Stereochemical substrate requirements of lecithin:cholesterol acyltransferase and its inhibition by enantiomeric lysolecithins

Battting and Best Departmetrt of Meclicol Research, Universitj of Toronto, I12 College Street, Toronto, Otrt., C(ittada h15G IL6

Keceived March 26, 1980

Smith. N. B. & Kuksis, A. (1980) Stereochemical substrate requirements of 1ecithin:choles- terol acjltransferase and its inhibition by enantiomeric lysolecithins. C(i11. J. Biochem 58, 1286--1291

The suitability of 1 -sn-phosphorylcholine- and 2-sn-phosphorylcholine-diacylglycerols as substrates for 1ecithin:cholesterol acyltransferase (LCAT) was assayed under standardized conditions using sonicated liposomes of radioactive phospholipids. The results bere compared with those obtained for the corresponding molecular species of the natural 3-sn-phosphoryl- choli~~ediacylglycerol. It was found that with the 2-str-phosphorylcholinediacylglycerol, LCAT had only 16'; of the activity that it showed for 3-stt-phosphorylcholinediacylg1jcerol, while its activity with 1-sti-phosphorylcholinediacq.lglycerol as the substrate was not significant. It is concluded that LCAT possesses highly specific steric and positiolial requirements for its phosphatidylcholine substrates and that, like phospholipase Az, it has a significant activity with the 2-stt-phosphorylcholinediacylglycerol. It was also shown that the activity of LCAT is inhibited by both e~~antiomers of ljsophosphatidylcholir~e as well as Triton X-100. In all instances the inhibition was reversed by the addition of lipid-free albumin. I t is concluded, therefore, that the inhibition of LCAT by lysophosphatidylcholine is a detergent rather than an end-product inhibition phenomenon.

Smith, N. B. & Kuksis, A. (1980) Stereochemical substrate requirements of lecithin :choles- terol acyltransferase and its inhibition by enantiomeric lysolecithins. Cun. J. Biochenl. 58, 1286-1 291

Nous avons examine la convenance des l-sn-phosphorylcholine- et 2-sn-phosphorylcholine- diacylglycerols comme substrats pour la 1ecithine:cholesterol acyltransferase (LCAT) dans des conditions standards, utilisant des liposomes traites aux ultrasons de phospholipides radioactifs. Nous comparons les resultats h ceux obtenus avec l'espkce moleculaire correspon- dante, le 3-stt-phosphorylcholinediacylglycerol. Avec le 2-sn-phosphorylcholinediacylg1ycerol, la LCAT n'a que 167; de l'activite qu'elle montre avec le 3-stt-phosphorylcholinediacylglycerol et son activite avec le I-stt-phosphorylcholinediacylglycerol comme substrat n'est pas signifi- cative. Nous concluons que la LCAT posskde des besoins positionnels et steriques trts speci- fiques pour ses substrats phosphatidylcholiniques et, qu'h l'exemple de la phospholipase A?, elle exerce une activite importante sur le 2-sn-phosphorylcholinediacylg1jcerol. Nous montrons aussi que l'activite de la LCAT est inhibee par les deux knantiomkres de la lysophosphatidyl- choline de mtme que par le Triton X-100. Dans tous les cas, l'inhibition est renversee par l'addition d'albumine delipidee. Nous concluons donc que l'inhibition de la LCAT par la lysophosphatidylcholine est un phenomkne d'inhibition par un detergent plut6t que par un produit terminal.

[Traduit par le journal]

Introduction rigidly established. Previous work has shown that Previous studies have extensively examined the role

of L C A T in the maintenance of the normal metabolism of plasma lipoproteins and in the regulation of plasma lipid levels (1-3), but the mechanism of action of the enzyme and its substrate requirements have not been

ABBREVIATIONS : LCAT, lecithin :cholesterol acyltransferase ; sp. act., specific activity; BSA. bovine serum albumin; TLC, thin-layer chromatography; DTNB, 5,5-dithiobis(2-nitroben- zoic acid); L-LPC, 1-palmitoyl-sn-gljcerol-3-phosphorylcho- line; D-LPC, 3-palmitoyl-sn-glycerol-1-phosphorylcholine.

