~ Charaterization of proteoliposomes ontaining apoprotein AI: a new substrate for the measurement of leithin: holesterol ayltransf erase ativity Chinghong Chen and John J. Albers' Department of Mediine, Harborview Medial Center, University of Washington Shool of Mediine, Seattle, WA 98104 Abstrat Proteoliposome vesiles ontaining apoai, leithin, and holesterol (inluding labeled holesterol) were prepared from various molar ratios of the three omponents by the holate dialysis tehnique. Comparative studies on the sensitivity and effiieny of these proteoliposomes to serve as substrate for leithin:holesterol ayltransferase (LCATase) indiated that the proteoliposome with apoa1:leithin:holesterol molar ratio of 0.8:250:12.5 was ideal for assaying LCATase ativity of both plasma and purified enzyme. This proteoliposome was shown to be omparable in size by gel filtration (radius, 131.9 t 4.8 A, n = 6) and by eletron mirosopy (radius, 123.4 f 5.1 A, n = 100). The proteoliposome preparation was stable as LCA Tase substrate for at least 3 and 5 weeks, respetively, when stored at 4OC and 2OoC, and was a better substrate for the enzyme ativity assay than were leithinholesterol liposomes inubated with apoai. Under the standardized assay system LCATase ativity was a linear funtion of plasma enzyme added and was independent of the amount of plasma holesterol added to the proteoliposomes in the range of 3 to 20 p1 of plasma. The mean LCATase ativity by this method was 95.1 f 14.0 (range 76.5122.5) nmol/hr per ml of plasma from fifteen normal human subjets.l This method of substrate formation using the holate dialysis tehnique permits the preparation of large amounts of stable, effiient, homogeneous, and welldefined substrate that is suitable for measuring low levels of enzyme ativity, omparative studies, and large sale inves tigations of plasma LCATase, as well as studies of the mehanism and regulation of LCATase reation.chen CH., and J. J. Albers. Charaterization of proteoliposomes ontaining apoprotein AI: a new substrate for the measurement of leithin:holesterol ayltransferase ativity. J. Lipid. Res. 1982. 23: 680691. Supplementary key words leithin holesterol eletron mirosopy Human plasma and that of many other mammals ontains an enzyme, 1eithin:holesterol ayltransferase (LCATase, EC 2.3.1.43), that reats preferentially with high density lipoproteins (HDL) to atalyze the transfer Of a fatty ayl group from leithin to unesterified holesterol, with the formation of lysoleithin and holesteryl (13)' A variety Of methods using and artifiial substrates have been employed for estimating the rate of LCATase reation. The existing assay methods, based on the nature and soure of the substrate, are the endogenous selfsubstrate method and the exogenous ommonsubstrate method. The endogenous selfsubstrate method, whih uses native plasma (the subjet's own plasma) as substrate and enzyme soure, measures the endogenous holesterol esterifiation rate either a) by measuring the rate of onversion of labeled holesterol to holesteryl ester after preinubation of fresh plasma with a reversible inhibitor (DTNB) and equilibration with radioative holesterol followed by inubation with meraptoethanol to reativate the enzyme (4), or b) by diretly measuring the derease in the mass of endogenous unesterified holesterol after inubation of the plasma sample itself, using gasliquid hromatography (5) or an enzymi method oupled with various kinds of hromogens (69). The measurement of holesterol esterifiation rate by the radioassay tehnique measures both enzyme and substrate effets. However, it may not reflet the true esterifiation rate, sine long preinubation and equilibration may alter the substrate properties of the plasma, and the labeled exogenous holesterol may not be in omplete equilibration with endogenous holesterol (10, 11). Therefore, the holesterol esterifiation rate by the radioassay proedure usually yields lower values than do the rates obtained by diret hemial measurement of unesterified holesterol (11). On the other hand, the diret measurement of the hange in unesterified holesterol by gasliquid hromatography (5) or by the enzymi method (69) may reflet the initial holesterol esterifiation rate, but it is not appropriate for the measurement of the intrinsi en Abbreviations: LCATase, 1eithin:holesterol ayltransferase; apoa I, apolipoprotein AI; HSA, human serum albumin; DTNB, 5,sdithiobisnitrobenzoi aid; DMPC, Ladimyristoylphosphatidylholine. ' Address orrespondene or reprint requests to John J. Albers, Northwest Lipid Researh Clini, Harborview Medial Center, 325 Ninth Avenue, Seattle, WA 98104. 680 Journal of Lipid Researh Volume 23, 1982
zyme ativity beause it does not distinguish qualitative or quantitative alterations of the enzyme from hanges of the substrate or ofators of the reation (10, 11). In addition, the latter method requires a large number of repliates and a highly sensitive method for holesterol quantifiation, sine it measures the rate of disappearane of substrate holesterol by the differene between test and ontrol samples (1 1). Consequently, this method is not suitable for largesale investigation or for measurement of very low levels of esterifiation rate. The ommonsubstrate method, whih uses heated plasma (1 2) or artifiial liposomes (1 3, 14) as ommon substrate, measures LCATase ativity by measuring the onversion of radiolabeled holesterol to holesteryl ester after inubation of fresh plasma as enzyme soure, with heated plasma equilibrated with radioative holesterol (1 2), or with syntheti leithinholesterol liposomes prepared by soniation (1 3), or by dissolving these lipids in ethanol (14). Although the use of heated plasma as a ommon substrate an minimize the effet of the substrate of the donors' plasma on the assay of LCATase ativity, this method is not ideal; heated plasma is a poor substrate for LCATase, usually resulting in very low levels of LCATase ativity (11, 15) and the results vary, depending on the soure of substrate (7, l l, 16). The soniated or ethanoli leithinholesterol liposomes