Chapter 10 Lecithin cholesterol acyltransferase

A . M . Cotto, J r . (Ed.) Plusma Lipoproleins 0 1987 Elsevier Science Publishers B . V . (Biomedical Division)

299 CHAPTER 10

Lecithin cholesterol acyltransferase ANA JONAS Department of Biochemistry, College of Medicine at Urbana-Champaign, University of Illinois, 506 South Mathews Avenue, Urbana, IL 61801, USA

1. Introduction Blood plasma of humans and other animal species contains an enzymatic activity which esterifies cholesterol. Although such an activity was first described by Sperry in 1935 [l], only in 1962 it was recognized by Glomset [2] that a single enzyme was responsible for the removal of an acyl chain from lecithin and its transfer to free cholesterol. The enzyme was called lecithin cholesterol acyltransferase (EC 2.3.1.43) (LCAT). The next major advance in LCAT research occurred in 1967 when Norum and Gjone [3, 41 described the first case of familial LCAT deficiency. Subsequent work on the pathology and lipoprotein abnormalities associated with this deficiency shed light on the role of LCAT in extracellular cholesterol transport. More recently, the first purification of LCAT to homogeneity, by Albers and coworkers (1976) [ 5 ] , opened the way for the detailed biochemical investigation of the substrate requirements and the mechanism of action of this enzyme. Lecithin cholesterol acyltransferase is synthesized by the liver and is secreted into plasma where it is present either free or in association with lipoproteins. Its preferred substrates are high density lipoproteins (HDL), especially in nascent disc form or as the smaller subclasses of spherical HDL particles. Apolipoproteins of HDL (particularly apolipoprotein A-I, apoA-I) activate LCAT, and the HDL core initially stores the cholesterol ester products of the enzymatic reaction; in addition, HDL are capable of transferring cholesterol esters to other lipoproteins, and of exchanging surface components with other lipoproteins and with cell membranes. Through its action on HDL, LCAT has the general effect of reducing the free (or unesterified) cholesterol content of lipoproteins and of cell plasma membranes; Abbreviations: LCAT, lecithin cholesterol acyltransferase; HDL, high density lipoproteins; apo,

apolipoprotein; apoA-I, apolipoprotein A-I; CE, cholesterol esters; PC, phosphatidylcholine; DMPC, dimyristoyl-PC; DPPC, dipalmitoyl-PC; POPC, palmitoyl oleoyl-PC; PE, phosphatidylethanolamine; PS, phosphatidylserine; PG, phosphatidylglycerol; PA, phosphatidic acid; DSPC, distearoyl-PC; SDS, sodium dodecyl sulfate; LDL, low density lipoproteins; VLDL, very low density lipoproteins.

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thus, LCAT participates in the maturation of HDL, changes the distribution of HDL subclasses and their composition, and modifies the lipid composition and overall structure of other lipoproteins. In decreasing the free cholesterol content of lipoproteins, LCAT affects the cholesterol content of membranes, and is involved in the maintenance of membrane structure, and indirectly in determining the levels of total cellular cholesterol. Much of our understanding of the function of LCAT was derived from investigations of individuals defective in LCAT. These patients show marked increases of free cholesterol in plasma, at the expense of cholesterol esters; have grossly abnormal lipoprotein patterns; and present erythrocytes with abnormal structures and elevated free cholesterol contents. In addition, LCAT deficient patients accumulate free cholesterol and phospholipids in various tissues, notably in the kidneys. Clearly, the physiological role of LCAT in the management of plasma cholesterol is extremely important. From the biochemical point of view, LCAT is particularly interesting as a watersoluble enzyme which acts on interfacial lipids and requires apolipoprotein activators. The pure enzyme has been partially characterized in its physical and chemical properties and has been used to produce specific antibodies. The substrate requirements of the enzyme are still under investigation, particularly the effects of particle properties and bulk lipid on the reactivity of LCAT with individual substrate molecules. Although apoA-I is recognized as the main apolipoprotein activator of LCAT, it is now known that several other apolipoproteins and amphipathic peptides can also activate LCAT to various extents. Numerous molecular and ionic activators and inhibitors of LCAT have been described, but the mechanisms of action of the enzyme and of its activation by apolipoproteins are not yet understood. The objective of this chapter is to summarize, in a critical and selective manner the current information (up to and including 1985 literature) on the physical and chemical properties of LCAT, its specificity for molecular and particulate substrates, its activation by apolipoproteins, the kinetics of the enzymatic reaction including the roles of various effectors of enzymatic activity, the function of LCAT in lipoprotein transformations, and its participation in ‘reverse cholesterol transport’. The emphasis in this chapter is on the biochemistry of LCAT; for more physiological or clinical approaches to this subject the reader can turn to several comprehensive review articles and chapters which have appeared since 1972 [6 - 111.

2. LCA T purification and assays After the original report by Albers and coworkers [ 5 ] on the purification of human LCAT to homogeneity, several other laboratories have published a variety of purification schemes for this enzyme [ 13 - 201. Most of the methods start either with

301 an ultracentrifugal or a precipitation step for the removal of lipoproteins; additional purification steps include anion exchange chromatography on DEAE columns and adsorption chromatography using hydroxylapatite columns. Numerous other steps have been described, but the ones mentioned above give the highest degrees of purification, and are most reproducible in different laboratories. Starting from plasma, purifications of LCAT from 11 000- to 35 000-fold have been reported. Since the average content of LCAT in plasma is 5 - 7 mg/l and the total plasma protein content is about 72 mg/ml, at least a 10 000-fold purification is required to produce homogeneous enzyme. The published yields are in the vicinity of 10%. Specific activities for the purified enzyme (from 200 to 200 000 nmol of cholesterol esterified/h/mg), and for LCAT in plasma (0.01 to 6 nmol of cholesterol esterified/h/mg protein) are not comparable for most of the reports because of the differences in assay procedures and in enzyme stability. Although all the known factors which affect LCAT activity will be covered in a subsequent section, a few comments about the assay of LCAT activity are in order here. Plasma LCAT activity can be measured using intrinsic or extrinsic substrate particles, i.e., lipoproteins present in the same plasma sample or exogenous particles added to the plasma (lipoproteins, vesicles, or micelles). In most cases formation of cholesterol esters (CE) or disappearance of unesterified cholesterol are measured as a function of time. The various particulate substrates, containing the cholesterol and phosghatidylcholine (PC) molecular substrates, have different reactivities with the enzyme, and respond differently to soluble components of the reaction mixture such as salts. Obviously, purified LCAT is assayed with exogenous substrates, and their choice as well as the choice of reaction conditions determines the observed specific activities. Another important point is that initial reaction velocities and linear responses to enzyme concentration are required conditions for the valid determination of enzyme activities. Such conditions are not always met in reports of enzyme purifications.

3. Physical and chemical properties Table 1 summarizes the known physical and chemical properties of human LCAT. Purified LCAT has a molecular weight of 60 000 as determined by sedimentation equilibrium in phosphate buffer, or in the presence of guanidine hydrochloride or mercaptoethanol [ 14, 211. By sodium dodecyl sulfate polyacrylamide electrophoresis the apparent molecular weight is around 67 000 [ 5 , 14, 17,211. The discrepancy between the two methods is due to the glycoprotein nature of the enzyme: 25% of its weight is carbohydrate. Hexose sugars (mannose and galactose) account for 13070, glucosamine for 6%, and sialic acid for 5% of the carbohydrate weight [14, 221. The partial specific volume of LCAT is 0.708 ml/g [14, 211. Isoelectric points of at least six isoforms range between 3.9 and 4.5 [23]. Circular dichroism spectra

302 in 1 mM phosphate buffer and in the same buffer containing 0.2 M NaCl indicate the presence of 18 - 24% a-helix, around 30 - 53% P-sheet, and 29 - 46% of other structure [21, 231. Removal of sialic acid residues decreases the molecular radius of LCAT from 40 to 36 A . The frictional coefficient ratio calculated from the molecular weight and the sedimentation coefficient of 3.9 S, is 1.57 [23], corresponding to a rather asymmetric and/or irregular protein. LCAT has a marked affinity for interfaces. It interacts with HDL and with vesicles containing phosphatidylcholine and cholesterol [24]. In addition, this enzyme adsorbs to air/water interfaces and becomes easily denatured, particularly when present in very low concentrations in small volumes of solution. Nishida and coworkers [25] investigated the stability of LCAT in solution as a function of buffer ionic strength, temperature, and the presence of albumin, apoA-I, and phosphatidylcholine vesicles. The enzyme was most stable in 0.4 mM phosphate (ionic strength, 0.001) buffer, pH 7.4, in the presence of substrate particles and of albumin or apoA-I. The latter proteins apparently prevented LCAT denaturation by adsorbing to the air/water interface. At an ionic strength of 0.1, corresponding to 40 mM phosphate buffer, at 37"C, LCAT was most active but also most easily denatured in the absence of substrate particles and of albumin or apoA-I. Apparently the active enzyme conformation present in 40 mM buffer exposes hydrophobic regions which can either interact with substrate or facilitate enzyme adsorption to other interfaces. TABLE 1 Physical and chemical properties of human LCAT

Molecular weight (sedimentation equilibrium) Molecular weight (SDS PAGE) Protein content Carbohydrate content mannose and galactose glucosamine sialic acid Partial specific volume Sedimentation velocity coefficient Frictional coefficient ratio (f/f,) Molecular radius lsoelectric points (six or more isoforms) Molar extinction coefficient (280 nm) Circular dichroism spectra or-helix 0-sheet random structure Normal concentration in plasma

