Effect of the human plasma apolipoproteins and phosphatidylcholine acyl donor on the activity of lecithin: cholesterol acyltransferase.

The human plasma apoproteins apoA-I and apoC-I enhanced the activity of partially purified lecithin: cholesterol acyltransferase five to tenfold with chemically defined phosphatidylcholine:cholesterol single bilayer vesicles as substrates. By contrast, apoproteins apoA-II, apoC-II, and apoC-III did not give any enhancement of enzyme activity. The activation by apoA-I and apoC-I differed, depending upon the nature of the hydrocarbon chains of phosphatidylcholine acyl donor. ApoA-I was most effective with a phosphatidylcholine containing an unsaturated fatty acyl chain. ApoC-I activated LCAT to the same extent with both saturated and unsaturated phosphatidylcholine substrates. Two of the four peptides obtained by cyanogen bromide cleavage of apoA-I retained some ability to activate LCAT. The efficacy of each of these peptides was approximately 25% that of the whole protein. Cyanogen bromide fragments of apoC-I were inactive. The apoproteins from HDL, HDL2, and HDL3, at low protein concentrations, were equally effective as activators of LCATand less effective than apoA-I. Higher concentrations of apoHDL, apoHDL2, and apoHDL3 inhibited LCAT activity. ApoC and apoA-II were both found to inhibit the activation of LCAT by apoA-I. The inhibition of LCAT by higher concentrations of apoHDL was not correlated with the aopA-II and apoC content.     

Phosphatidylcholine substrate specificity of lecithin: cholestrol acyltransferase-in high density lipoproteins and in lipids dispersions.

Lecithin: cholesterol acyltransferase (LCAT) was more highly activated by apolipoprotein A-I (apoA-I) with dimyristoyl phosphatidylcholine (DMPC) than with dilinoleoyl phosphatidylcholine (DLPC) when lipid dispersion of cholesterol and each phosphatidylcholine was used as a substrate. When the enzyme reactions were activated by whole apolipoproteins of high density lipoproteins (HDL), DLPC was more available to the LCAT reaction than DMPC with high concentrations of apoHDL in an incubation mixture. However, no detectable enzyme reaction was observed with dipalmitoyl phosphatidylcholine (DPPC) under both conditions. On the other hand, all of these phosphatidylcholines acted as substrates of LCAT when they were incorporated into HDL coupled to Sepharose. The order of their relative reactivities to cholesterol was DMPC, DPPC, AND DLPC under the conditions used.     

Characterization of proteoliposomes containing apoprotein A-I: a new substrate for the measurement of lecithin: cholesterol acyltransferase activity

Proteoliposome vesicles containing apoA-I, lecithin, and cholesterol (including labeled cholesterol) were prepared from various molar ratios of the three components by the cholate dialysis technique. Comparative studies on the sensitivity and efficiency of these proteoliposomes to serve as substrate for lecithin:cholesterol acyltransferase (LCATase) indicated that the proteoliposome with apoA-I:lecithin:cholesterol molar ratio of 0.8:250:12.5 was ideal for assaying LCATase activity of both plasma and purified enzyme. This proteoliposome was shown to be comparable in size by gel filtration (radius, 131.9 +/- 4.8 A, n = 6) and by electron microscopy (radius, 123.4 +/- 5.1 A, n = 100). The proteoliposome preparation was stable as LCATase substrate for at least 3 and 5 weeks, respectively, when stored at 4 degrees C and -20 degrees C, and was a better substrate for the enzyme activity assay than were lecithin-cholesterol liposomes incubated with apoA-I. Under the standardized assay system LCATase activity was a linear function of plasma enzyme added and was independent of the amount of plasma cholesterol added to the proteoliposomes in the range of 3 to 20 microliters of plasma. The mean LCATase activity by this method was 95.1 +/- 14.0 (range 76.5-122.5) nmol/hr per ml of plasma from fifteen normal human subjects. This method of substrate formation using the cholate dialysis technique permits the preparation of large amounts of stable, efficient, homogeneous, and well-defined substrate that is suitable for measuring low levels of enzyme activity, comparative studies, and large scale investigations of plasma LCATase, as well as studies of the mechanism and regulation of LCATase reaction.     

Inhibition of lecithin:cholesterol acyltransferase activity by synthetic phosphatidylcholine species containing eicosapentaenoic acid or docosahexaenoic acid in the sn-2 position.

