Cholesterol ester metabolism in the brain: Properties and subcellular distribution of cholesterol-esterifying enzymes and cholesterol ester hydrolases in adult rat brain

BIOCHIMICA ET BIOPHYSICA ACTA BBA

293

55881

CHOLESTEROL

ESTER

AND SUBCELLULAR ENZYMES

AND

METABOLISM

DISTRIBUTION

CHOLESTEROL

IN THE

BRAIN:

PROPERTIES

OF CHOLESTEROL-ESTERIFY

ESTER

HYDROLASES

IN ADULT

ING RAT

BRAIN *

YOSHIKATSU Department (Received

ET0

AND

KUNIHIKO

ofNeurology, University January

SUZUKI

ofPennsylvania

School of Medicine,

Philadelphia,

Pa. (U.S.A.)

rgth, rg7r)

SUMMARY

I. The properties and subcellular distribution of the enzymes directly related to the formation and degradation of cholesterol esters were investigated in the young adult rat brain. They were a cholesterol-esterifying enzyme, lecithin-cholesterol acyltransferase, and two distinct cholesterol ester hydrolases. 2. The cholesterol-esterifying enzyme incorporated exogenous free [I-%]oleic acid into cholesterol ester. The pH optimum was 5.5-5.6. Exogenous ATP or CoA did not stimulate the activity, and the presence of an excess amount of unlabelled oleyl-CoA did not diminish the incorporation of the radioactive oleic acid. Various bile acids and Tween 20 were inhibitory, particularly at higher concentrations. Under the optimum conditions, the total activity of the brain was approximately 40 ,ug cholesterol esterified per h per g. Nearly half of the activity was found in the crude mitochondrial fraction. Purified myelin contained no activity. 3. All attempts failed to demonstrate the activity of lecithin-cholesterol acyltranferase in the adult rat brain, using the specific substrate, lecithin labelled at the ,%position with [r-14C]oleic acid. The contribution of this enzyme to the formation of cholesterol ester in normal adult rat brain appears to be, if not nil, negligible. 4. Two distinct cholesterol ester hydrolases were present. One had the pH optimum of 4.2, was activated by deoxycholate, taurocholate, and cholate, and moderately inhibited by Tween 20. Under the optimum condition in the presence of taurocholate, the total activity of this enzyme was approximately 3opg of cholesteryl oleate hydrolyzed per h per g. This enzyme was primarily localized in the crude mitochondrial fraction, and the subfractionation of the crude mitochondrial fraction suggested that this enzyme might be localized in mitochondria, rather than in lysosomes. Purified myelin was devoid of the pH 4.2 cholesterol ester hydrolase. 5. The other cholesterol ester hydrolase had the pH optimum of 6.6. It was * The results of this investigation were presented in part at the second Meeting of the American Society for Neurochemistry, held in Hershey, Pa., March 15-19, 1971, and were published in an abstract form in the Transactions of the meeting. Biochim.

Biophys.

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Y. ETO, K. SUZUKI

strongly inhibited by deoxycholate and activated by tauro~holate. Cholate activated the enzyme at a low concentration, but was moderately inhibitory at a higher concentration. At the optimum condition with added taurocholate, the total activity of the brain was about 170 ,ug cholesteryl oleate hydrolyzed per h per g. The bulk of this enzyme appeared to be localized in microsomes. However, the highly purified myelin fraction was also enriched with this enzyme by more than z-fold, compared to the starting homogenate, and it retained ~0% of the total activity of thepH6.6 cholesterol ester hydrolase in the homogenate.

INTRODUCTION

Esterified cholesterol occurs only in minute amounts in nomlal adult mammalian brains, including humans1-5. Substantially higher concentrations of cholesterol esters are known to be present in the brain during development just prior to the onset of active myelination, but the concentration decreases to the low adult level during myelination and thereafter 3+10. On the other hand, there are frequent and drastic increases of esterified cholesterol in varieties of pathological demyelinating conditions, in which abnormal sudanophilic materials are demonstrated histologicallyi’-16. These phenomena suggest the possible importance of esterified cholesterol in the processes of normal myelination and pathological demyelination. There are extensive investigations on cholesterol esterifying enzymes in pancress?-18 liver20~21,intestine’@, adrenaWs*, aorta 25326,and plasma27-30, but relatively little is known about the enzymes concerning the formation of cholesterol esters in the brain. There are studies which suggested that cholesterol esters might be synthesized in the brainlIz; l*C-labelled cholesterol, when injected intraperitoneally, was incorporated into esterified cholesterol in rat brain. There are two possible pathways through which cholesterol may be esterified in the brain. Free fatty acids may serve as the fatty acid pool to esterify cholesterol by a cholesterol-esterifying enzyme. Alternatively, lecithin-cholesterol acyltransferase, well documented in plasma, may also be present in the brain, and cholesterol esters may be formed by transacylation of the /?-position fatty acid of lecithin. Our recent analytical study on the fatty acid composition of cholesterol esters in sudanophilic demyelinating conditions suggested the latter possibility, at least in those pathological conditions31. However, the fatty acid composition of cholesterol esters in normal brain is different from those in sudanophilic demyelination5, and therefore, a different metabolic mechanism of cholesterol esterification may be operating in the normal brain. The presence of cholesterol ester hydrolase has been reported for young and adult rat brain++3. PRITCHARD AND NICHOLLS found no increase in the activity of cholesterol ester hydrolase in rat brain during development between I and 40 days, and they reported predominant localization of the enzyme in microsomes. On the other hand, CLARENRURG et a1.33 found a substantial increase of the enzymatic activity during IO to 40 days of age, and they reported the enzyme to be localized equally in all particulate fractions. In view of the relative paucity of the data relevant to the brain, and some fundamental discrepancies among the existing data, we felt that it was necessary to investigate in detail the properties of the enzymes concerning the metabolism of brain cholesterol esters, before we could undertake meaningful studies on the developBiochim. Biofihys. Acta, zjg (1971) 293-311

CHOLESTEROL ESTERMETABOLISM

IN BRAIN

295

mental and pathological aspects of brain cholesterol ester metabolism. The present report describes the properties and subcellular distributions of cholesterol esterifying enzymes and cholesterol ester hydrolases in the adult rat brain. The findings differ, in some impo~ant respects, from those reported previously by other investigators3als3. MATERIALS AND METHODS

