Purification and properties of acyl-CoA:1-acyl-sn-glycero-3-phosphocholine-O-acyltransferase from bovine brain microsomes.
Acyl-CoA:1-acyl-sn-glycero-3-phosphocholine-O-acyltransferase has been purified approximately 3000-fold from bovine brain microsomes by detergent solubilization followed by ion-exchange and affinity chromatography. Polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate revealed a single protein of molecular weight 43,000. The specificity of the purified enzyme was studied by measuring the catalytic activity with various lysophospholipids and acyl-CoA derivatives. Of the lysophospholipids tested, only lysophosphatidylcholine was a substrate. Less specificity was exhibited toward the acyl-CoA derivatives, although the enzyme showed a clear preference for arachidonoyl-CoA and little or no activity with palmitoyl-CoA or stearoyl-CoA. High concentrations of arachidonoyl-CoA inhibited the enzyme. The velocity was a sigmoidal function of the concentration of lysophosphatidylcholine (LPC) with little activity obtained below 20 microM LPC. The specificity and kinetic properties of the enzyme were altered, however, by incorporation of the enzyme into liposomes composed of a mixture of phospholipids. Decanoyl-CoA and myristoyl-CoA, which were effective substrates for the soluble enzyme, did not serve as acyl donors for the liposome-bound acyltransferase. Furthermore, the liposome-bound enzyme, in contrast to the soluble form of the enzyme, was active at concentrations of LPC below the critical micelle concentration. The liposome-bound enzyme was also substantially less susceptible to thermal denaturation and proteolytic digestion. This modulation of the acyltransferase activity by interaction with phospholipids may relate to the kinetic properties and the regulation of the enzyme in vivo.
Deka
N
N
Sun
G Y
GY
MacQuarrie
R
R
D008243:1-Acylglycerophosphocholine O-Acyltransferase; D000217:Acyltransferases / Q000302:isolation & purification* / Q000378:metabolism; D000818:Animals; D001921:Brain / Q000201:enzymology*; D002417:Cattle; D006868:Hydrolysis; D008081:Liposomes; D008861:Microsomes / Q000201:enzymology*; D011485:Protein Binding; D012995:Solubility; D013379:Substrate Specificity; D014357:Trypsin
D008081:Liposomes; D000217:Acyltransferases; D008243:1-Acylglycerophosphocholine O-Acyltransferase; D014357:Trypsin