Identification of a protein complex between choline-phosphate cytidylyltransferase and a 112-kDa protein in rat liver.
Antisera raised against purified cytidylyltransferase (CT) immunoprecipitated CT activity from liver cytosol and detected the M(r) 45,000 subunit of CT on Western blots. Antisera detected a M(r) 112,000 protein on Western blots of liver cytosol. This protein was not detected in purified CT and was not detected by preimmune serum. The 112-kDa antibodies, isolated by affinity chromatography, did not immunoprecipitate CT activity. Antiserum raised against an N-terminal sequence of CT and antibodies raised against an internal sequence of CT immunoprecipitated CT activity but did not detect the 112-kDa protein. These results showed that the 112-kDa protein was not a form of CT. We concluded that the antiserum probably contained anti-idiotypic antibodies that recognized CT binding sites on the 112-kDa protein. Purified CT that was conjugated to horseradish peroxidase bound to crude 112-kDa protein immobilized on nitrocellulose. The binding was competitively reduced by purified CT and by affinity-purified antibodies to the 112-kDa protein. CT and 112-kDa protein coeluted from DEAE-Sepharose. When the putative 112-kDa protein-CT complex was chromatographed on a second DEAE-Sepharose column or on a Bio-Gel A-1.5m column, CT activity and 112-kDa protein were eluted together. Chromatography of the complex on hydroxylapatite dissociated most of the complex, producing CT free of the 112-kDa protein. We conclude that the 112-kDa protein is a CT-binding protein. The formation and/or dissociation of this complex may be important in the regulation of CT.
Veterans Affairs Medical Center, Ann Arbor, Michigan 48105.
D A
DA
Feldman
P A
PA
Weinhold
D000818:Animals; D000936:Antigen-Antibody Complex; D001667:Binding, Competitive; D015153:Blotting, Western; D019916:Choline-Phosphate Cytidylyltransferase; D002846:Chromatography, Affinity; D002852:Chromatography, Ion Exchange; D004591:Electrophoresis, Polyacrylamide Gel; D007106:Immune Sera; D008099:Liver / Q000201:enzymology*; D046911:Macromolecular Substances; D008970:Molecular Weight; D009713:Nucleotidyltransferases / Q000276:immunology / Q000302:isolation & purification / Q000378:metabolism*; D011485:Protein Binding; D011506:Proteins / Q000276:immunology / Q000302:isolation & purification / Q000378:metabolism*; D051381:Rats
D000936:Antigen-Antibody Complex; D007106:Immune Sera; D046911:Macromolecular Substances; D011506:Proteins; D009713:Nucleotidyltransferases; D019916:Choline-Phosphate Cytidylyltransferase