have been used as ommon substrate for plasma frations and purified enzyme, but have been used little for the measurement of plasma LCATase ativity. In this approah, the plasma onstituents ould alter the substrate properties of the artifiial substrate. Furthermore, an artifiial substrate has yet to be standardized or validated for the measurement of plasma LCATase ativity. Therefore, existing methods for measuring LCATase ativity are inadequate. In this report, we desribe a new method for preparation of a welldefined and standardized ommon artifiial substrate, a proteoliposome ontaining apoai, egg leithin, and [4'4C]holesterol, for a sensitive and preise determination of LCATase ativity. The validity of this new substrate was evaluated with both plasma and purified enzyme. Chemials MATERIALS AND METHODS Eggyolk leithin (50 mg/ml ethanol solution) was purhased from Applied Siene Laboratories, In. [4 ''C]Cholesterol (sp at, 54.0 mci/mmol) and [arboxyl ''C]holi aid (sp at, 52.0 mci/mmol) were obtained from New England Nulear Corporation. Cholesterol, holesteryl oleate, sodium hloride, Tris (hydroxymethyl) aminomethane, sodium holate, and human serum al bumin (essentially fatty aidfree) were supplied by Sigma Chemial Co. Ethylenedinitrilotetraaeti aid was a produt of MCB Manufaturing Chemists, In. Bovine serum albumin (BSA) was purhased from Armour Pharmaeutial Co. and Sepharose CL4B from Pharmaia Fine Chemials. TrisHC1 buffer solution whih ontained 10 mm TrisHC1, 140 mm NaCl, and 1 mm EDTA, ph 7.4, was used throughout this study. All hemials and proteins unless otherwise speified in this study were made in this TrisHC1 buffer solution, ph 7.4. Plasma samples Plasma samples used as enzyme soure for LCATase ativity study were obtained from adult volunteers without overt disease after a 1216hr overnight fast. Blood was drawn into vautainer tubes ontaining sodium EDTA (1 mg/ml) as antioagulant and plasma was obtained after entrifugation at 4 C. Protein determination Protein onentration was determined by the method of Lowry et al. (17) using BSA as a standard. Purifiation of apoai ApoAI was isolated from fresh normal human plasma as previously desribed (18). The final apoai preparation was in 10 mm TrisHC1 buffer ontaining 140 mm NaCl and 1 mm EDTA, ph 7.4. Purifiation of LCATase The enzyme was purified to homogeneity from fresh normal human plasma (in EDTA) by a ombination of density ultraentrifugation and hromatography on phenylsepharose, DEAESepharose, and hydroxylapatite as desribed (3, 19). The final enzyme preparation was stored in TrisHC1 buffer, ph 7.4 (10 mm Tris HCl, 140 mm NaCl, and 1 mm EDTA). Preparation of new artifiial substrate proteoliposomes Proteoliposomes were prepared by the holate dialysis tehnique similar to that desribed for reonstitution of membrane proteins into artifiial lipid vesiles (2022). A typial proteoliposome (apoa1:leithin:holesterol molar ratio 0.8:250:12.5), whih was found to be an ideal substrate for LCATase in this study, was prepared as follows. For every 40 assays, the substrate was made by pipetting 0.154 ml of 50 mg egg leithin/ml ethanol solution, 0. l 16 ml of l mg unlabeled holesterol/ml ethanol solution, and 0.108 ml of 1.8 mg [4'4C]holesterol/2.5 ml benzene solution (0.25 mci/2.5 ml benzene solution) into a glass vial. The lipid mixture was arefully evaporated to dryness under a stream of nitrogen at room Chen and Albers Proteoliposomes ontaining apoai, an LCAT substrate 681
temperature. To these dried lipids, 2.50 ml of TrisHC1 buffer solution, ph 7.4, 0.8 ml of 1.10 mg apoai/ml in TrisHC1 buffer, and 0.3 ml of 725 mm sodium holate solution in TrisHC1 buffer were added, mixed on a Vortex mixer for 1 min at room temperature, and then inubated in a 24 C shaking waterbath for 20 min. The mixture was dialyzed extensively against TrisHC1 buffer, ph 7.4, for 20 hr at 4OC to remove holate. The dialysate was then adjusted to 4 ml using TrisHC1 buffer, ph 7.4. One experiment with [14C]holate indiated that 99.4% and 99.9% of the holate was removed, respetively, after 10 and 18 hr of dialysis. Measurement of LCATase ativity with proteoliposomes The enzyme ativity assay was arried out by inubating the liposomes with apoai in the assay mixture as previously desribed (19, 24). Gel filtration of proteoliposomes The proteoliposome preparations were hromatographed on a Sepharose CL4B olumn (2.2 X 50 m) aording to the proedure of Huang (25). The Stokes' radius of the proteoliposome vesiles was determined by using the equation of Akers (26) as desribed by Huang (25). Ovalbumin (Stokes' radius = 27.3 A) and fibrinogen (Stokes' radius = 110.0 A) were used to determine the two alibration onstants a0 and bo of the olumn. Composition analysis of proteoliposomes For all assays using proteoliposomes as substrate Proteoliposomes were analyzed for leithin, holesthe following proedures desribed for a typial as terol, and apoai. Leithin was determined aording say, using the standardized proteoliposome (apoa to the method of Bartlett (27), holesterol by the enzymi 1:leithin:holesterol molar ratio 0.8:250:12.5) as sub proedure of Nagasaki and Akanuma (7), and protein strate, were followed unless otherwise speified. The by the method of Lowry et al. (17). assay was arried out in a srewapped ulture tube (16 X 125 mm) in whih 0.230 ml of TrisHC1 buffer, Eletron mirosopy of proteoliposomes ph 7.4, 0.125 ml of 2% HSA, and 0.1 ml of the pro Eletron mirosopy of the proteoliposomes negatively teoliposome were added. The substrate mixture was stained with 1% sodium phosphotungstate, ph 7.4, was preinubated at 37 C for 20 min, 0.025 ml of 100 mm arried out as previously desribed (28). meraptoethanol was added, and this was followed by addition of 0.015 ml of plasma (or purified enzyme). This assay mixture ontained 250 nmol leithin, 7.5 nmol unlabeled holesterol, 5 nmol [4'4C]holesterol, 0.8 RESULTS nmol (22 pg) apoai, 0.5% HSA, 5 mm meraptoethanol, and 0.015 ml of plasma (or purified enzyme) in Preparation of proteoliposomes a final volume of 0.5 ml. The assay