60 000 67 000 15 wt Vo 25 wt '70 13 wt 070 6.2 wt Yo 5.4 wt 070 0.708 ml/g 3.9 s 1.57 40 A 3.9-4.5 1.26 x lo5 M - '

. cm-'

18 - 24% 30- 53% 29 - 46% 6.0-6.4 ( + 1.03) mg/l

303 The 45 000 molecular weight polypeptide portion of LCAT is relatively rich in glutamic acid, aspartic acid, glycine, proline, and leucine residues. Each mol of the enzyme contains four half-cystine residues, two of them as cysteine groups which are easily reacted with a variety of sulfhydryl reagents, and are involved in the activity of LCAT. The enzyme is relatively rich in aromatic residues: 18 mol of phenylalanine, 17 mol of tyrosine, and 9 mol of tryptophan residues per mol of LCAT [5, 14, 221. As a consequence the molar extinction coefficient of LCAT at 280 nm is quite high, 1.26 x lo5 M - ' . cm-l (Elvo= 21 x l o 2 . g - ' cm2) [22]. Table 2 shows the average amino acid composition of LCAT taken from five independent reports found in the literature [5, 13, 14, 22, 231. Specific antibodies to LCAT have been raised in goats and rabbits and have been used to develop immunoassays for LCAT [26 - 281. Such assays allow the determination of LCAT mass in normal individuals (5.99 f 1.03 mg/l, n = 44, for males; and 6.44 t 0.79 mg/l, n = 22, for females) and in dislipoproteinemic patients. Some LCAT-deficient patients have from 0 to 0.89 mg/l of the enzyme in TABLE 2 Amino acid composition of human LCAT; mean 231

(k

SD) from five independent reports [5, 13, 14, 22,

Amino acid

Residues/mol (SD)

LYS

14 (0.7) 12 (0.5) 18 (0.9) 38 (4.0) 24 (2.7) 24 (2.3) 41 (3.3) 35 (2.6) 38 (3.7) 25 (2.5) 27 (3.2) 8 (1.0) 16 (2.4) 47 (4.0) 17 (2.3) 18 (2.3) 4 (0) 9 (1.7)

His Arg Aspa Thr Ser Glua Pro Gly Ala Val Met lle Leu Tyr Phe Cysb TrP Asn and Gln residues have been included. Half cystine residues. SD. standard deviation.

a

3 04 their plasma. Antihuman LCAT antibodies bound to labeled human LCAT can be displaced and competed for by the plasma of various animal species in the following order: man and sheep > nonhuman primates > cat or dog > pig > rabbit or guinea pig > mouse > rat [27].

4. LCA T reactions and substrate particles LCAT catalyzes the transfer of an acyl group from a donor lipid molecule to an acceptor. The best characterized and physiologically the most important reaction is the transesterification of an acyl group from lecithin to cholesterol, occurring on HDL particles in plasma [ 2 , 291. In competition with the transesterification reaction, or in the absence of lipid acceptors, LCAT may transfer acyl groups to water in a phospholipase reaction [13, 301. This reaction is distinct from the well-known phospholipase A, reactions because of the absence of the Ca2+ requirement, the dependence on apolipoprotein activators, and a less than perfect specificity for the hydrolysis of the sn-2 acyl chain by LCAT. Both the phospholipase and the transesterification reactions apparently involve the same functional residues of LCAT and are equally susceptible to inhibition with sulfhydryl reagents, heavy metals, and antiesterase inhibitors of active cysteine, serine, and histidine residues in enzymes [13, 301. In addition to the lecithin cholesterol acyltransferase and phospholipase activities, LCAT has been shown to transfer acyl groups from phosphatidylcholine molecules to lysolecithin acceptors [31- 331. The lysolecithin acyltransferase reaction is activated by LDL particles and does not require apoA-I nor HDL for activity. The physiological significance of this reaction is not clear. A reverse reaction, involving cholesterol ester transesterification to lysolecithin on HDL, has not yet been demonstrated. Most of the discussion of the LCAT reaction in this chapter will concentrate on the lecithin cholesterol acyltransferase function of this enzyme. Table 3 summarizes the known enzymatic activities of LCAT. Since the lipid substrates of LCAT are extremely water-insoluble molecules, they form part of large lipid aggregates or particles which present a lipid/water interface to the water-soluble LCAT molecule. The natural substrates of LCAT are HDL particles containing the lipid molecular substrates (phospholipids and cholesterol) in the surface monolayer of the lipoprotein. Common synthetic substrate particles include vesicles (i.e., liposomes which consist of a lipid bilayer enclosing a water compartment) and discoidal micelles (lipid bilayer discs stabilized by apolipoproteins). The phospholipid, cholesterol, and apoliproprotein components of these particles constitute their surface. Thus, with all of these substrate particles the LCAT reaction occurs at an interface, and is quite different from homogeneous enzymatic reactions where the chemical nature alone of the molecular substrates or effectors determines the rates of the reaction. With interfacial enzymes, such as LCAT, the nature of the

305

interface and the interaction of the enzyme with it, determine the activity of the enzyme in addition to the catalytic steps. Furthermore, LCAT activation by apolipoproteins implies the presence of apolipoprotein/iipid/enzyme interactions which are reflected in the reaction rates. The nature of the interface and the conformation of the apolipoprotein activator are important variables in any consideration of LCAT substrate specificity; therefore, a discussion of the characteristics of the available particulate substrates of LCAT is essential. Fig. 1 depicts the most common substrate particles for LCAT: (1) native spherical and nascent (discoidal) HDL, (2) vesicles of phospholipid and cholesterol with reversibly bound apolipoprotein, and (3) discoidal micelles of phospholipid and cholesterol with irreversibly bound apolipoprotein. Of course, native HDL are the natural substrates of the enzyme and any studies employing model particles should lead to a better understanding of LCAT action on HDL. However, a systematic investigation of LCAT molecular substrate requirements or of apolipoprotein activation are hindered by the complex and varied composition of native HDL. Lipid vesicles are chemically and physically defined synthetic substrate particles [ 14, 24, 34-36]. Under some circumstances these are very useful and easily prepared substrates; however, their binding properties for apolipoproteins and possible morphological changes in the presence of apolipoproteins must be evaluated very carefully in each case. Considerable information is now available on the spontaneous reaction of apolipoproteins with lipid vesicles to give rise to relatively small discoidal breakdown products [37, 381. The lytic reaction is kinetically controlled by the chemical nature of the phospholipids and the stability of the bilayer, by the TABLE 3 Reactions catalyzed by human LCAT

Reactants

Products

Substrate particles; activation

References

Phosphatidylcholinea cholesterol

Lysophosphatidylcholine + cholesterol ester

HDL, synthetic vesicles

[2, 61

Phosphatidylcholine + H,O

Lysophosphatidylcholine + fatty acid

Synthetic vesiclesc; activated by apoA-1

[13, 301

Phosphatidylcholine + lysophosphatidylcholine

Lysophosphatidylcholine + phosphatidylcholine

LDL; does not require apoA-1 for activation

[3 1 - 331

+

a

and discs; activated by apoA-lb

Other glycerophospholipids also serve as LCAT substrates [47]. Other HDL apolipoproteins may activate LCAT 161, 65 - 681. The phospholipase reaction has not been investigated with HDL nor with discoidal substrates.

306

reaction temperature relative to the gel/liquid crystalline phase transition temperature of the lipids, by the curvature of the lipid bilayer, and by the cholesterol content of the system. Thus dimyristoyl-PC (DMPC) and dipalmitoyl-PC (DPPC) unilamellar vesicles containing less than 20 mol 070 of cholesterol, can react with human apoA-I at the temperatures of their main phase transitions to produce discoidal products. O n the other hand, vesicles prepared with phosphatidylcholines such as palmitoyl oleoyl-PC (POPC) or egg-PC remain intact in the presence of apoA-I, in the normally accessible range of temperatures (0- 50°C). These mixed long chain PC molecules form rather stable vesicles which undergo phase transitions well below 0°C; therefore, vesicles of egg-PC or POPC bind apoA-I but are not disrupted by it. Apolipoproteins bind to the surface of these vesicles with an affinity and stoichiometry which depends on the chemical composition of the particles. For example, the affinity of apoA-I and the number of protein molecules bound increases with cholesterol content in small unilamellar vesicles of egg-PC up to a molar ratio of 411, PC/cholesterol [39]. Obviously the interpretation of experimen-

HDL

Veslcle

Disc

Fig. 1. Substrate particles for LCAT. (Left) Discoidal HDL and smaller spherical HDL are good LCAT substrates. They can be used as endogenous or exogenous substrates to measure LCAT activity in plasma [6, 101. When HDL are used as endogenous substrates, LCAT is first inhibited with reversible sulfhydryl reagents, radiolabeled cholesterol is introduced and equilibrated with plasma, the inhibition is reversed usually by adding excess P-mercaptoethanol or dithiothreitol, and the formation of radiolabeled cholesterol esters is measured with time [MI.Alternatively, changes in cholesterol concentration are measured with colorimetric or fluorometric methods. When used as exogenous substrates, HDL are isolated, radiolabeled, and are added to plasma to initiate the enzymatic assay. (Center) Vesicles of phospholipids and cholesterol, with added, reversibly bound apolipoproteins, are synthetic exogenous substrates [34 - 361. These particles are prepared from lipid dispersions by sonication or by organic solvent injection methods. They are most useful with purified enzyme preparations, and with lipids which form stable vesicles in the presence of apolipoproteins. (Right) Synthetic discs of phospholipids, cholesterol, and irreversibly bound apolipoproteins are excellent substrates for LCAT and close analogs of discoidal HDL [40- 431. These particles are prepared by the sodium cholate dialysis method and are used a s substrates for purified LCAT, but they may be adapted as standard, exogenous substrates for plasma LCAT.

