Stimulation of lecithin:cholesterol acyltransferase activity by apolipoprotein A-II in the presence of apolipoprotein A-I

Various combinations of incorporation and addition of apolipoprotein A-I (apo A-I) and apolipoprotein A-II (apo A-II) individually or together to a defined lecithin-cholesterol (250/12.5 molar ratio) liposome prepared by the cholate dialysis procedure were used to study the effect of apo A-II on lecithin:cholesterol acyltransferase (LCAT, EC 2.3.1.43) activity of both purified enzyme preparations and plasma. When apo A-I (0.1-3.0 nmol/assay) alone was incorporated or added to the liposome, apo A-I effectively activated the enzyme. By contrast, when apo A-II (0.1-3.0 nmol/assay) alone was incorporated into or added to the liposome, apo A-II exhibited minimal activation of LCAT activity, approximately 1% of the activity obtained by an equal amount of apo A-I. Addition of apo A-II (0.1-3.0 nmol/assay) together with apo A-I (0.8 nmol/assay) to the liposome reduced the LCAT activity to approximately 30% of the level obtained with addition of apo A-I alone. On the other hand, addition of apo A-II (0.1-3.0 nmol/assay) or addition of lecithin-cholesterol liposome containing apo A-II (0.1-3.0 nmol/assay) to lecithin-cholesterol liposome containing apo A-I (0.8 nmol/assay) did not significantly alter apo A-I activation of LCAT activity. However, when the same amounts (0.1-3.0 nmol/assay) of apo A-II were incorporated together with apo A-I (0.8 nmol/assay) into the liposome, apo A-II significantly stimulated LCAT activity as compared to activity obtained with incorporation of apo A-I alone. The maximal stimulation was obtained with 0.4 nmol apo A-II/assay for both purified and plasma enzyme. At this apo A-II concentration, approximately 4-fold and 1.8-fold stimulation was observed for purified enzyme and plasma enzyme, respectively. These results indicated that apo A-II must be incorporated together with apo A-I into lecithin-cholesterol liposomes to exert its stimulatory effect on LCAT activity and that apo A-II in high-density lipoprotein may play an important role in the regulation of LCAT activity.     

Selective extraction of lecithin:cholesterol acyltransferase (EC 2.3.1.43) from human plasma

A method for the rapid extraction of lecithin:cholesterol acyltransferase (LCAT) from human plasma or serum has been developed. The method is based on direct treatment of acidified plasma of fully conserved enzyme activity, with the strong ion exchanger Q-Sepharose, which under the experimental conditions bound all LCAT but only about 10% of the total protein content of the plasma, no albumin and essentially no lipoproteins. This corresponds to a 10-fold purification. Only traces of apolipoprotein A-I remained in the quantitatively desorbed LCAT preparation which, however, contained a residual fraction of apolipoprotein D and acidic plasma proteins.The present one-step procedure for extraction of LCAT in high yields from human plasma represents a simple and efficient alternative to the first step in previously described methods for preparation of the enzyme to homogeneity.     

Isolation and properties of porcine lecithin:cholesterol acyltransferase

Lecithin: cholesterol acyltransferase (LCAT, phosphatidylcholine: sterol O-acyltransferase, EC 2.3.1.43) was purified approximately 20 000-fold from pig plasma by ultracentrifugation, phenyl-Sepharose and hydroxyapatite chromatography. Purified LCAT had an apparent relative molecular mass of 69 000 +/- 2000. By isoelectrofocusing it separated into five or six bands with pI values ranging from pH 4.9 to 5.2. The amino acid composition was similar to that of the human enzyme. An antibody against pig LCAT was prepared in goat. The antibody reacted against pig LCAT and gave a reaction of partial identity with human LCAT. Incubation of pig plasma or purified enzyme with the antibody virtually inhibited LCAT activity. The same amount of antibody inactivated only 62% of the LCAT activity in human serum. Pig and human LCAT were activated to the same extent by either human or pig apolipoprotein A-I (apo-A-I) using small liposomes as substrate. Human apoA-I, however, caused a higher esterification rate for both enzymes. Using apoA-I and small liposomes as a substrate, the addition of apoC-II up to 4 micrograms/ml had no effect on the LCAT reaction, but above this concentration LCAT was inhibited. Small liposomes with phosphatidylcholine/cholesterol molar ratios of 3:1 up to 8.4:1 did not show any significant differences in the LCAT reaction, when used as substrates in the presence of various amounts of apoA-I and albumin. In contrast, the LCAT activity was significantly reduced by liposomes with phosphatidylcholine/cholesterol molar ratios below 3:1.     

