Purification and physicochemical characterization of a human cytotoxic factor produced by a human haemic cell line

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Abstract
A cytotoxic factor, produced by a human lymphoblastoid cell line (Karpas 160), was purified from the cell extracts and from the culture medium containing the cell lysate, by using ammonium sulfate precipitation, DEAE-cellulose chromatography, gel filtration and affinity chromatography on concanavalin [Con] A-Sepharose and on [3H]aminoethanol-glass beads. Two factors, Factor I and Factor II, were separated by DEAE-cellulose chromatography. Factor I was eluted from this column at 30 mM aminoethanol/HCl buffer, pH 8.0, whereas Factor II was bound strongly to DEAE-cellulose and was eluted only at 325 mM aminoethanol/HCl buffer, pH 8.0. The purified Factor I migrated as a single band on polyacrylamide gel electrophoresis. Its isoelectric point, pI, was 8.0 .+-. 0.3. Its sedimentation coefficient, s20,w, was 3.5 .+-. 0.1 S and its apparent MW was 65,000 .+-. 1000 as determined by sedimentation-velocity and sedimentation-equilibrium measurements. A linear relationship between MW and concentration was found in equilibrium runs, suggesting a non-spherical shape of the molecule. Factor I is not a glycoprotein, inasmuch as it does not bind to Con A-Sepharose. It consists of 2 subunits (MW 32,000 .+-. 4000), migrating on sodium dodecyl sulfate/polyacrylamide-gel electrophoresis as a single band. Factor II had pI 6.0 .+-. 0.4 and MW 75,000 .+-. 3000. Factors I and II are thus different proteins. [This cell line, established from a patient with non-Hodgkin''s lymphoma, may represent a proliferation of a population of cells that are involved in cell-mediated anti-tumor immunity.].