Studies on Hemagglutinins from Maackia amurensis Seeds

Abstract

Abstract Two phytohemagglutinins (strongly hemagglutinating Maackia amurensis hemagglutinin and strongly mitogenic Maackia amurensis hemagglutinin (MAH and MAM)), which have mitogenic activity against human peripheral lymphocytes, have been purified from the seeds of Maackia amurensis by affinity chromatography on porcine thyroglobulin glycopeptides-Sepharose columns followed by SESephadex chromatography and gel filtration on Sepharose 6B. The preparations were homogeneous by ultracentrifugal analysis and disc electrophoresis, and had s20,w values of 7.1 S (MAH) and 7.4 S (MAM), respectively. The approximate molecular weight of 130,000 was estimated by gel filtration for both hemagglutinins. From chemical analyses these hemagglutinins were found to be glycoproteins and only small differences in chemical composition were found between them. However, polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate revealed that MAH is a tetramer of subunits having an approximate molecular weight of 33,000 and MAM is a dimer of disulfide-containing subunits of an approximate molecular weight of 75,000. In hemagglutinating activity, MAH is much stronger than MAM, whereas MAM is a more potent mitogen than MAH. Binding experiments with 125I-labeled hemagglutinins indicate that normal human erythrocytes bind approximately 3.2 x 106 molecules of MAH and 7.8 x 105 molecules of MAM with apparent association constants of 2.9 x 107 m-1 and 1.2 x 107 m-1, respectively, and, furthermore, normal human lymphocytes bind approximately 1.1 x 107 molecules of MAH and 2.4 x 107 molecules of MAM with apparent association constants of 2.1 x 107 m-1 and 1.1 x 107 m-1, respectively. Inhibition assays using various sugars and glycoproteins as hapten inhibitors revealed that these hemagglutinins from M. amurensis seeds differ from each other in their specificities for sugars. Porcine submaxillary mucin (PSM) having blood group H activity was a potent hapten inhibitor against the hemagglutinating activity of MAH. On the other hand, a glycopeptide having N-linked oligosaccharide chains from porcine thyroglobulin was found to be a potent inhibitor of MAM in hemagglutination and mitogenic triggering. Although sequential enzymic degradation of this glycopeptide resulted in a gradual loss of inhibitory activity, the residual core glycopeptide still acted as a potent inhibitor against MAM and even against the mitogenic activity of MAH.