sn-1,2-diacylglycerol cholinephosphotransferase from pig liver: mixed micellar assay and kinetic analysis of the partially pure enzyme.
sn-1,2-Diacylglycerol cholinephosphotransferase from pig liver microsomes was partially purified through a procedure involving solubilization with sodium cholate and chromatography on Sepharose 6B. The resulting preparation was 19-fold enriched with respect to microsomes and was shown to be very sensitive to different detergents. Sodium cholate gave the best yields in activity. In a mixed micellar assay with Triton X-100 a strong dependence of the enzyme activity on the concentration of mixed micelles was observed, due to Triton X-100 acting as an inactivator. Soja phosphatidylcholine added exogenously protected the enzyme against detergent inactivation and stimulated the enzyme activity. Dioleoyl-phosphatidylcholine had a similar stimulatory effect, whereas didecanoyl- or dioctanoyl-phosphatidylcholine did not; thus long-chain phosphatidylcholines seem to be essential in the activation of cholinephosphotransferase. In a mixed micellar assay with sodium cholate no inactivation of the enzyme could be detected and it was found that soja phosphatidylcholine stimulates the activity in a greater extent than in Triton X-100 mixed micelles. The phospholipid activates the enzyme in a noncompetitive way with an activation constant of 176 mol%. Km was estimated as 1.54 mol% with a Vmax = 30 nmol/min/mg protein. Those results support an activation mechanism by phosphatidylcholine interacting at sites different from the active center. The high activation constant led to the conclusion that cholinephosphotransferase requires a lipidic boundary for full activation. No activation by substrate was observed. Short-chain diacylglycerides such as dihexanoyl-, dioctanoyl-, or didecanoylglycerol can be used as substrates although the enzyme in this case has only 5 to 10% of the activity it has for dioleoylglycerol or egg diglycerides.
Bru
R
R
Institut für Polymere, ETH-Zentrum, Zürich, Switzerland.
Blöchliger
E
E
Luisi
P L
PL
D000818:Animals; D019826:Cholic Acid; D002793:Cholic Acids; D002798:Diacylglycerol Cholinephosphotransferase / Q000302:isolation & purification / Q000378:metabolism*; D004075:Diglycerides / Q000378:metabolism; D004789:Enzyme Activation / Q000187:drug effects; D004795:Enzyme Stability; D007700:Kinetics; D008823:Micelles; D008862:Microsomes, Liver / Q000201:enzymology*; D008956:Models, Chemical; D017830:Octoxynol; D010713:Phosphatidylcholines / Q000494:pharmacology; D010743:Phospholipids / Q000378:metabolism; D012995:Solubility; D013379:Substrate Specificity; D013552:Swine
D002793:Cholic Acids; D004075:Diglycerides; D008823:Micelles; D010713:Phosphatidylcholines; D010743:Phospholipids; D017830:Octoxynol; D002798:Diacylglycerol Cholinephosphotransferase; D019826:Cholic Acid