mixture was mixed immediately on a Vortex mixer and inubated in a 37 C waterbath for 30 min. The enzymi reation was stopped, the holesterol and holesteryl ester were separated, and radioativity was ounted as previously desribed (19, 23). LCATase ativity was determined from the onversion of [414C]holesterol to labeled holesteryl ester. Control reations ontaining no enzyme were run simultaneously to orret for nonenzymi reation. The rate of LCATase reation is expressed as both frational ativity (the perentage of holesterol esterified per 30 min), and molar ativity (nmol holesterol esterified/hr per ml plasma or per mg purified enzyme). This latter value was obtained by multiplying the frational ativity with the onentrations of holesterol in the proteoliposomes. Measurement of LCATase ativity with liposomes Liposomes ontaining 1eithin:holesterol (inluding labeled holesterol) molar ratio of 250:12.5 and 4:l were prepared in the absene of apoai, respetively, by the holate dialysis tehnique used in this study and by the method of Batzri and Korn (14) as desribed (23, 24). In order to develop a welldefined, standardized, and effetive ommon artifiial substrate for LCATase, proteolipid vesiles ontaining apoai, leithin, and holesterol (inluding labeled holesterol) were prepared from various molar ratios of the three omponents by the holate dialysis tehnique. The proteoliposomes were then used as substrate for assaying LCATase ativity in both plasma and purified enzyme preparations in the presene of 0.5% HSA (unless otherwise speified) and 5 mm meraptoethanol. A series of studies was arried out to selet the relative proportions and absolute onentrations of the three vesile onstituents so that when the substrate was used the enzyme ativity would follow zeroorder kinetis and be independent of enzyme onentration within a given range, and yet the substrate would still be in exess in the assay mixture. The first step to selet the proteoliposomal omposition was to examine the effet of 1eithin:holesterol molar ratios on LCATase ativity. Assays were arried out using proteoliposomes formed from mixtures of onstant apoai (1 nmol/assay) and various proportions of leithin to holesterol (Figs. 1 and 2). Assay of plasma LCATase ativity in proteoliposomes, keeping leithin 682 Journal of Lipid Researh Volume 23, 1982
ontent onstant and varying the holesterol ontent (Fig. 1, top) indiated that inreasing the holesterol from 1 to 5 mol 70 resulted in only a slight derease in the frational LCATase ativity. However, inreasing the holesterol ontent to 10 mol % redued the frational LCATase ativity to approximately onehalf of that obtained with 5 mol % holesterol. Keeping holesterol ontent onstant and varying the leithin ontent (Fig. 1, bottom) showed that dereasing the leithin ontent from 500 to 100 nmol resulted in a slight inrease in the frational LCATase rate; but at a leithin ontent of 50 nmol (10 mol % holesterol), the frational LCATase ativity was redued to onehalf of that obtained with proteoliposomes ontaining 100 nmol of leithin. Similar results to those shown in Fig. 1 were obtained in four other plasma. Qualitatively similar trends in LCATase ativity were obtained when purified LCATase from three different preparations was used as enzyme soure (Fig. 2). These preliminary investigations suggested that proteoliposomes with 5 mol % holesterol would give the desired sensitivity (frational LCATase ativity > 5%) and produe a molar estifiation rate in the range obtained by 6 ; 4 0 m. D 2 P L '$LE (nmol) 250 250 250 250 250 I CH(nmo1) 25 5 125 25 50 u CH(mol%) 1 2 5 10 20 f? LL bhrh 2 250 100 50 25 LE (nmol) 50C CH(nmol) 5 5 5 5 5 CH (mol%) 1 2 5 10 20 IO0 z 80 60 a 40 2. 20 P L kl a I W 100 5 2, 80 E 60 i! U z V 40 J L 20 8 z Fig. 1. Effet of various molar ratios of 1eithin:holesterol in proteoliposomes on plasma LCATase ativity. Proteoliposomes prepared by the holate dialysis tehnique from mixtures of various 1eithin:holesterol molar ratios and onstant apoai (1 nmol/assay) were used for assay of plasma LCATase ativity by inubating the assay mixture at 37 C for 30 min. Eah assay mixture (0.5 ml) ontained 15 pl of fresh plasma, 0.5% HSA, 5 mm meraptoethanol,., 1 nmol apoai, and various amounts of leithin and holesterol as indiated. The number under eah pair of bars represents amounts of leithin and holesterol/assay, and mol?& holesterol. frational LCATase ativity; 0, molar LCATase ativity. Eah value is the mean of six determinations with an average error of less than 5.1%. E 30 20 x 10. D 9, 1. t 2 LE(nmol) 250 250 250 250 250 CH(nmo1) 25 5 125 25 50 6CH(mol%) 1 2 5 10 20 f v 2, 30 > e U 20 t V 1 0 0 10 & U e LL LE (nmol)500 250 100 50 25 CH(nmo1) 5 5 5 5 5 CH (md%) 1 2 5 10 20 120 ; X 80 + C 5 e Q OI E 40 ;. 9, r L al f I W 0 E 120 5 2, e t 80 : k Y 40 b Fig. 2. Effet of various molar ratios of 1eithin:holesterol in proteoliposomes on the ativity of purified LCATase. The substrate ompositions and assay onditions were the same as in Fig. 1, exept that 0.2 pg of purified enzyme was used instead of 15 pl of plasma. The number under eah pair of bars represents amounts of leithin and holesterol/assay, and mol 70 holesterol. a, frational LCATase ativity; 0, molar LCATase ativity. Eah value is the mean of six determinations with an average error of less than 5.6%. the endogenous substrate method. Therefore, we further investigated different onentrations of proteoliposomes ontaining 5 mol % holesterol (Fig. 3). With proteoliposomes ontaining 250 nmol of leithin and 12.5 nmol of holesterol, the frational LCATase ativity of plasma was maximal but it dereased signifiantly if higher onentrations of leithin and holesterol were used (Fig. 3, top). Proteoliposomes ontaining 125 nmol of leithin and 6.3 nmol of holesterol had the highest frational holesterol esterifiation if tested with purified enzyme preparations (Fig. 3, bottom). Similar results were obtained with three other plasma and three different LCATase preparations. In all previous experiments (Figs. 13) the apoai was kept onstant at 1 nmol and leithin and holesterol were varied. Therefore, we next investigated the effet of varying the amount of apoai in the proteoliposome while keeping the onentration and molar ratio of leithin to holesterol onstant (Fig. 4). Plasma LCATase ativity inreased as the apoai inreased, with maximal ativity obtained with 0.8 nmol of apoai. Further inreases in apoai did not hange the LCATase ativity. Similar findings were obtained with purified enzyme, exept that maximal ativity was reahed at 0.4 nmol P Chen and Albers Proteoliposomes ontaining apoai, an LCAT substrate 683