'Present address: Department of Biophysics, Health Science Centre, University of Western Ontario, London, Ont., Canada N6A 5C1.

2Authur to whom all correspondence is to be addressed.

LCAT will utilize a variety of free sterols as acyl ac- ceptors (4, 5 ) , but only phosphatidylcholine has been found to serve as acyl donor (6, 7 ) . It is known that the enzyme is specific for the 2-sil-acyl group of 3-sn- phosphatidylcholinc ( 8 ) and that it preferentially uti- lizes the unsaturated fatty acids ( 9 ) , but the cholesteryl esters formed are found to contain saturated fatty acids in excess of the proportions present in the 2 - ~ n position of phosphatidylcholine. Likewise, less than the stoichio- metric proportion of lysophosphatidylcholine is being formed during the L C A T action ( 3 ) . Recent work has suggested that L C A T may catalyze the transesterifica- tion of two molecules of l-acyllysophosphatidylcholine ( l o ) , and it has been postulated that the disaturated

0008-401 8/80/111286-06$0 1 .OO/O 1980 National Research Council of Canada/Conseil national de recherches du Canada

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SMITH AND I<UI<SIS 1287

phosphatidylcholines thus formed are subject to further transesterification with cholesterol, which might account fo r both the apparent excess of saturated fatty acids in the cholesteryl esters and for the missing lysophos- phatidylcholine. I t has been also demonstrated that purified preparations of LCAT release free fatty acids f rom the 2-.ni posit iol~ of phosphatidylcholine leading to the suggestion that this enzyme possesses a phos- pholipase A, like activity ( 1 1. 1 2 ) . Phospholipase A,, however, is activated by lysophosphatidylcholine (1 3. 1 4 ) , while LCAT is inhibited by lysophosphatidyl- choline (15, 1 6 ) . Neither the mechanism of the lyso- phosphatidylcholine inhibition no r the sin~ilari ty of ac- tion of LCAT and phospholipase A, has been estab- lished.

In the present study, we have utilized the unnatural enantiomer and the positional isomer of acylglycero- phosphorylcholine to probe further the steric and posi- tional specificity of LCAT as well as its susceptibility to the inhibitory action of the lysophosphatidylcholines. A preliminary report concerning these findings has ap- peared (1 7 ) .

Material and methods

[9,10-3H]Palmitic acid (sp. act. 230 mCi/rnmol. 1 Ci =

37 GBq) and [I-lT]linoleic acid (sp. act. 50 mCi/mmol) were purchased from New England Nuclear. Boston, MA. All other lipids were prepared in the laboratory from the above chemi- cals as described below. BSA (fatty acid free). Triton X-100. DL-dipalmitoylphosphatidj lcholine, and nonradioactive palmi- tic and linoleic acids were obtained from the Sigma Chemical Co., St. 1,ouis. MO. Sprague-Dawley rats (250-300 g) were supplied by Canadian Breeding Laboratories. Toronto, Canada.

Preparatiort of syrtthetic phosphatidvlcholines I -Palmitoyl-sn-glycerol-3-phosphorylcholine and 2.3-dipal-

mitoyl-sit-glycerol-1-phosphorylcholine were prepared by re- peated phospholipase A 2 hydrolysis of DL-dipalmitoylphospha- tidylcholine until no more release of the degradation products was observed. These phospholipids were then separated by silicic acid column chromatography and further purified by TLC. 3-Palmitoyl-sn-glycerol-I-phosphorylcholine was pre- pared by octylamine-catalyzed methanolysis of the pure 2,3-dipalmitoyl-stt-glycerol-1-phosphorylcholine, and similarly isolated and purified. These procedures have been described in detail previously (18) along with the nuclear magnetic re- sonance spectroscopy and optical rotation studies, which established the purity of the phospholipids. 1-Palmitoyl- 2-[ Wlpalmitoyl-sn-glycerol-3-phosphorylcholine and 1 -palmi- toyl-2-[14C]linoleoyl-Lsn-glycerol-3-phosphorylcholine were pre- pared by acylation of 1-palmitoyl-ai-glycerol-3-phosphoryl- choline with [9,10-3H]palmitic acid and [l-14C]linoleic acid, respectively. using a modification (18) of the method of Boss et al. ( 1 9). 3-Palmitoyl-2-[3~]palmitoyl-sti-glycerol- 1 -phos- phorylcholine and 3-palmitoyl-2-[lT]linoleoyl-sn-glycerol-1- phosphorylcholine were prepared similarly by acylation of 3-palmitoyl-stt-glycerol- 1 -phosphorylcholine. as were the cor- responding nonradioactive carriers for each of the four above radioactive molecular species of phosphatidylcholine. Each of these phosphatidylcholine and lysophosphatidylcholine prep- arations was chromatographically pure, migrating as a single spot on TLC. The 1-sn-phosphatidylcholines were completely