Separation of free and apolipoprotein D-associated human plasma lecithin: cholesterol acyltransferase

A high performance gel filtration method for the rapid and reproducible separation of free and apolipoprotein D-associated lecithin: cholesterol acyltransferase (LCAT) originating from human plasma has been developed. Starting from step 3 of a previously invented covalent chromatography procedure, free LCAT was obtained as a well separated fraction in a yield of 55% of that injected into the column. The free LCAT had a specific activity of over 34,000 units/mg and did not contain apolipoprotein D or any other contaminant in the injected sample. Further 28% of LCAT with fully retained activity was recovered in a second fraction, demonstrating a 66,000 u LCAT associated with all apolipoprotein D occurring as a mean 33,000 u and a minor 66,000 u species and with at least two unidentified proteins with apparent molecular masses of 76,000 u and 43,000 u, respectively. Both free and apolipoprotein D-associated LCAT accepted the free cholesterol of heat-inactivated plasma selectively depleted of VLDL and LDL (alpha-LCAT activity) and of HDL (beta-LCAT activity) as substrate.     

Activation of lecithin: cholesterol acyltransferase by apolipoproteins E-2, E-3, and A-IV isolated from human plasma

Apolipoprotein A-IV, apolipoprotein E-2 and apolipoprotein E-3 were individually incorporated into defined phosphatidylcholine/cholesterol liposomes for study of lecithin:cholesterol acyltransferase activation. Enzyme activities obtained with these liposomes were compared with that from liposomes containing purified apolipoprotein A-I. Apolipoprotein A-IV, apolipoprotein E-2, and apolipoprotein E-3 all activated lecithin:cholesterol acyltransferase. With purified enzyme and with egg yolk phosphatidylcholine as the acyl donor, maximal activation was obtained at a concentration of approximately 0.5 nmol for apolipoprotein A-IV and 0.4 nmol for the apolipoprotein E isoforms. Apolipoprotein A-IV was approximately 25% as efficient as apolipoprotein A-I for the activation of purified enzyme; apolipoprotein E-2 was 40% as efficient, and apolipoprotein E-3, 30%. Similar activation results were obtained using plasma as the enzyme source. Analysis of the plasma of patients with absence of apolipoprotein A-I or with only trace amounts of apolipoprotein A-I exhibited a reduced rate of cholesterol esterification and lecithin:cholesterol acyltransferase activity that was proportional to the reduced level of the enzyme's mass. These results indicate that apolipoprotein A-IV and apolipoprotein E may serve as physiological cofactors for the enzyme reaction.     

Activation of lecithin-cholesterol acyltransferase by apolipoprotein D: comparison of proteoliposomes containing apolipoprotein D, A-I or C-I

