Solubilization and reconstitution of cholinephosphotransferase from sarcoplasmic reticulum: stabilization of solubilized enzyme by diacylglycerol and glycerol.
Cholinephosphotransferase (CDPcholine: 1,2-diacylglycerol cholinephosphotransferase, EC 2.7.8.2), which catalyzes the terminal step in phosphatidylcholine synthesis via the CDPcholine pathway, is present in sarcoplasmic reticulum from rabbit skeletal muscle (Cornell, R. and MacLennan, D.H. (1985) Biochim. Biophys. Acta 835, 567-576). The conditions for solubilization and reconstitution of this enzyme were investigated as a preliminary step towards its eventual purification. The activity was not released by treatment of membranes with 1 M KCl, but was solubilized after dissolution of membranes with detergents. Cholinephosphotransferase was inactivated by cholate, deoxycholate, Triton X-100, octylglucoside, Tween-20 or SDS at concentrations which solubilize the membrane. However, the activity could be fully recovered after reconstituting the membrane by adding excess lipid (soybean) and removing detergent by gel filtration, dialysis or by absorption to Bio-Beads. When the membrane was solubilized with octylglucoside or cholate at weight ratios of detergent: membrane protein of at least 10, the activity was irreversibly lost unless stabilizers were added with detergent. The substrate diacylglycerol and glycerol were effective stabilizers.
D000818:Animals; D003902:Detergents / Q000494:pharmacology; D002798:Diacylglycerol Cholinephosphotransferase / Q000302:isolation & purification* / Q000378:metabolism; D004075:Diglycerides / Q000494:pharmacology*; D004355:Drug Stability; D005989:Glycerides / Q000494:pharmacology*; D005990:Glycerol / Q000494:pharmacology*; D007700:Kinetics; D009132:Muscles / Q000201:enzymology; D010770:Phosphotransferases / Q000302:isolation & purification*; D011817:Rabbits; D012519:Sarcoplasmic Reticulum / Q000201:enzymology*; D012995:Solubility
D003902:Detergents; D004075:Diglycerides; D005989:Glycerides; D010770:Phosphotransferases; D002798:Diacylglycerol Cholinephosphotransferase; D005990:Glycerol