resistant (>95( ,' recovered) and the 3-sn-phosphatidylcholines were completely susceptible (>99<; hydrolyzed) to the action of phospholipase An. These analyses have been fully de- scribed previously ( 18). I -[lT]Linoleoy 1-?-palmitoy l-sn-glyc- erol-2-phosphorylcholine and its corresponding nonradioactive carrier were made from 1,3-dipalmitoyl-ai-glycerol-2-phos- phorylcholine. The 1 .?-dipalmitoyl-sti-slycerol-2-phosphatidyl- choline (a gift from Dr. J . J. Myher) had been prepared by reacting 1,3-dipalmitoylglycerol with phosphorus oxychloride and choline chloride. The isomeric purity of the chromato- graphically pure 1,3-dipalmitoyl-sti-&cerol-2-phosphorylcho- line isolated after this reaction had been determined by phospholipase C hydrolysis and subsequent gas liquid chroma- tography - mass spectrometry of the liberated diacylglycerols (20). By both, this procedure and also by TLC of the intact 2-stt-phosphatidylcholine, isomeric purity of >99' ; had been confirmed. The 1,3-dipalmitoyl-sn-glycerol-2-phosphorylcho- line was subjected to hydrolysis with phospholipase AZ to yield 3-palmitoyl-sn-glycerol-2-phosphorylcholine. This lipid was immediately reacylated with [l-14C]linoleic acid and non- radioactive linoleic acid to yield I-[14C]linoleoyl-?-palmitoyl- sti-glycerol-2-phosphorylcholine and the corresponding non- radioactive carrier, respectively. The latter transformations were carried out using the procedures previously employed for work with enantiomeric phosphatidylcholines (1 8). The final product was found to be completely susceptible (>95'1 hydrolysis) to phospholipase An, confirming that the migration of the phosphorylcholine group from the 2-.st1 position to the 1-stt position of the derivative did not occur during this transformation. The presence of palmitic acid in the 3-.st1 position of the lyso derivative blocked any phosphorylcholine isomerization to that position. All starting materials, reaction intermediates, and final products were purified by TLC.

Tltitt-laver clrromatograplt~~ TLC of the total lipid extracts of the various chemical and

enzymic reaction mixtures were performed on silica gel H using heptane - isopropyl ether -acetic acid 64:40:3 as the developing solvent (21 ). The spots corresponding to cholesteryl ester and phospholipid were visualized by brief exposure to iodine vapour. After the stains disappeared by sublimation, the outlined areas were scraped into scintillation vials containing lOmL Aquasol and counted in a Searle liquid scintillation counter. Appropriate corrections were made for quenching.

Assa?, 0 /' LCA T activity The LCAT assay method was similar to that reported

previously (22), in which radioactive cholesterol was used as substrate. Radioactive and nonradioactive carrier phospha- tidylcholine of the same molecular species and enantiomer type were mixed in chloroform and, after evaporation of the solvent, were sonicated in a small volume of 0.9C; saline on ice for 3 min, using I-min bursts. This dispersion was then swirled rapidly at 37'C for 3 h with serum which had been heated at 55-60'C for 1 h to destroy LCAT activity. A 0.6-mL aliquot of a typical preparation contained 136 nmol radioactive phos- phatidylcholine (calculated to yield 1000-5000 dpm/nmol cholestery l ester), 0.05 mI, saline. and 0.55 mL heat-inactivated serum. In the LCAT assay each I-ml, sample contained 0.4 mL of untreated serum which served as the source of LCAT and 0.6 mi, of heat-inactivated substrate-serum preparation which contained the radioactive lecithin. Control samples contained 0.4 mL of 0.9"; saline instead of the active unheated serum. Any additions to be included in a sample were intro- duced into the substrate--serum preparation and mixed at 37'C for 10 min by rapid swirling immediately prior to the

