Regulation of CTP:phosphocholine cytidylyltransferase by lipids. 2. Surface curvature, acyl chain length, and lipid-phase dependence for activation.
We are investigating the mechanism of the activation of phosphocholine cytidylyltransferase by selective lipids. In the previous paper [Cornell, R. B. (1991) Biochemistry (preceding paper in this issue)] we found that the Triton X-100 present in the purified enzyme preparation interfered with activation of the enzyme by neutral lipid activators. The enzyme preparation was also contaminated with micromolar levels of phospholipids and fatty acids. To eliminate these interferences in our analysis, we removed 99.9% of the Triton and 99% of the endogenous lipid by DEAE-Sepharose chromatography. This preparation was inactive but could be reactivated in a lipid-specific manner up to 40-fold when added back to preformed liposomes. Small unilamellar vesicles were more effective than multilamellar vesicles of the same composition. The transferase showed a requirement for membrane acyl chains longer than 12 carbons and was poorly activated by lipids in the gel phase. Using PG-PC liposomes that undergo phase transitions within a useful temperature range (27 and 36 degrees C), we found an anomalous enhancement of enzyme activity around the phase transition temperature. These results support the idea that cytidyltransferase activation depends on intercalation into the membrane bilayer.
Cornell
R B
RB
Department of Chemistry/Biochemistry, Simon Fraser University, Burnaby, British Columbia, Canada.
D000818:Animals; D019916:Choline-Phosphate Cytidylyltransferase; D004789:Enzyme Activation; D007700:Kinetics; D008081:Liposomes; D008099:Liver / Q000201:enzymology; D009713:Nucleotidyltransferases / Q000302:isolation & purification / Q000378:metabolism*; D019301:Oleic Acid; D009829:Oleic Acids / Q000378:metabolism / Q000494:pharmacology*; D019308:Palmitic Acid; D010169:Palmitic Acids / Q000378:metabolism / Q000494:pharmacology*; D010713:Phosphatidylcholines / Q000378:metabolism / Q000494:pharmacology; D010743:Phospholipids / Q000378:metabolism / Q000494:pharmacology*; D011485:Protein Binding; D051381:Rats; D012710:Serum Albumin, Bovine / Q000494:pharmacology; D013816:Thermodynamics
D008081:Liposomes; D009829:Oleic Acids; D010169:Palmitic Acids; D010713:Phosphatidylcholines; D010743:Phospholipids; D012710:Serum Albumin, Bovine; D019301:Oleic Acid; D019308:Palmitic Acid; D009713:Nucleotidyltransferases; D019916:Choline-Phosphate Cytidylyltransferase