Interaction of rat lecithin-cholesterol acyltransferase with rat apolipoprotein A-I and with lecithin-cholesterol vesicles.

The interaction of rat plasma lecithin-cholesterol acyltransferase with lecithin-cholesterol vesicles and with rat apo-A-I was studied in comparison with that of human plasma lecithin-cholesterol acyltransferase to clarify the reaction mechanism of rat plasma lecithin-cholesterol acyltransferase. The interaction of both human and rat lecithin-cholesterol acyltransferase with lecithin-cholesterol vesicles was investigated by gel permeation chromatography on Superose 12. Both enzymes had almost the same affinity to the vesicles. The affinity of rat enzyme to rat apo-A-I was stronger than that of human enzyme to human apo-A-I when estimated on the apo-A-I-Sepharose 4B column. When human apo-A-I was added to the human enzyme/vesicle mixture which contained the enzyme-vesicle complex, the enzyme was effectively dissociated from the complex. But when rat apo-A-I was added to the rat enzyme/vesicle mixture, apo-A-I-enzyme-vesicle complex was still recognized by its elution pattern on gel permeation chromatography. This suggests that the mixture of rat enzyme, rat apo-A-I, and vesicles, which are the major components in the rat lecithin-cholesterol acyltransferase reaction, forms a stronger complex than do the components of the human reaction.     

Stimulation of serum lecithin-cholesterol acyltransferase activity by phenobarbital in the rat

The activity of serum lecithin-cholesterol acyltransferase was increased on administration of phenobarbital to the rat. This effect was dependent on dose and elapsed time after administration of the drug. Phenobarbital did not stimulate lecithin-cholesterol acyltransferase activity when added to serum from normal animals in vitro. Presumably, phenobarbital increased serum lecithin-cholesterol acyltransferase activity by induction of the microsomal enzyme and subsequent secretion by the liver.     

Isolation and characterization of lecithin-cholesterol acyltransferase from hog plasma

Lecithin-cholesterol acyltransferase (EC 2.3.1.43) was purified from hog plasma by a highly efficient procedure. The final enzyme preparation was purified 30,000-fold over the starting material and was homogeneous as indicated by polyacrylamide gel electrophoreses in the presence of both SDS and urea. The purified hog lecithin-cholesterol acyltransferase had an apparent molecular weight of 66 000 on SDS-polyacrylamide gel electrophoresis and HPLC and was found to contain about 21.4% (w/w) carbohydrate-hexose, 11.3%; hexosamine, 1.9%; sialic acid, 8.2%. The amino acid composition analysis showed that hog lecithin-cholesterol acyltransferase contains four half cystines per mol; two cysteines were titrated at neutral pH with 5,5'-dithiobis(2-nitrobenzoic acid). Nearly all the phenolic groups were unavailable to the solvent at neutral pH, while they become exposed at around pH 11. Hog lecithin-cholesterol acyltransferase was found to be associated with HDL in the plasma and it prefers HDL as a substrate. The physicochemical properties of hog lecithin-cholesterol acyltransferase were generally similar to those of the human and the rat enzyme.     

LCAT cholesterol esterification is associated with the increase of ApoE/ApoA-I ratio during atherosclerosis progression in rabbit

Apolipoprotein A-I and Apolipoprotein E promote different steps of reverse cholesterol transport, including lecithin-cholesterol acyltransferase stimulation. Our aim was to study the changes in the levels of Apolipoprotein A-I, Apolipoprotein E, and lecithin-cholesterol acyltransferase activity during atherosclerosis progression in rabbits. Quantitative echocardiographic parameters were analyzed in order to evaluate, for the first time, whether atherosclerosis progression in rabbit is associated to apolipoproteins changes and alteration of indices of cardiac function, such as systolic strain and strain rate of the left ventricle. Atherosclerosis was induced by feeding rabbits for 8?weeks with 2?% cholesterol diet. The HDL levels of cholesterol and cholesteryl esters were measured by HPLC. The lecithin-cholesterol acyltransferase activity was evaluated both ex vivo, as cholesteryl esters/cholesterol molar ratio, and in vitro. Apolipoproteins levels were analyzed by ELISA. The HDL levels of cholesterol and cholesteryl esters increased, during treatment, up to 3.7- and 2.5-fold, respectively, compared to control animals. The lecithin-cholesterol acyltransferase activity in vitro was halved after 4?weeks. During cholesterol treatment, Apolipoprotein A-I level significantly decreased, whereas Apolipoprotein E concentration markedly increased. The molar ratio Apolipoprotein E/Apolipoprotein A-I was negatively correlated with the enzyme activity, and positively correlated with both increases in the intima-media thickness of common carotid wall and cardiac dysfunction signs, such as systolic strain and strain rate of the left ventricle.     

