A cholesteryl ester transfer complex in human plasma.

Abstract

Immunoadsorption affinity chromatography has been used to define the structure of lipoproteins in human plasma containing lecithin:cholesterol acyltransferase (EC 2.3.1.43) (LCAT) and transfer protein (apo D). The whole of LCAT was adsorbed by antibodies specific for apo D and for apo A-1, indicating that the enzyme is present in plasma exclusively as a complex with its cofactor (apo A-1) and product transfer protein (apo D) About 80% of apo D (but no LCAT) was removed by antibody to apo A-2, indicating the presence of most of apo D in the form of an enzyme-free complex with apo A-1 and apo A-2. After removal of LCAT with antibody to apo D, plasma was unreactive as a substrate with isolated LCAT, but substrate activity was generated by ultracentrifugal flotation with either intact or adsorbed plasma. The apparent stoichiometry of the complex with LCAT (LCAT:apo A-1:apo D) was 1.0:0.9:1.8; that of the complex containing apo A-1, apo A-2, and apo D was 3.9:2.2:1.0. Lecithin:cholesterol acyltransferase (EC 2.3.1.43) (LCAT) in human plasma catalyzes the synthesis of almost the whole of plasma cholesteryl ester content. Its activity is mediated via an apoprotein cofactor (apo A-1) (1), which is a component of lipoproteins isolated within the high density (HDL) range (2). Most plasma cholesteryl esters are associated with low density lipoprotein (LDL), which contains little or no apo A-1 and is not a substrate for LCAT (3). These esters are delivered to LDL through the activity of a cholesteryl ester transfer protein (apo D) recently identified and isolated (4). The rates of cholesteryl ester synthesis and transfer in plasma are essentially equal (4), suggesting synergism between these reactions and possible structural association of the transferase and transfer proteins. However, after centrifugal flotation, LCAT was recovered in the p > 1.21 g/cm3 density fraction whereas the greater part of transfer activity was present in the 1.063 < p < 1.21 g/cm3 density range (4, 5). A complex containing LCAT and apo D, if present in whole plasma, would play a key role in the regulation of plasma cholesterol metabolism, yet centrifugation is effective in dissociating plasma lipoprotein complexes (6). In this research, immunoadsorption chromatography has been used to determine the properties and apoprotein stoichiometry of complexes containing LCAT and apo D. Although absence of binding of one protein to immobilized antibody of another may be only ambiguous evidence for lack of association (because the complex might have dissociated during chromatography), binding of one protein to antibody of another is strong evidence that a complex exists in that form in the medium. Such evidence is here obtained for the association of LCAT and apo D. MATERIALS AND METHODSPreparation and Assay of Lipoprotein Apoproteins. Apo D was isolated as described (4) from human plasma HDL. It was a single species by polyacrylamide gel electrophoresis and was immunologically and chemically identical to the active cholesteryl ester transfer prote...

Transfers and exchanges of esterified cholesterol between plasma lipoproteins

“…However, in the presence of the ECTEP the concentration gradient created by an incorporation into HDL drives a net mass transfer of esterified cholesterol into VLDL and LDL; ultimately, the newly synthesized esterified cholesterol will equilibrate between all fractions. Recently, however, this view has been challenged by a suggestion that previous conclusions about the site of action of LCAT may have been an artefact of the ultracentrifugal separation process (Fielding & Fielding, 1980a).…”

“…It has been proposed subsequently that apolipoprotein D and LCAT exist physiologically as components of a small molecular complex which picks up and esterifies free cholesterol from various lipoproteins; the subsequent delivery of esterified cholesterol from the complex to lipoproteins has been attributed to a transfer protein role of apolipoprotein D (Fielding & Fielding, 1980a).…”

“…It has been proposed that, physiologically, LCAT exists as a complex with two HDL apolipoproteins: apolipoprotein Al, its obligatory cofactor, and apolipoprotein D, to which has been ascribed the role of transfer protein (Fielding & Fielding, 1980a). According to this view, a 'transferase-transfer' complex interacts with all lipoprotein fractions, picking up free cholesterol, esterifying it and delivering it back in ester form.…”

Associations of lecithin: cholesterol acyltransferase (LCAT) mass concentrations with exercise, weight loss, and plasma lipoprotein subfraction concentrations in men

“…The removal of free cholesterol from the lipoprotein surface creates a concentration gradient that favors the net transfer of cholesterol from cell membranes to the lipoprotein surface. Particles within the high density lipoprotein 3 (HDL 3 ) density range are the preferred substrate for LCAT [3], and the newly synthesized esterified cholesterol is primarily incorporated into HDL [4]. Most of the cholesteryl ester formed by LCAT on HDL is transferred to the apolipoprotein B containing lipoproteins [4,5], which are then cleared by receptors for LDL or triglyceride-rich lipoprotein remnants [6].…”

Apolipoprotein A-I, Phospholipid Vesicles, and Cyclodextrins as Potential Anti-Atherosclerotic Drugs: Delivery, Pharmacokinetics, and Efficacy

“…This particle may be a particularly important factor for RCT (Fielding and Fielding 1980;Barbaras et al 1987;Castro and Fielding 1988;Francone et al 1989;Davidson et al 1995b). Transfer of carried cholesterol from this particle to a higher molecular weight pre-b apoA-I species, to LDL, and to the a -migrating apoA-I that makes up 96% of total apoA-I in plasma occurs (Castro and Fielding 1988).…”