307 tal results obtained with vesicles as substrates for LCAT must take into account the possible changes in the system upon addition of apolipoprotein activators. The second type of synthetic LCAT substrate particles consists of discoidal micelles of phospholipid, cholesterol, and apolipoprotein, prepared either by spontaneous reaction of apolipoproteins with lipid vesicles or by dispersion of lipids in sodium cholate and dialysis in the presence of apolipoprotein [37, 38, 40-431. The resultant discoidal particles are close analogs of nascent HDL, yet their size and chemical composition can be defined at will. The apolipoprotein activator is an integral component of the particles and is irreversibly bound to the periphery of the discoidal structure. During the initial stages of reaction with LCAT the morphology of these particles changes very little. Unlike vesicles which can only store about 3 mol Vo of cholesterol ester products, the discoidal substrate particles have a high capacity for cholesterol esters [ 191. Clearly, these substrates are especially well suited for the systematic investigation of the substrate specificity of LCAT. Recently, a cosonication method has been introduced for the preparation of spherical, chemically defined HDL analogs [44] (personal communication, M. Walsh, 1984). So far, only particles containing apoA-I, egg-PC, cholesterol oleate, and cholesterol have been prepared and characterized (unpublished results, A. Jonas, 1985). These particles, together with the discoidal HDL analogs, will be most useful in acquiring definitive information about the specificity and mechanism of action of LCAT. In the meantime, literature reports, particularly those describing studies with vesicle substrates, must be interpreted cautiously. Comparisons between different substrate particles are also dangerous because of the diverse effects of the interfaces on the enzymatic reaction. For example, vesicles of egg-PC, 20 mol Vo cholesterol, and apoA-I are only 1/4 as reactive as discoidal particles of the same relative composition [19]. Another very good example is the reactivity of DPPC with LCAT when the DPPC molecular substrate is present in different particles and lipid environments. In sonicated lipid dispersions DPPC, is essentially unreactive with LCAT at 37"C, probably as a result of poor binding of apoA-I activator and/or enzyme to the lipid vesicles [45]. However, when DPPC is incorporated into HDL it becomes a good substrate for the enzyme. This is probably due to the favorable lipid environment and to the presence of lipid bound apolipoprotein activator in the required conformation. Small discoidal particles (about 100 A in diameter) prepared with DPPC, apoA-I, and cholesterol are very good substrates for LCAT; yet analogous particles of larger diameters (about 200 A) are very poor substrates [46]. In this case the conformation of apoA-I changes in going from the smaller to the larger particle class. Finally, discoidal particles can be prepared from the ether analogs of phosphatidylcholines, apoA-I, cholesterol, and small amounts of test lipids [47]. DPPC incorporated into these particles is a good substrate for LCAT, but its reactivity depends on the nature of the matrix lipid, i.e., on the lipid environment and possibly on the apolipoprotein conformation. These examples illustrate the importance of the interface, in terms of the interaction of the enzyme

and apoA-I with it, its modulation of the conformation of apoA-I, and the effects of bulk lipid in determining the reaction rates of LCAT with one of its molecular substrates.

5. Molecular LCA T substrates (a) Acyl acceptors In the lecithin cholesterol acyltransferase reaction, LCAT uses cholesterol as the physiological acyl acceptor, but other molecules with hydroxyl functional groups may also serve as acyl acceptors. Employing stable vesicles of egg-PC which contained radiolabeled DPPC, various sterols, and bound apoA-I, Nishida and his coworkers [15, 481 examined the specificity of LCAT for the acyl acceptor. They found that a 3-0-hydroxy group and a trans configuration of the A/B rings are essential for activity. Cholesterol analogs with a modified side chain (e.g., campesterol, 0-sitosterol, desmosterol, and coprostanol) were less effective acyl acceptors than cholesterol; but androstenol, without a side chain, was slightly better than cholesterol as acceptor. The observed differences in reaction rates, however, could have been due to changes in the lipid/water interface as different sterols were incorporated into the vesicle phospholipid bilayer. In addition to sterols, LCAT can transfer acyl chains to long chain alcohols (12 carbons and longer) which partition effectively into phospholipid bilayers. In its phospholipase reaction LCAT transfers the acyl chains to water [13, 301. From the results of Nishida and coworkers [15, 481, the competing rates of a palmitoyl group transfer from DPPC in egg-PC vesicles to cholesterol, to water, and to long chain alcohols are approximately related as the ratios 12.2/ 1N0.9, respectively. Sterols incorporated into endogenous lipoprotein substrates of LCAT have a similar acceptor specificity as in vesicles, even if the relative reaction rates are somewhat different due to the different nature of the interfaces. Thus, desmosterol and 0-sitosterol added to human plasma become esterified at rates comparable to cholesterol, whereas cholecalciferol, which has an open B ring is not esterified [49]. (b) Acyl donors - head groups

The name ‘lecithin cholesterol acyl transferase’ suggests a strict specificity of the enzyme for the head group of phosphatidylcholine. Because of the abundance of phosphatidylcholine in HDL (74% PC, 12% sphingomyelin, 6.8% PE + PS) [50], and the use of sonicated lipid dispersions in the early investigation of head group specificity, it is not surprising that PC and N-methylated PE were thought to be the only acyl donors in the LCAT reaction [51, 521. The recent work of Pownall and coworkers [47] with discoidal particles containing apoA-I, ether-PC analogs,

309

cholesterol, and test phospholipids, has clearly shown that PE is an even better substrate for LCAT than PC, followed by dimethyl-PE, phosphatidylglycerol (PG), PA, and PS. The reactivity of a diglyceride and a triglyceride in the same particles was essentially nil, therefore, the phosphate group appears to be important in molecular substrate recognition by LCAT (See Fig. 2). Evidently in the early studies using sonicated lipid dispersions, the different hydration of PE and PS compared to PC, and the tendency of pure PE to form non-lamellar phases must have prevented effective apoA-I and/or enzyme interaction with the interface. Thus, LCAT has a broad specificity for phosphoglyceride molecular substrate head groups, with a preference for the basic ethanolamine, N-methyl-ethanolamine, and choline groups, but not to the exclusion of other phosphoglycerides.

(c) Acyl donors - glycerol backbone The natural sn-3 phosphorylcholine isomer of PC is the best substrate of LCAT, followed by the sn-2 isomer with only 16% relative reactivity, and the sn-1 isomer

A

B

I20 14-Q 160 IS0

C

Trans

Fig. 2. Molecular specificity of human LCAT for phospholipids. The test phospholipids represent 10 mol To, radiolabeled cholesterol (trace), apolipoprotein A-I 1 mol %, and the matrix lipid (an ether PC analog) 89 mol Yo of discoidal particles prepared by the sodium cholate method. Relative reaction velocities were obtained under conditions approaching enzyme saturation with substrates. Panel A represents the head group selectivity of LCAT using palmitoyl oleoyl test phospholipids in a DMPC ether matrix. Dipalmitin (DP) and tripalmitin (not shown) were essentially unreactive. Panel B shows the chain length effects using diacyl-PC test lipids in a palmitoyl oleoyl-PC ether matrix. Panel C represents the effects of unsaturation with distearoyl-PC (18 : O), dioleoyl-PC (18 : l), dilinoleoyl-PC (18 : 21, dilinolenoyl-PC (18 : 3), and the trans isomer of dioleoyl-PC (18 : 1, trans) in a palmitoyl oleoyl-PC ether matrix. (Adapted with permission from Pownall et al. [47])

with no activity at all [52].The enzyme is only capable of transferring acyl chains from and to compounds with ester bonds; amide-linked acyl chains are not transferred as indicated by the fact that sphingomyelin is not a substrate for LCAT 134,471. (d) Acyl donors - fatty acyl chains

Early work [6,29, 531 on the esterification of cholesterol in human plasma using endogenous lipoprotein substrates, indicated that the cholesterol ester species formed via the LCAT reaction decreased in the order: diunsaturated (56.6%), monounsaturated (20.6%), saturated (12.3%), and polyunsaturated (10.5%) cholesterol esters. Since the fatty acid composition in the sn-2position of HDL-PC was found to be: diunsaturated (56.5%), monounsaturated (14.5%), saturated (3.5%), and polyunsaturated (25.4%), it was concluded that acyl group transfer to cholesterol occurs from the sn-2position of PC, and that most of the cholesterol esters found in human plasma are produced by the LCAT reaction. The latter conclusion was based on the observation that circulating cholesterol ester species distribution is very similar to that produced by the LCAT reaction, and on the fact that intracellular cholesterol ester synthesis gives rise mostly to cholesterol oleate. Table 4 summarizes TABLE 4 Fatty acid chain composition of phosphatidylcholines and of cholesterol esters in human plasma

'7'0 Fatty acids in the sn-2 position of P C

Saturated Monoene Diene Triene Polyene

Subbaiah et al. [54Ib

Ueno et al. 1551'

3.5 14.5 56.5

4.9 16.1 45.8 10.4 18.5

3.0 13.0 44.2

25.4 070

Saturated Monoene Diene Triene Polyene

Glomset [6Ia

17.4

Fatty acids in cholesterol esters formed via the LCAT reaction 12.3 20.6 56.6 10.5

11.0

19.8 52.4 6.1

Measurements were performed on plasma, after removal of LDL. Measurements were performed o n LDL. Measurements were performed on plasma. In the fasting state the acyl chain composition and distribution in all lipoproteins is similar 1551.

