CTP:phosphorylcholine cytidylyltransferase from rat liver. Isolation and characterization of the catalytic subunit.
We reported previously the purification of CTP:phosphorylcholine cytidylyltransferase from rat liver (Weinhold, P. A., Rounsifer, M. E., and Feldman, D. A. (1986) J. Biol. Chem. 261, 5104-5110). The purified enzyme appeared to contain equal amounts of two nonidentical proteins, with Mr of about 38,000 and 45,000. We have now separated and purified these proteins. Polyacrylamide electrophoresis in the presence of sodium dodecyl sulfate indicated that each protein was homogeneous. The 45,000 protein contained the catalytic activity. Analysis by gel filtration chromatography and glycerol gradient centrifugation indicated that the 38,000 and 45,000 proteins in the purified cytidylyltransferase were independently associated with Triton X-100 micelles. The apparent Mr of the complexes suggested that a tetramer of each protein was bound to one Triton X-100 micelle. The isolated 45,000 catalytic protein had the same lipid requirement and kinetic properties as the purified cytidylyltransferase containing both proteins. Enzyme activity was stimulated to maximal values by phosphatidylcholine vesicles containing 9 mol % of either oleic acid, phosphatidylinositol, or phosphatidylglycerol. The amino acid compositions of the isolated 38,000 and 45,000 proteins were distinctly different. Overall, the results suggested that a tetramer of the 45,000 protein possessed nearly optimal catalytic activity. A functional role of the 38,000 protein as part of a cytidylyltransferase enzyme complex could not be documented. However, the need for stabilizing concentrations of Triton X-100 in the purified enzyme preparation may have prevented the association of the two proteins.
D000596:Amino Acids / Q000032:analysis; D019916:Choline-Phosphate Cytidylyltransferase; D004591:Electrophoresis, Polyacrylamide Gel; D007700:Kinetics; D008055:Lipids / Q000032:analysis; D008099:Liver / Q000201:enzymology*; D009682:Magnetic Resonance Spectroscopy; D008970:Molecular Weight; D009713:Nucleotidyltransferases / Q000378:metabolism*; D017830:Octoxynol; D019301:Oleic Acid; D009829:Oleic Acids / Q000494:pharmacology; D010713:Phosphatidylcholines / Q000494:pharmacology; D010715:Phosphatidylglycerols / Q000494:pharmacology; D011092:Polyethylene Glycols / Q000494:pharmacology
D000596:Amino Acids; D008055:Lipids; D009829:Oleic Acids; D010713:Phosphatidylcholines; D010715:Phosphatidylglycerols; D019301:Oleic Acid; D011092:Polyethylene Glycols; D017830:Octoxynol; D009713:Nucleotidyltransferases; D019916:Choline-Phosphate Cytidylyltransferase