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1288 CAN. J. BIOCHEM. VOL. 58, 1980

nmol 2 -PC] linoleoy I - L - LPC added

FIG. 1. Effect of adding increasing amounts of l-palmitoyl-2- [14C]linoleoyl-st~-glycerol-3-phosphorylcholine (2-[l4C]lineoyl- L-LPC) on the yield of cholesteryl linoleate (CL) formed by LCAT. 50-660 nmoles of pure I -palmitoyl-2[14C]linoleoyl-st~- glycerol-3-phosphorylcholine were preincubated with 0.6 mL heat-inactivated serum for 3 h and then incubated with 0.4 mL of active serum for 1 h. Each point is an average of two deter- minations.

addition of the unheated active serum. Lysophosphatidyl- choline was included as a film in the bottom of the tube, BSA was added as the dry powder, and Triton X-100 was pipetted in the form of an aliquot of a 100-mM stock solution in 0.9(;, saline. The LCAT assay mixture was then incubated with shaking at 37°C for 1-6 h.

The LCAT reaction was stopped by the addition of 20 vol- umes of chloroform-methanol 2: 1 (23). A Folch extract was then prepared, dried with anhydrous sodium sulfate, concen- trated, and an aliquot subjected to TLC. As a check on the method using heat-inactivated serum, an experiment was per- formed in which the sulfhydryl inhibitor DTNB was used to inhibit LCAT during the preincubation period while the labelled substrate was incorporated into the serum lipoproteins (24). Following the preincubation, the esterification reaction was started by the addition of mercaptoethanol, which reversed the effects of the DTNB.

Results and discussion

The LCAT activity with the various synthetic sub- strates and inhibitors was assayed with the total amount of substrates and (or ) inhibitor kept constant, while the ratio of the enantiomers and (or) isomers of the test compounds was varied. This allowed comparisons between enantiomers and (or) isomers to be made under conditions of constant detergent acivity.

Assay conditions The results of incubation of increasing amounts of

added 1 -palmitoyl-2-[14C]linoleoyl-sn-glycerol-3-phos- phorylcholine with LCAT are shown in Fig. 1. The yields of radioactive cholesteryl esters are linear up to 150 nmol. Further addition of the phosphatidylcholine results in only limited increases in the production of the cholesteryl esters. It is possible that this effect is due to an exhaustion of the readily available endogenous free cholesterol. Alternatively, the eventual decrease in cholesteryl ester formation may have been a result of inhibition of the reaction by the cholesteryl ester end products (15) . Still other explanations for the de- creased enzyme activity with high substrate loads may be advanced on the basis of a general inactivation of the enzyme by an excess of sonicated dispersions of lipid (25) . Therefore, all experiments were performed with the quantities of exogenous phosphatidylcholines being kept below the 150 nmol, where the yields of the esters were linear. In all experiments the free en- dogenous cholesterol served as the sterol substrate. This condition was chosen in order to avoid difficulties in dissolving both free sterol and saturated phospha- tidylcholines, as well as possible problems in subse- quent interpretation of results. Clearly, the free choles- terol did not become limiting under the conditions adopted for assaying the unnatural substrates and the potential inhibitors of LCAT. The results obtained with heat-inactivated serum and with DTNB were similar.