a

311 Glomset’s data [6,291 and those of other investigators [54,551. Although the correspondence of the proportion of diunsaturated acyl chains found in the cholesterol esters of human plasma and the sn-2chain composition of the PC donors is excellent, the correlation for the other acyl chain species is not as good: the saturated chains appear to be markedly overrepresented in the cholesterol esters, while polyunsaturated chains are less abundant than expected. Two possible explanations exist for this observation. First, the transfer of acyl chains from P C may not occur exclusively from the sn-2 position but may also involve the transfer of predominantly (83%) saturated chains from the sn-1 position; and second, the relative rates of saturated acyl chain transfers (from the sn-2 position) may be higher than for unsaturated chains [6].In fact, there is evidence in support of both of these alternatives. Studies using stable vesicles of P C labeled in the sn-1 or sn-2acyl chains show that from 1 to 40% of the acyl chains transferred in the LCAT reaction can be derived from the sn-1 position [13,561.Up to 10- 20070 acyl chain isomerization may occur in synthetic PC, but 40% transfers from the sn-1 position (for dilinoleoyl-PC) are most likely real. The efficiency of transfer from the sn-1 position depends on the nature of both acyl chains in the P C donor. For example, oleoyl or linoleoyl transfer from the sn-1 position is very efficient (60%) when a stearoyl chain (a very poorly transferred species) is present in the sn-2 position (unpublished results, Zorich and Jonas, 1985). In addition to the possible minor contribution to the formation of saturated cholesterol esters from the incomplete sn-2 positional specificity of LCAT, there is evidence that palmitoyl-cholesterol ester forms more efficiently than unsaturated cholesterol esters in HDL substrate particles or in discoidal particles [45,47, 551. Yokoyama et al. [45]showed that in sonicated lipid dispersions of DMPC, DPPC, and dilinoleoyl-PC, the latter phospholipid reacted most efficiently with LCAT. But when the radiolabeled test P C molecules were incorporated into HDL, DPPC became quite reactive, and the order of reactivity changed to DMPC > DPPC > dilinoleoyl-PC. Similarly, Pownall and coworkers [47],using discoidal apoA-I particles with ether-PC matrices including a variety of test lipids, observed that DMPC and DPPC are good substrates for LCAT, distearoyl-PC (DSPC) is very poor, and 18 carbon unsaturated PC’s are in several instances not as effective as the saturated PC’s (Fig. 2). From this work [47]it is clear that the nature of the matrix lipid (DMPC, DPPC, POPC ether analogs, or sphingomyelin) affects markedly the absolute reaction rates of individual test lipids, but influences much less their relative reactivities. The most favorable matrix is the POPC ether, which resembles most closely the P C chain composition and dynamic properties of the HDL interface. The poorest matrix is provided by sphingomyelin. Particles such as those described above (discoidal complexes with apoA-I, etherP C or sphingomyelin matrix, cholesterol, and test lipids) present an essentially uniform interface to the enzyme, and reveal the true molecular specificity of LCAT for acyl donors [47,571. Discoidal complexes containing apoA-I, cholesterol, and

312

the same PC species, as matrix and molecular substrate, give reaction rates with LCAT which are determined by the molecular selectivity of the enzyme but also by its interaction with the different interfaces. In general, mixed chain PC’s with a saturated chain in the sn-1 position and an unsaturated chain in the sn-2 position are most reactive with LCAT in these systems, whereas particles prepared with PC’s containing identical, long saturated or polyunsaturated acyl chains (DSPC or diarachidonyl-PC) are most unreactive (see Table 5 , unpublished results, Zorich and Jonas, 1985). Vesicular substrate particles prepared with pure PC acyl donors are of little use in studies of molecular specificity because of the differences in interface properties, complicated by the reversible association of the apolipoprotein activator

TABLE 5 Relative reactivity of discoidal apoA-I. PC . cholesterol substrates with human LCAT a

Phosphatidylcholine

Fatty acid chains

sn-1, sn-2

positions

vk

(nmol CE/min)

app. V,,

E: (Kcal/mol)

( x 10-6) nmol CE h.M

Palmitoyl linoleoyl-PC Palmitoyl oleoyl-PC Oleoyl palmitoyl-PC Palmitoyl arachidonyl-PC Stearoyl linoleoyl-PC Dioleoyl-PC (DOPC) Stearoyl oleoyl-PC Oleoyl stearoyl-PC Dipalmitoyl-PC (DPPC) Linoleoyl stearoyl-PC Stearoyl palmitoyl-PC Dilinoleoyl-PC (DLinPC) Stearoyl arachidonyl-PC Diarachidonyl-PC (DAPC) Distearoyl-PC (DSPC)

16:0, 16:0, l8:l, 16:0,

18:2 18:l 16:O 20:4 18:0, 18:2 l k l , 18:l 18:0, 18:l 18:1, 18:O 16:0, 16:O 18:2, 18:O 18:0, 16:O 18:2, 18:2 18:0, 20:4 20:4, 20:4 18:0, 18:O

2.52 1.44 1.32 1.01 0.85 0.80 0.62 0.60 0.52 0.48 0.44 0.30 0.28 0.04 0.03

19.2 18.2 10.8 14.0 2.9 9.8 8.2 7.3 1.6 1 .o 2.8 2.6 3.3 0.2 0.02

17.6 18.8 20.0 13.4 17.2 18.2 24.2 21.4 34.8 20.9 39.9 12.6 13.9 7.7 32.5

a Unpublished results, Zorich and Jonas, 1985. Uniform particles were prepared by the sodium cholate dialysis method [40]; the diameters were approximately 110 A . Initial reaction velocities (vo) were measured at 37°C in 10 mM Tris buffer (pH 8) + 0.15 M NaCI. All samples contained equal apoA-1 concentrations. The apparent kinetic constants were obtained from linear Lineweaver-Burk plots under the conditions in by varying substrate particle concentrations. Activation energies, were measured between 15 - 38°C under the conditions given in

’.

313

with the different vesicles. The use of a common matrix lipid with added test lipids could be adapted with vesicular substrates, but the binding of apoA-I to such particles would have to be characterized. In spite of the observation that saturated acyl chains, shorter than 18 carbons, are preferentially transferred by LCAT from molecular PC substrates with two identical acyl chains [47], little is known about the recognition and binding of PC molecules at the active site of LCAT. Acyl chain length and bulk may be important, since a branched chain PC (diphytanoyl-PC) is not an LCAT substrate (personal communication, H. Pownall, 1985) and DSPC is a poor substrate even in the optimal POPC-ether matrix. Regarding the sensitivity of LCAT to the matrix lipid, or to the nature of the interface, several factors could be involved: lipid fluidity, unsaturation, separation of head groups, acyl chain packing, and the effects of these factors on the structure of the activating apolipoprotein and on its contact with lipid. In the studies of Pownall et al. [47, 571, matrix lipid fluidity and larger head group separation favor the LCAT reaction; however, we have shown that the LCAT reaction is not sensitive to the phase state of the lipid (gel or liquid-crystalline phase) [41]. In discoidal substrates prepared with DMPC or DPPC there are no discontinuities in the reaction rates with LCAT at the transition temperatures of the matrix lipids. Furthermore, changing the fluidity of the particles by altering their cholesterol content has no effect on the reaction rates [58] with LCAT. Also, the relative reactivity of DMPC, DPPC, and POPC in their own matrices can be changed by simply altering the nature and the concentration of anions in the reaction medium [59]. We have established conclusively that the anion effects are exerted on the enzyme structure or on its interaction with the interface, and not on the substrate particles. Therefore, it appears that matrix lipid effects are not transmitted directly t o LCAT, rather that the matrix lipid modulates the apolipoprotein structure or the boundary between the apolipoprotein and the particle lipid, which in turn affect the conformation of the enzyme. To summarize the present status of our knowledge of the molecular acyl donor selectivity of the human LCAT reaction: acyl donors are sn-3 phosphoglycerides with the following order of reactivity PE > PC > dimethyl-PE > PG > PA > PS; sn-2 acyl chains are transferred preferentially, but some transfer may also occur from the sn-1 position; in HDL and in synthetic substrate particles with a comparable lipid matrix, saturated PC (DMPC, DPPC) are better molecular substrates of LCAT than unsaturated 18-C PCs, followed by the trans isomer of DOPC and DSPC as the least reactive species. In human plasma the composition of cholesterol ester species reflects the abundance and relative reactivity of the acyl chains in phosphoglyceride molecular substrates of LCAT. In rat plasma, the origin and the distribution of cholesterol ester species is different from that in human plasma. From the composition of the rat plasma PC acyl chains (sn-2 position), the circulating cholesterol ester composition, and the cholesterol ester species formed in the LCAT reaction, it is estimated that about

314 70% of the circulating cholesterol esters are not produced by LCAT [6, 551. Furthermore, isolated rat LCAT shows a 2-fold preference for arachidonyl transfer from the sn-2 position of PC over the human enzyme [57].