10 t 5 160 120 80 40 LE (nmo1)833 125 250 500 750 5 CH (nmol) 45 63 125 25 375 CH (mol %) 5 5 5 5 5 Fig. 3. Effet of various onentrations of leithin and holesterol. Proteoliposomes made by the holate dialysis tehnique from mixtures of onstant apoai (1 nmol/assay) and leithin and holesterol in a 2O:l molar ratio were used for the assay of enzyme ativity. The assay mixture was inubated at 37OC for 30 min. Eah assay mixture (0.5 ml) ontained 15 pl plasma (top), or 0.2 pg purified enzyme (bottom), 0.5% HSA, 5 mm meraptoethanol, 1 nmol apoai,., and various amounts of leithin and holesterol as indiated. The number under eah set of data points represents the amount of leithin and holesterol/ assay and mol 70 holesterol. Top: 0 0, frational LCATase ativity from two individual plasma samples; 0 0, 0 0, molar LCATase ativity from the two individual plasma samples. Bott6m: 0 0, frational LCATase ativity; 0 0, molar LCATase ativity. Eah value is the mean of six determinations with an average error of less than 5.4%. of apoai. Therefore, the minimal amount of apoai in the proteoliposomes needed for maximal plasma LCA Tase ativity is 0.8 nmol. Similar results were obtained with four other plasma and three other LCATase preparations. Charaterization of proteoliposomes Gel filtration of the proteoliposome vesiles (apoa 1:leithin:holesterol molar ratio 0.8:250:12.5) on a Sepharose CL4B olumn (2.2 X 50 m) gave only one symmetri peak whih ontained all vesiular onstituents initially introdued to the olumn (Fig. 5). Compositional analysis of the isolated proteoliposome vesiles from the peak of the olumn showed that the molar ratio of the three vesiular omponents was idential with that of the initial mixture (apoa1:leithin:holesterol molar ratio 0.8:250:12.5) from whih the proteoliposome was formed (data not shown). These results show that apoai, leithin, and holesterol in this proteoliposome preparation were fully assoiated with eah other as fairly uniform vesiles; this was onfirmed by eletron mirosopy using proteoliposome preparations negatively stained with sodium phosphotungstate (Fig. 6). The apparent vesile radius of this proteoliposome was found to be 131.8 f 4.8 %, (mean f SD, n = 6) and 123.4 + 5.1 %, (mean f SD, n = 100) by Sepharose CL4B olumn hromatography and by eletron mirosopy, respetively. Standardization and validation of the assay system The addition of apoai to the assay mixture that ontained the proteoliposomes (apoa1:leithin:holesterol molar ratio 0.8:250:12.5) was examined for its effet on LCATase ativity (Fig. 7). Addition of low onentrations of apoai (0.036 to 0.36 nmol/assay) to the assay mixture ontaining plasma and proteoliposomes did not affet LCATase ativity, but addition of a higher onentration of apoai (0.71 to 4.26 nmol/assay) redued it slightly (2 to 8%). In ontrast, addition of apoai to the assay mixture ontaining purified enzyme and proteoliposomes aused a marked derease in the ativity of the purified enzyme, with maximal inhibition at 1.43 nmol of added apoai. However, no further inhibition was observed by adding more than 1.43 nmol of apoa I to the assay mixture. Similar results were obtained with four other plasma and three different LCATase preparations. The effet of HSA on LCATase ativity assay was also examined by adding various amounts of HSA to assay mixture (Fig. 8). When plasma (15 pl) was used I I I I I I I 1 I I 04 08 12 16 Apo AI in proteoliposome (nmol) Fig. 4. Effet of apoai onentration in proteoliposomes ontaining a 1eithin:holesterol molar ratio 250:12.5. Proteoliposomes made by the holate dialysis tehnique from mixtures of various onentrations of apoai and onstant onentration of leithin and holesterol in a molar ratio 250:12.5 were used for assay of enzyme ativity by inubating the assay mixture at 37OC for 30 min. Eah assay mixture (0.5 ml) ontained 15 pl plasma or 0.2 pg purified enzyme, 0.5% HSA, 5 mm meraptoethanol, 250 nmol leithin, 12.5 nmol holesterol, and various amounts of apoai as indiated. 0 0, % Cholesterol esterified by plasma; X X, % holesterol esterified by purified enzyme. Eah value is the mean of six determinations with an average error of less than 4.7%. 684 Journal of Lipid Researh Volume 23, 1982
280 12 Fig. 6. Eletron mirograph of the proteoliposome (apoa1:leithin:holesterol molar ratio 0.8:250:12.5). The proteoliposome preparation was negatively stained with 1 % sodium phosphotungstate at ph 7.4. Bar marker represents 1000 A. 240 3 s!x.!oo 1 10 I a E 01 v 8 803 160 v L 6 a a v L U 60. 120. r L 0 6 0) 7 0 80 40 4 5.a g 20Q 0 20 40 60 80 Fration number 40 100 Fig. 5. Sepharose 4B olumn hromatography of the proteoliposomes (apoa1:leithin:holsterolmolar ratio 0.8:25012.5). Two ml of the proteoliposome preparations ontaining 0.440 mg apoai, 3.850 mg leithin, and 0.095 mg holesterol (molar ratio 0.8:250:12.5)was hromatographed on a Sepharose CL4B olumn (2.2 X 50 m) equilibrated with 10 mm Tris buffer, ph 7.4, ontaining 140 mm NaCl and 1 mm EDTA. The olumn was eluted with the same buffer, and 2ml frations were olleted at a flow rate of 30 ml/hr. Eah fration was analyzed for leithin, holesterol, and apoai as desribed under Materials and Methods. Vo and V,designate exluded olumn volume and total olumn volume, respetively. 