To study the activation of lecithin-cholesterol acyl transferase (LCAT) (phosphatidylcholine:sterol O-acyltransferase, EC 2.3.1.43) by apolipoprotein D in comparison to apolipoproteins A-I and C-I, proteoliposomes with a phosphatidylcholine/free cholesterol molar ratio of 24:1, containing 10-300 micrograms/ml of apolipoproteins were used. The proteoliposomes were prepared by the cholate dialysis technique. In all proteoliposome preparations we found rouleaux structures and stacked discs. The particles formed with apolipoprotein A-I were the most homogeneous, followed by apolipoprotein D- and apolipoprotein C-I-containing particles. Apolipoprotein A-I was the most potent LCAT activator in our system followed by apolipoproteins C-I and D. The fractional esterification rate observed with apolipoprotein D-containing substrates amounted to 15-48% that of apolipoprotein A-I-containing ones. Neither apolipoprotein A-I- nor C-I-containing proteoliposomes gave linear reaction kinetics with LCAT. Even during the first 15-30 min of incubation, the kinetics deviated strikingly from linearity at all apolipoprotein concentrations. In contrast, proteoliposomes containing apolipoprotein D exhibited linear reaction kinetics up to 60-90 min. At low apolipoprotein A-I concentrations (5 micrograms/ml), the addition of apolipoprotein D to the incubates resulted in significantly higher esterification rates as compared to substrates containing apolipoprotein A-I only. This was not the case using substrates with high apolipoprotein A-I concentrations (50 micrograms/ml). From our results we speculate that apolipoprotein D may have some stabilizing effect on the enzyme LCAT.     

A method for isolating human plasma lecithin:cholesterol acyltransferase without using anti-apolipoprotein D, and its characterization

A method for isolating human plasma lecithin:cholesterol acyltransferase (EC 2.3.1.43) purified more than 50 000-fold is described. The crude enzyme obtained by initial ammonium sulfate and citric acid treatment of 21 of human plasma is subjected to repeated DEAE-cellulose chromatography to yield a preparation purified more than 600-fold. Hydroxyapatite chromatography of concentrates from this fraction using 0.5 mM phosphate buffer, pH 6.8, yields enzyme preparations purified more than 50 000-fold. The enzyme isolated by this procedure was free of apolipoprotein D, as shown by the absence of an arc in immunodiffusion with anti-apolipoprotein D. The enzyme showed a single band by polyacrylamide gel electrophoresis in the presence and absence of SDS. Upon analytical isoelectrofocusing the enzyme separated into three iso forms with isoelectric points below that of egg albumin (pI 4.6). The enzyme was characterized by a high content of glutamic acid, leucine and glycine, and a lower content of tyrosine. The enzyme possessed both transferase and phospholipase A2 activities and both activities show absolute requirement for apolipoprotein A-I. The purified enzyme was injected into Balb/c mice and the antiserum reacted both with the purified enzyme and normal human serum in immunodiffusion, giving lines of complete identity. The antiserum gave no precipitation lines with albumin or apolipoprotein D, providing additional evidence for the absence of apolipoprotein D in the purified enzyme. The gamma-globulin isolated from the antiserum inhibited human lecithin:cholesterol acyltransferase activity.     

New substrate for determination of serum lecithin:cholesterol acyltransferase

Serum lecithin:cholesterol acyltransferase (LCAT) was estimated by enzymatically measuring the decrease in unesterified cholesterol after incubation of serum with liposomes. A high-performance liquid chromatography (HPLC) study showed the uptake of the lipids of liposomes by serum high density lipoprotein. Of all the examined liposomes prepared from cholesterol and various synthetic phosphatidylcholines, liposomes with dimyristoylphosphatidylcholine (DMPC) were found to be the most reactive in the LCAT reaction. When serum was used as an enzyme source, addition of purified apolipoprotein A-I, which is known to be an endogenous activator of LCAT, to the assay mixture resulted in a slight decrease in enzyme activity. Using DMPC-cholesterol liposomes as the substrate, the LCAT activities in 120 human sera showed a mean value of 485.4 +/- 64.6 nmol/hr per ml (mean +/- SD), which is 4.4- to 5.4-fold higher than the values obtained by self-substrate methods. LCAT activity was a linear function of the serum sample volume up to 670 nmol/hr per ml and coefficients of variation (CV) less than 4% were obtained under the standardized conditions. Moreover, when partially purified LCAT was added to various heat-inactivated sera, the activity was efficiently recovered. These results suggest that this method is sensitive, reproducible, and not greatly influenced by serum components.     

