Purification and biological activities of pokeweek (Phytolacca americana) mitogens.

Five mitogens, designated Pa-1 through Pa-5, were purified from the roots of pokeweed (Phytolacca americana) by means of ethanol fractionation, DEAE-cellulose column chromatography, affinity chromatography on a column of desialized human erythrocyte glycopeptide-Sepharose 4B, and gel filtration. Among these mitogens, only Pa-1 was mitogenic for both murine B-cells and T-cells, and the other mitogens were T-cell mitogens. Binding experiments with 125I-labeled Pa-1, a potent mitogen for B-cells, and with 125I-labeled Pa-2, the strongest T-cell mitogen, revealed that murine B-cells have more receptor sites for Pa-1 than for Pa-2 and murine T-cells have more receptor sites for Pa-2 than for Pa-1. The change of membrane fluidity within 30 min after binding of the mitogens to murine B- and T-cells was measured by fluorescence polarization of fluorescent hydrocarbon, 1,6-diphenyl-1,3,5-hexatriene, embedded in the membrane. Pa-1 induced increase of membrane fluidity of B-cells more markedly than Pa-2, whereas Pa-2 had a larger effect on the membrane fluidity of T-cells than Pa-1. Although both Pa-1 and Pa-2 stimulated the incorporation of 32 Pi into phosphatidylinositol of murine T-cells, neither Pa-1 nor bacterial lypopolysaccharide induced the activation of phospholipid metabolism of murine B-cells.