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

Abstract

A cytotoxic factor, produced by a human lymphoblastoid cell line [Karpas (1977) Br. J. Cancer 35, 152–160; Karpas (1977) Br. J. Cancer 36, 437–445], was purified both from the cell extracts and from the culture medium containing the cell lysate, by using ammonium sulphate precipitation, DEAE-cellulose chromatography, gel filtration and affinity chromatography on concanavalin A–Sepharose and on [3H]amino-ethanol–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 molecular weight, Mr, was 65 000 +/- 1000 as determined by sedimentation-velocity and sedimentation-equilibrium measurements. A linear relationship between molecular weight 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 concanavalin A–Sepharose. It consists of two subunits (Mr 32 000 +/- 4000), migrating on sodium dodecyl sulphate/polyacrylamide-gel electrophoresis as a single band. Factor II had pI 6.0 +/- 0.4 and Mr 75 000 +/- 3000. Factors I and II are thus different proteins.