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.     

The promotion of collagen polymerization by lanthanide and calcium ions.

Ca2+ (1-5 mM) and lanthanide (20-250 microM) ions enhance the rate of polymerization of purified calf skin collagen (1.5 mg/ml) at pH 7.0 in the presence of 30mM-Tris/HCl and 0.2 M-NaCl. Both the nucleation phase and the growth phase of polymerization are accelerated. The activation energy of the growth phase, 239.3 +/- 24.3 kJ/mol (57.2 +/- 5.8 kcal/mol), is decreased to 145.6 +/- 9.6 kJ/mol (34.8 +/- 2.3 kcal/mol) by 5 mM-Ca2+ and to 75.3 +/- 4.6 kJ/mol (18.0 +/- 1.1 kcal/mol) by 25 microM-Sm3+. In contrast, the activation energy of the nucleation phase, 191.6 +/- 23.4 kJ/mol (45.8 +/- 5.6 kcal/mol), is only slightly decreased by Ca2+ or Sm3+. Collagen fibrils formed in the presence of Sm3+ are thinner than control fibrils, and more thermoresistant.     

The importance of membrane-bound ribosomes during the activation of thymocytes by concanavalin A.

Concanavalin A (ConA) seleclively enhanced the incorporation of [³H]leucine into a range of proteins of thymocytes incubated in vitro. At the same time ConA seemed to selectively enhance the synthesis of proteins that occurred on membrane-bound ribosomes (extracted from the mitochondrial fraclion with 1% Triton X-100 buffer). The protein synthetic ‘commitment’ of ribosomes was assessed from the stability of ribosomes in 500 mM KC1 before and after puromycin treatment. This indirect method was necessary because of some polysome degradation in the case of membrane-bound ribosomes. Membrane-bound ribosomes were found to be more than 3 times as ‘committed’ as were free ribosomes and ConA increased their commitment by 37–54%. These observations indicate the potential importance of membrane-bound ribosomes in the regulation of thymocyte protein synthesis, particularly during ‘antigenic’ activation, even though this ribosome fraction constituted less than 20% of the total ribosome population.     

Modulation of Epstein-Barr virus-induced B-cell activation by concanavalin A

Direct addition of the T-cell mitogen, concanavalin A (Con A), to cultures of Epstein-Barr virus (EBV)-stimulated peripheral blood mononuclear cells (PBMC) resulted in a dose-dependent inhibition of immunoglobulin M (IgM) secreted in the supernatant, as measured by an enzyme-linked immunosorbent assay. Furthermore, Con A inhibited IgM secretion of isolated T-depleted cells stimulated with EBV, and both the proliferation and IgM secretion of EBV-driven lymphoblastoid cell lines. T-Enriched cells, precultured for 48 hr with Con A, were also able to suppress the IgM response of fresh autologous PBMC stimulated with EBV. This suppression was radiation sensitive (2000 rad), a procedure which resulted in enhancement of the IgM secretion of the responder cells in two out of three experiments. Studies on the long-term effects of Con A showed that the early suppression of IgM secretion was transient and that the mitogen prevented the development of the cytotoxic T-cell response normally seen with lymphocytes from EBV-seropositive donors after 5 weeks of culture. Thus, Con A appears to modulate human lymphocyte responses to EBV by multiple mechanisms.     

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.     

Evidence for the non-filamentous aggregation of actin induced by lanthanide ions.

We varied the molar ratio of added lanthanide ion to skeletal muscle actin (M3+/A) and observed their effects on the change in reduced viscosity (Nred) in the presence of polymerizing quantities of salt (0.1 M KC1). Once the concentration of the lanthanide ion exceeds the concentration of the nucleotide present (0.2 mM ATP), we noted that with M3+/A ratios up to 4: (a) there was a sharp peak in the observed Nred above the level achieved by control F-actin; (b) the magnitude of (a) was shown to be a function of the initial G-actin concentration. With an M3+/A ratio of greater than 4 we observed: (i) a sharp fall in the observed Nred; (ii) the formation of an insoluble aggregate of actin; (iii) the formation of (ii) was completely reversed by removal of the M3+; (iv) a complete inhibition of the ATP hydrolysis which always accompanies the G- to F-actin transition; (v) the number of mol of M3+ required to completely inhibit the rise in Nred (above the viscosity of G-actin) was a function of the ionic radii of the 11 lanthanide ions tested; and (vi) the effects described in (i) were not mimicked when the initial protein was in the F form. In the absence of added KCI, divalent cations (e.g. Mg2+) polymerize G-actin but this effect is not mimicked by the addition of the lanthanide ions. However, under these conditions the lanthanide ions cause the formation of an insoluble aggregate of actin. We conclude that with greater than 4 mol of lanthanide ions, G-actin aggregates in a form which contains little or no F-actin and that the lanthanide ion-induced aggregates are therefore different from the Mg2+-induced F-actin paracrystals.     

Hydrodynamic changes accompanying the loss of metal ions from concanavalin A.

The hydrodynamic changes which accompany the dissociation of metal ions, from concanavalin A at acid pH are a result of charge effects rather than of dissociation of metal ions as such. Measurements of the rotational relaxation time are discussed in terms of the hydration of the protein and its polymeric heterogeneity.     

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 sex hormones on human T cell activation by concanavalin A

The effect of sex hormones on concanavalin A (Con A)-activated human T cells was studied. We show that neither 17 beta-estradiol (E2) nor progesterone, in concentrations of up to 10(-6) M, alters the proliferative response of peripheral-blood mononuclear cells (PBMC) of healthy postmenopausal women. Furthermore, the hormones had no effect on the composition of T cell populations and on the expression of activation markers. We extended our study to a unique T cell population that is characterized by the ability to form rosettes with human erythrocytes, following Con A activation (designated autorosette-forming cells; ARFC) and known to manifest suppressive activity. Indeed, the in vitro addition of E2 (neither progesterone nor testosterone) to Con A-stimulated PBMC brought an about 2- to 4-fold increase in the frequency of ARFC. Tamoxifen, an antiestrogen drug, reduced the frequency of estrogen-stimulated ARFC to the original low level. Furthermore, the inhibitory effect of growth medium from ARFC cultures originally stimulated with Con A + E2 was found to be higher than that of ARFC cultures originally stimulated with Con A alone.