Formation of high density lipoproteins containing both apolipoprotein A-I and A-II in the rabbit

Human plasma HDLs are classified on the basis of apolipoprotein composition into those that contain apolipoprotein A-I (apoA-I) without apoA-II [(A-I)HDL] and those containing apoA-I and apoA-II [(A-I/A-II)HDL]. ApoA-I enters the plasma as a component of discoidal particles, which are remodeled into spherical (A-I)HDL by LCAT. ApoA-II is secreted into the plasma either in the lipid-free form or as a component of discoidal high density lipoproteins containing apoA-II without apoA-I [(A-II)HDL]. As discoidal (A-II)HDL are poor substrates for LCAT, they are not converted into spherical (A-II)HDL. This study investigates the fate of apoA-II when it enters the plasma. Lipid-free apoA-II and apoA-II-containing discoidal reconstituted HDL [(A-II)rHDL] were injected intravenously into New Zealand White rabbits, a species that is deficient in apoA-II. In both cases, the apoA-II was rapidly and quantitatively incorporated into spherical (A-I)HDL to form spherical (A-I/A-II)HDL. These particles were comparable in size and composition to the (A-I/A-II)HDL in human plasma. Injection of lipid-free apoA-II and discoidal (A-II)rHDL was also accompanied by triglyceride enrichment of the endogenous (A-I)HDL and VLDL as well as the newly formed (A-I/A-II)HDL. We conclude that, irrespective of the form in which apoA-II enters the plasma, it is rapidly incorporated into spherical HDLs that also contain apoA-I to form (A-I/A-II)HDL.     

High density lipoprotein subpopulations from galactosamine-treated rats and their transformation by lecithin:cholesterol acyltransferase

It is known that an acute hepatotoxicity is produced in rats by intraperitoneal administration of galactosamine; a consequence of this treatment is a marked deficiency of lecithin:cholesterol acyltransferase (LCAT) activity in the plasma compartment. In this study high density lipoprotein (HDL) from galactosamine-treated rats was isolated, resolved into subpopulations, and characterized. In contrast to HDL from control rats, which elutes from gel filtration columns as a single peak and has a diameter of 13.1 nm, HDL from the galactosamine-treated animals was found to elute in five major zones with diameters of 7.8-35 nm. Characterization of these subpopulations has revealed that the larger fractions are enriched in apolipoprotein E, phospholipid, and cholesterol, but contain little cholesteryl ester, while the smallest two fractions contain mainly apolipoprotein A-I, are enriched in phospholipid, and have 50-60% of their cholesterol in the ester form. Incubation of HDL from treated rats with a source of LCAT activity plus low and very low density lipoproteins caused transformation of these subpopulations into a species which, by size and composition, was essentially identical to control rat HDL. In addition, when the subpopulations were individually incubated with purified human lecithin:cholesterol acyltransferase and bovine serum albumin, there was a similar convergence toward a moderate particle size approximating control rat HDL. Cross-linking studies showed that incubation with LCAT activity reduced the heterogeneity of the treated rat HDL. We conclude that the galactosamine treatment induces a complex mixture of HDL that bears strong similarities to the small, apoA-I rich and large, apoE-rich particles seen in LCAT deficiency or secreted by hepatic cells in culture. Furthermore, these species appear to coalesce in the presence of the d greater than 1.21 g/ml fraction of control serum to yield a fairly homogeneous population that resembles control rat HDL in size, composition, and apoprotein content.     

Effect of apolipoprotein activators on the specificity of lecithin:cholesterol acyltransferase: determination of cholesteryl esters formed in A-I/C-III deficiency.

