ARCHIVES OF BIOCHEMISTRY AND BIOPHYSICS Vol. 197, No. 2, October 15, pp. 570-579, 1979
Microsomal Cholesterol Ester Hydrolase of Rat Brain: Lipids as Effector of Enzymatic Activity’ MYUNG-UN The
CHOI AND KUNIHIKO
Saul R. Korey Department of Neurology, Department of Neuroscience, for Research in Mental Retardation and Human Development, Albert Bronx,
New
York
SUZUKI and the Rose F. Kennedy Center College of Medicine,
Einstein
10461
Received February 26, 1979; revised May 15, 1979 Evidence is presented that lipid plays an important role in the function of the microsomal cholesterol ester hydrolase of rat brain. The catalytic activity was almost completely lost when most of cholesterol and up to 70% of phospholipids were removed from lyophilized microsomes by extraction with chloroform at -20°C. The activity was completely restored when the chloroform-extracted lipid was added back to the assay mixture in the amount equal to the original concentration. Cholesterol or individual phospholipid alone was not effective in reconstituting the lost enzymatic activity. Effective restoration of the activity required addition of cholesterol and a phospholipid. Among the phospholipids tested, phosphatidylserine was the most effective, followed by ethanolamine phospholipids and phosphatidylcholine. The apparent V was dependent on the amount of the lipid added, while the K, for the substrate, cholesteryl oleate, remained relatively constant, indicating that the effect of the added lipid was primarily on the reaction rate and not on the affinity of the enzyme to the substrate. The similar lipid dependence was observed with the Triton X-lOOsolubilized enzyme preparation. When the lipid phase of the microsomal membrane was perturbed, the enzyme became unstable when heated at 50°C and its activity showed a discontinuity in the Arrhenius plots. Therefore, not only the concentration of the added lipid but also the physical state of the lipid phase around the enzyme appeared to be important for the activity of the rat brain microsomal cholesterol ester hydrolase.
ately by sodium taurocholate (2). Exogenous lipids added to the assay system did not activate this microsomal cholesterol ester hydrolase as greatly as they did other two enzymes (4). However, during the course of our attempts at solubilization and purification of the rat brain microsomal cholesterol ester hydrolase, the enzyme exhibited the characteristic properties of an integral membrane protein (5). High concentrations of detergents were required for solubilization, and once solubilized, it appeared to associate with lipids during experimental manipulations, such as gel filtration. Therefore, by utilizing the commonly employed criteria to demonstrate lipid dependence of membranebound enzymes (6), we partially delipidated rat brain microsomes with organic solvents and examined the possible functional importance of lipids on the activity of the cholesterol ester hydrolase. The results,
At least three distinct cholesterol ester hydrolases (cholesterol esterase, EC 3.1.1.13) are present in rat brain (l-3). They are localized in the crude mitochondria, microsomes, and in the myelin sheath, respectively, and have different properties with respect to the pH optimum, requirements for detergents and/or exogenous lipids, and changes during brain development. One of the three, localized primarily in microsomes, has the pH optimum of 6.0, and is highly activated by Triton X-100 but only moder1 This investigation was supported by Research Grants NS-13578, NS-03356, and HD-01799 from the United States Public Health Service. A part of the content of this article was presented at the 10th annual meeting of the American Society for Neurochemistry, March 12- 16,1979, in Charleston, S. C. and was published in the form of an abstract (M. Choi and K. Suzuki, 1979, Trans. Amer. Sot. Neurochem. 10, 69). 0003-9861/79/120570$02.00/0 Copyright 8 1979 by Academic Press, Inc. All rights of reproduction in any form reserved.