A rapid radioassay procedure for plasma lecithin-cholesterol acyltransferase

A rapid and accurate single step procedure is described for the assay of lecithin-cholesterol acyltransferase activity. After incubation, using radiolabeled cholesterol as the substrate, an ethanolic solution of digitonin is added directly to the incubation mixture to extract the lipids. Excess cholesterol is then added, and the labeled cholesterol-digitonide along with denatured proteins are sedimented by low speed centrifugation, leaving the labeled esterified cholesterol in solution. An aliquot of the supernatant is counted in an aqueous scintillation mixture. The method correlates well with the established thin-layer chromatographic procedure using either lecithin-cholesterol vesicles or heat-inactivated plasma as the substrate for lecithin-cholesterol acyltransferase.     

Aromatic boronic acids as probes of the catalytic site of human plasma lecithin-cholesterol acyltransferase

We have recently proposed a catalytic mechanism for human plasma lecithin-cholesterol acyltransferase (EC 2.3.1.43) (J. Biol. Chem. (1986) 261, 7032-7043), implicating single serine and histidine residues in phosphatidylcholine cleavage and two cysteine residues in cholesterol esterification. We now confirm the involvement of serine and histidine in catalysing the phospholipase A2 action of lecithin-cholesterol acyltransferase by demonstrating the inhibition of this activity by phenylboronic acid (Ki = 1.23 mM) and m-aminophenylboronic acid (Ki = 2.32 mM), inhibitors of known serine/histidine hydrolases. The specificity of the interaction of aromatic boronic acids with catalytic serine and histidine residues and the putative formation of a tetrahedral adduct between boron and the lecithin-cholesterol acyltransferase serine hydroxyl group which is similar to the transition-state intermediate formed between phosphatidylcholine and the catalytic serine residue was suggested by: substrate protection against inhibition by phenylboronic acids; a much reduced incorporation of phenylmethane[35S]sulphonyl fluoride into the enzyme in the presence of phenylboronic acid; the lack of interaction of histidine- or serine-modified enzyme with immobilized phenylboronic acid in the presence of glycerol (Ve/Vo = 2.7 and 2.3 respectively) when compared to the native enzyme (Ve/Vo = 5.25). Fatty acyl-lecithin-cholesterol acyltransferase, produced by incubation of the enzyme with a lecithin-apolipoprotein A-I proteoliposome substrate, was not retarded upon the sorbent column (Ve/Vo = 1.5). Modification of the enzyme's two free cysteine residues with 5,5'-dithiobis(2-nitrobenzoic acid) or potassium ferricyanide reduced (Ve/Vo = 3.5) but did not abolish retardation on the sorbent column, indicating that these modifications resulted in steric hinderance of the interaction of the boron atom with the lecithin-cholesterol acyltransferase serine hydroxyl group. These data suggest that the serine and histidine residues are proximal within the enzyme catalytic site and that both cysteine thiol groups are close to the serine hydroxyl group. The presence of significant amino-acid sequence homologies between lecithin-cholesterol acyltransferase, triacylglycerol lipases and the transacylases of fatty acid synthase is also reported.     