Eflect of enantioi.tzers and positioncll isomers The effect of steric configuration of the phosphatidyl-

choline on LCAT activity was assayed by means of I -palmitoyl-2-[14C]linoleoyl-sn-glycerol - 3 - phosphoryl-

choline and its enantiomer using 1-h incubations, and of 1-palmitoyl - 2 - [%H]palmitoyl-sn-glycerol-3-phospho- rylcholine and its enantiomer using 6-h incubations. The results of incubating a total of 132 nmol of the di- palmitoylphosphatidylcholine are shown in Fig. 2. The amount of radioactive cholesteryl ester formed in- creased linearly with the proportion of the 1,2-di- palmitoyl -sn -glycerol - 3 - phosphorylcholine increasing from 0-100% in a mixture with the corresponding nonradioactive enantiomer. Figure 2 suggests that the presence of the unnatural enantiomer had no apparent effect on the transacylation of the natural enantiomer. A parallel incubation with 132 nmol of radioactive 3- pal mi toyl-2-[3H]palmitoyl-.~iz-glycerol- 1 -phosphorylcho- line gave only 5 % of the yield of radioactive steryl esters seen with the natural enantiomer. Similar results are shown in Table 1 for incubations with l-palmitoyl- 2-[14C]linoleoyl-.~n-glycerol-3-phosphorylcholine and its unnatural enantiomer. These results are expressed rela- tive to those obtained with the corresponding natural species of phosphatidylcholine as 100%. The relative activities of 5-6% recorded for the dipalmitoyl and palmitoyllinoleoyl species may possibly include some activity due to cross-contamination with the natural enantiomers during chemical synthesis, which would not be detected by our physicochemical techniques. In any case, it is apparent that 1-sn-phosphatidylcholine

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SMITH AND ICUKSIS 1289

nmol 2 - ~ ~ ] ~ o l r n i t o ~ l -L-LPC added

FIG. 2. Effect of varying the proportions of 2,3-dipalmitoyl- sn-glycerol-1 -phosphorylcholine and 1,2-dipalmitoyl-sn-glyc- erol-3-phosphorylcholine in a total of 132 nmol of dipalmitoyl- phosphatidylcholine on the amount of radioactive cholesteryl palmitate (CP) formed by LCAT from I-palmit0yl-2-[~H]- palmitoyl-sn-glycerol-3-phosphorylcholine (2-[3H]palmitoyl-~- LPC). X-1,2-Dipalmitoylphosphatidylcholine (1 32 nmol) con- taining increasing proportions of enantiomeric l-palmitoyl- 2-[3H]palmitoyl-.~rr-glycerol-3-phosphorylcholine were prein- cubated with 0.6 mL heat-inactivated serum for 3 h and then incubated with 0.4 mL of active serum for 6 h. Each point is an average of two to three determinations.

neither effectively reacts with the enzyme nor interferes with its activity on the natural enantiomer. In this connection it is pertinent to point out that the un- natural ~-st~-~hos~hatid~lcholi& has been previously shown (26) to complex with cholesterol much less readily than the natural 3-sn-phosphatidylcholine, de- spite effective bulk uptake of cholesterol into the lipo- somes. Therefore, it is possible that the 1-sn enantiomer failed to form the proper cholesterol-phosphatidyl- choline complex, which may be the real substrate of LCAT. Addition of the unnatural enantiomer of phos-

phatidylcholine may be ineffective in either diluting or disrupting this cholesterol-phosphatidylcholine com- plex and thus be unable to compete for the active site on the enzyme surface. There may be similar steric requirements for a possible association of phosphatidyl- choline with LCAT-activating peptides such as apo- protein A-1 (27) or with the LCAT itself, but as yet there is no evidence to support or refute these possi- bilities. It is obvious that the reasons for the apparent inertness of 1-sn-phosphatidylcholine in the LCAT re- action may be multiple, and a more detailed under- standing requires more experimental work.

Table 1 also shows the utilization of the 2-sn-phos- phatidylcholine by LCAT, which averages about 16%. This increased activity compared with that for the 1-sn isomer may be due to a more favourable steric relationship between the 1-sn fatty acid and 2-sn- phosphorylcholine moiety. Whether these steric differ- ences are important in phosphatidylcholine-cholesterol, or phosphatidylcholine-protein (activating peptides, and (or) enzyme) interactions is not known. Phospho- lipase A, has been shown to react comparably with both the 2-sn and 3-sn isomers but not with the 1-sn isomer of phosphatidylcholine (28). T o our knowl- edge, there have been no nuclear magnetic resonance studies on the interaction of free cholesterol with the 2-sn-phosphatidvlcholine. Like snake venom phospho- lipase A,, LCAT, therefore, acts on the 1-sn position of the 2-.\n-phosphatidylcholine but fails to attack the 2-rtz position of the 1-sn-phosphatidylcholine. These results appear to provide two other criteria supporting the phospholipase A, like nature of LCAT activity pre- viously suggested on the basis of the observed release of free fatty acids in the presence of apoprotein A, (1 1. 12) .

