Preparation and Purification of Tritiated Phytohemagglutinin and Studies of Cellular Localization in Human Leukocyte Cultures

Abstract

Abstract A tritiated bean extract was obtained from bean plants (Phaseolus vulgaris) grown hydroponically in nutrient media containing various concentrations of tritium water. Purification was accomplished by ammonium sulfate precipitation and column chromatography using DEAE, CM-52 cellulose columns, and finally by Sephadex G-100 gel filtration. The purified product showed greatly increased mitogenic activity and electrophoretically showed a single labeled protein band on electrophoresis. Radioactivity of the purified PHA varied between 0.1-0.5 µCi/mg. which, though not as high as desirable, was sufficient for autoradiographic and subcellular fractionation studies in human leukocyte cultures. Within a few hours cytoplasmic localization of the label was noted in most of the cells in culture, including neutrophils and larger mononuclear cells. Breakdown of many of these cells during the first 24 hr. resulted in labeled amorphous basophilic staining masses of cell particles. Though agglutination of cells was commonly seen, the lack of label in red cells or surface label of leukocytes was notable. Blast forms which appeared by the second day showed prominent labeling which appeared to be largely cytoplasmic as evidenced by grain distribution. Several reasons were discussed which made it seem likely that the mitogenic molecule was represented in the cell label. Assay of subcellular fractions showed the major portion of radioactivity in the mitochondrial fraction. Specific activity, however, was too low to permit electron microscopic localization of label in individual organelles. Lack of label of RNA and DNA proteins was demonstrated by cellular distribution of the label and chemical extraction of RNA and DNA. Interesting speculation arises as to whether the PHA may stimulate mitochondrial activity and induce RNA synthesis and blastogenesis. Further experiments are in progress to obtain a purer PHA with higher specific radioactivity in order to more precisely define the site and mechanism of action of the mitogen.