Distinctive structure and function of human apolipoprotein variant ApoA-IV-2

We have investigated the molecular structure, phospholipid binding, and lecithin-cholesterol acyltransferase catalytic activity of pure apoA-IV-2, a basic variant isoform of apoA-IV which is inherited as a classical Mendelian allele with a gene frequency of 0.09. Circular dichroism spectroscopy established that the alpha-helical content of apoA-IV-2 was 75% in the native state (versus 56% for apoA-IV-1), and increased to 88% in the presence of phospholipid. Fluorescence titration established that apoA-IV-2 bound to egg phospholipid vesicles with a Ka of 3.3 x 10(6) liter/mol, 2.4-fold greater than the affinity of apoA-IV-1. Fluorescence quenching studies revealed that, unlike apoA-IV-1, binding of apoA-IV-2 to phospholipid vesicles induced strong shielding of the amino-terminal tryptophan against iodide quenching. Enzyme kinetic studies using both saturated and unsaturated phospholipid substrates demonstrated that apoA-IV-2 was 36-71% more efficient in activating lecithin-cholesterol acyltransferase than apoA-IV-1. We conclude that apoA-IV-2 has more alpha-helical structure, is more stable in solution, and is more hydrophobic than apoA-IV-1, and that these distinctive structural features are associated with a higher affinity for phospholipid surfaces and an increased catalytic efficiency of lecithin:cholesterol acyltransferase activation. The biophysical basis for this latter characteristic may be the ability of apoA-IV-2 to penetrate phospholipid surfaces to a greater depth than apoA-IV-1. These molecular properties may be responsible for the increased levels of high density lipoproteins which have been observed in apoA-IV-2 heterozygotes.     

Antigenic relatedness between human lecithin-cholesterol acyltransferase and phospholipases of the A2 family

A monoclonal antibody, B10, generated against pure human lecithin-cholesterol acyltransferase (EC 2.3.1.43) caused the inhibition of the esterolytic and cholesterol esterifying activities of the enzyme. This antibody also reacted with a number of pancreatic and snake venom phospholipases A2 species but not phospholipase A1. A concentration-dependent inhibition of phospholipase A2 was also seen in the presence of B10. Treatment of lecithin-cholesterol acyltransferase or B10-reacting phospholipases with phenacyl bromide, a reagent known to interact with the active site of phospholipase A2, inhibited both their esterolytic activity and their capacity to bind to B10. A dimeric phospholipase A2 species with a known occluded active site did not cross-react with B10. Thus, lecithin-cholesterol acyltransferase and some enzymes of the phospholipase A2 family share a common antigenic determinant which is probably located near or at their esterolytic active site.     

Apolipoprotein A-I structure and lipid properties in homogeneous, reconstituted spherical and discoidal high density lipoproteins

We prepared a spherical reconstituted high density lipoprotein (rHDL) particle in pure form and compared it with its homogeneous discoidal rHDL precursors, in terms of the structure and stability of the apolipoprotein A-I (apoA-I) component, the dynamics of the surface lipids, and the relative reactivity with lecithin-cholesterol acyltransferase. The apoA-I-structure was examined in the rHDL particles by circular dichroism and fluorescence spectroscopic methods, and the binding of monoclonal antibodies specific for apoA-I epitopes. The stability of apoA-I on the rHDL particles was assessed by the effects of guanidine hydrochloride on the wavelength of maximum intrinsic fluorescence of the apolipoprotein. Lipid dynamics in the acyl chain region and the polarity of the lipid-water interface were investigated by means of fluorescence probes. The conformation of apoA-I in the spherical 93-A rHDL particles was found to be very similar to that in the 96-A rHDL discs but distinct from the apoA-I structure in the 78-A rHDL discs. The stability of apoA-I to denaturation by guanidine hydrochloride was highest in the 93-A rHDL spheres. The experiments on the lipids indicate somewhat more ordered and motionally restricted acyl chains in the spheres, relative to the discs, but a similar surface polarity. These results suggest that the folding and organization of apoA-I on the three particles include protein domains consisting of interacting alpha-helical segments in the carboxyl-terminal region and a globular domain in the amino-terminal region of each apoA-I molecule. The reactivity with lecithin-cholesterol acyltransferase was highest for the 96-A rHDL disc, and 16- and 34-fold lower for the 78-A rHDL disc and the 93-A rHDL sphere, respectively, possibly as a result of differences in apoA-I structure and product inhibition in these particles.     

Effects of dietary cholesterol and fat, sex and sire on lecithin-cholesterol acyltransferase activity in baboons

