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Poly (ADP-ribosylated) histones appear to be intermediates in nuclear processes that involve DNA strand breaks. We have studied histone ADP-ribosylation in cellular lysates from activated human lymphoid cells in culture. Modified histones differing in the number of ADP-ribose groups gave separate bands upon two-dimensional gel electrophoresis. Cellular lysates from control cells contained histones modified with 1 to 15 ADP-ribose groups. Stimulation of the cells during culture with phytohemagglutinin (PHA) or a phorbol ester (TPA) as well as combinations of these two reagents led to a significant increase in the upper limit number of ADP-ribose groups attached to histones in the presence of divalent metal ions. Hyper (ADP-ribosylated) H2B carrying at least 32 ADP-ribose groups gave a distinctly characteristic pattern on two-dimensional gels showing that highly ordered enzymatic steps are followed for its synthesis. Moreover, it was found that PHA and/or TPA induces branching of the poly (ADP-ribose) on H2B. The increase in histone poly (ADP-ribosylation) following lymphocyte activation was less dramatic during incubation of cellular lysates in the absence of divalent metal ions. The increased histone modification observed in this study may result from an increase in cell proliferation during activation of lymphoid cells. The finding that the number of ADP-ribose groups on H4 equals or exceeds by one the number of acetyl groups suggests that the two modifications may share common functions.

We describe the isolation of pig spleen lymphocyte glycoproteins that interact with phytohemagglutinin (PHA), the lectin from Phaseolus vulgaris. Purification was achieved by affinity chromatography of a Nonidet P-40 extract of the cells on a PHA--Affi-Gel 10 column. The retained glycoproteins were eluted with an acidic (pH 3.0) glycine buffer and represented 1.9-2.4% of the amount of protein applied to the column. They contained 20 +/- 1.3% hexose and 1.7 +/- 0.7% fatty acids, on a weight basis. Electrophoretic analyses (sodium dodecyl sulfate--polyacrylamide gel electrophoresis) showed the presence of major Coomassie blue positive bands with apparent molecular masses of 50-55, 75, 95, 130, and 155 kdaltons along with minor bands of 20-40, 42, 45, 60-65, 175, and 200-250 kdaltons. The purified PHA-receptor glycoproteins inhibited, in a dose-dependent manner, the incorporation of [3H]thymidine in pig lymphocytes cultured at a concentration of 10(6) cells/mL in the presence of PHA. A 50% inhibition was observed when 20 micrograms/mL of the glycoproteins was added to the lymphocyte cultures containing 0.5 microgram/mL of PHA. Scatchard analysis of the binding of 125I-labelled PHA, in the presence of increasing amounts of the purified glycoproteins, showed a suppression of the binding of the lectin to high affinity sites of the cells, as evidenced by a change from biphasic to a linear profile. Results of binding suggested a competitive inhibition by a population of purified glycoproteins with a similar affinity for the lectin. The purified glycoproteins decreased PHA-dependent interleukin 2 (IL-2) production by pig lymphocytes as assayed with a IL-2 dependent murine cell line.(ABSTRACT TRUNCATED AT 250 WORDS)