6. Apolipoprotein activators of LCA T Since HDL are the best natural substrates of LCAT, HDL structure or composition must be responsible for the observed activity of the enzyme. In fact, studies of isolated HDL apolipoproteins in conjunction with egg-PC/cholesterol vesicles, have shown that apoA-I, the major protein component of HDL, activates the enzyme [60]. Subsequent investigations indicated that apoC-I is also an activator of LCAT; however, in the vesicle systems used in those studies, the effects of other HDL apolipoproteins (apolipoproteins A-11, C-11, C-111, D) appeared nil by themselves, or inhibitory in the presence of apoA-I or apoC-I [61- 631. The latter effect was evidently due to the competition of these apolipoproteins with the main activating polypeptides (apoA-I or apoC-I) for the vesicle surface [14, 641. The same apolipoproteins incorporated into discoidal complexes with egg-PC and cholesterol were recently shown to activate LCAT over the control lipids, in the order: apolipoproteins A-I > E > C-I > C-I11 > C-I1 > A-I1 [65, 661 (See Table 6). ApoA-I1 is capable of displacing apoA-I or apoC-I from lipoproteins or discoidal complexes, and in that sense can be considered an inhibitor of the LCAT reaction; but apoA-I1 in discoidal complexes still activates LCAT about 6-fold over control lipids [65].. ApoA-IV, another of the minor HDL apolipoproteins, activates about 20% of the apoA-I level when incorporated into vesicles prepared with unsaturated phosphatidylcholines [67, 681. ApoD has been reported to be an activator of LCAT in one study [69], but ineffective as activator in another [63]. In any event, it is clear that in vitro apoA-I is the best but not sole activator of LCAT; other apolipoprotein, notably E, A-IV, and C-I have significant activating capacity with lipids (e.g., egg-PC) analogous to the physiological HDL phospholipids. Therefore, it is not surprising that cholesterol ester synthesis proceeds in the plasma of patients with Tangier disease or with hypo-alpha-lipoproteinemia, albeit at reduced levels compared with normal plasma [70, 711. An important point is that several apolipoproteins (A-I, C-I, and A-IV) have different relative LCAT-activating capacities depending on the nature of the phospholipid in the substrate particles [61, 671. Part of the differences may be ascribed to differences in apolipoprotein binding to the vesicle substrate particles and to the possibility of particle structural changes; however, apoA-IV and apoA-I have also been incorporated into analogous lipid complexes by the cholate dialysis method and they still exhibit a preferential activation of LCAT by apoA-IV over apoA-I, when DMPC-containing particles are used. When egg-PC vesicles are used as substrates, apoA-I is by far the better activator of the two apolipoproteins [67].

315

Apparently different lipid environments may affect the structure and function of various apolipoproteins in different ways. Several peptides have been prepared by cleavage of activating apolipoproteins (apoA-I and apoC-I) or by chemical synthesis, which have activating properties for LCAT in egg-PC vesicles or in discoidal complexes with DMPC [39, 61, 72 - 751. Up to 20% of the activating capacity of apoA-I can be obtained with a structurally unrelated 24 amino acid peptide [39, 741 and 65% with a structural analog of 20 amino acids [73]. Up to 50% of the activating capacity of apoC-I is displayed by a 25 residue peptide from its C-terminus [72]. The common characteristics of all these peptides are a length of about 20 amino acid residues or longer, potential for TABLE 6 Apolipoprotein activators of human LCAT

Apolipoprotein

Relative LCAT activation To

Substrate particles; PC

References

A-I E

100

Discs, 100-200 A in diameter; egg-PC

[65, 661

Prepared by the cholate dialysis methoda; egg-PC

[671

18

c-I

12.1 5.4 4.0 3.0

c-111-1 c-111-2 c-I1 A-I1

1.5

A-1

100 38

A-IV A-I1

3.5

A-I A-IV E-2 E-3

100 25 40 30

Prepared by the cholate dialysis methoda; egg-PC

[681

A-I

100

Vesicles; egg-PC

[77 - 791

A-I (Pro'43

- Arg)

A-I (Lysio7

- 0)

pro-apoA-I

60 - 70 40 - 60

100

[791

Although the references indicate that proteoliposornes were prepared by the sodium cholate method, it is very likely that the products were discoidal. Other laboratories have observed discs under similar preparation conditions [47, 581.

a

316 forming amphipathic a-helices, and ability to bind to lipid interfaces. However, the different degrees of activation by these peptides or by apolipoproteins must be due to additional factors. Considering that apolipoproteins A-I, A-11, and C-111 in discoidal complexes with egg-PC and cholesterol all have high contents of a-helix and are essentially irreversibly bound to the particles, their relative activation of LCAT of 100, 1, and 5% must be attributed to a more favorable structure of apoAI. The question of why apoA-I is the best activator of LCAT, and how its structure differs from other apolipoproteins, is still unresolved. A-I apolipoproteins isolated from various animal species activate human LCAT very significantly: from 50 to 100% relative to the homologous apoA-I [76]. Evidently apoA-I has conserved the structural features required for very effective LCAT activation through evolution. Several well-characterized mutant forms of human apoA-I tested in eggPC/cholesterol vesicles have from 60 to 100% of the reactivity of normal apoA-I with LCAT. The Giessen variant, where Pro-143 is substituted with an Arg residue, has 60-70% of the activating capacity of normal apoA-I [77]. Residue 143 is located at a putative @-turnbetween two amphipathic helical segments of apoA-I. The apoA-I variant with the Lys-107 residue deleted (Marburg or Miinster 2), but with the rest of the primary structure retained, has 40 - 60% of the LCAT activating capacity of normal apoA-I [78]. Other mutants of apoA-I (Milano, Miinster 3) or pro-apoA-I do not show, in vitro, any differences from apoA-I in terms of LCAT activation [79, 801. Chemical modification of apoA-I in discoidal substrates of LCAT (complexes containing apoA-I, egg-PC, and cholesterol), using Lys residue reagents, indicates that individual Lys are probably not involved, and that charge interactions are not particularly important in LCAT activation by apoA-I [81]. Modification of 80 - 90% of Lys residues to N-dimethyl-Lys has no effect on the structure of the particle substrates nor on their reactivity with LCAT. On the other hand, extensive modification of apoA-I-Lys residues with reagents (citraconic anhydride and diketene) which alter the charge of apoA-I, affects its LCAT-activating function when significant structural changes in the apolipoprotein are evident [81]. Similarly, structural changes in apoA-I, incorporated into discoidal particles of different diameters containing DPPC, may lead to different levels of reactivity with LCAT [46]. Small discs ( - 100 A in diameter) are most reactive with LCAT, the largest discs show an intermediate level of reactivity, while the particles of intermediate size (-200 A in diameter) are the least reactive. Evidently, a specific structural organization of apoA-I is required for optimal activation of LCAT; it may involve the amino acid sequence of apoA-I between residues 100 to 150, but does not depend on specific Lys residues nor on extensive electrostatic interactions. The role of apoA-I in LCAT activation in plasma has been probed with antibodies to human apoA-I. After precipitation of antibody-antigen complexes, about 50% of LCAT activity was found in the supernatant and the rest in the precipitate. The

317 precipitated lipoproteins were reactive with LCAT even in the presence of blocking antibodies [82]. This observation is consistent with the ability of other apolipoproteins, besides apoA-I, to activate LCAT, but it may also mean that the antibodies against apoA-I do not interfere completely with the LCAT-activating function of apoA-I. Recent studies have revealed a marked immunochemical heterogeneity of apoA-I in HDL, which can be attributed to differences in apoA-I structure in particles with different sizes and net charges [83].

7. Modulators of LCAT activity A general summary of the major modulators of LCAT activity is given in Table 7. The sensitivity of LCAT to sulfhydryl reagents is very well documented. It is due to the two cysteine groups in the enzyme which must be in the reduced state for LCAT to be active. Mercaptoethanol and dithiothreitol stimulate enzyme activity at 10 mM concentrations by maintaining it in the reduced state. Covalent modification of the cysteine residues with 5 , 5 ‘-dithiobis-2-nitrobenzoic acid (DTNB) or p hydroxymercuribenzoate inactivates the enzyme, but the modification and inhibition can be reversed by excess mercaptoethanol or dithiothreitol [2, 6, 22, 841. This approach has been used in the assay of LCAT in plasma by following cholesterol esterification in endogenous substrates, as LCAT inhibition by DTNB is reversed [85]. Other sulfhydryl reagents such as cysteine, glutathione, N-ethylmaleimide, and metal ions (Cu2+, Hg2+, C d 2 + , Zn2+) inhibit LCAT by reaction with the same residues. In some cases the inhibition has been reversed by using competing reagents, such as serum albumin and EDTA with the metal ions [86, 871. Serine reagents, diisopropylfluorophosphate, and a specific inhibitor of serinehistidine hydrolase activity, diethyl@-nitropheny1)phosphate (E-600), inactivate LCAT very efficiently [6, 13, 221. Substrates of LCAT (HDL or vesicles) fail to protect the enzyme against inactivation by the serine or cysteine reagents [22]. This suggests that free and substrate-bound LCAT equilibrate during the comparatively slow enzymatic reaction. Salt and buffer concentration effects have been implicated in the stability of LCAT. Low concentrations of phosphate buffer (0.4 mM) promote stability, while 40 mM concentrations are required for maximal activity but also lead to most rapid inactivation of LCAT in the absence of substrates. Apparently, a conformational change occurs in the enzyme going from a low to a higher salt concentration which exposes the active site [25]. At still higher salt/buffer concentrations ( > 0.2 M) there may be inactivation by added salts, even in the presence of substrates [59]. These effects are due to the anions rather than to the monovalent cations in the buffer, and follow the Hofmeister or lyotropic series of ions in the sequence: (most activating) F - , C1-, Br-, NO, I - , and SCN- (most inactivating). One of the effects of the anions (e.g., C1-) is an alkaline shift in the optimum pH of the LCAT

