DISSOCIATION OF LIPID-FREE APOLIPOPROTEIN-A-I FROM HIGH-DENSITY-LIPOPROTEINS

Conditions under which apolipoprotein (ape) A-I dissociates from human high density lipoproteins (HDL) during incubation in vitro have been investigated. Dissociation of apoA-I was demonstrated by non-denaturin g gradient gel electrophoresis followed by immunoblotting for apoA-I a nd by size-exclusion chromatography. It was quantitated after ultracen trifugation 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 3 7 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 prot ein (CETP) in the absence of other lipoprotein fractions or with other lipoproteins in the absence of CETP. However, when mixtures of HDL an d CETP were incubated for 24 h in the presence of physiological concen trations 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 o f apoA-I coincided with a time-dependent reduction in HDL particle siz e. The percentage of apoA-I that dissociated from HDL correlated posit ively with the concentrations of VLDL, LDL, and CETP in the incubation but negatively with the concentration of HDL. When lecithin:cholester ol acyltransferase was added to mixtures at the completion of 24 h of incubation with CETP, the size of the HDL increased and the dissociate d apoA-I returned to the fraction of d<1.25 g/ml. Analysis of the lipo protein-deficient fraction of d >1.25 g/ml isolated by ultracentrifuga tion 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, phosphol ipids, or other apolipoproteins. When the dissociated apoA-I was subje cted to agarose gel electrophoresis, it migrated to a prebeta position comparable to that of purified, lipid-free apoA-I. This contrasted wi th the original HDL that exhibited alpha migration. Thus, CETP-mediate d transfers of cholesteryl esters from HDL to VLDL and LDL are accompa nied not only by a reduction in HDL size but also by the progressive d issociation from HDL of a pool of prebeta-migrating, essentially lipid -free apoA-I.