Young adult male Sprague-Dawley rats, weighing 250-300 g, were killed by decapitation, and the brains, including cerebellum and medula oblongata, removed immediately, weighed and rinsed with ph~iolo~cal saline. The brain was homogenized in 5 vol. of ice-cold physiological saline or 0.32 M sucrose in a hand-operated Dounce homogenizer with a tight-fitting pestle {Kontes Glass Co, Vineland, N.J.). In the standard assay systems for the activity of the cholesterol-esterif~ng enzyme and cholesterol ester hydrolases, there were no differences in the measured activities between the saline and sucrose homogenates. Sztbcelldar fractianatim Survey fractionation. To investigate the subcellular distribution of the enzymes, the brain tissue was fractionated essentially by the method of CLENDENON AND ALLEN~~,which was a modification of the procedure originally developed by DERoBERTIS et aJ.36q3a.Two brains were pooled and homogenized in 4 vol. of 0.32 M sucrose in a Potter-Elvehjem homogenizer with a motor-driven Teflon pestle (A. H. Thomas Co., Plliladelphia, Pa.). Small portions of this 20% honlogenate were used for the determination of enzyme activities of the whole homogenate. The remainder of the homogenate was diluted I :I with 0.32 M sucrose and subjected to the fractionation procedure which was carried out at 4’ throughout. The ~entrifugation at goo x g was carried out in a Sorvall RC3 refrigerated centrifuge with an HG-4 rotor, and all other high speed centrifugations were in a Spinco Lz-65B centrifuge with an SW-27 rotor for density gradient centrifugations, and with a 5oT rotor to obtain high-speed precipitates. The diluted homogenate (ro%) was first centrifuged at 1054~0 x g for 60 min, and the soluble supernatant fraction was obtained (Fraction S). The remainder of the homogenate was reconstituted to the original volume by resuspension in 0.32 M sucrose. The resuspended homogenate was centrifuged at goo x g for IO min, and the pellet was washed twice with 0.32 M sucrose. The final pellet was the nuclei-cell debris fraction (Fraction N). The combined supe~atant and washings of the lowspeed centrifugation were then centrifuged at IIOOO x g for 20 min. The resultant pellet was washed once; the crude mitochondrial fraction, The combined supernatants were centrifuged at 105400 x g for 60 min to obtain the microsomal pellet (Fraction Mic). The crude mitochondrial fraction was fractionated on a discontinuous sucrose density gradient of 0.32 M and 0.8 M at ~OOOOO x g for 120 min. At the end of the centrifugation, the white and fluffy myelin-rich fraction was obtained at the interface of 0.8 M and 0.32 M sucrose (Fraction My). The nerve-ending-rich fraction was collected as a diffuse grayish layer below the myelin layer {Fraction NE). The pellet consisted mainly of mitochondria (Fraction Mit). Fraction Mit was subfractionated further by layering it on a discontinuous sucrose gradient of 0.32, 0.8, 1.0, 1.2 and 1.4 RI, and by ~iOChim.

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centrifugation at 50000 x g for 120 min. Subfractions were obtained at each interface of the gradient, and as a pellet at the bottom (Subfractions A, B, C, D and E, in the order of increasing density). All of the subfractions were diluted with 0.32 M sucrose and spun at IOOOOOx g for 30 min, and obtained as pellets. A very small portion of each pellet was removed for electron microscopic examination, according to the procedure previously described 37. Finally, all subcellular fractions were suspended in appropriate volumes of 0.32 M sucrose to give protein concentrations of 0.2-1.5 mg/o.r ml. The amount of protein was determined by the method of LOWRY et aL3a. Isolation of mydin. The subcellular fractions obtained by the survey fractionation procedure were suitably enriched by respective components to obtain information as to the probable localization of the enzyme. However, they were always significantly contaminated by other subcellular components. Since the possible localization of the pH 6.6 cholesterol ester hydrolase in the myelin sheath became an important question, mo~holo~ca~y and chemic~ly highly purified myelin was isolated by the method of NORTON~~, which has been outlined previously37@. The high purity of myelin isolated by this method has been amply demonstrated 3Qy41. Successive myelin fractions with increasing purity were examined: Myelin I was the fraction obtained at the interface of 0.32 M and 0.85 M sucrose after the first sucrose density gradient centrifugation and before the osmotic shock; Myelin II was obtained as the same interface material after the second density gradient centrifugation following the three osmoticshocks of Myelin I; Myelin III was the final purified myelin after having been washed with distilled water five times. All myelin fractions were suspended in 0.32 M sucrose. Chemicals Radioactive substrates. Oleic acid was chosen as the fatty acid of choice for the study of cholesterol ester metabolism in the brain. This was based on the data that oleic acid always constitutes the largest proportion (30-507~) of the fatty acids of cholesterol esters in normal brain5, varieties of pathologic~ brain+, and in chick embryo brains.&% [I-14C]OIeic acid (specific activity, 53.5 mC/mmole) and [7-3H]cholesteryl oleate (specific activity, IO C/mmole) were purchased from New England Nuclear Corporation, Boston, Mass. The radioactive oleic acid and cholesteryl oleate were diluted with unlabelled compounds to the final specific activities of 0.1 mC/mmole and 0.6 mC/mmole, respectively. Both compounds were then purified by the silicic acid column chromatography of HORNINGet al. 43. This purification was necessary to reduce the radioactivity of blank tubes to a minimum, particularly for the radioactive oleic acid, which originally contained significant radioactivity in the region of cholesterol esters on thin-layer chromatograms. Lecithin labelled specifically at the B-position with [I-W]oleic acid was prepared enzymatically according to ROBINSONANDLANDS44. Briefly, the procedure involved isolation and purification of egg lecithin, conversion to Iysolecithin with phospholipase A, and enzymatic synthesis of /?-labelled lecithin from lysolecithin and [r-l*C]oleic acid by rat liver microsomes. The final purified radioactive lecithin had a specific activity of 0.083 mC/mmole. More than 97% of the total radioactivity co-chromatographed with lecithin by thin-layer chromatography in chloroform-methanol-cont. ammonia (70:30:5, by vol.). Treatment with phospholipase A removed more than Biochim. Biophys. Acta, 239 (1971)293-31 I