Phospholipids isolated from the plasma of monkeys fed a diet enriched in fish oil were poor substrates for cholesteryl ester (CE) synthesis by the lecithin:cholesterol acyltransferase (LCAT) reaction relative to those from animals fed a lard containing diet when the phospholipids were used for the preparation of recombinant particles by cholate dialysis (Parks, J. S., B. C. Bullock, and L. L. Rudel. 1989. J. Biol. Chem. 264: 2545-2551). The purpose of the present study was to directly test the influence of eicosapentaenoic acid (20:5 n-3) and docosahexaenoic acid (22:6 n-3) in the sn-2 position of phosphatidylcholine (PC) on the activity of LCAT. PC species containing 1-palmitoyl-2-oleoyl PC (POPC), 1-palmitoyl-2-linoleoyl PC (PLPC), 1-palmitoyl-2-arachidonoyl PC (PAPC), 1-palmitoyl-2-eicosapentaenoyl PC (PEPC), or 1-palmitoyl-2-docosahexaenoyl PC (PDPC) were purchased or synthesized and made into recombinant particles of uniform size and composition with [14C]cholesterol and apoA-I using the cholate dialysis procedure. The recombinant particles (PC:cholesterol:apoA-I molar ratio = 42:1.9:1) exhibited the following order of reactivity towards purified human LCAT in vitro: POPC greater than PLPC greater than PEPC = PAPC greater than PDPC. The apparent Vmax/Km for recombinant particles containing PEPC and PDPC was 17% and 7% that of particles containing POPC, respectively. There was a linear decrease in CE formation when the percentage of PEPC or PDPC was increased from 0 to 100% relative to POPC in recombinant particles with a constant PC:cholesterol:apoA-I molar ratio, suggesting that the PEPC and PDPC were competitive inhibitors of the LCAT reaction.(ABSTRACT TRUNCATED AT 250 WORDS)     

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.     

Studies on the substrate specificity of human and pig lecithin: cholesterol acyltransferase: role of low-density lipoproteins

The substrate properties of low-density lipoprotein (LDL) fractions from human and pig plasma and of lipoprotein a [Lp(a)] upon incubation with either pig or human lecithin:cholesterol acyltransferase (LCAT, EC 2.3.1.43) were investigated and compared with those of pig high-density lipoproteins (HDL) or human HDL-3. The cholesterol esterification using purified native pig LDL-1, human LDL, or Lp(a) as a substrate was approximately 36-42% that of pig HDL or human HDL-3, while cholesteryl ester formation with pig LDL-2 was 41-47%. No significant difference was found in the substrate activity between pig HDL and human HDL-3, and between human LDL and Lp(a), respectively. After depletion of pig LDL-1, pig LDL-2, and human LDL from apolipoprotein A-I (apoA-I), cholesteryl ester formation decreased to about 22-28% of the value found with pig HDL. Depletion of human LDL from apolipoprotein E (apoE) did not result in significantly different esterification rates in comparison to native LDL. Total removal of non-apoB proteins from human LDL resulted in esterification rates of approximately 10-15% that of HDL. Readdition of apoA-I to all these LDL fractions produced solely in apoA-I-depleted LDL fractions an increase of cholesteryl ester formation, whereas in those LDL fractions that were additionally depleted from apoE and/or from apoC polypeptides, a further decrease in the esterification rate occurred.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effects of detergents on the lecithin:cholesterol acyltransferase reaction

This paper describes the effect of an ionic (sodium dodecyl sulfate; SDS) and a nonionic detergent (Triton X-100) on the substrate and enzyme components of the lecithin: cholesterol acyltransferase (LCAT) reaction. When the enzyme sources (purified or partially purified) or the respective substrates [high-density lipoproteins (HDL) or proteoliposomes] were preincubated with detergents, a consistent trend in LCAT activity was only seen when partially purified LCAT was used as the enzyme source. This trend indicated an approximately 25% increase in enzyme activity over the control when 10(-4) M SDS and 2 X 10(-3)% Triton X-100 were present in the preincubation mixtures, respectively. Those observations suggested that, during the preincubations and subsequent assays, the enzyme (in the presence of detergents) was allowed to dissociate from the endogenous substrate and subsequently interact with the exogenous substrate molecules. Additional experiments utilizing molecular-sieve chromatography with whole plasma and partially purified enzyme also showed that dissociation of LCAT/lipoprotein complexes occurred in the presence of detergent. SDS was also shown to enhance the reaction of LCAT in whole plasma with anti-LCAT antibody in an enzyme-linked immunoassay system, indicating that the detergent treatment facilitated the exposure of additional antigenic sites, perhaps via dissociation of the enzyme from plasma lipoproteins.     