0 0, Leithin; 0 0,holesterol; X X, apoai. esterifiation rate and the plasma LCATase ativity determined by the proteoliposome method on four plasma samples, two normolipidemi (1 and 2) and two hyperlipidemi (3 and 4) (Table 1). Furthermore, the plasma LCATase ativity agreed well with the holesterol esterifiation rate of the plasmaproteoliposome mixture with the esterifiation rate of the mixture approximately 9% higher (range 8 to 10%) than the plasma LCATase ativity. This result suggests that there is little equilibration between plasma unesterified holesterol and proteoliposome labeled unesterified holesterol. Furthermore, it suggests that plasma is not a signifiant soure of substrate for LCATase under onditions of the proteoliposome assay. T o further examine the effet of holesterol on the assay system, 7.5 p1 of heated normal plasma, or 7.5 pl of plasma from eah of two subjets (S.F.and D.H.) with no detetable LCATase ativity from a Canadian kindred (lo), or 7.5 p1 of assay buffer was added to the assay system ontaining the proteoliposomes and 7.5 pl of normal fresh plasma as enzyme soure. Normal fresh plasma diluted with heated plasma or LCATasedefi Chen and Albers Proteoliposomes ontaining apoai, an LCAT substrate 685 as enzyme soure, a slight inrease in LCATase ativity was observed with the addition of 0.130.5% HSA in the assay mixture; however, no further enhanement of enzyme ativity was obtained in the presene of more than 0.5% of HSA. When purified enzyme was used for the assay, the enzyme ativity again reahed its maximal value at 0.5% HSA, whih was approximately double the enzyme ativity obtained without HSA. Therefore, the presene of 0.5% HSA in the assay mixture is essential for obtaining maximal LCATase ativity using proteoliposomes as substrate. T o validate the assay system, the proteoliposome (apoa1:leithin:holesterol molar ratio 0.8:250: 12.5) was inubated with various amounts of plasma or purified enzyme (Fig. 9). T h e plasma LCATase ativity was a linear funtion of plasma enzyme added and was independent of the amount of plasma holesterol added to the proteoliposomes in the range of 3 to 20 p1 of plasma (Fig. 9, top). Similar results were obtained with two normal plasma and two hyperlipidemi plasma. Also, LCATase ativity was linear with the amount of purified enzyme up to approximately 2.5 pg/assay or until approximately 40% of the holesterol was onverted to holesteryl ester (Fig. 9, bottom). Similar results were obtained with three other LCATase preparations. There was exellent agreement between the plasma holesterol
I 1 14 I I z 201. 1 2 3 4 Apo AI added to assay mixture (nmoll Fig. 7. Effet of addition of apoai to assay mixture on LCATase ativity. Various amounts of apoai were added to the assay mixture, whih ontained the proteoliposomes, and the enzyme ativity was assayed by inubating the mixture at 37OC for 30 min. Eah assay mixture (0.5 ml) ontained 15 p1 plasma or 0.2 pg purified enzyme, 0.5% HSA, 5 mm meraptoethanol, 250 nmol leithin, 12.5 nmol holesterol, and 0.8 nmol apoai in proteoliposomes, plus various amounts of added apoai as indiated. 00, % LCATase ativity remaining in plasma; X X, 70 LCATase ativity remaining in purified enzyme. Eah value is the mean of six determinations with an average error of less than 4.5%. ient plasma gave the same frational LCATase rate as plasma diluted with buffer. The assay system was further validated when addition of 520 p1 of heated normal plasma or 520 p1 eah of plasma from the same two LCATasedefiient subjets to the assay mixture ontaining 0.2 pg of purified enzyme as enzyme soure failed to influene the ativity of the purified enzyme (Table 2). Furthermore, the addition of 520 p1 of heated plasma from five other normolipidemi subjets and three hypertriglyeridemi subjets (triglyeride range 600 to 1600 mg/dl) to normal plasma or purified enzyme had no effet on the measurement of LCATase ativity using the proteoliposomes. E t i 0.5 1.0 1.5 20 HSA added to assay mixture (%) Fig. 8. Effet of HSA onentration in the assay mixture on LCATase ativity. Various amounts of HSA were added to the assay mixtures, whih ontained the proteoliposomes, and the enzyme ativity was assayed by inubating the mixtures at 37OC for 30 min as desribed under Materials and Methods. Eah assay mixture (0.5 ml) ontained 15 p1 plasma or 0.2 pg purified enzyme, 0.8 nmol apoai, 250 nmol leithin, 12.5 nmol holesterol, and various amounts of HSA as indiated. 00, % Cholesterol esterified/30 min by plasma; X X, % holesterol esterified/30 min by purified enzyme. Eah value is the mean of six determinations with an average error of less than 5.3%. I 0 f Purified LCAT preparation (Ng) Fig. 9. LCATase ativity as a funtion of plasma volume or amount of purified enzyme assayed. Various amounts of plasma or of purified enzyme were added to assay mixtures whih ontained the proteoliposomes and the enzyme ativity was assayed by inubating the assay mixtures at 37 C for 30 min as desribed under Materials and Methods. Eah assay mixture (0.5 ml) ontained 0.8 nmol apoai, 250 nmol leithin, 12.5 nmol holesterol, 5% HSA, and 5 mm meraptoethanol, plus various amounts of fresh plasma as enzyme soure (top) or various amounts of purified enzyme (bottom). Eah value is the mean of six determinations with an average error of less than 5.0%. The time ourse of LCATase ativity in the presene of either 15 pl of plasma or 0.2 pg of purified enzyme is shown in Fig. 10. With all seven plasma samples examined, LCATase ativity was linear for up to 60 min, whereas with purified enzyme from three different LCATase preparations it was linear for approximately 120 min. Effet of storage of plasma on LCATase ativity assayed by the proteoliposome method The effet of storage of plasma at 4 C and 20 C was examined over a period of 5 weeks by using the proteoliposome (apoa1:leithin:holesterol molar ratio 0.8:250:12.5), whih was made fresh at eah time of assay. Table 3 shows that there is no visible hange in plasma LCATase ativity during storage of plasma at either 4OC or 2OOC for 5 weeks. Stability of the proteoliposome The stability of the proteoliposome (apoai: 1eithin:holesterol molar ratio 0.8:250: 12.5) was studied by storing the substrate at two temperatures over a period of 5 weeks. At weekly intervals, one portion of the substrate was used for LCATase ativity assay, using frozen plasma as enzyme soure. It was found that at 2OOC the proteoliposome is stable for at least 5 weeks, whereas 686 Journal of Lipid Researh Volume 23, 1982