CHOLESTEROL

ESTER METABOLISM

297

IN BRAIN

go”/ of the radioactivity, and all of the liberated radioactivity was recovered from the area of free fatty acid by thin-layer chromatography in chlorofo~-methanol-water (70:30:4, by vol.). Other ~~~~~c~~sa& enzyncas. Coenzyme A, cytochrome c, sodium taurocholate, sodium deoxycholate, cholic acid, Tween 20, Tween 80, Triton X-100, sodium salt of ATP, and unlabelled oleic acid were purchased from Sigma Chemical Company, St. Louis, MO. Unlabelled cholesteryl oleate and cholesterol was from Applied Science Laboratories, State College, Pa. Snake venom phospholipase A was supplied by Boehringer-~~annheim Corporation, N.Y. Mann Research Laboratories, N-Y., was the source of $-nitrophenyl-B-D-glucopyranoside and $-nitrophenyl-N-acetyl$-D-glucosaminide. The thixotropic gel powder (Cab-0-Sil) was purchased from Packard Instrument Company, Downers Grove, Ill. Unlabelled oleyl-CoA was purchased from Supelco, Incorporated, Bellefonte, Pa. Assay procedwes CkoZestero2-este~fying enzyme. Benzene solutions of 250 pg of unlabelled cholesterol and 400 ,ug of, [I-%]oleic acid (specific activity, 0.1 mC/mmole) were dried in a IO ml glass centrifuge tube with a screw cap. They were dissolved in 0.05 ml of acetone, and dispersed by immediately adding 1.0 ml of 0.15 M citrate-phosphate buffer (pH 5.5). The dispersion was sonicated for one min in a bath-type ultrasonicator. Then, 0.1 ml of either homogenate (1-3 mg protein) or a suspension of a subcellular fraction (o.z-1.5 mg protein) was added. The final incubation volume was r.15 ml with the final pH 5.6. The tubes were incubated for 3 h at 37” with gentle shaking. For each experiment, blank tubes without the tissue components or with boiled homogenate (2 min at x00’) were included and examined at both o-time and after 3 h incubation. The reaction was stopped by adding 5 vol. of chloroform-methanol (z : I, v/v). After mixing and centrifugation, the upper water-methanol phase was discarded. The lower chloroform phase and the insoluble residue at the interface were filtered through Whatman No. 41 filter paper, and the tube and the residue were washed by three 3-ml portions of chloroform-methanol (2: I, v/v). The combined extracts were evaporated to dryness at 37” under a stream of nitrogen. The dry lipid was dissolved in a known amount of chloroform-methanol, an aliquot was spotted as a band on a o.z5-mm thick silica gel plate, and thin-layer chromatography carried out in hexaneether (95:5, v/v). The spots of standard cholesteryl oleate, included in each plate, were visualized by exposing the plate to iodine vapor, and the area corresponding to the standard was scraped into the counting vial. The vial was filled with the thixotropic gel powder by gentle tapping, and xz ml of scintillation solvent (7 g PPO and 0.6 g dim~thyl-POPOP per 1 toluene) were added to form stable gel. The radioactivity was measured with a Packard Tricarb Model 3320 liquid scintillation counter. The blank tubes were processed in exactly the same way as experimental tubes, and the radioactivity of experimental samples was corrected for the blank counts. There never was a significant difference between the blank samples with or without boiled homogenate, or between the blank samples of o-time and 3 h incubation. Since the brain contains very little free fatty acids and since the added labelled oleic acid was in great excess compared to the expected endogenous oleic acid, no correction was necessaryfor the radioactive dihrtion of the added oleic acid by the endogenous oleic acid. Biochinz.

Biophys.

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z3g (1971) 293-311

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298

Le~~t~~~-C~~~~t~Y~~ a~y~t~a?~sfe~~se.hiumerous unsuccessful attempts were made to measure the activity of lecithin-cholesterol acyltransferase in the brain. The starting system was based on the assay system developed by GLOMSET AND KAPLAN~~. However, these authors used radioactive cholesterol as the precursor, while we used lecithin, labelled at the @-position by [I-W]oleic acid, the specific substrate for this enzyme. System I: Human whole plasma or albumin, dissolved in o.r ;?I sodium phosphate buffer (pH 7.4) at a concentration of IO mg/ml, was preheated at 60” for 30 min. One ml of this preheated plasma or albumin solution was added to a tube containing 400-700 pg of the ~-position-IabeIled lecithin (specific activity, 0.083 mC/ mmole), IOO fig of unlabelled oleic acid and 250 ,ug of unlabelled cholesterol. The mixture was sonicated in a water-bath type ultrasonicator. Various amounts of whole brain homogenate were added (the protein content up to 15 mg/tube). The mixture was incubated for 6-12 h at 37”. The four types of blank tubes were included as described for the cholesterol-esterifying enzyme, and the radioactivity of the cholesterol ester fraction was assayed similarly. Since there is a report to indicate that tecithincholesterol acyltransferase content was high in the cell sap fraction in the IiverPe, the homogenate was replaced by the high-speed supernatant in several experiments. The pH of the incubation was varied from 5.1 to 7.4. The following detergents were tried at the final concentrations of 0.1 to o.z~/o; Tween 20, Tween 80, Triton x-100, deoxycholate, taurocholate and cholate. In some experiments, lipids were dissolved in 0.05 ml acetone before dispersion. System II: This system was based on that of SHAHet al.4’. Solutions of 700 pg B-position-labelled lecithin, TOOpugunlabelled oleic acid and 250 pg cholesterol were dried in a tube. They were dispersed by adding 1.0 ml of 0.1 M sodium pllaspllate buffer (pH 7.4) containing 0.3% of Tween 20. One ml of preheated plasma was added, and also 0.5 ml whole brain homogenate, or 1.0 ml of the high-speed supernatant, or 1.0 ml of fresh human plasma, as the source of enzyme. The tubes were incubated for 12 h at 37”. At the end of incubation, the radioactivity incorporated into cholesterol esters was assayed and corrected for the blanks, as described above. &~la~est~~o~ ester ~y~~ol~s~s. A benzene solution of 250 pg /7-3Hjcholesteryl oleate (specific activity, 0.6 mC/mmole) was dried in a IO ml centriftrge tube. It was dissolved in 0.05 ml of acetone and dispersed immediately by adding 1.0 ml of either 0.1 M sodium phosphate buffer (pH 6.6) or 0.15 M citrate-phospll~te buffer (pH 4.z}. The mixture was sonicated for I min, and then 0.2 ml of whole homogenate or the suspension of a subcellular fraction was added. The mixture was incubated for 3 h at 37”. Since taurocholate was found to be highly stimulative, the later experiments, such as those on subcellular distribution, were carried out in the presence of 6 !&moles of sodium taurocholate per incubation. In these instances, acetone was omitted from the above standard system because taurocholate and sonication were sufficient to achieve uniform dispersion of the substrate. The reaction was stopped by the addition of 5 vol. of chloroform-methanol (2: I, v/v). Lipids were extracted in the same way as for the assay of the cholesterol-esterif~~ing enzyme. Dry lipid was subjected to silica gel G thin-layer chromatography in the solvent system of hexane-ether (60:40, v/v). The cholesterol band was located by exposure to iodine vapor. After complete sublimation of iodine, the area of cholesterol was scraped directly onto a small funnel-shaped Whatman No. 41 filter paper, and cholesterol was extracted directly into the counting vial Aiochim.. Biehys.

