THE NUMBER OF AMPHIPATHIC ALPHA-HELICAL SEGMENTS OF APOLIPOPROTEIN-A-I, APOLIPOPROTEIN-E, AND APOLIPOPROTEIN-A-IV DETERMINES THE SIZE AND FUNCTIONAL-PROPERTIES OF THEIR RECONSTITUTED LIPOPROTEIN PARTICLES

The objective of this work was to determine the role of the amphipathic alpha-helical structural units of human apolipoproteins A-I, E, and A-IV in defining the sizes and reactivities with lecithin:cholesterol acyltransferase (LCAT) of their reconstituted lipoprotein particles. We prepared reconstituted high density lipoprotein (rHDL) particles with each of the three apolipoproteins in two weight ratios with lipid: 2.7/0.07/1 and 1.35/0.04/1, palmitoyloleoylphosphatidylcholine/cholesterol/apolipoprotein, by the sodium cholate dialysis procedure; and examined the rHDL product sizes and distributions by nondenaturing gradient gel electrophoresis. The rHDL particles were also incubated with low density lipoprotein (LDL), and with LDL plus LCAT, to observe any structural modifications due to phospholipid transfers to LDL and to cholesterol esterification by LCAT. In addition, we examined the average structural properties of the original rHDL by several fluorescence methods and circular dichroism spectroscopy, and determined their reaction kinetics with LCAT. The results indicate that the diameters of the largest rHDL particles, containing two apolipoproteins per particle, correlate with the maximum number of putative amphipathic alpha-helical segments in their sequences, and that smaller particles of this class may arise from the removal of one or more alpha-helical segments from contact with lipid. Furthermore, the larger particles may be converted into the smaller ones upon loss of phospholipid to LDL, and may form one or two well defined products when reacted with LCAT. In general, the subclasses of particles have distinct spectroscopic properties, consistent with a different apolipoprotein folding in particles containing different proportions of phospholipid to apolipoprotein. Furthermore, the different apolipoprotein structures lead to significant differences in reactivity with LCAT.