Organization of unesterified cholesterol in high density lipoproteins probed by filipin.
The initial rate of filipin association with unesterified cholesterol in high density lipoproteins (HDL) was measured by stopped-flow spectrophotometry to assess the roles played by apolipoproteins and phospholipids in modulating the surface exposure of cholesterol. The initial rate of filipin-unesterified cholesterol association was enhanced upon hydrolysis of the glycerophospholipids of human HDL3 by phospholipase A2. Rate enhancements were also observed following trypsin-catalyzed hydrolysis of apolipoprotein A-I in canine HDL and of apolipoproteins A-I and A-II in human HDL3. However, the initial rate of filipin-unesterified cholesterol association was not altered upon incubation of HDL3 with polymorphonuclear cells, which causes hydrolysis of apolipoprotein A-II but leaves apolipoprotein A-I intact. These results are consistent with the general structural model of HDL in which unesterified cholesterol, apolipoproteins and glycerophospholipids are presumed to be localized at the surface of the HDL particle. From these studies and from results indicating that the initial rate of filipin-unesterified association was enhanced in canine HDL hybrids in which 50% of the apolipoprotein A-I had been replaced by apolipoprotein A-II, we also conclude that apolipoprotein A-I in HDL is in closer proximity to unesterified cholesterol than apolipoprotein A-II. Thus, it appears that rapid kinetic measurements of filipin-cholesterol association may be useful in assessing the organization of unesterified cholesterol in serum lipoproteins.
D000818:Animals; D016632:Apolipoprotein A-I; D016633:Apolipoprotein A-II; D001053:Apolipoproteins / Q000097:blood; D002784:Cholesterol / Q000097:blood*; D008076:Cholesterol, HDL; D004285:Dogs; D005372:Filipin / Q000494:pharmacology*; D006801:Humans; D007700:Kinetics; D008075:Lipoproteins, HDL / Q000097:blood*; D008297:Male; D009504:Neutrophils / Q000378:metabolism; D010741:Phospholipases A; D054467:Phospholipases A2; D011090:Polyenes / Q000494:pharmacology*; D011485:Protein Binding
D016632:Apolipoprotein A-I; D016633:Apolipoprotein A-II; D001053:Apolipoproteins; D008076:Cholesterol, HDL; D008075:Lipoproteins, HDL; D011090:Polyenes; D005372:Filipin; D002784:Cholesterol; D010741:Phospholipases A; D054467:Phospholipases A2