Isolation from human chronic-lymphocytic-leukaemia cells of membrane glycoproteins associated with Fc-receptor functions. Physical parameters and production of polyclonal antibodies.
Two membrane glycoproteins that bound immune complexes and inhibited Fc-receptor- (FcR-)mediated functions in vitro were purified from human FcR+ chronic-lymphocytic-leukaemia cells. A multi-step purification was developed, consisting essentially in: (i) Tween 40 extraction of crude cell membranes; (ii) solubilization of membrane fragments by Renex-30; (iii) isolation of glycoproteins by affinity chromatography on Lens culinaris haemagglutinin-Sepharose; (iv) papain treatment of the eluted glycoproteins followed by gel-filtration chromatography; (v) purification by polyacrylamide-gel electrophoresis of two molecular species from the protein-size fraction enriched for immune-complex-binding activity. The two electrophoretically isolated components displayed apparent molecular masses of 70 and 45 kDa by SDS/polyacrylamide-gel electrophoresis and restricted charge heterogeneity by two-dimensional analysis. Two-dimensional peptide mapping revealed the presence of many peptides in common between the two proteins and the absence of a number of peptides in the 45 kDa component. These two polypeptides were used as immunogens to produce polyclonal antibodies that cross-reacted with both proteins and specifically inhibited FcR-mediated reactions in vitro. Furthermore, FcR-related components from detergent-extracted lysates of the human K562 and U937 cell lines or human placental membranes were revealed by the putative anti-FcR antibodies adsorbed on Protein A-Sepharose.
Gorini
G
G
Laboratory of Pathology, ENEA, C.R.E., Rome, Italy.
Medgyesi
G A
GA
Garavini
M
M
Dorrington
K J
KJ
Down
J
J
D000936:Antigen-Antibody Complex / Q000276:immunology; D004591:Electrophoresis, Polyacrylamide Gel; D006023:Glycoproteins / Q000276:immunology* / Q000302:isolation & purification; D006801:Humans; D007106:Immune Sera / Q000276:immunology; D007945:Leukemia, Lymphoid / Q000276:immunology*; D008214:Lymphocytes / Q000276:immunology; D008565:Membrane Proteins / Q000276:immunology* / Q000302:isolation & purification; D010449:Peptide Mapping; D011961:Receptors, Fc / Q000276:immunology*
D000936:Antigen-Antibody Complex; D006023:Glycoproteins; D007106:Immune Sera; D008565:Membrane Proteins; D011961:Receptors, Fc