It1 hibition by l?~sophosphatidylcholine and Triton X- ZOO The effect of the lysophosphatidylcholines and Triton

X-100 on LCAT activity was assessed in the presence of 137 nmol of I -palmitoyl-2-[14C]linoleoyl-sn-glycerol- 3-phosphorylcholine. The results of the experiments with several levels of the inhibitor in the presence and absence of albumin are shown in Fig. 3. The reference incubation mixture contained onlv the radioactive phos-

TABLE 1 . Relative activity of LCAT with enantiomeric phosphatidylcholinesa

Substrate Relative activity

- -

aphosphatidylcholine (1 36 nmol) was preincubated with 0.6 m L heat-inactivated serum for 3 h and then incubated with 0.4 m L active serum for I o r 6 h. The L C A T activities for each 1.2-diacyl-sn-glycerol-3-phosphorylcholine enantiomer were normalized a t 100% for comparison with the activity against the corresponding 2,3-diacyl-sn- glycerol- l -phosphorylcholine species.

bMean of six determinations + S E M , 6-h L C A T incubation. CMean of six determinations + SEM, I-h L C A T incubation. dMean of three determinations + SEM, I-h L C A T incubation.

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FIG. 3. Effect of enantiomeric lysolecithins and Triton X-100 on L,CAT activity. l-Palmitoyl-2-[14C]1inoleoq.l-sr~-glyc- erol-3-phosphorylcholine ( 1 3 6 nmol) was preincubated with 0.6 mL heat-inactivated serum for 3 h and then incubated for 1 h with 0.4 mL active serum along with various additives. BSA; L-LPC. L-isomer of lysophosphatidylcholine; D-LPC, D-isomer of lysophosphatidylcholine; and TX-100, Triton X-100 detergent. Each point is an average of two to four determinations.

TABLE 2. Effect of enantiomeric lysolecithins on LCAT activity0

Additions Concentration Relative activity

None 100 L-LPC 1 mM 46 f 8 b

D-LPC 1 rnM 44 f 8 b

BSA 40 mg/rnL 136, 136c L-LPC f BSA As above 85,90C D-LPC f BSA As above 92, 92C

a1 -16:0,2+H) 16 :0-SIT-gl) cerol-3-phosphorylcholine (1 36 nniol) was pre- incubated with 0.5 niL heat-inactivated serum for 3 h and then incubated with 0.4 mL active serum for 6 h.

bMran + SEM of three experiments. CResults of two experiments.

phatidylcholine. T h e relative activities observed for the different inhibitors were compared with the activity of the reference incubation set at 100%. It is seen that both 1-sn and 3-sn enantiomers of lysophosphatidyl- choline inhibited the LCAT reaction similarly and that