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.     

Structural and functional properties of reconstituted high density lipoprotein discs prepared with six apolipoprotein A-I variants

Six apolipoprotein A-I (apoA-I) variants containing the following amino acid changes: Pro3----Arg, Pro4----Arg, Lys107----0 (Lys deletion) Lys107----Met, Pro165----Arg, and Glu198----Lys, and the corresponding normal allele products, were isolated by preparative isoelectric focusing from heterozygous individuals. The apoA-I samples were reconstituted with palmitoyloleoyl phosphatidylcholine (POPC) or dipalmitoyl phosphatidylcholine (DPPC), and small amounts of cholesterol, into discoidal high density lipoprotein (HDL) complexes in order to examine their lipid binding and structural properties as well as their ability to activate lecithin:cholesterol acyltransferase (LCAT). Starting with initial molar ratios around 100:5:1 for phosphatidylcholine-cholesterol-apolipoprotein, all the normal and variant apoA-Is were completely incorporated into reconstituted HDL (rHDL). The rHDL particle sizes and their distributions were examined by nondenaturing gradient gel electrophoresis, before and after incubation with LDL, to assess the folding of apoA-I in the complexes. Intrinsic Trp fluorescence properties of the rHDL were measured, as a function of temperature and guanidine hydrochloride concentration, to detect conformational differences in the apoA-I variants. In addition, the LCAT reaction kinetics were measured with all the rHDL, and the apparent kinetic constants were compared. In terms of the structure of the rHDL particles, all the normal variant apoA-Is had similar sizes (94, 96 A) and size distributions, and indistinguishable fluorescence properties, with the exception of the Lys107----0 mutant. This variant formed slightly larger particles that were resistant to rearrangements in the presence of LDL, and had an altered apoA-I conformation in the vicinity of the Trp residues. The kinetic experiments with LCAT indicated that the apoA-I variants, Lys107----0 and Pro165----Arg, in rHDL particles had statistically different (30 to 90%) kinetic constants from the corresponding normal allele products; however, the variability in the kinetic constants among the normal apoA-I products was even greater (40 to 430%). Therefore, we conclude that the effects of these six mutations in apoA-I on the activation of LCAT are minor, and that the structural effects on rHDL, and possibly native HDL, are insignificant with the exception of the Lys107----0 mutation.     

Interaction of N-methyl-anthraniloyl-labelled porcine apolipoprotein A-I with porcine lecithin: Cholesterol acyltransferase: An energy transfer study

• 1.1. To investigate whether a direct protein-protein interaction between apoA-I and lecithin: cholesterol acyltransferase (LCAT) is necessary for the activation of the enzyme, apoA-I was labelled with N-methylisatoic anhydride at lysine residues. The intermolecular resonance energy transfer from tryptophan residues of LCAT (donor) to N-methyl-anthraniloyl (NMA)-labelled apoA-I (NMA-apoA-I) (acceptor) was used as a sensitive fluorescence method for studying molecular interactions.
• 2.2. In the absence of lipids no fluorescence energy transfer was measurable.
• 3.3. Fluorescence energy transfer occurred from LCAT to NMA-apoA-I in the presence of liposomes with phospholipid/cholesterol ratios ranging from 5:1 to 18:1 and regardless whether only 1 or up to 5 NMA-apoA-I molecules resided at the liposome surface.
• 4.4. This indicates a preferred binding of the enzyme directly to or in spatial proximity to the activator protein NMA-apoA-I even if enough space at the liposome surface is available to allow LCAT binding at a distance, where no energy transfer is measurable.