570
MICROSOMAL
CHOLESTEROL
taken collectively, indicated strongly that lipids, particularly cholesterol and phosphatidylserine, act as effecters for the activity of the enzyme, when brain microsomes were delipidated by cold chloroform and acetone. EXPERIMENTAL
PROCEDURE
Materials [4-‘%]Cholesteryl oleate (54.0 mCi/mmol) and Econofluor were purchased from New England Nuclear Corporation (Boston, Mass.). The labeled cholesteryl oleate was diluted IOO-fold with unlabeled cholesteryl oleate (Sigma Chemical Co., St. Louis, MO.). Its radiopurity was examined by thin-layer chromatography, and if necessary, it was purified by silicic acid (Unisil, Clarkson Chemical Co., Williamsport, Pa.) column chromatography (1). Cholesterol and Triton X-100 were from Sigma Chemical Company. Phosphatidylserine, sphingomyelin, and galactosylceramide (all from bovine brain) were products of Koch-Light Laboratories, purchased through Research Products International (Elk Grove Village, Ill.). Synthetic distearoyl phosphatidylcholine was from Applied Science Laboratories (State College, Pa.) and precoated silica gel G thin-layer plates were from Analtech (Newark, Del.). Digitonin and all of the organic solvents used were supplied by Fisher Scientific Company (Fairlawn, N. J.). The Bio-Rad dye-binding protein assay kit was from Bio-Rad Laboratories (Richmond, Calif.). All commercial lipids were tested by thin-layer chromatography. When 100 pg of the material was spotted, all preparations were without detectable impurities, except for phosphatidylserine, which contained detectable impurity that cochromatographed with cholesterol in two solvent systems, chloroform-methanol-coned ammonia (‘70:30:5, by volume) and chloroform-methanol (9:1, v/v).
Methods Preparation of rat brain microsomes. Young adult Sprague-Dawley rats were used throughout. The animals were decapitated and the whole brains were removed and homogenized in 9 vol of ice-cold 0.32 M sucrose solution in a hand-operated Dounce homogenizer (Kontes Glass Co., Vineland, N. J.). The homogenate was centrifuged at 11,OOOg x 20 min. The pellet was resuspended in 0.32 M sucrose and centrifuged once more. The combined supernatants were then centrifuged at 100,OOOg x 60 min to obtain the microsomal fraction. The microsomal pellet was washed once with ice-cold distilled water by suspension and recentrifugation. The final pellet was suspended in water and lyophilized. Lyophilization did not significantly affect the enzymatic activity and the lyophilized microsomes could be
ESTER
HYDROLASE
571
stored at -20°C in a desiccator for several months without loss of the activity. Extraction with organic solvents. Lipid-depleted microsomes were prepared by extractions with organic solvents. Lyophilized microsomes, 100 mg, were placed in a glass centrifuge tube, and 10 ml of the extracting solvent, chilled to -20°C was added. This ratio of the microsomes to the solvent was maintained for all routine extractions. After 5 min with gentle shaking, the mixture was centrifuged at -20°C and the organic solvent phase was transferred. When chloroform was the extracting solvent, special care was required so as not to collect floating microsomes. The extraction was repeated once more with the same solvent. Finally, the residue was extracted with 10 ml of acetone at -20°C. All of the solvent phases including the final acetone extract were combined and used as the source of the extracted lipids for reactivation studies. The final acetone extraction was not used when microsomes were extracted with other organic solvents other than chloroform. The lipid-depleted microsomes were dried gently under a stream of nitrogen and kept at -20°C in a desiccator. For studies of the effect of the extraction conditions, variations were introduced to the above standard procedure, such as different numbers of extractions. Characterization of extracted microsomal lipid. The extracted lipid in the combined organic solvent phase was recovered by vacuum evaporation of the solvent. The resulting residue was dissolved in a mixture of chloroform-methanol (21, v/v). The insoluble material was eliminated by centrifugation and transfer of the soluble material to a tared tube. The amount