Acta,

239 (1971) 2?3-31~

CHOLESTEROLESTER METABOLISMIN BRAIN

299

with five portions of 3 ml chloroform-methanol (2 : I, v/v). Preliminary experiments had indicated the recovery of cholesterol to be go-95% with this procedure. The extracted cholesterol was dried, dissolved in 12 ml of the scintillation solvent described above, and the radioactivity measured. As in other enzyme assays, blank tubes were included for both o-time and incubated samples with and without boiled homogenate, and the counts for experimental tubes were corrected for the blank counts. Less than I pug of endogenous cholesterol ester was present in the assay system, and consequently no correction was necessary for the radioactive dilution of the added cholesteryl oleate. Other enzymes. p-Glucosidase was assayed essentially by the method of GATT as the substrate. NAND RAPPORT*~, and with p-nitrophenyl-@-n-glucopyranoside acetyl-P-n-glucosaminidase was measured according to FROHWEIN AND GATT*~, also with a p-nitrophenyl derivative as the substrate. Succinate-cytochrome c oxidoreductase (EC 1.3.99.1) was measured by the method of SOTTOCASAet a1.50. RESULTS Properties of the enzymes Cholesterol-esterifying

in whole homogenate

enzyme. The incorporation of the added [I-%]oleic acid into cholesterol ester was linear for at least 5 h in the standard incubation system, and the 3-h incubation period was adopted for routine assays. There was no non-enzymatic chemical transacylation, as ascertained by the incubated blank tubes with or without boiled homogenate. In one experiment, the thin-layer chromatography scraping of the region of cholesterol esters was extracted by five portions of 3 ml chloroformmethanol (2: I, v/v) and dried. The sample was methanolyzed in 5% methanolic HCl for 4 h at IOOO,and fatty acid methyl esters were extracted by hexane. All of the radioactivity in the initial extract was recovered from the fatty acid methyl ester band on a thin-layer chromatogram developed in hexane-ether (95:5, by vol.). This finding provided further evidence that we were measuring the radioactivity of newly formed cholesterol esters, and not of unknown metabolites of oleic acid which happened to co-chromatograph with cholesterol esters. When the amount of homogenate was increased in the standard system, the formation of cholesterol ester increased linearly, up to 3 mg of homogenate protein, and then it reached a plateau between 4 to 8 mg of protein (Fig. I). When the concentration of labelled oleic acid was varied in the standard system, there was an optimum concentration of 400 ,ug per incubation. Higher concentrations were inhibitory (Fig. 2). Exogenous cholesterol, added to the system, did not affect the incorporation in the range of 250 to 500 pg. Exogenous cholesterol of 250 ,ug was added to the standard system, however, to minimize the variation in the total cholesterol in the svstem, particularly in experiments with subcellular fractions. In the standard system, there was a relatively sharp pH optimum at the pH 5.5-5.6 region, and this pH was chosen for all assays (Fig. 3). The addition of ATP, CoA, or various amounts of unlabelled oleyl-CoA to the standard system did not affect the incorporation of [I-%]oleic acid into cholesterol esters (Table I). Bile acids and Tween 20 were inhibitory, particularly at higher concentrations (Table II). Lecithin-cholesterol acyltransferase. Despite extensive trials, as described in the MATERIALSAND METHODSsection, we were unable to demonstrate any activity of the B&him.

Biophys.

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Y. ETO, K. SUZUKI

0

2.0

4.0

6.0

protein/tube

8.0

(mg)

0

0.2

0.4

0.6

0.8

oleic

acid/tube

(mg)

1.0

Fig. I. Effect of the protein concentration on the incorporation of [I-Wjoleic acid into cholesterol esters by rat brain homogenate. The experiment was carried out with the standard assay system for the cholesterol-esterifying enzyme, as described in the text, but with varying amounts of homogenate protein. Routine assays were carried out with protein concentrations within the linear range.

Fig. 2. Effect of the concentration of [G*C]oleic acid on its incorporation into cholesterol esters by rat brain homogenate. The experiment was carried out with the standard assay system for the cholesterol-esterifying enzyme, as described in the text, except that the amounts of the radioactive substrate were varied. The optimum amount of 400 pugper tube was chosen for routine assays

PH Fig. 3. Effect of pH on the incorporation of [I-‘C]oleic acid into cholesterol esters by rat brain homogenate. The experiment was done with the standard system for the cholesterol-esterifying enzyme, as described in the text, except that the pH of the system was varied. a---a, 0.15 M citrate-phosphate buffer; x---x, 0.1 M sodium phosphate buffer. The optimum pH of 5.6 was adopted for the standard system. BiOChi?,Z. B@hJG.

z&U,

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301

CHOLESTEROL ESTER METABOLISM IN BRAIN TABLE

I

EFFECT

OF COFACTORS

ESTERS

BY

RAT BRAIN

ON THE

INCORPORATION

OF ADDED

[I-‘4c]OLEIC

ACID

INTO

CHOLESTEROL

HOMOGENATE

Incorporation of [I-%]oleic acid into cholesterol ester was assayed as described in the text, with the standard system and with the addition of IO ,umoles of ATP, 2 pmoles of CoA, or the indicated amounts of unlabelled oleyl-CoA. This table is a composite of two separate experiments in which the activities of the standard system without additions agreed within IO%. Each value is the average of duplicate determinations. Incubation