     

The in vitro formation of HDL2 during the action of LCAT: the role of triglyceride-rich lipoproteins

We examined the effects of lecithin:cholesterol acyl transferase (LCAT) and of lipoprotein lipase (LPL) on the conversion of high density lipoproteins (HDL) towards fractions of lower densities using the analytical ultracentrifuge. Freshly isolated whole plasma was incubated for 24 h at 37 degrees C in the presence or absence of active enzyme systems. In some cases, lipoproteins were removed by selective precipitations; alternatively, we added triglyceride-rich lipoproteins (TGRLP) or Intralipid to the incubations. The results are as follows. 1) The incubation of whole plasma containing active LCAT leads to a conversion of HDL3 to a fraction of lower density, notably HDL2a. If LCAT is inhibited, the conversion is far less pronounced. 2) If very low and low density lipoproteins are removed by phosphotungstate precipitation and the supernatant is incubated with LCAT, HDL3 shifts towards higher densities. 3) The presence of phosphatidylcholine/cholesterol liposomes or the presence of blood cells as a source of additional LCAT substrate had only little influence on the HDL conversion in our system. 4) The addition of TGRLP or of Intralipid at minimal ratios of 2.5:1 caused an almost complete conversion of HDL3 to HDL2b. This conversion was dependent on active LCAT. 5) LPL also caused a shift of HDL3 to HDL2a if TGRLP was present. HDL2b, however, was not formed by LPL unless LCAT was active.     

Lecithin:cholesterol acyltransferase regulation. II. Effect of fluidity of egg phosphatidylcholine vesicles

The regulation of human plasma lecithin:cholesterol acyltransferase (LCAT) by changes in bilayer fluidity of substrate egg phosphatidylcholine (egg PC) unilamellar vesicles was investigated using pyrene excimer fluorescence to measure fluidity. Fluidity was decreased by adding up to 20% cholesterol or increased by adding up to 10% egg 2-lysophosphatidylcholine (lysoPC). The fluidizing effect of lysoPC was suppressed by the addition of cholesterol. LCAT activity with 10% cholesterol vesicles was decreased by adding 5% lysoPC, yet activity with 5% cholesterol vesicles was unaffected by adding 5% lysoPC. This difference may be explained by a balance between the known LCAT inhibitory effect of lysoPC and its ability to increase bilayer fluidity and thereby increase LCAT activity. LCAT esterification of up to 37% of vesicle cholesterol failed to alter the lysoPC/cholesterol balance sufficiently to influence activity in this system. The findings of our studies are in keeping with modulation of LCAT activity by bilayer fluidity, but fluidity changes caused by enzyme action are not sufficient to regulate that activity.     

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.     

Influence of hypoxanthine and other purines on the lecithin:cholesterol acyltransferase reaction in the plasma of rat and other species.

1. Esterification of radiolabelled cholesterol in the plasma of rat, mouse, pig, ox and, to a lesser extent, guinea pig was partially inhibited by hypoxanthine, xanthine and guanine; esterification in human plasma and in plasma from 12 other vertebrate species was unaffected by purines. 2. Esterification of endogenous cholesterol and the formation of lysolecithin in rat plasma were decreased in the presence of purines indicating that it was the lecithin:cholesterol acyltransferase (LCAT) reaction that was inhibited rather than the isotopic equilibration of labelled cholesterol with the endogenous substrate lipoproteins. 3. Maximum inhibition of the LCAT reaction in rat plasma occurred at 1.4 mM hypoxanthine or xanthine; inhibition was not dependent upon the concentration of LCAT or plasma lipoproteins but increased with the amount of lipoprotein depleted rat plasma (LDRP) present in the incubation mixture. 4. Partial inhibition of the LCAT reaction in rat or mouse plasma by purines had no significant effect on the fatty acyl composition of the cholesteryl esters (CE) formed by LCAT. 5. In the presence of heated rat plasma, LDRP or, to a lesser extent, rat high density lipoproteins (HDL) prepared from heated plasma, the LCAT reaction in human plasma was inhibited by hypoxanthine. 6. Rat HDL and LDRP prepared from plasma pre-incubated at 37 degrees C for 4 hr before heating increased and decreased, respectively, the inhibitory effect of hypoxanthine on human plasma LCAT compared with HDL and LDRP prepared from unincubated rat plasma.(ABSTRACT TRUNCATED AT 250 WORDS)     

Continuous fluorescence assay for lecithin:cholesterol acyltransferase using a water-soluble phosphatidylcholine.

