The purpose of this study was to test the hypothesis that lipid fluidity regulates lecithin:cholesterol acyltransferase (LCAT) activity. Phosphatidylcholine (PC) species were synthesized that varied in fluidity by changing the number, type ( cis vs. trans ), or position of the double bonds in 18 or 20 carbon sn -2 fatty acyl chains and recombined with [ 3 H]cholesterol and apolipoprotein A-I to form recombinant high density lipoprotein (rHDL) substrate particles. The activity of purified human plasma LCAT decreased with PC sn -2 fatty acyl chains containing trans versus cis double bonds and as double bonds were moved towards the methyl terminus of the sn -2 fatty acyl chain. The decrease in LCAT activity was significantly correlated with a decrease in rHDL fluidity (measured by diphenylhexatriene fluorescence polarization) for PC species containing 18 carbon ( r 2 ‫؍‬ 0.61, n ‫؍‬ 18) and 20 carbon ( r 2 ‫؍‬ 0.93, n ‫؍‬ 5) sn -2 fatty acyl chains. rHDL were also made containing 10% of the 18 carbon sn -2 fatty acyl chain PC species and 90% of an inert PC ether matrix ( sn -1 18:1, sn -2 16:0 PC ether) to normalize rHDL fluidity. Even though fluidity was similar among the PC ether-containing rHDL, the order of PC reactivity with LCAT was significantly correlated ( r 2 ‫؍‬ 0.71) with that of 100% PC rHDL containing the same 18 carbon sn -2 fatty acyl chain species, suggesting that PC structure in the active site of LCAT determines reactivity in the absence of measurable differences in bilayer fluidity.We conclude that PC fluidity and structure are major regulators of LCAT activity when fatty acyl chain length is constant. -Parks,

The goal of this study was to determine whether apolipoprotein A-I (apoA-I) is lipidated before secretion by HepG2 cells. ApoA-I was extracted from microsomes after radiolabeling with [ 35 S]Met/Cys. After ultracentrifugal flotation, d Ͻ 1.25 g/ml and d Ͼ 1.25 g/ml fractions were immunoprecipitated and analyzed by SDS-PAGE. Under steady state radiolabeling conditions, 20% of extracted microsomal apoA-I floated at d Ͻ 1.25 g/ml. Pulse-chase experiments demonstrated that the percentage of microsomal apoA-I associated with lipid peaked between 2 and 8 min postsynthesis. Density gradient ultracentrifugation, and nondenaturing gradient gel electrophoresis of HepG2 cell medium, indicated that 50% of secretory apoA-I existed as small HDL particles with a diameter of ϳ 7.5 nm. These and additional data suggested that ϳ 20% of newly secreted apoA-I is lipidated intracellularly and another 30% is secreted in lipid-free or lipid-poor form but acquires sufficient lipid to become small HDL within 1 h of secretion, with little further maturation over the time course of the incubation (2 h). These results indicate that a process exists for the presecretory intracellular assembly of apoA-I with lipid in HepG2 cells and that apoA-I is secreted in both lipid-poor and lipidated forms. -Chisholm, J. W., E. R. Burleson, G. S. Shelness, and J. S. Parks. ApoA-I secretion from HepG2 cells: evidence for the secretion of both lipid-poor apoA-I and intracellularly assembled nascent HDL.

No abstract

We have previously described a novel pathway for the metabolism of HDL subfractions in which small [2 apolipoprotein (apoA-I) molecules per particle] HDL particles are converted in a unidirectional manner outside the plasma compartment to medium (3 apoA-I molecules per particle) or large (4 apoA-I molecules per particle) HDL particles, which are subsequently removed from the circulation by the liver (Colvin et al. 1999.