Isolation and characterization of a mitogen from pokeweek (Phytolacca americana). (1967)

Previous studies'-3 in this laboratory have shown that a lectin derived from the plant Phytolacca americana (pokeweed) possesses three distinct biological activities: hemagglutination, leukagglutination, and mitogenicity, i.e., the capacity to trans- form resting peripheral blood lymphocytes into "blast-like" cells in vitro.Lym- phocyte transformation by the pokeweed mitogen (PWM) involved distinctive biochemical, histochemical, and fine structural changes, distinguishable from those reported for phytohemagglutinin (PHA).1-3A recent report4 on the natural history of pokeweed poisoning attests to the analogous action of this mitogen in vivo; ingestion of this mitogen resulted in plasmacytosis.The specific role and interrelationships of the cell types designated as lympho- cytes, plasma cells, and precursor "blasts" in immunological states have been the subject of intensive investigation and controversy in recent years.5' 6The findings of distinctive alterations in immunocompetent cells produced by PWM in vivo and in vitro has given special impetus to its further purification and characterization.This communication reports the isolation of PWM in homogeneous form and the characterization of its physical and chemical properties.Methods.-Preparation of pokeweed mitogen (PWM): Roots from the plant Phytolacca americana, growing in the vicinity of Bethesda, Maryland, were harvested in late summer, and PWM was isolated from this source as described previously.'The fractionation procedure consisted essentially of saline extraction of the roots, heat coagulation of the extract, centrifugation following trichloracetic acid (TCA) precipitation of the resultant supernatant, and chromatography of the dissolved and dialyzed TCA-precipitate on a calcium phosphate (hydroxylapatite) column.Multiphase zone electrophoresis on polyacrylamide gel: Preparative gel electrophoresis was performed using the apparatus and method essentially as described by Jovin, Chrambach, and Naughton.7The "Poly Prep" apparatus manufactured by Buchler Instruments, Fort Lee, N.J., was used for all preparative electrophoreses.The buffer system described by Jovin, Chrambach, and Naughton7 was modified as described previously,8 and both analytical and preparative electro- phoresis were performed at pH 9.4 and 25°C in the absence of urea in gels with a 13% acrylamide concentration.The relative mobility of protein bands obtained by analytical acrylamide gel electrophoresis were expressed as Rf values which were computed from the position of each band with reference to the bromophenol blue tracking dye front.In a typical experiment, 20 mg of PWM obtained by chromatography on hydroxylapatite was dissolved in 3 ml of upper gel buffer, containing 5% (w/v) sucrose (0.046 M Tris, 0.032 M H3PO4, pH 6.9) and thoroughly dialyzed against this same buffer.The sample was carefully layered on top of a preparative acrylamide gel column (50 ml lower gel, 30 ml upper gel), and a trace of bromophenol blue dye was added.A constant current of 15 ma and 100 v was applied until the sample had completely entered the upper gel.The current was then increased to 60 ma and 280 v.The acrylamide concentration of the upper gel was 2.5% and that of the lower gel 13%.The elution rate was held constant at a 0.8 ml per minute; fractions were collected at 6-min intervals.Amino acid analyses: The amino acid composition of PWM was determined using the Beck- man-Spinco model 120 B analyzer modified for high sensitivity9 and rapid elution schedules.'0Samples were hydrolyzed in racuo in 5.7 N HCO at 110°C for 24, 48, and 72 hr, respectively.Tryptophan was determined spectrophotometrically, using a method described by Edelhoch."The cystine content of native PWM was expressed as half-cystine.Cystine was also determined