Purification and analysis of an 80-kDa carcinoembryonic antigen-binding protein from Kupffer cells.
The receptor-mediated interaction of Kupffer cells with carcinoembryonic antigen (CEA) has led to the identification of an 80-kDa CEA-binding Kupffer cell protein. This study is aimed at the isolation and analyses of this protein from rat Kupffer cells. The binding protein was purified using a combination of gel filtration, preparative polyacrylamide gel electrophoresis (PPAGE), and affinity chromatography using a CEA-Sepharose column. Fractions obtained from the gel filtration produced two major and few minor peaks with CEA-binding activity. Maximum reactivity was detected in the first major peak. The first major peak protein was partially precipitated following fractionation with 30% loss of activity in the precipitate. Fractions with CEA-binding activity were pooled and separated on the basis of molecular weight (MW) in PPAGE. The fractions between MW 70 and 90 kDa were pooled and affinity purified using CEA-Sepharose affinity chromatography. The purity of the 80-kDa protein was demonstrated by a single protein band on SDS-polyacrylamide gel. The protein was further identified by an anti-80-kDa binding protein antibody in Western blot analysis. The pI of the 80-kDa protein is 4.95. Amino acid analysis demonstrated no histidine; higher percentages of glutamine (13.3%), leucine (11.2%), asparagine and alanine (10.4%), and lysine (9.2%) were observed. Protein microsequencing revealed two unique sequences, one with 16 amino acids and the other with 11 amino acids. The 16-amino-acid sequence has less than 50% homology with a large sample of unrelated proteins, whereas the sequence containing 11 amino acids has 60-70% homology with the alpha chain of collagen from a variety of species but no significant homology with other known proteins, suggesting the presence of collagen-like domains in the 80-kDa receptor.
Gangopadhyay
A
A
Laboratory of Cancer Biology, Deaconess Hospital and Harvard Medical School, Boston, Massachussetts 02115, USA.
Lazure
D A
DA
Kelly
T M
TM
Thomas
P
P
D000595:Amino Acid Sequence; D000596:Amino Acids / Q000032:analysis; D000818:Animals; D000918:Antibody Specificity; D015153:Blotting, Western; D002272:Carcinoembryonic Antigen / Q000378:metabolism*; D002352:Carrier Proteins / Q000737:chemistry* / Q000276:immunology / Q000378:metabolism*; D007525:Isoelectric Focusing; D007728:Kupffer Cells / Q000737:chemistry* / Q000378:metabolism; D008099:Liver / Q000737:chemistry* / Q000166:cytology / Q000378:metabolism; D008297:Male; D008969:Molecular Sequence Data; D011485:Protein Binding; D051381:Rats; D017207:Rats, Sprague-Dawley; D017421:Sequence Analysis
D000596:Amino Acids; D002272:Carcinoembryonic Antigen; D002352:Carrier Proteins