Activity*

system

Standard system @US ATP plus CoA plus ATP and plus ATP and plus ATP and plus ATP and

0.89 0.87 0.85

CoA 0.5 ,~g oleyl-CoA I .o ,ug oleyl-CoA ro ,~g oleyl-CoA

* Expressed as nmoles cholesterol

0.94 0.95 0.97 I.01

esterified per h per mg protein.

direct transfer of the ,9-position fatty acids of lecithin to cholesterol in the adult rat brain. This is unlikely to be due to technical difficulties because, when we assayed fresh human plasma in the standard System II, we obtained the enzyme activity similar to those reported in the literature. Our assay systems used a specific substrate, lecithin labelled with [r-l*C]oleic acid at the /?-position, and they contained sufficient free unlabelled oleic acid to exclude the possible false activity as the result of the two step reaction: lecithin + lysolecithin+oleic acid; oleic acid+cholesterol --f cholesteryl oleate. Cholesterol ester hydrolase. When hydrolysis of added [7-3H]cholesteryl oleate

6.0 PH

Effect of pH on the hydrolysis of [7-3H]cholesteryl oleate by rat brain homogenate. The standard assay system for cholesterol ester hydrolase was used without added taurocholate, as described in the text, except that the pH of the system was varied. W--w, 0.15 M citratephosphate buffer; O-O, 0.1 M sodium phosphate buffer. The curve was qualitatively similar, when the hydrolysis was activated by the addition of sodium taurocholate. The two pH optima, 4.2 and 6.6, were adopted for subsequent assays of cholesterol ester hydrolases. Biochim.

Biofihys.

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Y. ETO, K. SUZUKI

302 TABLE

II

EFFECT OF DETERGENTSON THE INcoRPoRATIoN ESTERS

BY

RAT

BRAIN

OF ADDED

[I-‘4c]OLEIC

ACID

IiYTO CHOLESTEROL

HOMOGENATE

Formation of cholesterol ester was determined as described in the text, with the standard system with or without the designated amounts of detergents. Incubation

Activity*

system

Standard system plus sodium taurocholate, 5 pmoles plus sodium taurocholate, 10 pmoles plus sodium deoxycholate, 5 pmoles plus sodium deoxycholate, 10 pmoles plus sodium cholate, 5 pmoles plus sodium cholate, IO pmoles @us Tween 20, I oh * * plus Tween 20, 2 o/0* *

0.61 0.24 0.05 0.39 0.06 0.48 0.01 0.40 0.28

* Expressed as nmoles cholesterol esterified per h per mg protein. X’alues are averages of duplicate samples in a single experiment. * * Final concentrations.

was measured in the standard system but with varying pH, two pH optima were observed (Fig. 4). One was at pH 4.2 and the other at pH 6.6, and only negligible hydrolysis occurred between the two peaks. Because of this finding, subsequent assays were always carried out at both pH 4.2 and 6.6. At both pH, the activity was linear against the incubation time, at least for 3 h, and against the protein concentration of up to 8 mg per tube. At least roopg of [T-~H]cholesteryl oleate was necessary to saturate the system at pH 4.2, and at least zoo ,ug

cholerteryl

oleate/tube

(mg)

Fig. 5. Effect of concentration of [7-3Hlcholesteryl oleate on its hydrolysis by rat brain homogenate. The hydrolysis of cholesteryl oleate was measured at both pH 4.2 and 6.6 with the standard assay system without sodium taurocholate, as described in the text, except that the amounts of the labelled substrate were varied. For the standard assay system, 250 ,ug of [7-3H]cholesteryl oleate were used to saturate the system both at pH 4.2 and 6.6. E--M, pH 4.2; O------O, pH 6.6. The amounts necessary to saturate the system were the same when assays were carried out in the presence of sodium taurocholate. Biochim.

Biofihys.

Acta,

239 (1971) 293-3”

CHOLESTEROL TABLE

303

IN BRAIN

III

EFFECT BRAIN

ESTER METABOLISM

OF FREEZE-THAWING

AND

ULTRASONICATION

ON CHOLESTEROL

ESTER

HYDROLASES

OF RAT

HOMOGENATE

Rat brain homogenate was subjected to one of the following treatments: no treatment; freezethawing five times; and freeze-thawing five times followed by r-min sonication. Hydrolysis of [7-3H]cholesteryl oleate was then measured in the standard system without taurocholate, as described in the text, at both pH 4.2 and pH 6.6. -___ ..--____. Activity

TWatWWtt ~_ No treatment

Freeze-thawing Freeze-thawing

and ultrasonication

*

PH 4.2

pH

0.054 0.049 0.026

0.064 0.066 0.051

* Expressed as nmoles of cholesteryl oleate hydrolyzed of duplicate samples in a single experiment.

TABLE EFFECT

4.6

per h per mg protein. Values are averages

IV OF DETERGENTS

OX CHOLESTEROL

ESTER

HYDROLASES

OF RAT BRAIN

HOMOGENATE

Hydrolysis of [7-3Hlcholesterol oleate was measured for rat brain homogenate at pH 4.2 and pH 6.6 in the standard system, as described in the text, but with designated amounts of detergents added. ____Imubatimz

system

Activity

*

-6.6

pH

PH 4.2

o.osz Standard system 0.302 pEus sodium deoxycholate, 5 @moles plus sodium deoxycholate, IO ymoles 0.173 plus sodium taurocholatc, 5 pmoles 0.257 0.1g* plus sodium taurocholatc, IO ,umoles 0.206 plus sodium cholate, 5 ,umoles 0.326 pks sodium cholate, IO jcmoles 0.038 plus Tween 20, I yO* * 0.01g plats Tween 20, 2% * * .--______ * Expressed as nmoles of cholesteryl oleate hydrolyzed of duplicate samples in a single experiment. * * Final concentrations.