Although it is known that plasma lecithin:cholesterol acyltransferase (LCAT) is activated by several apolipoproteins (apo) including A-I, C-I, D, A-IV, and E, it is not clear what the physiological importance of having different apolipoprotein activators is. One possible explanation is that the activation by different apolipoproteins may result in the utilization of different species of phosphatidylcholine (PC), leading to the formation of different species of cholesteryl esters (CE). In order to determine this possibility, we analyzed the molecular species composition of PC and CE in two patients with familial deficiency of apoA-I and apoC-III. The LCAT activity, assayed by three different procedures, was found to be 36-63% of the control value. The lower LCAT activity, however, was due to deficiency of the enzyme rather than the absence of apoA-I. The patients' plasma was relatively enriched with sn-2 18:2 PC species reflecting the partial deficiency of LCAT activity. The fatty acid composition of plasma CE was not significantly different from that of controls. HPLC analysis of labeled CE formed after incubation of plasma with [C14]cholesterol showed no significant difference in the species of CE synthesized by the LCAT reaction. The transfer of pre-existing as well as newly formed CE from HDL to the apoB-containing lipoproteins was accelerated compared to control plasma. These results show that the absence of apoA-I does not significantly affect either the activity or the specificity of LCAT, and that the other apolipoprotein activators can substitute adequately for it.     

Physical and chemical characteristics of apolipoprotein A-I-lipid complexes produced by Chinese hamster ovary cells transfected with the human apolipoprotein A-I gene

Chinese hamster ovary cells transfected with the human apolipoprotein A-I gene linked to the human metallothionein gene promoter region secrete large quantities of apolipoprotein A-I (7.1 +/- 0.4% total secreted protein) in the presence of zinc. Approx. 16% of the secreted apolipoprotein A-I is complexed with lipid and can be isolated ultracentrifugally at d less than or equal to 1.21 g/ml. The latter complexes are composed of discs and vesicles as judged by electron microscopy and can be further separated by column chromatography into three fractions: fraction I, mostly vesicles (60-260 nm) and large discs (18-20 nm diameter); fraction II, discs 14.2 +/- 2.6 nm diameter; and fraction III, nonresolvable by electron microscopy. The latter fraction is extremely lipid-poor (94% protein, 6% phospholipid); in contrast, the protein, phospholipid and unesterified cholesterol content for the other fractions are 43, 33 and 24%, respectively, for fraction I and 53, 33 and 14%, respectively, for fraction II. Fraction II particles contain three and four apolipoprotein A-Is per particle as determined by protein crosslinking while large structures in fraction I contain primarily six to seven apolipoprotein A-Is per particle. Following incubation with purified lecithin: cholesterol acyltransferase, discoidal particles were transformed into apparent spherical particles 12.9 +/- 3.4 nm diameter; this transformation coincided with 19-21% conversion of unesterified cholesterol to esterified cholesterol. The apolipoprotein A-I-lipid complexes isolated from Chinese hamster ovary cell media are similar to nascent HDL found in plasma of lecithin:cholesterol acyltransferase-deficient patients and those secreted by the human hepatoma line, Hep G2. The ability of the Chinese hamster ovary cell nascent HDL-like particles to undergo transformation in the presence of purified lecithin:cholesterol acyltransferase indicates that they are functional particles.     

Synthesis and secretion of lecithin-cholesterol acyltransferase by the human hepatoma cell line HepG2