of the extracted lipid was determined after drying under a stream of nitrogen. It was then redissolved in a known volume of chloroformmethanol (21, v/v) for further analysis. For comparison, the total microsomal lipid fraction was prepared by extractingintact microsomes with chloroform-methanol (21, v/v), essentially according to Folch et al. (7). The lipid remaining after chloroform extraction was also extracted similarly from the “lipid-depleted” microsomes with chloroform-methanol (2:1, v/v). The extracted lipids were fractionated into seven groups by silicic acid column chromatography according to the elution scheme of Norton and Autilo (8), which involved application of the lipid onto the column in chloroform and stepwise elution with increasing proportion of methanol in chloroform. Each fraction was semiquantitatively examined by thin-layer chromatography and was dried for gravimetric determination of the amount. Semiquantitative comparison of various lipid fractions was carried out by thin-layer chromatography in the solvent system of chloroform-methanolwater (65254, by volume), followed by sulfuric acid spray and charring at 135°C. Several lipid standards were chromatographed simultaneously to aid identification of the spots,
572
CHOI AND SUZUKI
Solubilization of the enzyme. Rat brain microsomal cholesterol ester hydrolase could be solubilized with reasonable recovery and stability by a medium consisting of 0.75% Triton X-100, 120 mM KCl, 40 mM Tris-HCl buffer, pH 7.6, and 10% glycerol. This formula was arrived at after extensive examinations of various solubilizing conditions (Choi and Suzuki, unpublished). The lipid-depleted microsomes, extracted twice with chloroform and once with acetone as described above (chloroform-extracted microsome), was suspended in this solubilizing medium at the ratio of 10 mg dry chloroform-extracted microsome to 1 ml medium in a glass homogenizer and was kept in ice for 30 min with occasional brief homogenization. After centrifugation at 100,OOOgfor 60 min, the clear supernatant was dialyzed overnight against 10 mM Tris-maleate buffer, pH 6.2, containing 0.01% Triton X-100, 1 mM EDTA, and 20% glycerol. The turbidity that developed during dialysis was eliminated by another centrifugation at 100,OOOg x 60 min. The final solubilized enzyme was stable for at least several days at 4°C. Enzyme assay. The assay procedure for the rat brain microsomal cholesterol ester hydrolase was essentially as described previously (2, 4). For assays of microsome-bound activity, 125 pg of [4-W]cholesteryl oleate (specific activity, 0.54 mCi/mmol), 1.25 mg of Triton X-100, and when included, appropriate amounts of lipid, were dried together in a 13 x loo-mm screw-capped test tube under a stream of nitrogen. The mixture was dispersed by the addition of 25 ~1 of ethanol and sonication of a water-bath type sonicator, followed by further addition of 0.5 ml of 0.1 M sodium phosphate buffer, pH 6.0 and sonication. When solubilized enzyme was assayed, the amount of Triton X-100 was reduced to 0.12 mg/tube. The reaction was initiated by addition of 0.1 ml of microsomal suspension in water (10 mg dry wt/ml) (0.25-0.4 mg protein), or 0.1 ml of the solubilized enzyme source (0.4-0.45 mg protein). The incubation was at 37°C for 60 min. All assays were carried out at least in duplicate, and blank tubes containing boiled enzyme source were included. The reaction was terminated by the addition of 5 ml of acetone -ethanol (1: 1, v/v) containing 100 pg of unlabeled cholesterol as carrier. The radioactivity of the cholesterol liberated by the enzymatic reaction was determined by the digitonin precipitation procedure (4). The results of duplicate assays always agreed within 10%. Analytical methods. Phospholipid was determined by its phosphorus content by the method of Chen et al. (9). The average molecular weight of phospholipids was assumed to be 775. The amount of cholesterol was determined by the method of Searcy and Bergquist (10). The Bio-Rad dye-binding assay system was used for most protein determination. This is a procedure developed commercially by Bio-Rad Laboratories based on the method of Bradford (11). Bovine serum albumin was used as the standard.