TABLE 1. Comparison of plasma LCATase ativity determined by the proteoliposome method to plasma esterifiation rate and to the esterifiation rate of the plasmaproteoliposome mixture Plasma Plasma LCATase Unesterified Ativity Plasma PlasmaProteoliposome Plasma Cholesterol Proteoliposome Method Esterifiation Rate Esterifiation Rate mg/dl nmol holesterol esten$ed/hr per ml plasma 1 63.70 f 1.82 97.32 f 4.06 96.42 f 6.41 106.32 f 6.27 2 66.52 f 1.25 104.20 f 3.82 108.74 f 9.38 116.03 f 8.68 3 104.44 f 1.65 147.21 f 4.65 149.00 f 8.74 159.81 f 9.78 4 112.51 f 1.97 151.73 f 4.34 152.26 f 7.86 166.57 f 8.05 The holesterol esterifiation rate of plasma and the plasmaproteoliposome mixture was determined by measuring the derease in unesterified holesterol in sextupliate by an enzymi method (7, 11). The determination of plasma LCATase ativity by the proteoliposome method was arried out in sextupliate and alulated as desribed under Materials and Methods. at 4 C the substrate is stable for the first 3 weeks, but its substrate effiieny appeared to derease slightly thereafter (Table 4). This slight derease in LCATase ativity after storage at 4 C for a period longer than 3 weeks ould not be due to a loss of plasma LCATase ativity, beause we have shown that plasma LCATase ativity does not hange if plasma is stored at 20 C for 5 weeks (Table 3). Comparison of substrate effiieny of the standardized proteoliposome and leithinholesterol liposomes The effiieny of the standardized proteoliposome (apoa1:leithin:holesterol molar ratio 0.8:250:12.5) was ompared with that of leithinholesterol liposomes made in the absene of apoai by the holate dialysis tehnique and by the BatzriKorn proedure (14, 19, 23). The liposomes prepared by the holate dialysis tehnique had the same 1eithin:holesterol molar ratio (250: 12.5) as the standardized proteoliposome, whereas the liposomes prepared by the BatzriKorn proedure had a 1eithin:holesterol molar ratio of 4: 1. Twentytwo jig of apoai/assay was added to the assay mixture ontaining either kind of liposome as substrate, but no apoa I was added to the assay mixture using the standardized proteoliposome that already ontained 22 pg apoai/ assay. The results show that the standardized proteoliposomes were 610 times more effiient for measurement of LCATase ativity than were liposomes prepared by the holate dialysis tehnique or by the BatzriKorn proedure (Table 5). DISCUSSION The existing methods for assaying plasma LCATase ativity using the ommon substrates, heated plasma, or artifiial leithinholesterol liposomes have been shown to be far from ideal primarily beause of their inferior substrate properties or lak of standardization and val idation. If a welldefined ommon artifiial substrate ould be made to possess substrate properties similar to those of native plasma substrate, it would be the preferred substrate for the measurement of LCATase ativity. Investigations arried out in this study with a new syntheti proteoliposome substrate, in whih apoai is inorporated into leithinholesterol vesiles by the holate dialysis tehnique (2022), have shown that preparing suh a substrate for LCATase is possible. It was found that when apoai was kept onstant, the LCATase ativity varied with the molar ratio of leithin to holesterol, and that proteoliposome with 5 mol % holesterol yielded the desired substrate sensitivity, with frational ativity greater than 5% (Figs. 13), and the neessary substrate effiieny with molar LCATase ativity approximately TABLE 2. Effet of addition of heated plasma or LCATasedefiient plasma on LCATase ativity of plasma and purified enzyme assayed using the proteoliposome method Assay Constituents Fresh plasma (7.5 pl) assay buffer (7.5 pl) heated plasma (7.5 pl) S.F. plasma (7.5 pl) + D.F. plasma (7.5 pl) Purified enzyme (0.2 pg) assay buffer (20 pl) + heated plasma (5 pl) (10 rl) (20 rl) + S.F. plasma (5 pl) (10 4) (20 4 ) + D.F. plasma (5 pl) (10 4 ) (20 PI) Frational LCATase Ativity perent holesterol esterified/30 min 2.27 f 0.10" 2.32 f 0.13 2.41 f 0.16 2.19 f 0.08 14.65 f 0.43 14.56 f 0.50 14.63 f 0.62 14.60 f 0.65 14.75 f 0.51 14.72 f 0.46 15.03 f 0.58 14.78 f 0.45 14.81 f 0.63 14.92 f 0.55 a Mean & SD, n = 6. Heated normal plasma or plasma from two LCATasedefiient subjets (S.F. and D.F.) was added to the assay system ontaining the proteoliposomes and fresh plasma or purified enzyme as enzyme soure. The assay was arried out in sextupliate as desribed under Materials and Methods. Chen and Albers Proteoliposomes ontaining apoai, an LCAT substrate 687
60 120 180 240 Inubation time (min) Fig. 10. Time ourse of LCATase ativity assayed by the proteoliposome method. Plasma of purified enzyme was assayed for LCATase ativity using the proteoliposomes as substrate. The assay mixtures were inubated at 37 C and the enzymi reation was stopped at various time intervals as indiated. Eah assay mixture (0.5 ml) ontained 0.8 nmol apoai, 250 nmol leithin, 12.5 nmol holesterol, 0.5% HSA, and 5 mm meraptoethanol, plus 15 jd plasma or 0.2 pg purified enzyme. 0 0, % Cholesterol esterified by plasma; X X, 70 holesterol esterified by purified enzyme. Eah value is the mean of six determinations with an average error of less than 5.8%. 