PURIFICATION AND ANALYSIS OF AN 80-KDA CARCINOEMBRYONIC ANTIGEN-BINDING PROTEIN FROM KUPFFER CELLS

The receptor-mediated interaction of Kupffer cells with carcinoembryon ic antigen (CEA) has led to the identification of an 80-MDa CEA-bindin g Kupffer cell protein, This study is aimed at the isolation and analy ses of this protein from rat Kupffer cells, The binding protein was pu rified using a combination of gel filtration, preparative polyacrylami de gel electrophoresis (PPAGE), and affinity chromatography using a CE A-Sepharose column, Fractions obtained from the gel filtration produce d two major and few minor peaks with CEA-binding activity. Maximum rea ctivity was detected in the first major peak, The first major peak pro tein was partially precipitated following fractionation with 30% loss of activity in the precipitate, Fractions with CEA-binding activity we re pooled and separated on the basis of molecular weight (MW) in PPAGE , The fractions between MW 70 and 90 kDa were pooled and affinity puri fied using CEA-Sepharose affinity chromatography, The purity of the 80 -kDa protein was demonstrated by a single protein band on SDS-polyacry lamide gel, The protein was further identified by an anti-80-kDa bindi ng protein antibody in Western blot analysis, The pI of the 80-kDa pro tein is 4.95, Amino acid analysis demonstrated no histidine; higher pe rcentages of glutamine (13.3%), leucine (11.2%), asparagine and alanin e (10.4%), and lysine (9.2%) were observed, Protein microsequencing re vealed two unique sequences, one with 16 amino acids and the other wit h 11 amino acids, The 16-amino-acid sequence has less than 50% homolog y with a large sample of unrelated proteins, whereas the sequence cont aining 11 amino acids has 60-70% homology with the alpha chain of coll agen from a variety of species but no significant homology with other known proteins, suggesting the presence of collagen-like domains in th e 80-kDa receptor, (C) 1996 Academic Press, Inc.