Dissociation of lipid-free apolipoprotein A-I from high density lipoproteins

Conditions under which apolipoprotein (apo) A-I dissociates from human high density lipoproteins (HDL) during incubation in vitro have been investigated. Dissociation of apoA-I was demonstrated by non-denaturing gradient gel electrophoresis followed by immunoblotting for apoA-I and by size-exclusion chromatography. It was quantitated after ultracentrifugation as the loss of apoA-I from the fraction of d < 1.25 g/ml. ApoA-I did not dissociate from HDL when they were incubated alone at 37 degrees C for up to 24 h. Nor was there dissociation of apoA-I when the HDL were incubated either with the cholesteryl ester transfer protein (CETP) in the absence of other lipoprotein fractions or with other lipoproteins in the absence of CETP. However, when mixtures of HDL and CETP were incubated for 24 h in the presence of physiological concentrations of either very low density lipoproteins (VLDL) or low density lipoproteins (LDL), there was a dissociation of up to 36% of the apoA-I from the HDL fraction that was linear with time. The dissociation of apoA-I coincided with a time-dependent reduction in HDL particle size. The percentage of apoA-I that dissociated from HDL correlated positively with the concentrations of VLDL, LDL, and CETP in the incubation but negatively with the concentration of HDL. When lecithin:cholesterol acyltransferase was added to mixtures at the completion of 24 h of incubation with CETP, the size of the HDL increased and the dissociated apoA-I returned to the fraction of d < 1.25 g/ml. analysis of the lipoprotein-deficient fraction of d > 1.25 g/ml isolated by ultracentrifugation and of the lower molecular weight fractions recovered after size-exclusion chromatography revealed that the dissociated apoA-I was not associated with significant quantities of either cholesterol, phospholipids, or other apolipoproteins. When the dissociated apoA-I was subjected to agarose gel electrophoresis, it migrated to a prebeta position comparable to that of purified, lipid-free apoA-I. This contrasted with the original HDL that exhibited alpha migration. Thus, CETP-mediated transfers of cholesteryl esters from HDL to VLDL and LDL are accompanied not only by a reduction in HDL size but also by the progressive dissociation from HDL of a pool of prebeta-migrating, essentially lipid-free apoA-I.