95 f 14 nmol/hr per ml plasma for 15 normal human subjets, whih was omparable to the plasma holesterol esterifiation rate of 97 f 15 nmol/hr per ml by diret measurement of the derease of unesterified holesterol (11). LCATase ativity was linear with the amount of plasma added as enzyme soure up to 20 pl/assay (Fig. 9, top) and with purified enzyme up to 2.5 pg/ assay (Fig. 9, bottom). Within this range, the plasma onstituents, other than LCATase, have little effet on the proteoliposome substrate or on the measurement of LCATase ativity (Tables 1 and 2). On the average, the plasma LCATase ativity by the proteoliposome method is nearly idential to the plasma molar esterifiation rate TABLE 3. determined by measuring the derease in the mass of endogenous unesterified holesterol. On the other hand, in our previous study (1 l), plasma LCATase ativity in 25 adult volunteers, determined by the older method of Glomset and Wright (12) was only 25 f 8% of the molar esterifiation rate. Also, the plasma esterifiation rate by the radioassay method of Stokke and Norum (4) was only 59 & 9% of the molar esterifiation rate determined by enzymi measurement of the derease of the mass of unesterified holesterol. Thus, the proteoliposome method, with its better sensitivity, reproduibility, and substrate effiieny is far superior to the Glomset and Wright method. It should be emphasized that the measure of the plasma LCATase ativity by this exogenous ommon substrate method is not synonymous with a measure of the endogenous holesterol esterifiation rate of plasma. LCATase ativity as measured by the proteoliposome method is an index of the amount of ative enzyme in plasma and is largely independent of the nature or amount of the LCATase substrates or ofators in the plasma. In ontrast to this ommon substrate method whih uses plasma as only the enzyme soure, the endogenous selfsubstrate method of measurement of the LCATase reation, usually referred to as the plasma holesterol esterifiation rate, is an index of not only the amount of LCATase but also the nature and amount of substrate and ofators in the plasma. We reommend, therefore, that the term "LCATase ativity" refer only to the exogenous ommon substrate method. Although our preliminary studies (11) suggest that most of the variation in plasma esterifiation rate among individuals is due to variation in LCATase levels, two plasma ould have idential LCATase ativity, e.g., idential levels of Effet of storage of plasma on plasma LCAT ativity measured by the proteoliposome method Frational LCATase Ativity Plasma A" Plasma B Plasma Storage Time 4o 2oo 4o 2oo weeks perent holesterol estenfied/30 mm 0 5.68 f 0.25' 5.81 f 0.14 5.67 f 0.22 5.67 f 0.29 1 5.50? 0.20 5.85 f 0.26 5.56 f 0.14 5.60 _+ 0.19 2 5.41? 0.17 5.70 f 0.18 5.53 f 0.29 5.58 _+ 0.25 3 5.53 k 0.28 5.79 f 0.30 5.70 k 0.19 5.64 f 0.24 4 5.53 f 0.23 5.79 f 0.12 5.54 k 0.29 5.59 f 0.17 5 5.48 f 0.26 5.83 f 0.17 5.60 f 0.30 5.63 f 0.22 Plasmas A and B denote two individual plasma samples. Mean f SD, n = 6. A pool of fresh plasma sample was subdivided into 0.2ml portions and stored at either 4OC or 2O'C in sealed vials. At weekly intervals a portion of plasma at eah storage temperature was assayed for LCATase ativity using freshly made proteoliposomes (apoa1:leithin:holesterol molar ratio 0.8:250:12.5) as substrate. The assay was arried out in sextupliate as desribed under Materials and Methods. 688 Journal of Lipid Researh Volume 23, 1982
TABLE 4. Stability of the proteoliposome as LCATase substrate Substrate Frational LCAT Ativity Storaae Time Substrate at 4'C Substrate at 2O'C weeks perent holeslerol esleri'ed/30 min 0 5.70 f 0.23" 5.70 f 0.23 1 5.66 f 0.29 5.65 f 0.16 2 5.64 f 0.09 5.72 f 0.26 3 5.62 f 0.21 5.61 f 0.22 4 5.50 f 0.26 5.70 f 0.19 5 5.40 f 0.23 5.64 f 0.17 a Mean f SD, n = 6. A pool of freshly made proteoliposomes (apoa1:leithin:holesterol molar ratio 0.8:250:12.5) was subdivided into 0.5ml portions and stored at either 4OC or 20 C in sealed vials. At the same time, a pool of fresh plasma was subdivided into 0.2 mlportions and stored frozen at 20 C as an enzyme soure. At weekly intervals for a period of 5 weeks, substrate from one portion stored at eah temperature was used for assaying LCATase ativity using freshly thawed plasma from one portion of the frozen plasma. The assay was arried out in sextupliate as desribed under Materials and Methods. ative LCATase, but signifiantly different holesterol esterifiation rates if the substrates of the plasma differ signifiantly (see referene 11). Charaterization and properties of the proteoliposome preparation The proteoliposome preparation was shown by both Sepharose CL4B olumn hromatography (Fig. 5) and eletron mirosopy (Fig. 6) to be fairly uniform in size. ApoAI, leithin, and holesterol in the proteoliposome are fully assoiated with eah other in the form of a stable vesile, sine all the protein and lipids are oeluted in a single symmetri peak from the olumn (Fig. 5). The omposition of the proteoliposome isolated from the olumn was idential to that of the initial proteoliposome mixture. The vesile radius obtained by gel filtration (131.8 k 4.8 A) and by eletron mirosopy (123.4 k 5.1 A) are omparable. However, this vesile size is slightly smaller than that of pure leithin vesiles, whih have a 150 A radius when prepared by solubilizing the leithin with 30 mm holate and removing the detergent by gel filtration (29). Other investigators have prepared stable omplexes of apoaidmpcholesterol either by vortexing (30) or by soniating (31) the proteinlipid mixture in a buffer solution. Bathtobath variation of egg leithin and apoai did not lead to variation in the substrate effiieny of the proteoliposome preparation. When a freshly made proteoliposome preparation was used at eah time of assay, the plasma LCATase ativity in plasma stored at either 4 C or 20 C for 5 weeks was observed to be stable (Table 3). This observation is in aord with the result of Heller, Desager, and Harvengt (32), who have reently reported that plasma LCATase ativity is not altered by storage of the plasma for 17 weeks at 25 C. Furthermore, the proteoliposome preparation was shown to be stable as an LCATase substrate for at least 3 and 5 weeks, respetively, when stored at 4 C and 20 C (Table 4). The ability to prepare stable proteoliposomes is espeially useful for omparative studies and large sale investigations of plasma LCATase ativity. Standardization and validation of the proteoliposome assay system TABLE 5. Comparison of substrate effiieny of the proteoliposomes and leithinholesterol liposomes The proteoliposome ontains suffiient apoai for maximal enzyme ativity and the presene of additional apoai in the assay mixture is not neessary. Although Frational LCATase Ativitf Molar LCATase Ativityb Purified Purified Substrates Plasma Enzyme Plasma Enzyme Proteoliposomes 5.80 f 0.22' 29.34 f 1.72 96.72? 