318

reaction from 7.4, in low salt, to 8.2 in 0.15 M salt, accompanied by a marked inhibition of the ionization of the functional groups on the basic side of the pH activity curve. The groups involved in this effect are probably free cysteine residues. Other effects of anions, particularly at concentrations > 0.2 M, are exerted on the structure of LCAT and/or on its interactions with the substrate particles, since the TABLE 7 Modulators of human LCAT activity

Sulfhydryl reagents

Modulators

Effect(s)

References

0-Mercaptoethanol Dithiothreitol 5 3 ’ -Dithiobis-2-nitrobenzoic acid (DTNB) p-Hydroxymercuribenzoate

Activation at 10 mM Activation at 10 rnM Covalent, reversible inhibition Covalent, reversible inhibition Inhibition Inhibition Inhibition Inhibition

12, 6, 22, 84, 851

Cysteine Glutathione N-Ethylrnaleimide Cu2+, H g 2 + , Cd2+, Zn2+ Active serine reagents

Diisopropyl fluorophosphate (DW Diethylb-nitropheny1)phosphate (E-600)

186, 871

Inhibition

-

Anions

F - , C1-, Br-, NO, I - , SCNPhosphate

50- 100 mM; [25, 591 Activation to inhibition at 2 200 mM; shift in pH optimum from 7.4 (no salt) to 8.2 ( 3 50 mM)

Detergents

SDS Triton X-100

Activation in plasma Activation in plasma; inhibition of pure LCAT Inhibition reversed by serum albumin

[881 [89]

Lyso-PC Fatty acids

[52, 91 -931

Substrate analogs

Ether PC analogs Sphingomyelin

Inhibition due to substrate dilution, competition, and matrix effects

[47, 901

Products

Cholesterol esters

Inhibition in vesicular substrates

[3 1, 921

319

physical properties of the substrate particles themselves are not affected u p to 1 M neutral salt concentrations [59]. There are some reports of detergent effects on the LCAT reaction [88, 891. In plasma, sodium dodecyl sulfate (SDS) stimulates the LCAT reaction at 0.5 mM concentrations; this effect has been attributed to the dissociation of the enzyme from endogenous lipoprotein substrates which makes it available for reaction with exogenous substrates. The effects of Triton X-100 in plasma are similar to those of SDS; however, with purified enzyme Triton X-100 is inhibitory. Substrate analogs, such as ether analogs of PC or sphingomyelin, inhibit the LCAT reaction by several mechanisms: by dilution of the PC substrates at the interface, by competition for the active site, and also by matrix effects if the analogs are sufficiently abundant, and different in properties from the molecular substrates themselves [47, 901. The lysophosphatidylcholine (lyso-PC) products of the LCAT reaction are inhibitory. Strong binding at the active site does not seem to be the reason for the inhibition because the sn-1 and sn-3 phosphorylcholine enantiomers of lyso-PC are equally inhibitory, whereas only the sn-3 enantiomer is an end product of the enzymatic reaction [52]. It appears that the inhibitory effects of lyso-PC are general detergent effects on the enzyme or the substrate particles, which can be relieved by addition of serum albumin to the reaction mixtures [91- 931. Free fatty acids exert a similar inhibitory effect on the LCAT reaction, an effect which may also be neutralized by serum albumin [93]. Inhibitory effects of cholesterol ester reaction products have been described in systems containing vesicle substrates [31, 921. However, vesicles are only capable of accommodating 3 mol To of cholesterol esters in their bilayer structure. Discoidal substrates, on the other hand, readily store 10 - 20 mol Yo cholesterol esters and perhaps even more [ 191. Upon extensive reaction of discoidal substrates the reaction rates decrease, but it is not yet known whether product inhibition, substrate depletion, or changes in the interface are responsible. To complete the discussion of the known modulators of LCAT activity, the reported effects of PC/cholesterol molar ratios should be considered. Using vesicle substrates, several laboratories reported that optimal LCAT reaction rates occur at 3/1 to 4/1 molar ratios of PC/cholesterol [34, 35, 92, 941. With discoidal substrates there is no dependence of reaction rates over a wide range of molecular substrate ratios: 3/1 to 12/1, PC/cholesterol [58]. The earlier results, using the vesicle substrates, can be explained by the different affinity and stoichiometry of the apoAI activator binding to vesicles containing different amounts of cholesterol. In fact, optimal apoA-I binding coincides with the PC/cholesterol molar ratios that give the highest reactivity with LCAT [39]. The independence of LCAT reaction rates from the cholesterol content and from PC/cholesterol molar ratios, in discoidal substrate particles, is due to the irreversible binding of apoA-I to these particles. In addition, cholesterol (up to 15 mol To) does not dilute significantly the PC concentration at the interface, and does not participate as a variable in the enzyme kinetics [58].

320

8. Kinetics and mechanism (a) Enzyme kinetics

The kinetics of the LCAT reaction have only been examined systematically using discoidal substrate particles [ 19, 65, 901. Initial velocity measurements as a function of particle concentration give apparent Michaelis-Menten kinetics and linear double reciprocal plots. These plots can be used to extract apparent V,, and K , values; however, the usual meaning of these kinetic terms does not apply in the case of interfacial enzymes such as LCAT. Several kinetic models have been developed for interfacial enzymes, particularly the phospholipases. The model of Verger and deHaas [95, 961 can be applied effectively to the kinetics of LCAT if two equilibria are assumed: the binding and activation of the enzyme on the interface, and the subsequent binding of molecular substrates at the active site followed by the catalytic steps of the reaction: Kd

E-E*

+

kl S=ES+E k- 1

kcat

iP.

In the presence of interfacial inhibitors an additional equilibrium expression is included: E*

+ =.I

Ki*

EI.

The rate expressions in the absence of inhibitors lead to:

where E, E * , and E, are enzyme concentrations free in solution, activated on the interface, and total, respectively; S is interfacial substrate concentration expressed as molecules/unit surface; ES, P, and I are interfacial concentrations of enzymesubstrate complex, products, and inhibitors as molecules/unit surface; Kd is the dissociation constant of the enzyme from the interface; k,,k - 1 , and kcatare rate constants; K; is the intrinsic Michaelis-Menten constant = (k-' + kcat)/kl;and V,,, and K , are the apparent, experimental kinetic constants determined from double reciprocal plots of initial velocities as a function of substrate particle concentrations. Additional kinetic information may be obtained by diluting the molecular substrate, PC, in the interface. This can be accomplished using discoidal complexes

321 containing PC and an ether-PC analog in increasing proportions [90].In such a system, several different experimental variables may be chosen: the particle concentration, the interfacial concentration of PC at constant bulk PC concentration, or interfacial PC concentration at constant particle concentration. The kinetic model of Verger et al. [95,961, including the inhibitor equilibrium, is applicable in this case. For DPPC discoidal particles with added DPPC-ether analog, the results indicate that the ether analog is not just an inert diluent of the interfacial substrate, rather that it acts as a competitive inhibitor at the active site [90]. The interpretation of apparent V,,, and K , results in terms of the intrinsic kinetic constants (k,,, and K z ) requires the independent determination of enzymesubstrate particle binding constants (i.e., Kd values). Such data are not yet available. Nevertheless, V,,,/K, ratios can be a useful measure of relative substrate effectiveness (See Table 5 ) . The kinetics of the LCAT reaction have not been studied with vesicle substrates; however, it can be anticipated that a more complex kinetic model would be required to account for the equilibration of the apolipoprotein activator with the interface. Kinetic models developed for lipoprotein lipase reacting with lipid emulsions or vesicles to which the apoC-I1 activator had been added [97],would probably be applicable to this case. (b) LCAT active site

From the effects of sulfhydryl reagents and active serine-histidine inhibitors, LCAT appears to have functional cysteine, serine, and histidine residues, analogous to the active site residues of some esterases [98].Compared to the well-known pancreatic or venom A, phospholipases [99],LCAT has a similar but not absolute specificity for sn-2acyl chain cleavage of interfacial phosphoglycerides; LCAT does not require Ca2+ for activity, but depends on apolipoprotein activators. By analogy with other acyltransferases [loo],it is possible, but not yet proven, that LCAT may go through an acyl intermediate during the acyl transfer reaction. Binding of phosphoglyceride substrates to the active site and the cleavage of the acyl-glycerol ester bond by LCAT do not show a marked specificity for the head group [47],but discriminate in favor of sn-3phosphoryl isomers [52]and of sn-2 acyl chain cleavage [6,131. In addition, the enzyme is sensitive to the acyl chain length, bulk, and unsaturation of the phosphoglycerides [41,47, 591. Therefore, a large active site, capable of accommodating the glycerol-phosphate region and the hydrophobic acyl chains is probably present in LCAT. If an acyl enzyme intermediate exists then it should be accessible to the various acyl acceptors: 3-0hydroxy sterols, water, lyso-PC, and long chain alcohols [48,49].The nature of the enzyme interaction with the acyl acceptors is not known, but it is probably not rate limiting, since the unesterified cholesterol concentration in discoidal substrates does not affect the reaction rate with LCAT [58].

3 22

The rate-limiting step of the LCAT reaction has not yet been identified; however, judging from the changes in activation energy of the LCAT reaction with discoidal substrates of different compositions, it can be narrowed down to the phosphoglyceride binding at the active site or to an acyl-enzyme formation step. Discoidal particles containing different PC have activation energies ranging from 7.7 kcal/mol for diarachidonyl-PC to 39.9 Kcal/mol for stearoyl palmitoyl-PC [41] (unpublished results, Zorich and Jonas, 1985) (See Table 5). On the other hand, discoidal complexes prepared with various HDL apolipoproteins, but containing the same PC (egg-PC), all have activation energies around 18 Kcal/mol [65, 661. Thus, the events of LCAT activation by apolipoproteins are probably not rate limiting.