0.073 0.043 0.01 r 1.110

0.556 0.127 o-035 0.060 0.056 per h per mg protein. Values are averages

at pH 6.6 (Fig. 5). No inhibitory effect of excess substrate was observed up to 500 ,ug per tube. The activity of the pH 6.6 cholesterol ester hydrolase was not affected appreciably by freeze-thawing up to five times, or by additions sonication. However, combined freeze-thawing and sonication appeared to be slightly inhibitory to the pH 4.2 cholesterol ester hydrolase, although freeze-thawing alone showed little effect (Table III). The two enzymes with the different pH optima also showed clear differences in regard to the effect of added detergents (Table IV). Deoxycholate was stimulatory to the pH 4.2 enzyme, but strongly inhibitory to the pH 6.6 enzyme. Taurocholate was an effective activator for both enzymes. Cholate at the concentration of 5 ,umoles/ tube moderately increased the hydrolysis of cholesterol ester at both pH, but a higher concentration (IO pmoles) was inhibitory to the pH 6.6 enzyme, while the pH 4.2 enzyme was further activated. Tween 20 was moderately inhibitory to both enzymes. The optimum concentration of taurocholate for the activation of both enzymes was found to be 6 pmoles/tube (Fig. 6). At this concentration of taurocholate in the Biochim.

Biophys.

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0 P

nmclescholerteryl

abate

hydrolyzed/h

/mg protein

CHOLESTEROL

ESTER METABOLISM

3%

IN BRAIN

L‘ 5

2.0

$ ,g 1.0 P f $ 0

S

Mic

N

S

Mic

N

My

My NE

Mit

Fig. 7. Subcellular distribution of (a) fatty acid-choiesterol acyltrausferase, (b) pH 6.6 cholesterol ester hydrolase, and (c) pH 4.4 cholesterol ester hydrolase. The nature of the fractions is described in detail in the text. The width of each fraction is proportional to its protein content, and therefore, the area of each fraction is proportional to the total activity present in the fraction.

Less than I$$, of the total activity was in the microsomal fraction. The soluble fraction contained little activity. ~ho~~s~e~~~esder hy~~~~~ses. The subcellular distribution was clearly different between the two cholesterol ester hydrolases. The pH 6.6 cholesterol ester hydrolase was localized primarily in the microsomal fraction and myelin-enriched fraction (Fig. 7b). The recovery of the activity after the subcellular fractionation was 86%. Fraction Mic and Fraction My contained 56% of the total recovered activity, and both fractions had relative specific activity greater than 2. Only 11% of the activity was found in the crude mitochondrial fraction, and the soluble fraction had no activity. On the other hand, the bulk of the pH 4.2 cholesterol ester hydrolase activity was found in the crude mitochondriai fraction, 66% of the total recovered activity, which was 73% of the activity in the starting homogenate (Fig. 7~). Only the crude mitochondri~ fraction had a specific activity greater than I. In contrast to the pH 6.6 hydrolase, only 13% of the activity was present in both the microsomal and myelinrich fractions combined. When the crude mitochondri~ fraction was further subfractionated to Fractions A-E, the distribution of the pH 4.2 cholesterol ester hydrolase was similar to that of the typical mito~hondrial enzyme, succinate~ytochrome c oxidoreductase, and different from that of the typical lysosomal enzyme, N-acetyl73%.

Biochina. Biophys.

ilcta, 239 (x971) 293-31

I

306

Y. ETO,

K. SUZUKI

Subfractions

Fig. 8. The distribution of pH 4.2 cholesterol ester hydrolase, succinate-cytochrome c oxidoreductase and N-acetyl-,%wglucosaminidase among the subfractions of the crude mitochondrial fraction. The nature of the subfractions is described in the text. O---O, pH 4.2 cholesterol ester hydrolase; m-.-m, succinate-cytochrome c oxidoreductase; x . . . . . x , N-acetyl-@-D-glucosaminidase.

@-D-glucosaminidase

(Fig. 8). The recoveries

of the activities

of pH 4.2 cholesterol

ester hydrolase, succinate-cytochrome c oxidoreductase and N-acetyl-j?-D-glucosaminidase, compared to Fraction Mit, were 67%, 81% and 88% respectively. Presence of pH 6.6 cholesterol ester hydrolase in myelin. Since the myelin-rich

Adult rat brain myelin was prepared by the method of NORTON~~.Small portions of myelin were removed at different purification steps and assayed for enzymatic activities. The nature of the three myelin fractions is described in the text in detail. Enzyme activities are expressed as nmoles per h per g wet weight. Relative specific activity (R.S.4.) = ratio of the specific activity of the fraction to the specific activity of whole homogenate, the specific activity being defined as the activity per unit amount of protein. n.d. = not detectable. Values are the averages of duplicate determinations in a single experiment, except for the data for the pH 6.6 cholesterol ester hydrolase, which are averages of two separate experiments.

Homogenate

EPlZpiW Cholesterol-esterifying

Myelin II ___-

11.1

n.d.

n.d.

0.106 0.02

n.d.

enzyme

Activity R.S.A. Cholesterol ester hydrolase, pH 4.2 Activity R.S.A. Cholesterol ester hydrolase, pH 6.6 Activity R.S.A. Succinate-cytochrome c oxidoreductase Activity R.S.A. ,&Glucosidase Activity R.S.A.

N-Acetyl-/J-glucosaminidase Activity R.S.A. Protein content,

Mydin III -

Myelin I .__.~~

78.6 1.00

mg/g wet weight

Biochim. Biophys. Acta, 239 (1971) 293-311

0.653 0.07

52.5 1.00 272 I .oo

223 3.88

2090 I.00

24.8 0.06

3570 1.00

82700 I.00

-..

0.77

131

557 0.85

8110

0.53 24.2

61.5 I .98

56.9 3.38

n.d.

n.d.

