Vol.

THE JOURNAL OF BIOLOGICAL CHEMISTRY 252, No. 6, Issue of March 25, pp. 2095-2100, Prmted in U.S.A

1977

Mechanisms
BINDING KINETICS

of Lymphocyte
OF PHYTOHEMAGGLUTININ

Activation
TO
(Received

HUMAN

LYMPHOCYTES*
April 12, 1976, and in revised form, August 19, 1976)

for publication,

GUY

B. FAGUET the Department of Medicine, Medical College of Georgia, Augusta, Georgia 30902

From

The interactions of phytohemagglutinin (PHA) with normal human lymphocytes were studied utilizing radioiodinated leukoagglutinin (“$I-LPHA) over a concentration spectrum encompassing the entire range of lymphocyte metbinding was temperature-, pH-, abolic responses. “jI-LPHA and time-dependent. Ligand association was rapid …ver más…

Lymphocyte suspensions, virtually free of phagocytes, platelets, and red cells, were obtained by a modified Ficoll-Hypaque gradient centrifugation technique as previously described (2). PHA Purification - Leukoagglutinin (LPHA) was prepared from PHA-P (Difco. Batch No. 577755) according to a modification of the method of Weber et al. (20). Briefly, PHA-P was layered over a sulfoethyl (SE)-Sephadex C-50 (Pharmacia) column equilibrated with I/X M KH,PO,, pH 4.5. After elution of the initial peak with the starting buffer, the LPHA containing second peak was eluted with ‘115 M KH,PO,, Na,HPO,, pH 6.0. This peak was further purified by gel filtration on Sephadex G-200 (Pharmacia), dialyzed against 0.9% NaCl, rechromatographed on SE-Sephadex C-50 (Pharmacia), and concentrated by pressure ultracentrifugation and lyophylized. The LPHA so obtained was found to be homogenous by the criteria of chromatography on SE-Sephadex C-50, e&lusion chromatography on G-200 and by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. It is known to be completely devoid of erythroagglutinating activity (EPHA) (20). lodination of LPHA - LPHA was iodinated (with reductant-free ‘9; specific activity, 17 Ci/mmol, ICN, or with Y), by the chloramine-T method of Hunter (21) preceding each experiment. Iodinated I The abbreviations used