A water-soluble fluorescent phosphatidylcholine, 1,2-bis[4-(1-pyreno-butanoyl]-sn-glycero-3-phosphocholine (DPybPC) has been used to develop a sensitive, continuous assay for pure lecithin:cholesterol acyltransferase (LCAT) in solution. The monomeric substrate allowed us to examine the reaction of LCAT in the absence of a lipid/water interface in terms of the sensitivity of the enzymatic reaction to anions, ionic strength, apolipoproteins A-I and A-II, and a series of lysophosphatidylcholines and fatty acids. In contrast to the reaction of LCAT with aggregated phosphatidylcholines, the reaction of DPybPC with LCAT was not significantly affected by anions, ionic strength, nor apolipoproteins, indicating that these are only effectors of the interfacial reaction. Lysophosphatidylcholines and fatty acids inhibited LCAT in a chain-length-dependent manner below the critical micellar concentrations of these amphiphiles, indicating that the products of the LCAT reaction can bind to the enzyme and affect its kinetics even in the absence of an interface.     

Synthesis of reconstituted high density lipoprotein (rHDL) containing apoA-I and apoC-III: the functional role of apoC-III in rHDL

Apolipoprotein (apo) C-III is a marker protein of triacylglycerol (TG)-rich lipoproteins and high-density lipoproteins (HDL), and has been proposed as a risk factor of coronary heart disease. To compare the physiologic role of reconstituted HDL (rHDL) with or without apoC-III, we synthesized rHDL with molar ratios of apoA-I:apoC-III of 1:0, 1:0.5, 1:1, and 1:2. Increasing the apoC-III content in rHDL produced smaller rHDL particles with a lower number of apoA-I molecules. Furthermore, increasing the molar ratio of apoC-III in rHDL enhanced the surfactant-like properties and the ability to lyse dimyristoyl phosphatidylcholine. Furthermore, rHDL containing apoC-III was found to be more resistant to particle rearrangement in the presence of low-density lipoprotein (LDL) than rHDL that contained apoA-I alone. In addition, the lecithin:cholesterol acyltransferase (LCAT) activation ability was reduced as the apoC-III content of the rHDL increased; however, the CE transfer ability was not decreased by the increase of apoC-III. Finally, rHDL containing apoC-III aggravated the production of MDA in cell culture media, which led to increased cellular uptake of LDL. Thus, the addition of apoC-III to rHDL induced changes in the structural and functional properties of the rHDL, especially in particle size and rearrangement and LCAT activation. These alterations may lead to beneficial functions of HDL, which is involved in anti-atherogenic properties in the circulation.     

Characterization of lecithin:cholesterol acyltransferase expressed in a human lung cell line

Lecithin:cholesterol acyltransferase (LCAT) is a key enzyme for the transfer of mammalian cholesterol from peripheral tissues to the liver. In patients deficient in LCAT, serum cholesterol levels rise and can lead to corneal opacity, proteinuria, anemia, and kidney failure. As early as 1968, relatively low volume transfusion of normal plasma was shown to temporarily correct the abnormal lipoprotein profiles in LCAT-deficient patients. However, despite the cloning, study, and extensive expression of LCAT in mammalian cell lines, there is still no viable, clinical therapy for LCAT deficiency. The current study was initiated to provide a source of recombinant human LCAT for enzyme replacement therapy. Accordingly, human LCAT has been cloned and expressed for the first time in a human cell line. The recombinant LCAT secreted by these cells was purified by phenyl-Sepharose chromatography, analyzed to determine the nature of its glycosylation, and tested for its enzymatic properties. The activity and basic kinetic parameters for the enzyme were determined using both a fluorescent water-soluble substrate and a macromolecular (proteoliposome) substrate. The enzymatic properties and the carbohydrate components of the recombinant LCAT were all sufficiently similar to those of the circulating human plasma enzyme, suggesting that this source of LCAT may be appropriate for use in some form of enzyme replacement therapy.     

Behavior of human apolipoprotein A-I: phospholipid and apoHDL:phospholipid complexes in vitro and after injection into rabbits