this inhibition could be simulated by the addition of a comparable molar amount of the nonionic detergent Triton X-100. The inhibition was abolished by the in- clusion of 0.5 rnol albumin for each mole of added lysophosphatidylcholine. A similar, although not as marked, effect was realized when albumin was added to samples incubated with Triton X-100. Albumin alone markedly stimulated the reaction presumably by binding the lysophosphatidylcholine produced by it, as already claimed in the literature ( 1 5 , 16) . Comparable results were obtained using radioactive 1,2-dipalmitoyl- sn-glycerol-3-phosphorylcholines as the substrate. These results are given in Table 2. In the latter incubations, the additions of the lysophosphatidylcholines were re- stricted to 1 mM, with appropriate concentrations of albumin. The close similarity in the results obtained with the enantiomeric lysophosphatidylcholines and the Triton X-100 suggests that the inhibiting effect is due to the detergent action of the additives. Previous work- ers ( 15, 16) have claimed that the inhibition of LCAT by lysophosphatidylcholine is due to product inhibi- tion. These investigators, however, did not perform any control experiments for the detergent activity of the lyso compounds. Other studies have shown that lysophosphatidylcholine can form a stable complex with cholesterol (29) and that it is primarily taken u p by the lipid rather than the protein moiety of lipoproteins (30, 3 1 ). Thus, while these investigations do not rule out the involvement of direct lysophosphatidvlcholine- protein interactions in the regulation of LCAT activity by this phospholipid, the possible involvement of non- specific detergent effects appears to be quite real. Although the exact nature of Triton X-100 - lipopro- tein lipid micelles and lysophosphatidylcholine-lipo- protein lipid micelles remains to be established and may not necessarily be the same, it is of interest to compare the micellar characteristics of the pure compounds. Triton X-100 has physical properties comparable with those of lysophosphatidylcholine. T h e molecular weight of the monomer is 623, micellar weight 9 0 000, and the aggregation number 140 mol/micelle, while the cor- responding values for lysophosphatidylcholine are 495. 9 2 000. and 18 1, respectively ( 3 2 ) . The finding that albumin abolished the inhibitory effect when included in the incubation medium in a ratio of 0.5 rnol albumin/mol lyso compound correlates well with the previous obsel-vation that albumin binds 2 rnol lyso- phosphatidvlcholinei rnol protein ( 16) . This binding of lysophosphatidylcholine apparently is not stereospecific, as the addition of albumin abolished the inhibitory effect of both enantiomers, and of Triton X-100, as well.

The utilization of the metabolically inert I-srr-lyso- phosphatidylcholine as a control for the detergent ac- tivity of the natural 3-sn-lysophosphatidylcholine has previously demonstrated that the stimulation of the hepatic and mucosal phosphocholine cytidyltransferase (33) and the mucosal glycosyltransferase (34) may be accounted for largely by the detergent properties of

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these molecules. I n contrast, the stimulation of triacyl- glycerol transport by the intestinal mucosa has been observed to be largely a metabolic effect of the natural lysophosphatidylcholine, which is not mimicked by the unnatural enantiomer (35) .

I n view of the successful recent isolation. purification, and assay in vitro of L C A T using single bilayer vesicles ( 2 7 ) , it ought to be most instructive to include the un- natural metabolically inert phosphatidylcholines as controls in any further determinations of the mecha- nism of action of the enzyme in geometrically and chemically defined systems.

Acknowledgments This work was supported by funds f rom the Ontario

Hear t Foundation, Toronto, Ont.. and the Medical Research Council of Canada, Ottawa, Ont. T h e authors wish to thank Dr. J. J. Myher for a generous gift o f 1,3-dipalmitoyl-sn-glycerol-2-phosphorylcholine.

Glomset, J. A. (1968) J. Lipid Res. 9. 155-166 Glomset, J. A. & Norum, K. R. ( 1973 ) Adv. Lipid Res. 11, 1-65 Glomset, J. A. (1979) Prog. Biochem. Pharmacol. 15, 41-66 Nordby, G. & Norum, K. R. (1975) Scand. J. Clin. Lab. Invest. 35.677-682 Piran, U . & Nishida, T. (1979) Lipids 14, 478-482 Nichols. A. V. & Gong, E. L. ( 197 1 ) Biochirn. Biophps. Acra231, 175-184 Fielding, C. J. (1974) Scnnd. J. Clin. Lab. Illvest. 33 Suppl. 137, 15-17 Portman, 0. W. & Sugano, M. (1964) Arch. Biochem. Biophps. 105, 532-540 Sgoutas, D. S. (1972) Biochemistry 11, 293-296 Subbaiah, P. V. & Bagdade, J. D. ( 1978) Life Sci. 22, 1971-1977 Piran, U. & Nishida, T. (1976) J. Biochem. (Tokyo) 80,887-889 Kitabatake, K., Piran, U., Kamio, Y., Doi, Y. & Nishida, T. (1979) Biochirn. Biophys. Acta 573, 145- 154

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16. Nakagawa, M. & Nishida, T. (1973) J. Biochem. (Tokyo) 74, 1263-1266

17. Smith, N. B. & Kuksis, A. ( 1976) Proc. Can. Fed. Biol. SOC. 19 ,97

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