9. LCAT in plasma LCAT is synthesized in the liver and is secreted into circulation [6]. Although the majority of the synthesis and tissue distribution studies have been performed in the rat [101, 1021, there is some evidence suggesting that the liver is also the source of LCAT in humans [ 1031. Liver disease in humans is frequently associated with a deficiency of LCAT [6]. Control of LCAT synthesis has not yet been investigated, but a dietary fat load results in increased LCAT activity (37%) following the increase of triglyceride-rich lipoproteins in plasma [104]. It is not clear whether the effect is due to increased LCAT mass in plasma or t o the availability of better substrates [85]. In circulation, LCAT is present in complexes with lipoproteins and perhaps as free protein [6]. Its catabolism has not been studied, but the fact that LCAT is a glycoprotein containing sialic acid residues, suggests that it may be removed by the liver via the asialoglycoprotein receptor mechanism. In the plasma of normal individuals the average mass of LCAT is 5.9 -t 1.8 pg/ml and the average LCAT activity with endogenous lipoprotein substrates, measured by three different methods, is 25 f 5, 61 k 17, and 106 k 30 nmol cholesterol esterified/h/ml [ 1051. Studies using isolated lipoproteins indicate that most of the mass and activity (94- 99O70)of LCAT are present in the HDL or d > 1.21 g/ml fractions; low density lipoproteins (LDL) bind the remainder of LCAT and express 1 - 2% of the activity; very low density lipoproteins (VLDL) do not associate with the enzyme nor do they act as substrates [28, 1061 (see Fig. 3). Regarding the distribution of LCAT between HDL (18-92%) and the d > 1.21 g/ml fraction (11 - 8O%), the proportions vary in different studies, probably as a result of the different separation procedures, handling of the plasma, and physiological state of the donors. Apparently, ultracentrifugal separation methods give higher levels of LCAT in the d > 1.21 g/ml fractions [106]. The nature of LCAT in the non-lipoprotein fraction (d > 1.21 g/ml) is not certain, although LCAT may be associated with apolipoproteins and perhaps even with small amounts of lipid. That apoD and apoA-I may interact with LCAT

323 is suggested by the complete adsorption of LCAT activity to antibodies specific for these apolipoproteins [ 1071, and by the observation that apoD, in particular, copurifies with LCAT through several isolation steps [ 5 , 131. On the other hand, LCAT does not appear to have a high affinity for free apoA-I in vitro, compared to its interaction with HDL [24, 108, 1091. This observation, however, does not rule out binding of the enzyme to apoA-I in lipid complexes or in lipoproteins, where the apolipoprotein has a very different conformation from the lipid-free form. A physiological role for an LCATIapoA-I/apoD complex, containing small amounts of lipid, as the main substrate for LCAT and as a cholesterol ester transport complex [lo71 has not been substantiated by more recent work. Several laboratories have shown that the plasma lipid transfer protein is distinct from apoD [110- 1121, and numerous reports indicate that recombined apoA-I complexes with lipids, especially the discoidal particles, are excellent substrates for LCAT in the absence of any lipid transfer and of apoD [19, 37, 40-431. Exposed to isolated subclasses of HDL, LCAT shows a marked preference for the smaller HDL, particles over HDL, particles as substrates [113, 1141. It is clear, however, that HDL, binds substantial amounts of LCAT [28, 1151. Therefore, conflicting reports which indicate that HDL, is an inhibitor of LCAT [114], on the one hand, or that it is required to express the full activity of LCAT in plasma [116], on the other, may be rationalized on the basis of competing equilibria and of differential reactivities of the two major HDL subclasses with LCAT.

i 1.6

w m

8

1 . 2-

'

;0.851 n 4

0.4 -

Fraction number ( 2 rnl /fraction)

Fig. 3. Distribution of LCAT activity and mass in human plasma. Plasma was fractionated by gel filtration on a Bio-Gel A-5 m column. LCAT activity was measured using radiolabeled substrate particles prepared by the sodium cholate procedure and LCAT mass was determined by a double antibody radioimmunoassay. Elution positions 1, 2, 3, and 4 correspond to VLDL, LDL, HDL, and plasma proteins, respectively. (Reproduced with permission from Chen and Albers [ 1061)

324 Abnormal lipoproteins, discoidal HDL from LCAT-deficient patients [ 1 131, and Lp-X from patients with obstructive jaundice [117],have been studied in vitro as substrates for LCAT. Discoidal HDL, particularly fractions enriched in apoA-I over apoE, are very good substrates for LCAT - considerably better than spherical HDL [113]. Lp-X can be shown to react with LCAT, but at much lower rates than discoidal particles or spherical HDL. The estimated rates of reaction appear to be several thousand times slower for Lp-X [117]. Although the reaction of LCAT in normal and abnormal plasma occurs primarily on the HDL particles, it is clear that all other lipoprotein classes undergo transformations as a result of this activity (see Fig. 4). At the root of these transformations is the ability of lipoproteins to exchange and transfer lipids and apolipoproteins

LIVER

INTESTINE

--- -- +VLDL

---- t L D L

---- +CHYLOS --- +REMNANTS

Fig. 4. Role of LCAT in HDL transformation and in the modification of other lipoprotein classes [118]. Nascent discoidal and small spherical HDL arise in the liver, intestine, and in the circulation as a result of the lipolysis of VLDL and chylomicrons. Excess surface phospholipids (PL), cholesterol ( C ) , and apolipoproteins (A and C) produced during the action of lipoprotein lipase on the triglyceride-rich lipoproteins appear in the HDL density region. Nascent HDL are the preferred substrates of LCAT. During the transformation of the nascent particles into spherical HDL, and HDL, subclasses by LCAT, further lipid and apolipoprotein transfers take place. ApoE and apoC are transferred to nascent triglyceriderich lipoproteins. The transfer of apoC to chylomicrons, and CE and triglyceride (TG) transfers from chylomicrons to HDL are not shown in this diagram. Cholesterol esters and phospholipids are transported from HDL to VLDL and LDL by the action of the lipid transfer protein(s). In turn, triglycerides, phospholipids, and cholesterol flow into HDL (cholesterol by aqueous diffusion and triglycerides and phospholipids via the lipid transfer protein(s)). In addition to lipid exchanges and transfers among lipoproteins, cell membranes exchange lipids (phospholipids and cholesterol) with HDL and other lipoproteins (not shown). Although HDL, appears in the diagram only as a product of HDL, transformation by LCAT, it too can act as a donor and acceptor of lipids in conjunction with other lipoproteins and membranes. In LCAT deficiency, nascent HDL as well as a variety of triglyceride-rich lipoprotein lipolysis products appear in plasma. Furthermore, cholesterol and phospholipids accumulate in cell membranes and in tissues. The role of LCAT in ‘reverse cholesterol transport’ is also illustrated in this diagram. The direction of unesterified cholesterol flow is from cells and various lipoproteins to HDL, esterification on HDL by LCAT, transfer of cholesterol esters to VLDL and to LDL, and removal by the liver or steroidogenic tissues as the lower density lipoproteins, or as HDL. The latter, catabolic steps, are not yet completely defined, nor are they depicted here.

325 among themselves [118, 1191. Free cholesterol from VLDL and LDL transfers spontaneously to HDL and is used in the LCAT reaction [ 1201. Chylomicron and VLDL surface components, including phospholipids, cholesterol, and apolipoproteins A-I and C, appear in the HDL fraction during lipolysis and participate in the LCAT reaction [ 1 181. This transfer process may involve formation of HDL particles directly from the surface components, or the incorporation of individual molecules into preexisting HDL, either by spontaneous transfer (cholesterol, apolipoproteins) or by lipid transfer protein-mediated movement (phospholipids, cholesterol esters, triglycerides). The cholesterol esters formed in HDL during the LCAT reaction can be exchanged with triglycerides via the lipid transfer protein; the donors of the triglycerides and acceptors of cholesterol esters in this exchange are primarily VLDL. Most of the cholesterol esters of VLDL and LDL, therefore, originate in HDL during the LCAT reaction [118, 1191. In addition to cholesterol ester transfers from HDL to VLDL, apoE redistributes from discoidal HDL (nascent or from LCAT-deficient patients) to VLDL in the course of the LCAT reaction [121]; and in rat plasma apoA-IV changes from the lipid-free to the lipoprotein-bound state when LCAT is active [122]. Aside from the lipid transfers that occur among lipoproteins, phospholipids and cholesterol transfer readily between cell membranes and lipoproteins. Several reports indicate that the LCAT reaction in isolated plasma leads to the conversion of HDL, to particles with the flotation characteristics of HDL,. This conversion is especially efficient in the presence of triglyceride-rich lipoproteins, and does not occur when LCAT is inactivated [123 - 1251. However, other activities must be considered in human and animal plasma, in vivo and in vitro, such as the activity which reportedly converts HDL, into a larger and a smaller particle in the absence of known enzymes and transfer proteins [126]; and the activity of lipoprotein lipase which, in vitro, gives rise to HDL2-like particles from HDL, during the lipolysis of VLDL [127, 1281.