The human liver cell line HepG2 was investigated for its synthesis and secretion of lecithin-cholesterol acyltransferase. The cells were grown to confluency in Eagle's minimal essential medium plus 10% fetal bovine serum. At the onset of the study, fetal bovine serum was removed and cells were grown in minimal essential medium only. At 6, 12, 24, and 48 h the cells were harvested, and the culture medium collected at each time point was assayed for lecithin-cholesterol acyltransferase mass and activity, cholesterol esterification rate, and apolipoprotein A-I mass. The rate of the enzyme secretion measured by both mass and activity was linear over 24 h of culture. The enzyme mass by radioimmunoassay was 1.7, 4.1, 7.9 and 13.7 ng/ml culture medium (or 8.3, 19.9, 38.5 and 66.7 ng/mg cell protein), respectively, and enzyme activity using an exogenous source of phosphatidylcholine/cholesterol liposomes containing apolipoprotein A-I as substrate was 85, 170, 315, and 402 pmol cholesterol esterified/h per ml culture medium (or 414, 828, 1534 and 1957 pmol cholesterol esterified/h per mg cell protein) for 6, 12, 24, and 48 h of culture, respectively. The endogenous cholesterol esterification rate of the culture medium was 47, 104, 224 and 330 pmol/h per ml and apolipoprotein A-I mass was 305, 720, 2400 and 3940 ng/ml culture medium over the same time frame. In contrast to culture medium, low levels of enzyme activity (approximately 10% of that in culture medium at 24 and 48 h) were observed in the extracts of HepG2 cells. The enzyme secreted by HepG2 was found to be similarly activated by apolipoprotein A-I, apolipoprotein E, or apolipoprotein A-IV, and was similarly inhibited by phenylmethylsulfonyl fluoride, dithiobisnitrobenzoate, p-hydroxymercuribenzoate, or iodoacetate as compared to human plasma enzyme. High-performance gel filtration of the culture medium revealed that the HepG2-secreted enzyme was associated with a fraction having a mean apparent molecular weight of approximately 200,000. We concluded that human hepatoma HepG2 cells synthesize and secrete lecithin-cholesterol acyltransferase, which is functionally homologous to the human plasma enzyme.     

A fluorescence method to detect and quantitate sterol esterification by lecithin:cholesterol acyltransferase

We describe a simple but sensitive fluorescence method to accurately detect the esterification activity of lecithin:cholesterol acyltransferase (LCAT). The new assay protocol employs a convenient mix, incubate, and measure scheme. This is possible by using the fluorescent sterol dehydroergosterol (DHE) in place of cholesterol as the LCAT substrate. The assay method is further enhanced by incorporation of an amphiphilic peptide in place of apolipoprotein A-I as the lipid emulsifier and LCAT activator. Specific fluorescence detection of DHE ester synthesis is achieved by employing cholesterol oxidase to selectively render unesterified DHE nonfluorescent. The assay accurately detects LCAT activity in buffer and in plasma that is depleted of apolipoprotein B lipoproteins by selective precipitation. Analysis of LCAT activity in plasmas from control subjects and sickle cell disease (SCD) patients confirms previous reports of reduced LCAT activity in SCD and demonstrates a strong correlation between plasma LCAT activity and LCAT content. The fluorescent assay combines the sensitivity of radiochemical assays with the simplicity of nonradiochemical assays to obtain accurate and robust measurement of LCAT esterification activity.     

Cholesterol esterification by lecithin-cholesterol acyltransferase in A-I-free plasma

Lecithin-cholesterol acyltransferase (LCAT) mass, activity and endogenous cholesterol esterification rate were measured in plasma and apolipoprotein A-I-free (A-I-free) plasma from two normolipidemic and two hyperlipidemic subjects, and from a patient with Tangier disease. A-I was removed from plasma by an anti-A-I immunosorbent. LCAT activity was measured using an exogenous substrate. The plasma LCAT concentration of the four non-Tangier subjects was 4.63 +/- 0.64 micrograms/ml (mean +/- S.D.); means of 26 +/- 7% of total LCAT mass and 22 +/- 11% of plasma LCAT activity were found in their A-I-free plasma. The plasma LCAT concentration of the Tangier subject was 1.49 micrograms/ml. About 95% of LCAT mass and all LCAT activity were found in the A-I-free plasma. Thus, the LCAT mass (1.4 micrograms/ml) and activity (43.1 nmol/h per ml) in Tangier A-I-free plasma were not significantly different from that found in the four non-Tangier A-I-free plasmas (mass = 1.21 +/- 0.44 micrograms/ml; activity: 27.3 +/- 18.4 nmol/h per ml). Although the LCAT activity per unit mass of the enzyme in plasma and A-I-free plasma were comparable (24.9 +/- 2.8 vs. 22.8 +/- 7.8 nmol/h per micrograms LCAT, n = 5), the plasma cholesterol esterification rate of A-I-free plasma from all subjects was lower than that found in plasma (7.5 +/- 2.7 vs. 13.0 +/- 3.8 nmol/h per micrograms LCAT). In conclusion, although A-I-containing lipoproteins are the preferred substrates of LCAT, other LCAT substrates and cofactors are found in A-I-free plasma along with LCAT. Thus, non-A-I-containing particles can serve as physiological substrates for cholesterol esterification mediated by LCAT.     

