HIGH-DENSITY-LIPOPROTEIN SUBPOPULATIONS AS SUBSTRATES FOR THE TRANSFER OF CHOLESTERYL ESTERS TO VERY-LOW-DENSITY LIPOPROTEINS

Human total HDL (high-density lipoprotein), HDL2 and HDL3 were labelled in vitro by incubation with lipoprotein-deficient serum (LPDS) which contained either [3H]cholesteryl oleate or [14C]cholesterol under different conditions. The lipoproteins were then subfractionated by heparin-Sepharose column chromatography, and three subfractions (A, B and C) were successively eluted from each preparation of HDL, HDL2 and HDL3. When the labelling was done at 37°C for 17 h, the subfractions were homogeneously labelled with [3H]cholesteryl oleate. However, when it was performed for only 30 min at 4°C, the subfractions showed marked differences in the 3H specific radioactivity, which was much higher in the C fractions than in the others. 3H-labelled HDL2 and HDL3 subfractions behaved differently under the precipitant action of heparin-Mn2+; fraction C (the richest in apolipoprotein E) produced the largest amount of radioactive and chemical precipitate. More 3H radioactivity, but not the cholesterol, was precipitated from HDL2 or HDL3 by the reagent, demonstrating that 3H-labelled HDL2 and HDL3 behave like their fraction C, which becomes labelled to the highest specific radioactivity despite having the smallest mass. The incubation of 3H-labelled HDL subfractions with human LPDS and very-low-density lipoprotein (VLDL) at 37°C increased the quantity of 3H radioactivity that was precipitated, in proportion to the amount of VLDL present in the media. These changes were attributable to the action of cholesterol ester transfer protein, since they did not occur at 4°C or when human LPDS was replaced with rat LPDS. Kinetics of the transfer of HDL [3H]cholesteryl oleate to VLDL showed a greater apparent V(max.) for fractions A than for fractions B from either HDL2 or HDL3, whereas the apparent K(m) values were very similar, which suggest that this transfer process is influenced by the apoprotein composition of the donor lipoprotein.