13 0.04

n.d. -

773 0.09 13.1

3’9 0.05 9.7

CHOLESTEROL

ESTER METABOLISM

307

IN BRAIN

fraction in the survey subcellular fractionation showed relative enrichment of the cholesterol-esterifying enzyme and pH 6.6 cholesterol ester hydrolase, and since the ultimate goal of our study is the relationship between cholesterol ester and myelin metabolism, highly pure myelin was prepared, and, at different stages of purification, samples were taken and assayed for the cholesterol-esterifyining enzyme, both pH 6.6 and pH 4.2 cholesterol ester hydrolases, succinate-cytochrome c oxidoreductase, @glucosidase and N-acetyl-/I-glucosaminidase (Table V). In Myelin I, which was still heavily contaminated primarily by microsomes, all of the enzymes were detectable, but only pH 6.6 cholesterol ester hydrolase showed enrichment over the whole homogenate. Both of the lysosomal enzymes showed a moderate decline in specific activity, but succinate-cytochrome c oxidoreductase and pH 4.2 cholesterol ester hydrolase showed a drastic decrease from whole homogenate. The activities of these last two enzymes became undetectable in Myelin II, which was obtained following the second density gradient centrifugation, after three osmotic shock treatments of Myelin I. The two lysosomal enzymes decreased drastically from Myelin I to Myelin II, in which less than 1% of the activity of whole homogenate remained. Myelin II, however, was still highly enriched with pH 6.6 cholesterol ester hydrolase, retaining about 20% of the whole homogenate activity. Myelin III was obtained after five washes of Myelin II with distilled water. p-Glucosidase became undetectable, in addition to the cholesterol-esterifying enzyme and succinate-cytochrome c oxidoreductase. The activity of N-acetyl-p-p-glucosaminidase was still detectable, 0.04% of the whole homogenate activity being found in Myelin III. In contrast, the relative specific activity of pH 6.6 cholesterol ester hydrolase was even higher than that in Myelin II, because the distilled water washes eliminated some protein but not the activity of the enzyme. Myelin III was more than 3 times enriched with this enzyme, containing 20% of the total activity of pH 6.6 cholesterol ester hydrolase in the starting homogenate. DISCUSSION

Thepresent series of studies demonstrated the presence of at least three different enzymes in the young adult rat brain, which are directly involved in the formation and degradation of cholesterol esters. They were a cholesterol-esterifying enzyme, which is capable of incorporating extraneously added oleic acid into cholesterol esters, and two distinct cholesterol ester hydrolases. Despite the extensive search, we were unable to demonstrate the activity of lecithin-cholesterol acyltransferase in the adult rat brain. The properties of the rat brain cholesterol-esterifying enzyme were similar to those in other organs. Exogenous CoA or ATP was not required for full activity, as shown also for the pancreas 18, in the mitochondrial fraction of the adrenal24, or peritoneal macrophage+. While these data alone do not rigorously exclude the possibility that there are sufficient endogenous cofactors in the whole homogenate to meet the requirement of the enzyme, the inability of excess unlabelled oleyl-CoA to reduce the incorporation of radioactive free oleic acid into cholesterol esters clearly excluded the participation of oleyl-CoA as an intermediate. The possibility of a same enzyme catalyzing both esterification and hydrolysis can be excluded by the quite dissimilar subcellular distribution of the cholesterol-esterifying enzyme and the two cholesterol ester hydrolases. The optimum pH of 5.6 fell precisely at the lowest point of the pH Riochim. Biophys.

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239 (1971) 293-311

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curves of the two cholesterol ester hydrolases. Since the activities of cholesterol ester hydrolases at the respective pH optima, 4.2 and 6.6, were less than 10% of the activity of the cholesterol-esterifying enzyme at these pH in the absence of taurocholate, and since the concentration of newly formed cholesterol ester was extremely small compared to the necessary amount (250 ,L& for the full activity of cholesterol ester hydrolases, we can exclude the influence of cholesterol ester hydrolase activities in the pH curve of the cholesterol-esterifying enzyme. CLARENBURG et al.33 did not detect activities of cholesterol-esterifying enzyme in rat brain, but our findings clearly established its presence in adult rat brain. The activity was substantial: approx. 1.0 nmole/h per mg protein, or 40 pg cholesterol esterified per h per g wet weight. The subcellular localization of this enzyme could not be clearly defined. Nearly half of the recovered activity was in the crude mitochondrial fraction. Since the microsomal fraction had a relative specific activity smaller than one, and contained only 9% of the total activity, the primary localization is probably not microsomal. The high relative specific activity of the crude myelin in the survey fractionation was of interest. However, the experiments with purified myelin clearly excluded the localization of this enzyme in myelin. Since the crude myelin in the survey fractionation was contaminated by both microsomes and mitochondria, it was not possible to determine which non-myelin component was the site of the enzyme activity. The entire data suggest that the cholesterol-esterifying enzyme in adult rat brain is primarily localized in mitochondria but some activity may also be localized in microsomes. We were unable to detect any activity of lecithin-cholesterol acyltransferase in rat brain, using the specific substrate, lecithin labelled at ,!?-position with [r-X]oleic acid. GLOMSET AND KAPLAN~~reported some activity of this enzyme in rat brain. However, they used labelled cholesterol as the precursor, and the system contained a relatively large amount of free fatty acids, because a large amount of heat-inactivated plasma was present. In view of the highly active cholesterol-esterifying enzyme in rat brain, at least some, if not all, of the activity measured by GLOMSET AXD KAPLAN~~ may have been the result of free fatty acid incorporation into cholesterol esters, rather than the direct transfer of the lecithin p-fatty acids. In fact, the activity reported was only a few percent of the activity of the cholesterol-esterifying enzyme at the optimum pH. Even at the higher pH of 7.4, where their assays were done, the activity of the cholesterol-esterifying enzyme was still higher than the activity reported by GLOMSET AND KAPLAN~~ as that of lecithin-cholesterol acyltransferase. OUr assay system did not exactly duplicate that of GLOMSET AND KAPLAN~~ (it did not contain the partially purified lipoprotein fraction), and we may have failed to detect the activity because of this. However, we think it is unlikely, since we were able to obtain the activity of lecithin-cholesterol acyltransferase in the plasma, which was similar to those reported in the literature. In any event, the activity of lecithin-cholesterol acyltransferase in adult rat brain appears to be, if not zero, negligible, compared to the cholesterol esterification through free fatty acids. This finding was somewhat disappointing, because our earlier analytical study strongly suggested contribution of this enzyme in the cholesterol ester formation in sudanophilic demyelination31. The alternative possibility we suggested then, the participation of exogenous macrophages in cholesterol ester formation in these pathological conditions,must now be considered. Two distinct cholesterol ester hydrolases exist in rat brain. They are different from each other in the pH optimum, effects of bile salts, and the subcellular distribuBiochim.