Apolipoprotein A-I was purified from human high density lipoprotein and complexed with polyunsaturated phosphatidylcholine (PC) in deoxycholate (Lipostabil); the bile salt was removed subsequently by dialysis. The behavior of the resultant apoA-I/PC complexes was compared with that of Lipostabil in vitro and after injection into rabbits. In vivo apoA-I/PC complexes had the density of HDL throughout but had both alpha and pre beta electrophoretic mobility, the latter probably reflecting dissociation of apoA-I from PC. Lipostabil initially behaved like LDL but gradually acquired the density of HDL after incubation with plasma and in vivo. Both preparations increased plasma total phospholipids in normolipidemic rabbits to a similar extent, but, increments in HDL phospholipid were greater after apoA-I/PC complexes were injected. ApoHDL/PC complexes, prepared in a similar manner, appeared to promote efflux of cholesterol from perfused rabbit aortas in the presence of lecithin:cholesterol acyltransferase (LCAT) activity, consistent with a stimulatory effect on cholesterol mobilization. Injection of apoHDL/PC complexes into hyperlipidemic rabbits decreased plasma cholesterol but increased HDL cholesterol, whereas Lipostabil decreased both. These findings suggest that human apoA-I/PC complexes resemble HDL in their behavior more closely than does Lipostabil, and show that both types of liposome undergo modification upon interaction with plasma. It remains to be shown whether they possess any therapeutic potential.     

Serum activity and hepatic secretion of lecithin:cholesterol acyltransferase in experimental hypothyroidism and hypercholesterolemia

Lecithin:cholesterol acyltransferase (LCAT), the major cholesterol esterifying enzyme in plasma, plays an important role in the removal of cholesterol from peripheral tissues. This study in rat focuses upon the effects of hypothyroidism and cholesterol feeding on serum activity and hepatic LCAT secretion. To obviate the effect that inclusion of high concentrations of cholesterol in the rat serum may have on the proteoliposome used in the assay of LCAT, very low and low density lipoproteins (VLDL and LDL) were removed by ultracentrifugation at d 1.063 g/ml. The molar esterification rate in the euthyroid VLDL + LDL-free serum was found to be 0.94 +/- 0.06 compared to 0.67 +/- 0.05 in hypothyroid rats and 1.56 +/- 0.14 in hypercholesterolemic rats. LCAT secretion by suspension cultures of hepatocytes from hypercholesterolemic rats was found to be significantly depressed when compared to that for euthyroid and hypothyroid animals. Secretion by hepatocytes from hypothyroid rats was depressed for the first 0-4 hr, but rapidly recovered. The depressed secretion of LCAT by hepatocytes from hypercholesterolemic rats correlates with the appearance in the media of apoE-rich, discoidal HDL. Discoidal HDL was six times more effective as a substrate for purified human LCAT than HDL from hypercholesterolemic serum, and twice as effective as serum and nascent HDL from euthyroid animals. It is concluded that the depressed LCAT activity in serum from hypothyroid rats is due to a depressed hepatic secretion of the enzyme and that the elevated serum activity of hypercholesterolemic rats may be related to a defect in LCAT clearance. Finally, the appearance of discoidal HDL in the medium upon culture of hepatocytes from hypercholesterolemic rats appears to be due to an inhibition of LCAT secretion by these cells.     

Utilization of diacylglycerol in phospholipid bilayers by pig brain diacylglycerol kinase and Rhizopus arrhizus lipase

Diacylglycerol kinase purified from pig brain cytosol could use sonication-dispersed diacylglycerol in the presence of its activator, phosphatidylcholine vesicles. However, the kinase failed to significantly use diacylglycerol cosonicated with phosphatidylcholine. Similarly, the kinase could not use diacylglycerol generated in microsomes by the back reaction of diacylglycerol choline phosphotransferase, though phospholipase C treatment of microsomes yielded effective substrate for the kinase. In order to elucidate the mechanism of these discrepant findings, we studied the activity of the purified kinase and Rhizopus arrhizus lipase utilizing dioleoylglycerol incorporated into various phospholipid vesicles. The inaccessibility of diacylglycerol contained in phospholipid vesicles was observed similarly for the two different enzymes. We considered that the apparent enzymic latency of diacylglycerol could be best accounted for by an extremely limited solubility of diacylglycerol in the outer leaflet of phospholipid bilayers. The experimental bases for this interpretation are: 1) diacylglycerol cosonicated with dihexanoyl phosphatidylcholine was exceptionally effective as substrate for the kinase; 2) the enzyme activities with cosonicated and separately sonicated lipids became similar when bile salts were present; 3) both enzymes could use diacylglycerol generated on phosphatidylcholine vesicles by a limited phospholipase C hydrolysis; and 4) phosphatidylcholine diacylglycerol vesicles at widely different molar ratios (from 1:0.014 to 1:0.2) were similarly ineffective as substrate for both enzymes.