Abstract
To determine how substrate fluidity and molecular structure independently regulate cholesteryl ester formation, the substrate specificity of lecithin:cholesterol acyltransferase with respect to a number of model reassembled high density lipoproteins (R-HDLs) is reported. The R-HDLs are composed of 1 mol % apolipoprotein A-I, 89 mol % of sphingomyelin or a nonhydrolyzable diether analog of phosphatidylcholine (PC) plus 10 mol % of test lipids that are potential acyl donors; a trace of [3H]cholesterol, which permits quantification of cholesteryl ester formation is also included. With respect to the lipid class of the acyl donor, the rate of ester formation decreases in the order phosphatidylethanolamine greater than phosphatidylcholine greater than N,N,-dimethylphosphatidylethanolamine greater than phosphatidylglycerol - phosphatidic acid greater than phosphatidylserine greater than dipalmitin greater than tripalmitin. Within an R-HDL composed of 90% PC ether or sphingomyelin, the relative rates of ester formation are greatest for dipalmitoyl and dimyristoyl PC, with distearoyl PC being almost unreactive; in a solid lipid environment, the rate with respect to unsaturation of the PC is greatest for oleate. In a fluid lipid environment, all unsaturated PCs were utilized nearly equally. All lipids tested were most reactive within an R-HDL composed of an unsaturated PC ether and least reactive within an R-HDL composed mostly of sphingomyelin. These results suggest that the rates of ester formation by lecithin:cholesterol acyltransferase are separate functions of the identity and the microscopic environment of the acyl donor. This is the first example of the use of diether analogs for the separation of the effects of macromolecular and molecular structure on the specificity of lecithin:cholesterol acyltransferase.
Highlights
To determine how substrate fluidity andmolecular by Hamilton et al [1]in the perfused rat liver, while another structure independently regulate cholesteryl ester for- is believed to be composed,in part, of the surface components mation, the substratespecificity of 1ecithin:cholesterol of the triglyceride-rich lipoproteins that are released during acyltransferase with respect to a number of model lipolysis [2, 3]
These results suggest that the reassemble lipoproteins and pure lipids have occurred since rates of ester formation by 1ecithin:cholesterol acyltransferase are separate functions of the identity and the microscopic environment of the acyl donor
Massey et a1.' haveshown that the physical properties of reassembled high density lipoproteins (R-HDLs) composed of DMPC and DMPC ether arenearly indistinguishable and thatwhen increasing amounts of DMPC replace the DMPC ether in RHDL, the K, for the 1ecithin:cholesterolacyltransferase-catalyzed reaction remains constant but the V, increases linearly with the amount of DMPC
Summary
Dylethanolamine; PC, phosphatidylcholine; PA, phosphatidic acid; PS, phosphatidylserine; DPPC, dipalmitoyl phosphatidylcholine; DSPC, distearoyl phosphatidylcholine; DMPC, dimyristoyl phospha-. The specific activity of 1ecithin:cholestirol acyltransferase was 13 nmol of cholesteryl ester/pg of 1ecithin:cholesterolacyltransferase/h when a substrate composedof complexes of POPC (93mol %), cholesterol These data showed that the per cent conversion of cholesterol to its esters was nearly independentofcholesterol content between 0 and 18mol %. Human plasma sphingomyelin was obtained from the extracted lipids of human low density lipoproteins and purified by silica gel chromatography on a Waters PrepLC500 chromatograph using 65:25:4 ch1oroform:methanol:waterT. The substrates were composed of POPC, cholesterol, and apo-A-I with a total lipid to protein ratio of 100
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