We analyzed the effects of dietary cholesterol, type of dietary fat, sex and sire progeny family on lecithin-cholesterol acyltransferase activity in 80 adult baboons. The animals were the progeny of 80 dams and 6 sires and were randomly assigned at birth to breast feeding or to one of three formulas containing 0.02, 0.30 or 0.60 mg cholesterol/ml. After weaning at 4 months of age the animals were fed one of four diets that were either high or low in cholesterol with 40% of the calories from either saturated or unsaturated fat. The fractional and molar rates of lecithin-cholesterol acyltransferase activity were measured at 7-8 years of age by an HPLC method. Infant diet (breast vs. formula feeding or level of cholesterol in formula had no effect on enzyme activity later in life. The adult diets that were high in cholesterol decreased the fractional lecithin-cholesterol acyltransferase rate by 20% / compared to diets low in cholesterol (7.89 vs. 9.84%/h, P less than 0.002), but dietary cholesterol did not affect the molar activity. Animals fed the high cholesterol diets had higher unesterified cholesterol concentrations compared to those fed the low cholesterol diets (38.1 mg/dl vs. 31.6 mg/dl, P less than 0.0001). The molar lecithin-cholesterol acyltransferase rate was increased 13% by saturated compared to unsaturated fat (83.3 vs. 73.6 nmol/h per ml plasma, P less than 0.07), but no effect of dietary fat was observed on the fractional enzyme activity. Females compared to males had significantly higher fractional (10.9 vs. 7.14%/h, P less than 0.0001) and molar lecithin-cholesterol acyltransferase activities (99.3 vs. 61.7 nmol/h per ml plasma, P less than 0.0001). After adjustment for the effects of diet and sex we observed differences in the fractional activity (range, 7.2-10.8%/h, P less than 0.04) and in the molar rate (range, 63.6-99.8 nmol/h per ml plasma, P less than 0.07) among the six sire progeny groups. The differences among sire progeny groups are evidence for genetic differences in lecithin-cholesterol acyltransferase activities among the baboon families.     

Defective enzyme causes lecithin-cholesterol acyltransferase deficiency in a Japanese kindred

Lecithin-cholesterol acyltransferase mass levels and activity and apolipoproteins A-I, A-II, B and D were measured in a Japanese family who have a familial lecithin-cholesterol acyltransferase deficiency. This analysis was performed to gain insight into the molecular basis of the enzyme deficiency and to compare findings in this family with other families with familial lecithin-cholesterol acyltransferase deficiency. The mass of the enzyme in plasma was determined by a sensitive double antibody radioimmunoassay, and enzyme activity was measured by using a common synthetic substrate comprised of phosphatidylcholine, cholesterol and apolipoprotein A-I liposomes prepared by a cholate dialysis procedure. The lecithin-cholesterol acyltransferase-deficient subject had an enzyme mass level that was 35% of normal (2.04 micrograms/ml, as compared with an average normal level of 5.76 +/- 0.95 micrograms/ml in 19 Japanese subjects) and an enzyme activity of less than 0.1% of normal (0.07 nmol/h per ml, as compared with normal levels of 100 nmol/h per ml). This subject also had lower levels of apolipoproteins: apolipoprotein A-I was 53 mg/dl (42% of normal), apolipoprotein A-II was 10.6 mg/dl (31% of normal), apolipoprotein B was 68 mg/dl (68% of normal), and apolipoprotein D was 3.6 mg/dl (60% of normal). The three obligate heterozygotes had enzyme mass levels ranging from 65% to 100% of normal and enzyme activity levels ranging from 23% to 65% of normal (23.4, 56.8, and 64.7 nmol/h per ml, respectively). The proband's sister had an enzyme mass level of 6.55 micrograms/ml (114% of normal) and an enzyme activity of only 64.8 nmol/h per ml (65% of normal), suggesting that she was also a heterozygote for lecithin-cholesterol acyltransferase deficiency. The obligate heterozygotes and the sister had normal apolipoprotein levels. We conclude that the lecithin-cholesterol acyltransferase deficiency in this family is due to the production of a defective enzyme that is expressed in the homozygote as well as in the heterozygotes, and, further, that this family's mutation differs from that reported earlier for other Japanese lecithin-cholesterol acyltransferase-deficient families.     

Cloning and structure analysis of the rat apolipoprotein A-I cDNA

Apolipoprotein A-I, the major protein in mammalian high-density lipoprotein, acts as a cofactor for lecithin-cholesterol acyltransferase during the formation of cholesterol ester and as such, is thought to promote cholesterol efflux from peripheral cells to the liver. In this paper, we report the partial purification of rat liver apolipoprotein A-I mRNA by a polysome immunoadsorption technique, and its cDNA cloning. Isolation of two overlapping cDNA clones enabled us to derive the whole rat apolipoprotein A-I cDNA coding sequence. Comparison of the deduced protein sequence with its human counterpart reveals a striking homology between the prepropeptide precursors. Both mature protein amino-terminal regions are very homologous, suggesting that this particular domain could be involved in lipid/protein binding or lecithin-cholesterol acyltransferase activation.     