3.67 14670 f 860 Liposomesd 1.03 f 0.07 4.53 f 0.24 17.18 f 1.17 2265 f 120 Liposomes' 0.58? 0.06 1.80 f 0.12 15.48 f 1.60 1440 f 96 a Perent holesterol esterified/30 min. nmol Cholesterol esterified/hr per ml plasma or mg protein of purified enzyme. Mean f SD, n = 6. Made by holate dialysis tehnique. Made by BatzriKorn proedure (14). The proteoliposomes (apoa1:leithin:holesterol molar ratio 0.8:250: 12.5) were prepared by the holate dialysis tehnique. Liposomes ontaining 1eithin:holesterol in molar ratios of 250:12.5 and 4:l were prepared in the absene of apoai, respetively, by the holate dialysis tehnique and the BatzriKorn proedure (14) as desribed (23, 24). For LCATase ativity assay, using both kinds of liposomes, an amount of 22 pg apoai was added to eah assay mixture; no apoai was added to the assay mixture when the optimized proteoliposome, whih ontains 22 pg apoai assay, was used. The assay was arried out in sextupliate as desribed under Materials and Methods. Onehalf pg of purified enzyme was used per assay. Chen and Albers Proteoliposomes ontaining apoai, an LCAT substrate 689
addition of apoai to the assay mixture did not ause any signifiant effet on plasma LCATase ativity, it markedly redued the ativity of purified enzyme (Fig. 7). The reason for this differene is unlear, but the purified destabilized enzyme may be more sensitive to altered onformation of the apoai ativator. We showed in other studies that apoai added to liposomes is a less effetive ativator than is apoai inorporated into the liposome by the holate dialysis proedure. When the proteoliposome was used as substrate, the presene of a 0.5% onentration of HSA in the assay mixture is essential for maximal LCATase ativity (Fig. 8). The ontribution of HSA from 15 pl plasma added as enzyme soure is approximately 1 mg (33), whih yields a final onentration of about 0.2% in an assay volume of 0.5 ml. This amount of HSA from the plasma sample is therefore not suffiient to obtain the maximal enzyme ativity. Addition of HSA to the assay mixture is essential for optimal measurement of LCATase ativity. These observations are in agreement with our previous findings using artifiial leithinholesterol liposomes as substrate (19, 23, 24). It is possible that HSA enhanes apoaistimulated LCATase ativity by stabilizing the enzyme or, alternatively, by binding lysoleithin or holesteryl ester and thereby minimizing produt inhibition. The ability of the proteoliposomes to serve as substrate for LCATase ativity was assessed by omparing their effiieny to that of leithinholesterol vesiles inubated with apoai. The liposomes were made in the absene of apoai either by the holate dialysis tehnique used in the present study or by the BatzriKorn proedure (14). We found that the proteoliposome used in this study is 6fold and 10fold more effiient than the holate liposome and the BatzriKorn liposome, respetively. This suggests that holate permits apoai to assoiate with leithin and holesterol in a speifi arrangement whih serves as a superior substrate for LCATase. The holate dialysis tehnique has been used extensively to reonstitute membrane proteins with lipids in order to regenerate their biologial ativity (20, 21, 34, 35). This method of substrate formation using the holate dialysis tehnique permits the preparation of large amounts of stable, effiient, homogeneous, and welldefined proteoliposome vesile substrate for a rapid and preise determination of plasma or serum LCATase ativity. The availability of this method will permit omparative studies and largesale investigations of plasma LCATase ativity to be arried out as well as studies of the mehanism and regulation of the LCATase reation. Reent appliation of this proteoliposome substrate to the measurement of plasma LCATase ativity has enabled us to demonstrate that the LCATase ativity of heterozygous arriers of the LCATase defiieny is ap proximately halfnormal (10). Earlier studies using onventional methods for measuring LCATase ativity failed to distinguish between normal subjets and heterozygotes (3638). In addition, the proteoliposome substrate has also enabled us to demonstrate that familial LCATase defiieny in the four Norwegian kindreds is due to the presene of low levels of a funtionally defetive enzyme (39). Thus the proteoliposome prepared by the holate dialysis method promises to be an effetive tool in understanding LCATase regulation, metabolism, and geneti ontro1.l We thank students Chris Chapman and Sue Rafhiek for expert tehnial assistane. We also thank Dr. Henry Pownall of the Baylor College of Mediine, Houston, Texas, for initially reommending the use of an apoaiontaining proteoliposome prepared with holate for the determination of LCATase ativity. This researh was supported, in part, by grant HL 15263, ontrat NOlHVI2157L from the Lipid Metabolism Branh, NHLBI, and a grant from R. J. Reynolds, In. This work was done during the tenure of an Established Investigatorship from the Amerian Heart Assoiation (Dr. Albers) and with funds ontributed in part by the Amerian Heart Assoiation of Washington. Manusript reeived 2 Otober 1981 and in revised form 29 January 7 982. 1. 2. 3. 4. 5. 6. 7. 8. 9. REFERENCES Glomset, J. A., and K. R. Norum. 1973. The metaboli role of 1eithin:holesterol ayltransferase. Adv. Lipid Res. 11: 165. Lako, A. G., H. L. Rutenberg, and L. A. Soloff. 1974. Serum holesterol esterifiation in speies resistant and suseptible to atheroslerosis. Atheroslerosis. 19: 297305. Albers, J. J., J. L. Adolphson, and C. H. Chen. 1981. Radioimmunoassay of human plasma leithinholesterol ayltransferase. J. Clin. Invest. 67: 141148. Stokke, K. J., and K. R. Norum. 1971. Determination of 1eithin:holesterol ayltransferase in human blood plasma. Sand. J. Clin. Lab. Invest. 27: 2127. Marel, Y. L., and C. Vdina. 1973. 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