The purification and characterization of CTP:phosphorylcholine cytidylyltransferase from rat liver.
We have purified CTP:phosphorylcholine cytidylyltransferase from rat liver cytosol 2180-fold to a specific activity of 12,250 nmol/min/mg of protein. The purified enzyme was stable at -70 degrees C in the presence of Triton X-100 and 0.2 M phosphate. The purified enzyme gave a single protein and activity band on nondenaturing polyacrylamide electrophoresis. Separation by sodium dodecyl sulfate-polyacrylamide electrophoresis indicated that the purified enzyme contained subunits with Mr of 39,000 and 48,000. Gel filtration analysis indicated that the native enzyme was a tetramer containing two 39,000 and two 48,000 subunits. The purified enzyme appeared to bind to Triton X-100 micelles, one molecule of tetramer/micelle. Maximal activity was obtained with 100 microM phosphatidylcholine-oleic acid vesicles (8-10-fold stimulation). Phosphatidylglycerol produced a 4-5-fold increase in activity at 10 microM. The pH optimum and true Km values for CTP and phosphorylcholine were similar to those reported previously for crude preparations of cytidylyltransferase. The overall behavior of cytidylyltransferase during purification and subsequent analysis suggested that it has hydrophobic properties similar to those exhibited by membrane proteins.
D000818:Animals; D011232:Chemical Precipitation; D019916:Choline-Phosphate Cytidylyltransferase; D002845:Chromatography; D003600:Cytosol / Q000201:enzymology; D004355:Drug Stability; D004591:Electrophoresis, Polyacrylamide Gel; D005260:Female; D008099:Liver / Q000201:enzymology*; D046911:Macromolecular Substances; D008823:Micelles; D008970:Molecular Weight; D009713:Nucleotidyltransferases / Q000037:antagonists & inhibitors / Q000302:isolation & purification / Q000378:metabolism*; D017830:Octoxynol; D010743:Phospholipids / Q000494:pharmacology; D011092:Polyethylene Glycols / Q000494:pharmacology; D051381:Rats; D013439:Sulfhydryl Reagents / Q000494:pharmacology
D046911:Macromolecular Substances; D008823:Micelles; D010743:Phospholipids; D013439:Sulfhydryl Reagents; D011092:Polyethylene Glycols; D017830:Octoxynol; D009713:Nucleotidyltransferases; D019916:Choline-Phosphate Cytidylyltransferase