Substitution in vitro of lecithin-cholesterol acyltransferase. Analysis of changes in plasma lipoproteins

Lecithin-cholesterol acyltransferase (EC 2.3.1.43) was purified 15 000-fold from human plasma. The active material was homogeneous in different gel electrophoretic systems but separated into three major bands with apparent pI values of 4.28, 4.33 and 4.37 in isoelectrofocusing. The apparent Mr of the enzyme is 67 000 +/- 2000. An antiserum prepared against the purified enzyme specifically inhibited the activity of lecithin-cholesterol acyltransferase in whole serum. Serum from a patient with familial deficiency of lecithin-cholesterol acyltransferase was substituted in vitro with the highly purified enzyme. The serum from this patient did not contain immunochemically detectable enzyme protein. Substitution of enzyme resulted in the following major changes. 1. Cholesteryl ester content in serum increased by 36-89 mg/100 ml depending on the experimental conditions. The enzyme-mediated formation of cholesteryl ester led to an increase of cholesteryl ester content in high-density and very-low-density lipoproteins and in low-density lipoproteins containing apoprotein-B. No increase occurred in fractions containing very large flattened structures and the abnormal lipoprotein-X and in lipoprotein-E. Incubation of isolated fractions with lecithin-cholesterol acyltransferase led to significant cholesterol esterification only in high-density lipoproteins. 2. The characteristic disc-shaped rouleaux-forming high-density lipoproteins of enzyme-deficient serum disappeared. Instead a single homogeneous population of high-density lipoproteins formed. The particles generated were spherical and had the electrophoretic properties, density (1.080 g/ml), diameter (12.5 nm) and apoprotein composition of normal high-density lipoproteins-2. 3. The concentration of spherical particles containing apolipoprotein E (density 1.040-1.080 g/ml) and the lamellar lipoprotein-X-like structures in the low-density lipoprotein fraction were not affected by the enzyme substitution. 4. A single homogeneous population of spherical lipoprotein-B particles of 26.5-nm diameter occurred at density 1.029 g/ml. The data suggest that the discoidal high-density lipoproteins are the major site of cholesteryl ester formation that apolipoprotein-E is not involved in an undirectional transport of newly formed cholesteryl ester from high-density lipoproteins to other lipoproteins and that lipoprotein-X and lipoprotein-E are not preferential substrates for the acyltransferase.     

Secretion of the arginine-rich and A-I apolipoproteins by the isolated perfused rat liver.

Rates of secretion of the arginine-rich and A-I apolipoproteins into perfusates of rat livers were measured by specific radioimmunoassays. Livers were perfused for 6 hr in a recirculating system in the presence or absence of 5,5'-dithionitrobenzoic acid, an inhibitor of lecithin-cholesterol acyltransferase. Arginine-rich apoprotein (ARP) was secreted at a constant or increasing hourly rate of about 40 micro g/g liver, whereas the rate of accumulation of apoprotein A-I decreased progressively from about 12 to less than 5 micro g/g liver. These rates were not affected by inhibition of lecithin-cholesterol acyltransferase. The distribution of these two apolipoproteins was also measured in ultracentrifugally separated lipoprotein fractions from perfusates and blood plasma. Apoprotein A-I was mainly in high density lipoproteins, with the remainder in proteins of density > 1.21 g/ml. The percent of apoprotein A-I in the latter fraction was lowest in plasma (5%); in perfusates it was greater when the enzyme inhibitor was present (33%) than in its absence (11%). By contrast much less ARP was in proteins of d > 1.21 g/ml in perfusates than in blood plasma. Discoidal high density lipoproteins, recovered from perfusates in which lecithin-cholesterol acyltransferase was inhibited, contained much more arginine-rich apoprotein than apoprotein A-I (ratio = 10:1). The ratio in spherical plasma HDL was 1:7 and that in perfusate high density lipoproteins obtained in the absence of enzyme inhibitor was intermediate (2:1). It is concluded that: 1) the arginine-rich apoprotein is a major apolipoprotein whereas apoprotein A-I is a minor apolipoprotein secreted by the perfused rat liver; 2) the properties of the high density lipoproteins produced in this system are remarkably similar to those found in humans with genetically determined deficiency of lecithin-cholesterol acyltransferase.     

