Fatty acids promote the formation of complexes between choline-phosphate cytidylyltransferase and cytidylyltransferase binding protein.
We previously identified a 112-kDa protein (CTBP) that binds CTP:choline-phosphate cytidylyltransferase (CT) (D.A. Feldman and P.A. Weinhold, 1993, J. Biol. Chem. 268, 3127-3135). In this study we show that fatty acids promote the binding of cytidylyltransferase to CTBP. Gel filtration chromatography separated CTBP from CT in liver cytosol. CTBP was eluted slightly slower than the thyroglobulin standard. The addition of oleate to cytosol followed by incubation at 37 degrees C resulted in the formation of aggregates containing both CT and CTBP. The aggregates eluted in the void volume of the gel filtration column, sedimented to the bottom of glycerol density gradients, and precipitated at concentrations of polyethylene glycol lower than required to precipitate CT and CTBP in untreated cytosol. Immunoprecipitation by CT antibodies of both CT and CTBP in aggregate preparations provided further evidence for the formation of CT-CTBP complexes. A smaller CT-CTBP complex was isolated by glycerol density centrifugation from cytosol incubated with oleate at 4 degrees C. Overall, these results suggest that oleate promotes the binding of CT to CTBP. The results suggest that a dissociable complex is formed at 4 degrees C in the presence of oleate which is polymerized by incubation at 37 degrees C to a large aggregate that is more resistant to dissociation. Complex formation at 37 and 4 degrees C was dose dependent at oleate concentrations between 50 and 200 microM. Complex formation at 4 degrees C was specifically promoted by long-chain, unsaturated fatty acids. These results suggest that CTBP may be involved in the fatty acid-induced translocation of cytidylyltransferase.
Weinhold
P A
PA
Veterans Affairs Medical Center, Ann Arbor, Michigan 48105, USA.
Feldman
D A
DA
D000818:Animals; D002352:Carrier Proteins / Q000302:isolation & purification / Q000378:metabolism*; D002499:Centrifugation, Density Gradient; D011232:Chemical Precipitation; D019916:Choline-Phosphate Cytidylyltransferase; D002850:Chromatography, Gel; D003600:Cytosol / Q000187:drug effects / Q000378:metabolism; D005227:Fatty Acids / Q000737:chemistry / Q000378:metabolism* / Q000494:pharmacology; D066298:In Vitro Techniques; D008099:Liver / Q000187:drug effects / Q000378:metabolism; D008297:Male; D009713:Nucleotidyltransferases / Q000302:isolation & purification / Q000378:metabolism*; D011092:Polyethylene Glycols; D011233:Precipitin Tests; D051381:Rats; D017207:Rats, Sprague-Dawley; D013329:Structure-Activity Relationship
D002352:Carrier Proteins; D005227:Fatty Acids; D011092:Polyethylene Glycols; D009713:Nucleotidyltransferases; D019916:Choline-Phosphate Cytidylyltransferase