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CHOLESTEROLESTER METABOLISMIN BRAIN

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tion. The properties of these hydrolases differ in many respects from those previously reported in rat brain32?. However, comparison of our data with others is not appropriate because the activities of these enzymes in our system were approx. 30 pg cholesteryl oleate hydrolyzed per h per g for the pH 4.2 hydrolase, and 170 ,ug per h per g for the pH 6.6 enzyme, while both PRITCHARDANDNICHOLLSand CLARENBURG et aZ.33obtained only I pg or less cleavage per h per g in their systems. These authors used linoleic acid in their studies, and the discrepancies might conceivably be due to the different specificity of the enzyme toward the nature of fatty acidss2. The pH curve with two pH optima was not the result of the effect of cholesterolesterifying enzyme, the pH optimum of which fell exactly on the valley of the two pH optima of the hydrolases. The same pH optima were observed with the system activated by taurocholate, which was simultaneously inhibitory for the synthetic enzyme. Since the synthetic system requires substantial amounts of free oleic acid for full activity, resynthesis of once-cleaved cholesterol ester should be negligible in the assay systems of cholesterol ester hydrolases. Although far from conclusive, the data suggest that the pH 4.2 cholesterol ester hydrolase might be localized in mitochondria rather than lysosomes. The distribution of specific activities in the subfractions of crude mitochondria was similar to succinatecytochrome c oxidoreductase and different from N-acetyl$-n-glucosaminidase. The enzyme was not activated by freeze-thawing or ultrasonication. In the crude myelin fraction of NORTON~~ (Myelin I), only about 1% of the total homogenate activity was found for both pH 4.2 cholesterol ester hydrolases and succinate-cytochrome c oxidoreductase, while 10% or more still remained for fi-glucosidase and N-acetyl-p-nglucosaminidase. The subcellular localization of the pH 6.6 cholesterol ester hydrolases was even more intriguing. The experiment with the survey fractionation indicated that it is primarily a microsomal enzyme. But a substantial activity was also found in the myelin-rich fraction. In the experiment with purified myelin, it became clear that, although the bulk of the enzyme was in microsomes, the myelin fraction, prepared by a series of osmotic shocks and two density gradient centrifugations, retained approx. 20% of the total homogenate activity, while all other enzymes assayed were less than 1% of the total. These residual activities of other enzymes could be further reduced to non-detectable levels by five additional washings with water, but no change was observed in the activity of the pH 6.6 cholesterol ester hydrolase. The pH 6.6 cholesterol ester hydrolase is particulate-bound. Therefore, we can exclude adsorption of a soluble enzyme to the myelin fraction. The myelin fraction prepared by the Norton method is highly pure, and this fraction was enriched more than 3-fold with this enzyme. If the activity of the pH 6.6 cholesterol ester hydrolase in the purified myelin fraction is due to any adsorbed particulate fractions, such particulates must be specifically and very highly enriched with the enzyme. Although the data do not rigorously exclude this possibility, it seems to be highly unlikely. Recently, RIEKKINEN AND RUMSBY~~reported residual activities of nonspecific esterase in purified myelin. The specific cholesterol ester hydrolase with pH optimum of 6.6 may account for some of the activity. The data therefore strongly suggest that a substantial portion (20%) of the pH 6.6 cholesterol ester hydrolase, but not the pH 4.2 hydrolase, in the adult rat brain may be localized in myelin. Since our ultimate interest is the relationship between cholesterol esters and the metabolism of myelin, this finding is particularly Biochim.

Biofihys.

Acta,

239 (1971)

293-311

Y. ETO, K. SUZUKI

3ro

significant. Attempts to solubilize the myelin-bound pH 6.6 cholesterol ester hydrolase with detergents have not been satisfactory. The present data will serve as the basis for our further exploration of the biochemistry of myelination and demyelination. In view of the known relatively high concentration of esterified cholesterol in the premyelination stage of development and its subsequent disappearance, the changes of these enzymes in developing rat brains are presently under investigation. The relationship among pathological demyelination, formation of cholesterol esters, and activities of these enzymes, will be studied in detail in a suitable experimental model. ACKNOWLEDGEMENTS

This investigation was supported by the Inex. J. Warriner Memorial Grant for Research on Multiple Sclerosis (670-A-1) from the National Multiple Sclerosis Society, and by Research Grants NS-08420 and NS-08075 from the U.S. Public Health Service. REFERENCES I Ii. CLARENBURG, I. L. CHAIKOFF AND M. D.MoRRIs,J. Neurochem., 10(1g63) 735. 2 E. T. PRITCHARD,J. Neurochem., IO (1963) 495. 3 L. SVENNERHOLM. J. Neurochem., 11 (1964) 839. 4 J. TICHY,Neurology, 16(1966)121g. 5 C._~LLINGAND L. SVENNERHOLM,J. Neurochem., 16(196g)751. 6 P.MANDELL, R. BIETH AND R. STOLL,Compt.Rend. Soc.Biol., 143 (1948)1224. 7 C. W. M. ADAMS AND A. N. DAVISON,J. Neurochem., 4 (1959) 282. 8 R. FUMAGALLI AND R.PAOLETTI,L@ Sci.,5(1g63)2gr. g A. N. DAVISON,Advan. Lipid Res., 3 (1965) 171. IO D.GRAFNETTER, E. GROSSIAND P.MORGANTI, J. Neurochem., 12 (1965)145. II G. BRANTE, Acta Physiol. Stand.,18,Suppl.63 (1949)p. I. 12 A. C. JOHNSON, A. R. MCNABB AND R. J. ROSSITER,Biochem. J., 45 (1949)500. 13 A. N. DAVISON AND W. WAJDA, Biochem. J., 82 (1962) 133. 14 W. T. NORTON, S. E. PODUSLO AND K. SUZUKI,J. Neuropathol.Exptl.Neurol., 25(1966)582. 15 K. SUZUKI,K. SUZUKI AND S. KAMOSHITA,J.Neuropathol.Exptl.Neurol., 28(196g) 25, 16 Y. SUZUKI,S. H. TUCKER, L. B. RORKE AND K. SUZUKI,J. Neuropathol. Exptl. Neural., 29 (1970) 405.

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