Formation of spherical, reconstituted high density lipoproteins containing both apolipoproteins A-I and A-II is mediated by lecithin:cholesterol acyltransferase

Previous studies have provided detailed information on the formation of spherical high density lipoproteins (HDL) containing apolipoprotein (apo) A-I but no apoA-II (A-I HDL) by an lecithin:cholesterol acyltransferase (LCAT)-mediated process. In this study we have investigated the formation of spherical HDL containing both apoA-I and apoA-II (A-I/A-II HDL). Incubations were carried out containing discoidal A-I reconstituted HDL (rHDL), discoidal A-II rHDL, and low density lipoproteins in the absence or presence of LCAT. After the incubation, the rHDL were reisolated and subjected to immunoaffinity chromatography to determine whether A-I/A-II rHDL were formed. In the absence of LCAT, the majority of the rHDL remained as either A-I rHDL or A-II rHDL, with only a small amount of A-I/A-II rHDL present. By contrast, when LCAT was present, a substantial proportion of the reisolated rHDL were A-I/A-II rHDL. The identity of the particles was confirmed using apoA-I rocket electrophoresis. The formation of the A-I/A-II rHDL was influenced by the relative concentrations of the precursor discoidal A-I and A-II rHDL. The A-I/A-II rHDL included several populations of HDL-sized particles; the predominant population having a Stokes' diameter of 9.9 nm. The particles were spherical in shape and had an electrophoretic mobility slightly slower than that of the alpha-migrating HDL in human plasma. The apoA-I:apoA-II molar ratio of the A-I/A-II rHDL was 0.7:1. Their major lipid constituents were phospholipids, unesterified cholesterol, and cholesteryl esters. The results presented are consistent with LCAT promoting fusion of the A-I rHDL and A-II rHDL to form spherical A-I/A-II rHDL. We suggest that this process may be an important source of A-I/A-II HDL in human plasma.     

High density lipoprotein particle size distribution in subjects with obstructive jaundice

High density lipoproteins (HDL) from 14 patients with obstructive jaundice were examined by gradient gel electrophoresis to determine the effect of obstruction on particle size distribution. HDL from 7 of these patients were fractionated by gel permeation chromatography and further characterized by electron microscopy, SDS gel electrophoresis, apolipoprotein A-I and apolipoprotein A-II immunoturbidimetry, and analysis of chemical composition. In addition, lecithin:cholesterol acyltransferase (LCAT) activity was measured and correlated with plasma apolipoprotein A-I concentration and particle size distribution. HDL were abnormal in all patients regardless of severity, cause, or duration of obstruction. The major HDL subfraction in normal subjects, HDL3a (radius 4.1-4.3 nm) was either absent or considerably diminished, and HDL2b (radius 5.3 nm) was also frequently absent. Very small particles comparable in size to normal HDL3c (radius 3.8 nm) were prominent. In patients with a bilirubin concentration greater than 250 mumol/l, normal HDL had totally disappeared and were replaced by large discoidal particles of radius 8.5 nm and small spherical particles of radius 3.6-3.7 nm. Both populations of particles were markedly depleted of cholesteryl ester and enriched in free cholesterol and phospholipid. The discoidal particles were rich in apolipoproteins E, A-I, A-II, and C, while the small spherical particles contained predominantly apolipoprotein A-I. LCAT activity was diminished in all subjects to 8-54% of normal, and was strongly positively correlated (r = 0.91 P less than 0.05) with plasma apolipoprotein A-I levels.     