Anion effects on the reaction of lecithin-cholesterol acyltransferase with discoidal complexes of phosphatidylcholines . apolipoprotein A-I . cholesterol

Discoidal complexes of phosphatidylcholine (PC) . apolipoprotein A-I . cholesterol were prepared with egg PC, palmitoyloleoylPC, dipalmitoylPC, or dimyristoylPC, and were used as substrates of purified lecithin-cholesterol acyltransferase to investigate the effects of neutral salts on the enzymatic reaction. Sodium fluoride, chloride and bromide concentrations up to 1 M, did not affect the properties of the substrate particles, but caused marked and distinct changes in the activity of the enzyme with the various PC particles. The effects of salts were largely due to the anions, which followed the order of the lyotropic series in their inactivating capacity: F- less than Cl- less than Br- less than NO3- less than I- less than SCN-. Sodium salts (F-, Cl-, and Br-) produced a very large increase in the pH optimum of the enzymatic reaction (7.4 to at least 8.5) essentially obliterating the ionization of a functional group with pK of 8.1. The kinetics of the enzymatic reaction revealed major differences among the PC particles, and different responses of their kinetic parameters with increasing salt concentrations. The conclusions reached in this work are the following: (1) The relative reactivity of PC substrates, in discoidal particles, with lecithin-cholesterol acyltransferase depends strongly on the concentration and type of salts in the medium. (2) Anions (in lyotropic series) rather than cations affect the enzymatic reaction. (3) There are functional groups with pK of 8.1 which are affected markedly in their ionization behavior by anion binding. (4) The active site of lecithin-cholesterol acyltransferase and its interaction with anions are affected by the exact nature of the PC-apolipoprotein interface.     

Inhibition of lecithin-cholesterol acyltransferase by diphytanoyl phosphatidylcholine

Model high density lipoproteins containing human apolipoprotein A-I, cholesterol, and a variety of phosphatidylcholines (PCs) have been prepared and tested. The PCs included 1-palmitoyl-2-oleoyl PC (POPC) and its diether analog 1-O-hexadecyl-2-oleyl PC (POPC ether), 1,2-diphytanoyl PC (DPhPC), 1-palmitoyl-2-phytanoyl PC, and 1-phytanoyl-2-palmitoyl PC. All ester PCs were good acyl donors for the transesterification of cholesterol catalyzed by human lecithin-cholesterol acyltransferase except DPhPC, which showed no reactivity. The PCs containing one phytanoyl chain donated an acyl chain to cholesterol as fast as non-branched fatty acyl chains. However, the competitive inhibition of lecithin-cholesterol acyltransferase by POPC ether and DPhPC was similar, and both lipids formed a macromolecular matrix that supported the reactivity of other ester PC substrates. The bulk of physicochemical properties of model high density lipoproteins composed of DPhPC were indistinguishable from those of POPC ether. These properties included 1) alpha-helical content of the apoprotein as assessed by circular dichroism, 2) microviscosity as determined from the fluorescence polarization and lifetime of the probe 1,6-diphenyl-1,3,5-hexatriene, 3) macromolecular weight based upon analytical gel filtration chromatography, and 4) surface polarity revealed by the fluorescence of 6-propionyl-2(dimethylamino)naphthalene. The only major difference in a physicochemical property was that the molecular surface area of DPhPC (area = 69 A2 at collapse pressure) determined by monolayer methods was 17 A2 greater than that of POPC (area = 53 A2 at collapse pressure) at all surface pressures measured. We suggest that the properties of DPhPC in being enzymatically nonreactive but a competitive inhibitor are due to its much larger size and that the active site of lecithin-cholesterol acyltransferase cannot bind phospholipid substrates in a catalytically productive way if they have surface areas of 70 A2 or more.     

