Human apolipoprotein A-I liberated from high-density lipoprotein without denaturation.
Apolipoprotein A-I (apoA-I) was liberated from human high-density lipoprotein (HDL) without exposure to organic solvents or chaotropic salts by the action of isolated insect hemolymph lipid transfer particle (LTP). LTP-catalyzed lipid redistribution results in transformation of HDL into larger, less dense particles accompanied by an overall decrease in HDL particle surface area:core volume ratio, giving rise to an excess of amphiphilic surface components. Preferential dissociation of apolipoprotein versus phospholipid and unesterified cholesterol from the particle surface results in apolipoprotein recovery in the bottom fraction following ultracentrifugation at a density = 1.23 g/mL. ApoA-I was then isolated from other contaminating HDL apolipoproteins by incubation with additional HDL in the absence of LTP, whereupon apolipoprotein A-II and the C apolipoproteins reassociate with the HDL surface by displacement of apoA-I. After a second density gradient ultracentrifugation, electrophoretically pure apoA-I was obtained. Sedimentation equilibrium experiments revealed that apoA-I isolated via this method exhibits a tendency to self-associate in an aqueous solution while its circular dichroism spectrum was indicative of a significant amount of alpha-helix. Both measurements are consistent with that observed on material prepared by denaturation/renaturation. The ability of apoA-I to activate lecithin:cholesterol acyltransferase was found to be similar to that of apoA-I isolated by conventional methods. The present results illustrate that LTP-mediated alteration in lipoprotein particle surface area leads to dissociation of substantial amounts of surface active apoprotein components, thus providing the opportunity to isolate apoA-I without the denaturation/renaturation steps common to all previous isolation procedures.(ABSTRACT TRUNCATED AT 250 WORDS)
Ryan
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Department of Biochemistry, University of Alberta, Edmonton, Canada.
Yokoyama
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Liu
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Czarnecka
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Oikawa
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Kay
C M
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D000818:Animals; D016632:Apolipoprotein A-I / Q000737:chemistry / Q000302:isolation & purification*; D002352:Carrier Proteins / Q000737:chemistry; D002846:Chromatography, Affinity; D002942:Circular Dichroism; D004789:Enzyme Activation; D006458:Hemolymph / Q000737:chemistry; D006801:Humans; D008075:Lipoproteins, HDL / Q000737:chemistry*; D009036:Moths; D007862:Phosphatidylcholine-Sterol O-Acyltransferase / Q000737:chemistry; D011489:Protein Denaturation; D013816:Thermodynamics
D016632:Apolipoprotein A-I; D002352:Carrier Proteins; D008075:Lipoproteins, HDL; C030363:lipid transfer protein; D007862:Phosphatidylcholine-Sterol O-Acyltransferase