Phytohemagglutinin (PHA), concanavalin A (Con A), pea lectin, and wheat germ agglutinin (WGA) have been used to investigate their binding properties to Jurkat 77 6.8 leukemic human T cells and their ability to induce these cells to produce interleukin-2 (IL-2). Binding studies showed that the Jurkat cells fixed 0.82 +/- 0.11 microgram pea lectin, 2.02 +/- 0.17 micrograms Con A, 1.85 +/- 0.07 micrograms PHA and 8.88 +/- 0.61 micrograms WGA. Scatchard plots were linear, indicating that the binding process was homogeneous with respect to the binding constant. PHA and Con A bound with the highest affinity [Kass (apparent) approximately equal to 9 x 10(9) M-1], followed by pea lectin and WGA [Kass (apparent) approximately equal to 3 x 10(9) M-1]. The number of lectin binding sites was in agreement with the results of saturation experiments. We also evaluated the effect of the presence of 12-O-tetradecanoylphorbol-13-acetate (TPA) on the binding process. Results show that there were no gross alterations in the value of (apparent) Kass in the case of PHA and WGA. In contrast, the presence of TPA decreased the affinity of Con A and modified the Scatchard profile for pea lectin, which was curvilinear with a concavity turned upward. In this case, data were (apparent) K1 = 17.7 x 10(9) M-1 ("high-affinity" sites) and (apparent) K2 = 2.6 x 10(9) M-1 ("low-affinity" sites). The four lectins shared the ability to stimulate Jurkat 77 6.8 cells to secrete IL-2. Optimal lectin concentrations were 20 micrograms/ml (PHA) and 50 micrograms/ml (WGA and Con A). Pea lectin failed to display a dose-response relationship, and IL-2 production increased proportionally with lectin concentration. Con A was the most efficient stimulator (250 U/ml), followed by WGA (160 U/ml) and PHA (108 U/ml). Addition of TPA potentiated Jurkat cell response. In this case, Con A and pea lectin were the most efficient stimulators (470 U/ml), followed by PHA (316 U/ml) and WGA (271 U/ml). Here, also, pea lectin failed to show a dose-response relationship. Our data do not reveal an obvious relationship between the binding parameters and the ability of the lectins to induce IL-2 production. We suggest that the efficiency of the four lectins to stimulate IL-2 production in the case of the Jurkat 77 6.8 variant is most likely related to the nature of their specific cell surface receptor(s).

We have studied the binding of 125I-labeled phytohemagglutinin (PHA) to porcine splenic lymphocytes (10(7) cells . mL-1) at 37 degrees C. Our data show that PHA binding is dependent on the incubation period and that a maximum quantity of 4.53 +/- 0.13 micrograms is bound when 200 micrograms of PHA is added. The binding process is rapid and saturation can readily be achieved in less than 2 min. These observations suggest, in accordance with the mass-action principle, that the binding equilibrium can be rapidly displaced towards PHA-receptor complex formation when sufficient amounts of PHA are added. Our data further suggest that receptor sites are exposed at the cell surface and that putative cryptic receptor sites are unlikely to play a major role in the initial part of the binding process. We have studied this aspect by comparing Scatchard plots for PHA binding to receptors in intact lymphocytes and to lymphocyte-derived receptors incorporated into liposomes. In one case, partially purified plasma membranes were solubilized and incorporated into phosphatidylcholine-phosphatidylserine (PC-PS) vesicles prepared by detergent dialysis. In another case, PHA-receptor glycoproteins were purified by affinity chromatography and reassembled into PC-PS vesicles, using the same technique. Scatchard plot analyses showed nonlinear profiles with a concavity turned upward for PHA binding to receptors of intact cells or of PC-PS vesicles with plasma membranes, and a concavity turned downward for vesicles with purified glycoproteins. These observations suggest that PHA-receptor environment plays a determining role in the binding process of the lectin. Numerical data from binding experiments were obtained by computer-assisted nonlinear regression analysis, using a one ligand--two sites model. The (apparent) high-affinity constant (K1) for intact lymphocytes or partially purified plasma membrane components reassembled into liposomes was 4.6 X 10(7) M-1 and the (apparent) low-affinity constants (K2) were 4.4 X 10(6) +/- 1.5 X 10(6) M-1 (intact lymphocytes) and 1.7 X 10(6) +/- 0.6 X 10(6) M-1 (plasma membranes constituents reassembled into liposomes). The value obtained for reconstituted PHA-glycoprotein receptors (K) was 0.70 X 10(7) +/- 0.10 X 10(7) M-1. The apparent number of sites was n1 = 0.19 X 10(6) +/- 0.05 X 10(6) (high affinity) and n2 = 2.50 X 10(6) +/- 0.50 X 10(6) (low affinity) sites per intact lymphocyte cell.(ABSTRACT TRUNCATED AT 400 WORDS)