essential for its interaction with various glycoproteins. Even though metal ions are known to control the sugar binding ability of legume lectins, the interplay between sugar and metal ion binding to ConA has not been elucidated in a detailed manner at the atomic level. We have carried out long, explicit solvent molecular dynamics simulations for tetrameric, dimeric, and monomeric forms of ConA in both the presence and absence of trimannoside and metal ions. Detailed analyses of these trajectories for various oligomeric forms under different environmental conditions have revealed dynamic conformational changes associated with the demetalization of ConA. We found that demetalization of ConA leads to large conformational changes in the ion binding loop, with some of the loop residues moving as far as 17 Å with respect to their positions in the native trimannoside and metal ion-bound crystal structure. However, the β-sheet core of the protein remains relatively unperturbed. In addition, the high mobility of the ion binding loop results in drifting of the substrates in the absence of bound metal ions. These simulations provide a theoretical rationale for previous experimental observations regarding the abolition of the sugar binding ability upon demetalization. We also found that the amino acid stretches of ConA, having high B-factor values in the crystal structure, show relatively greater mobility in the simulations. The overall agreement of the results of our simulations with various experimental studies suggests that the force field parameters and length of simulations used in our study are adequate to mimic the dynamic structural changes in the ConA protein.     

Structural changes accompanying the removal of pyridoxal 5'-phosphate from phosphorylase b.

The changes in physical properties accompanying the removal of pyridoxal 5'-phosphate from glycogen phosphorylase b have been examined. The apoenzyme retains a high degree of structural rigidity, as determined from the time decay of anisotropy. The bulk of the secondary structure remains intact, although a significant change in circular dichroism indicates some degree of alteration. The mobility of a sulfhydryl-linked spin label increases. The restoration of pyridoxal 5'-phosphate reverses this effect, with indication of interaction between subunits. One or more new binding sites for 1-anilinonaphthalene-8-sulfonate appear for the apoenzyme. The kinetics of the recombination of pyridoxal 5'-phosphate with the apoenzyme, as monitored by difference spectra, indicate a high activation energy for the process. The apoenzyme is a reversibly associating system at 20-30 degrees C, pH 7.0.     

Acid proteases from species of Mucor. III. Interaction with concanavalin A and concanavalin A Sepharose.

The reaction of Mucor miehei protease with concanavalin A was followed by a turbidimetric assay in the pH range 5-8. At pH 4.0, no turbidity developed but binding of the enzyme to concanavalin A could be demonstrated by gel filtration. Two fractions of apparent molecular weight 65000 and 52000 were isolated, the 65000 molecular weight species apparently representing a protomer of concanavalin A (24000) bound to the enzyme. An analysis of the circular dichroism spectrum of this complex suggested that protomer binding results in a conformational change in the enzyme which is associated with a 30% increase in proteolytic activity. At pH 6.0, the enzyme was strongly bound to columns of concanavalin A Sepharose but could be removed by including alpha-methyl D-glucoside and NaC1 in the elution buffer. Some column degradation occurred at room temperature but was not detectable at 4 degrees C where rapid elution of the enzyme resulted in a greater than 90% yield of highly active protein. Periodate-oxidized Mucor miehei protease and Mucor renin did not react with concanavalin A and were not bound to the affinity column.     

Metal ion induced conformational changes in concanavalin A: evidence for saccharide binding to one metal free structure.

The addition of Mn²⁺, Zn²⁺, Co²⁺, Ca²⁺ or Pb²⁺ to apo-concanavalin A results in a slow conformational conversion of the protein to the active saccharide binding form. The rates of conversion are dependent upon the sample pH and identity of the ions which occupy the native transition metal and calcium ion sites yet the affinity of each metalloform for the fluorescent sugar, 4-methylumbelliferyl-α-D-mannopyranoside, is independent of these same parameters (above pH 5.6). EDTA quickly removes all metal ions from the active Mn²⁺ or Co²⁺-concanavalin A samples leaving a metastable metal free structure which retains its high saccharide affinity for several hours at room temperature. This form of apo-concanavalin A and the metallized derivatives have equally high saccharide binding affinities in 1M NaCL but the former dramatically loses its sugar affinity as the ionic strength is lowered.     

Developmental changes of proteins and concanavalin A reactive glycoproteins in growth cones from rat forebrain.

Growth cones were isolated from the forebrains of 1, 5 and 9 days-old rats. The ultrastructural characterization of the obtained subcellular fractions reveals that two of them (GC1 and GC2) contain predominantly growth cones. It was found that the protein content of the membranes contained in these fractions increases 7.5 times, while in whole forebrain the increase is only 3 times, showing that during the studied developmental period there is a predominant protein enrichment of the specialized brain structures (e.g. growth cones). Electrophoretic studies show that there are characteristic changes of the Coomassie Brilliant Blue R250 staining and concanavalin A reactive protein profiles. Comparison of the protein patterns of growth cones to those of synaptosomes from mature forebrain reveal a number of bands, which appear to be characteristic for one of these structures. The possible roles of the developmentally controlled proteins in the processes of synaptogenesis is discussed.