10. Physiological role of LCA T The physiological significance of the cholesterol esterification reaction carried out by LCAT in plasma, is best illustrated by the studies of LCAT-deficient patients and their lipoprotein and tissue abnormalities. Familial LCAT deficiency was described first in 1967 [3, 41 and has been investigated intensively since then [7, 111. There is less information on LCAT deficiency due to parenchymal liver diseases, such as alcoholic hepatitis, but similarities with the hereditary deficiency are evident, particularly in the lipoprotein patterns. The hereditary form of LCAT deficiency has been described in families throughout the world [129- 1321. As of January 1981, 12 families had been reported in nine countries [ l l ] . It is now clear that some of the mutants lack any measurable amounts of LCAT in plasma, whereas others con-

3 26 tain reduced levels of inactive enzyme 11321. Thus, as with other proteins, diverse mutations can result in the absence or reduced levels of LCAT activity in plasma. Familial LCAT deficiency is characterized by diffuse corneal opacities, normochromic anemia with a reduced erythrocyte life span, and proteinuria often followed by renal insufficiency [133]. Foam cells are observed in the bone marrow and the kidney glomeruli, and ‘sea-blue histiocytes’ are detected in the spleen and the bone marrow. Most patients have turbid or milky plasma, and a tendency to develop premature atherosclerosis. The tissue and intracellular changes are due to lipid deposits, particularly of unesterified cholesterol and phospholipids, which are also present in excessive amounts in erythrocyte membranes and in plasma. In view of these pathological manifestations of familial LCAT deficiency and the wellknown cholesterol esterification reaction of LCAT involving HDL, Glomset has suggested that the general role of LCAT is to reduce the levels of free cholesterol in plasma and in tissues [6, 1341. Important functions of LCAT in humans would be the removal of excess unesterified cholesterol and phospholipids from remnants of chylomicrons and VLDL, and participation in the transport of cholesterol from peripheral tissues to the liver. The following discussion will be limited to the lipoprotein abnormalities in LCAT deficiency, the effect of LCAT on the abnormal lipoproteins, and the effects of LCAT on the efflux of cholesterol from cells, as illustrations of the physiological role of LCAT in humans. The lipoproteins of LCAT-deficient individuals, separated by a variety of means, show major differences from the normal lipoprotein patterns [ 1 1 , 135, 1361. VLDL have abnormal beta-mobility on electrophoresis, high levels of unesterified cholesterol, and low amounts of total protein. Some of the larger VLDL may, in fact, be chylomicrons since they disappear after several days of lipid-free diets. The LDL, density fraction frequently includes very large particles. Upon gel filtration through 2% agarose, LDL, usually yields three subfractions, whereas normal plasma only gives one peak. The largest abnormal particles (90 nm) have a multilamellar appearance and contain very high free cholesterol/PC ratios in the bilayer structures. Albumin is the principal protein in this subfraction. The intermediate size LDL, contain particles with all the characteristics of Lp-X. They appear as flattened vesicles on electron micrographs, have high contents of free cholesterol and PC, and contain apoC’s and albumin as the major protein components. In the same size range, there are also some spherical particles resembling remnants. The smallest LDL, of LCAT-deficient patients have a size comparable to normal LDL (20- 22 nm), but contain large amounts of triglycerides in place of cholesterol esters (CE). Apolipoprotein B is the main protein component of these particles; however, it is only present at levels 1/2 to 1/3 of the apoB content of normal plasma. It is thought that all these LDL, particles result from the action of lipoprotein lipase on triglyceride-rich lipoproteins [ 135, 1361. Patient HDL is also abnormal. There are two major fractions: one disc-shaped,

327 and another spherical but unusually small (4 - 6 nm). The latter particles contain apoA-I as the only apolipoprotein, and small amounts of free cholesterol, CE, and phospholipids [ 1371. The discoidal HDL consist largely of free cholesterol and phospholipids, and can be further fractionated according to their apolipoprotein distribution into particles enriched in apoE or in apoA-I plus apoA-I1 [113]. Similar particles have been described in alcoholic liver disease [138, 1391 and in rat or monkey liver perfusates, where the activity of LCAT had been inhibited or diminished [140, 1411. The discoidal particles have diameters in the range from 15-20 nm and widths around 4.5 nm [140]. It is generally agreed that the apoEenriched particles in LCAT deficiency are mostly of hepatic origin and represent nascent HDL [135, 1361; however, recent studies show that apoE and discoidal particles of HDL size and density can be synthesized by cholesterol-enriched macrophages [142, 1431, and can be isolated from the interstitial lymph of cholesterol-fed dogs [144]. In vitro lipolysis of VLDL also gives rise to discoidal products containing C apolipoproteins [118, 1451; it is plausible that segments of VLDL or chylomicron excess surface materials could break off giving rise to HDL density, discoidal particles [118, 1461. The origin of the small spherical HDL is still unknown. The plasma of LCAT-deficient patients fractionated by conventional ultracentrifugal methods contains significant amounts of lipoprotein free apoA-I. It is not certain what proportion of this free apoA-I is due to dissociation during the separation procedures. In any event, only about 1/3 of the normal apoA-I levels can be found in patient plasma [ 1361. A number of studies indicate that discoidal HDL of any origin are considerably more reactive with LCAT than spherical HDL of comparable apolipoprotein composition [113, 136, 1401. When LCAT is added to plasma from deficient patients or when liver function is restored in alcoholic liver disease patients, marked changes occur in lipoprotein patterns, shapes, and apolipoprotein distribution [17, 136 - 1381. There is a general decrease in unesterified cholesterol and phospholipid levels and an increase in CE; LDL of normal size increase; and discoidal and small spherical HDL are replaced by normal HDL of the HDL, and HDL, subclasses. ApoA-I content in HDL increases while apoE decreases and appears in VLDL. In short, there is a normalization of the lipoproteins as a result of LCAT action. Indeed, Glomset [6, 71 suggests that the lipoproteins in LCAT-deficient patients are not abnormal, rather that they have not been exposed to LCAT. If this is the case, then in normal plasma LCAT is essential for the maturation, interconversion, and rearrangements of all lipoprotein classes. The lipoproteins isolated from normal plasma, particularly the HDL, probably represent the products, rather than the substrates of the LCAT reaction. The involvement of LCAT in ‘reverse cholesterol transport’ (i.e., in removal of cholesterol from peripheral tissues and transport to the liver) [6, 291, is supported by studies of cholesterol efflux from cells. Normal erythrocytes may exchange or transfer free cholesterol to HDL, but the extent of the net transfer may be increased dramatically in the presence of LCAT activity [6, 1471. The excess cholesterol ap-

pears in HDL as cholesterol esters. Similar studies with cells which are able to synthesize cholesterol (e.g., fibroblasts in culture), show in most cases that LCAT activity in plasma, or previous exposure of plasma to LCAT, results in net efflux of cholesterol from cells [ 148 - 1511. The nature of the physiological acceptors of cholesterol is not known precisely, but very high density small particles containing apoA-I, normal HDL, or even discoidal HDL are possible candidates. All these particles may react directly with LCAT or may supply unesterified cholesterol to the best LCAT substrate particles. The transfers of free cholesterol between lipoproteins and between lipoproteins and cell membranes are determined by the physicochemical activity of cholesterol in each of the particles and membranes, and proceed spontaneously through solution [150- 1551. In the fasted state, plasma cholesterol transfers must be near equilibrium in all interfaces except those where the LCAT reaction is taking place. Thus, LCAT reaction on its preferred substrate particles should increase free cholesterol incorporation into these lipoproteins at the expense of all other lipoproteins or membranes. Much of the cholesterol esters formed in the LCAT reaction are subsequently transferred into VLDL via the lipid transfer protein of plasma, and also appear in LDL, mostly as a result of VLDL catabolism [118]. In the original hypothesis of ‘reverse cholesterol transport’ the postulated final steps involved cholesterol transport in HDL particles, HDL uptake by the liver, and excretion of free cholesterol or its metabolism to bile salts [6, 291. To date, direct evidence for such a process is lacking, and it appears that HDL metabolism is more complex than originally expected [155]. Whether or not human HDL particles are taken up intact or as separate components by the liver, kidney, and steroidogenic tissues, and whether or not specific or non-specific uptake mechanisms are involved, are still unresolved problems. However, a significant part of the cholesterol esters produced by the LCAT reaction on HDL, after transfer to VLDL and LDL, may enter the liver via the apoB/E receptor pathway [ 155 - 1571.

I I . Conclusions and future directions The availability, in the last decade, of pure and stable LCAT preparations, led to significant progress in the research on LCAT physical and chemical properties, kinetics, and substrate requirements. Further advances in the determination of LCAT structure - amino acid sequence, 3-dimensional structure, and carbohydrate sequences and linkages to protein - will require sizable amounts of enzyme. Current preparations of LCAT yield about 0.5 mg of enzyme per liter of plasma. Repeated purifications from several liters of plasma could be performed, but LCAT c-DNA cloning and expression in prokaryotic cells is a possible, very attractive alternative to the classical amino acid sequencing and purification approaches. Since antibodies to LCAT are available, and human liver c-DNA libraries exist in several

329

laboratories, the isolation and characterization of LCAT c-DNA clones can be expected in the near future. The c-DNA probes will be also useful in the determination of LCAT m-RNA tissue distribution, and in the investigation of the chromosomal localization, structure, and regulation of the LCAT gene. At the cellular level, the synthesis, processing, and secretion, as well as the catabolism of LCAT, remain to be investigated, particularly in systems of human origin. In the area of LCAT mechanism of action and substrate specificity, larger amounts of enzyme will be required for substrate-enzyme binding studies. Equilibrium parameters will be essential for the elucidation of the apolipoprotein activation mechanism, for the full description of the LCAT interaction with interfaces, and for the interpretation of kinetic parameters.

A ckno wledgements I wish to thank N. Zorich for her critical reading of the manuscript, and t o acknowledge the support of NIH Grants H L 16059 and H L 29939 for our research on LCAT.

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