The role of apolipoprotein A-I and apolipoprotein A-II in high-density lipoprotein binding to human adipocyte plasma membranes

Adipocyte plasma membranes purified from omental fat tissue biopsies of massively obese subjects possess specific binding sites for high-density lipoprotein (HDL3). This binding was independent of apolipoprotein E as HDL3 isolated from plasma of an apolipoprotein E-deficient individual was bound to a level comparable to that of normal HDL3. To examine the importance of apolipoprotein A-I, the major HDL3 apolipoprotein, in the specific binding of HDL3 to human adipocytes, HDL3 modified to contain varying proportions of apolipoproteins A-I and A-II was prepared by incubating normal HDL3 particles with different amounts of purified apolipoprotein A-II. As the apolipoproteins A-I-to-A-II ratio in HDL3 decreased, the binding of these particles to adipocyte plasma membranes was reduced. Compared to control HDL3, a 92 +/- 3.1% reduction (mean +/- S.E., n = 3) in maximum binding capacity was observed along with an increased binding affinity for HDL3 particles in which almost all of the apolipoprotein A-I had been replaced by A-II. The uptake of HDL cholesteryl ester by intact adipocytes as monitored by [3H]cholesteryl ether labeled HDL3, was also significantly reduced (about 35% reduction, P less than 0.005) by substituting apolipoprotein A-II for A-I in HDL3. These data suggest that HDL binding to human adipocyte membranes is mediated primarily by apolipoprotein A-I and that optimal delivery of cholesteryl ester from HDL to human adipocytes is also dependent on apolipoprotein A-I.     

Properties of phosphatidylethanolamine-containing phospholipid-apolipoprotein complexes modified by lecithin-cholesterol acyltransferase

The effect of the inclusion of phosphatidylethanolamine (PE), a phospholipid with unusual packing properties, on the substrate properties of protein-lipid complexes toward lecithin-cholesterol acyltransferase (LCAT) has been studied. Recombinant particles of apolipoprotein A-I with dimyristoylphosphatidylcholine (DMPC), dilauroylphosphatidylethanolamine (DLPE) and cholesterol were prepared at a molar ratio of 1:140:14 (A-I/DMPC/cholesterol) or 1:70:70:14 (A-I/DMPC/DLPE/cholesterol); the efficiency of cholesterol incorporation into complexes containing phosphatidylethanolamine was found to be very pH-dependent, with enhanced cholesterol incorporation at elevated pH values. By incubating the complexes with either purified human LCAT or the d greater than 1.21 g/ml fraction of rat serum as a source of LCAT activity, it was found that a high degree of cholesterol esterification could be achieved with either complex; however, the DLPE-containing complex possessed a much smaller Stokes' diameter than the DMPC-only particle despite compositional similarities between these complexes. With respect to particle diameter the DLPE-containing particles behaved more like complexes prepared with egg yolk lecithin than did complexes prepared with DMPC alone. When human LDL was added to the incubations to provide a source of additional cholesterol, the products were markedly different. Concomitant with an increased cholesteryl ester core was an increase in the protein stoichiometry in both types of particles, from 2 to 3 or 4 apo A-I per particle. The proportion of DLPE to DMPC in the products was reduced from 1:1 to 0.3:1, reflecting a preferential hydrolysis of PE by LCAT, and the Stokes' diameters of the DMPC-only and the DLPE-containing complexes were closely similar. We conclude that the presence of elevated proportions of certain phospholipid species may significantly alter both the physical properties of the particles and their substrate properties with regard to reactions with enzymes of lipid metabolism.