A purification method for apolipoprotein A-I and A-II

Apolipoproteins A-I and A-II were isolated from precipitates obtained by cold ethanol fractionation of human plasma. The starting material used in this report was precipitate B of the Kistler and Nitschmann method which corresponds approximately to fraction III of the Cohn and Oncley procedure. Through the use of urea, chloroform, and ethanol in appropriate concentrations, apolipoproteins A-I and A-II were isolated by a simple extraction technique avoiding time-consuming ultracentrifugation. Starting from 10 g of centrifuged precipitate B, approximately 100 mg of apolipoprotein A-I and 10 mg of apolipoprotein A-II were obtained. When incubated with normal human or rabbit plasma, both apolipoproteins were readily incorporated into high-density lipoproteins. Apolipoprotein A-I obtained by the cold ethanol method activated lecithin-cholesterol acyltransferase to the same extent as apolipoprotein A-I prepared by the classical flotation method. Apolipoprotein A-II had no such properties by itself, but was capable of potentiating lecithin-cholesterol acyltransferase activity of apolipoprotein A-I.     

Distribution of lecithin-cholesterol acyltransferase (LCAT) in human plasma lipoprotein fractions. Evidence for the association of active LCAT with low density lipoproteins

The distribution of lecithin-cholesterol acyltransferase (LCAT) in human plasma was assessed by measuring both LCAT mass and activity in plasma fractions separated by sequential flotation ultracentrifugation, single-spin gradient ultracentrifugation, dextran sulfate-Mg²⁺ precipitation or agarose gel filtration. Although most of the LCAT was found to be associated with the high density lipoprotein fraction, a small amount of active LCAT (approximately 1% of the plasma LCAT mass and activity) was consistently associated with the low density lipoprotein fraction. LCAT was not found in the very low density lipoprotein fraction. The LDL-associated LCAT may play an important role in the acylation of lysolecithin by lysolecithin acyltransferase activity of LCAT.     

Amphiphilic and Nonamphiphilic Forms of Bovine and Human Dopamine β-Hydroxylase

We show that human and bovine dopamine beta-hydroxylases (DBH) exist under three main molecular forms: a soluble nonamphiphilic form and two amphiphilic forms. Sedimentation in sucrose gradients and electrophoresis under nondenaturing conditions, by comparison with acetylcholinesterase (AChE), suggest that the three forms are tetramers of the DBH catalytic subunit and bind either no detergent, one detergent micelle, or two detergent micelles. By analogy with the Gna4 and Ga4 AChE forms, we propose to call the nonamphiphilic tetramer Dna4 and the amphiphilic tetramers Da4I and Da4II. In addition to the major tetrameric forms, DBH dimers occur as very minor species, both amphiphilic and nonamphiphilic. Reduction under nondenaturing conditions leads to a partial dissociation of tetramers into dimers, retaining their amphiphilic character. This suggests that the hydrophobic domain is not linked to the subunits through disulfide bonds. The two amphiphilic tetramers are insensitive to phosphatidylinositol phospholipase C, but may be converted into soluble DBH by proteolysis in a stepwise manner; Da4II----Da4I----Dna4. Incubation of soluble DBH with various phospholipids did not produce any amphiphilic form. Several bands corresponding to the catalytic subunits of bovine DBH were observed in sodium dodecyl sulfate-polyacrylamide gel electrophoresis, but this multiplicity was not simply correlated with the amphiphilic character of the enzyme. In the case of human DBH, we observed two bands of 78 and 84 kDa. As previously reported by others, the presence of the heavy subunit characterizes the amphiphilic forms of the enzyme. We discuss the nature of the hydrophobic domain, which could be an uncleaved signal peptide, and the organization of the different amphiphilic and nonamphiphilic DBH forms. We present two models in which dimers may possess either one hydrophobic domain or two domains belonging to each subunit; in both cases, a single detergent micelle would be bound per dimer.     

The degradation of platelet-activating factor by high-density lipoprotein in rat plasma. Effect of ethynyloestradiol administration.

The degradation of platelet-activating factor (PAF; 1-O-alkyl-2-acetyl-sn-glycero-3-phosphocholine) in human plasma is brought about by a specific acetylhydrolase that is associated with low-density lipoprotein. In this study, the presence of a similar activity on high-density lipoprotein (HDL) of rat plasma has been demonstrated which is independent of lecithin-cholesterol acyltransferase (LCAT) activity. Furthermore, PAF acetylhydrolase activity may be decreased by 66% by treatment of rats with 17 alpha-ethynyloestradiol (1 mg/kg body wt.; 5 days). This treatment also decreased HDL cholesterol concentrations by 90%, but did not affect LCAT activity. Thus rat LCAT and PAF acetylhydrolase are associated with different subspecies of HDL.