PURIFICATION OF MITOGENIC PROTEINS DERIVED FROM Phaseolus vulgaris : ISOLATION OF POTENT AND WEAK PHYTOHEMAGGLUTININS POSSESSING MITOGENIC ACTIVITY

Abstract

Abstract.-Commercially obtained phytohemagglutinin (PHAP) derived from Phaseolus vulgaris contains 17 different protein bands when analyzed by acrylamide gel electrophoresis. When it is subjected to CM-Sephadex chromatography followed by molecular sieving on Sephadex G150, several species of potent mitogenic proteins, which differ greatly in their hemagglutinating capacity, are obtained. A low hemagglutinating mitogen (L-PHAP), homogeneous by several different criteria, is the most potent mitogen isolated, and also possesses potent leukoagglutinating activity. It is a glycoprotein with a molecular weight of 115,000, containing glucosamine, mannose, xylose, and fucose or arabinose. Also isolated is a mixture of at least two closely related proteins possessing high hemagglutinating capacity, with hemagglutination titers 250 times more potent than L-PHAP. This material is a slightly less potent mitogen than L-PHAP and also possesses leukoagglutinating capacity, although of a lower order of magnitude. Its amino acid and carbohydrate composition are similar to L-PHAP, but it contains approximately twice as much carbohydrate and is slightly larger as determined by molecular sieving.In order to study in detail the mechanism whereby phytohemagglutinin, a protein derived from the kidney bean Phaseolus vulgaris, causes lymphocyte transformation, it was deemed necessary to obtain a highly purified transforming factor. The present report describes procedures by which such a factor may be isolated from commercially available starting materials, characterizes the fractions obtained, and clarifies the relationship between the serologic and mitogenic properties of several closely related protein species encountered.Materials and Methods. -Bacto-phytohemagglutin P (PHAP) was obtained from Difco Laboratories (Detroit, Michigan; cat. #3110-57). Four different batches of starting material prepared by the method of Rigas and Osgood' were used, and no significant differences were encountered. The contents of 1-3 vials (41-125 mg) of PHAP were dissolved in 5-10 ml of 0.15 M NaCl and dialyzed for 6 hours at 4VC against several liters of 0.01 M phosphate buffer, pH 5.5. A precipitate that constituted 5-10% of the original material was spun down and discarded. The supernatant solution was applied to a 1 X 25 cm column of CM-Sephadex C-50 (Pharmacia Fine Chemicals Inc., capacity 4.5 i 0.5 meq/gm, particle size 40-120 /u) previously equilibrated with 0.01 M phosphate, pH 5.5, and eluted with the same buffer at a flow rate of 24 ml/hour in 8-ml aliquots. After elution of the initial peak, stepwise elution was carried out with 0.0167 M phosphate, pH 6.6, followed by 0.03 M phosphate, pH 8.1. Finally, a linear gradient was developed using 250 ml 0.03 M phosphate, pH 8.1, containing 0.5 M NaCl and an equivalent volume of 0.03 M phosphate, pH 8.1. In other experiments the gradient was developed, using 0.2 M NaCl in 0.03 M P04, pH 8.1, as the limit buffer. The resultant fractions were monitored for protein content (OD 280 mu), hemagglutination t...

Studies in a Murine Model Confirm the Safety of Griffithsin and Advocate Its Further Development as a Microbicide Targeting HIV-1 and Other Enveloped Viruses

“…All flow cytometry data were acquired and analyzed using CellQuest Pro from BD, counting 10,000 events per sample. ConA (0.37 μM) and PHA (10 μg/mL) were used as positive controls [12,18], and PBS as the negative control.…”

The Sézary Syndrome Lymphoid Cell: Abnormal Surface Properties and Mitogen Responsiveness

“…A single animal was used as the source of SRBC throughout the course of these studies. The pokeweed mitogen (P WM), concanvalin A (Con-A), and phytohaemagglutin (L-PHAP) utilized in these studies have been characterized in earlier reports (Allen et al, 1969;Yachnin, 1972). Antithymocyte antiserum (ATS) was prepared in New Zealand white rabbits by five or six successive weekly i.v.…”

“…Peripheral blood lymphoid cells, free of erythrocytes and containing 50-100 platelets per IOO lymphocytes, were obtained by means of gravity sedimentation of heparinized whole blood, nylon fibre column passage, osmotic lysis of RBC, and centrifugation through a plasma -saline gradient (Allen et al, 1969). The yield of cells so isolated from patients with S S ranged from 40 to 88% with a mean of 67%, and contained less than 1% monocytes.…”

Human alpha-fetoprotein as a modulator of human lymphocyte transformation: correlation of biological potency with electrophoretic variants.

“…The techniques for the isolation of peripheral blood lymphocytes from normal human adults, as well as methods for their culture, have been described previously (6,7). The potency of HAFP preparations in suppressing human lymphocyte transformation was derived from dose-response curves plotted on semilogarithmic graph paper and is expressed as the concentration of HAFP required to produce 50% inhibition.…”