The values for membrane-bound protein obtained with the dye-binding assay gave approximately 55% of the values obtained by the more conventional Lowry method (12). Comparison of the results obtained by the two different protein assays could be made with satisfactory accuracy using the above conversion factor. The dye-binding assay was relatively insensitive to interference from Triton X-100 but equivalent amounts of Triton X-100 were added to the standard bovine serum albumin when the sample contained Triton X-100. RESULTS
Effect of Organic Solvent Extraction on the Microsowu~l Cholesterol Ester Hydrolase When the lyophilized microsomes were extracted with varieties of organic solvents, the activities of the microsomal cholesterol ester hydrolase decreased in varying degrees. The residual activities of organic solvent extracted microsomes correlated well with the amount of lipid left unextracted in these preparations (Fig. 1). When only a small fraction of the total lipid was extracted, such as single extraction with acetone or diethylether, the reduction of the cholesterol esterase activity was minor, while the microsomes from which 75% of the lipid was extracted with chloroform completely lost the hydrolytic activity. Extraction with chloroform-methanol (2:1, v/v) resulted essentially in complete loss of both lipid and the cholesterol esterase activity. Unlike in other extraction conditions, however, the chloroform-methanol extraction resulted in partial but irreversible inactivation of the enzyme, as described below. Restoration of the Enzyme Activities Extraction with Organic Solvents
After
With the exceptions of extractions with chloroform-methanol (2:1, v/v) and with iso-butanol, the cholesterol ester hydrolase activities of the organic solvent-extracted microsomes could be restored to the original level by adding back the extracted lipid fraction to the assay mixture (Fig. 2). When varying amounts of the lipid mixture obtained by cold chloroform extraction of dry microsomes were added to the assay system, the cholesterol ester hydrolase in the native intact microsomes was activated by 30% at low concentration of the added
MICROSOMAL
CHOLESTEROL
FIG. 1. Effect of partial delipidation of microsomes. The lyophilized rat brain microsomal fraction was extracted with organic solvents and then assayed for activity of the cholesterol ester hydrolase with the standard procedure in the absence of exogenous lipids. The amounts of cholesterol and phospholipids remaining in the microsomes were determined as described under Methods. The vertical and horizontal bars at “a” represent the means and the standard deviations of the lipid content and hydrolase activity of untreated microsomes (n = 5). The bars at “b” give the same data for the microsomes extracted twice with cold chloroform (n = 6) followed by a single extraction with acetone. 0 Single extraction with acetone; 0 single extraction with diethylether; n double extraction with acetone; 13 double extraction diethylether; 0 double extraction with iso-butanol; 0 double extraction with 2-propanol; A single extraction with chloroform followed by single extraction with acetone; A single extraction with chloroformmethanol (2:1, v/v). Except for the chloroformmethanol extraction, there was a good correlation between the amount of the residual lipid and the enzymatic activity.
lipid. The acetone- or chloroform-extracted microsomes required larger amounts of lipid for optimal activation. Despite the almost complete loss of the activity after the extraction, the cholesterol ester hydrolase activity in the chloroform-extracted microsomes could be restored completely (Fig. 2 and Table I). The amounts of the chloroform-extracted lipids for the optimal restoration of the hydrolase activity differed, depending on how the microsomes had been depleted of lipid. However, in each instance, the optimal amount of the lipid to be added to the assay system was approximately equal to the amount of lipid that had been removed by the solvent extraction. The cholesterol ester hydrolase activity in the chloroform-extracted microsome, restored
ESTER HYDROLASE
573
with the optimal amount of added lipid, showed general characteristics similar to those in the untreated microsomes with respect to the linearity against the incubation time and the enzyme source, the pH optimum, and the effect of Triton X-100 (2). The hydrolase activity in the chloroform-methanol-extracted microsomes could not be restored to more than 25% of the original activity, suggesting that substantial hydrolase activity was irreversibly inactivated by the chloroform-methanol extraction (Fig. 2). Nature of Chloroform-Extracted Microsomal Lipid Since the cold chloroform extraction of lyophilized microsomes appeared to provide
FIG. 2. Effect of added lipid mixture on the cholesterol ester hydrolase. Untreated and variously delipidated preparations of rat brain microsomes were assayed with or without additional lipid in the assay mixture. Each assay tube contained 1 mg of delipidated dry enzyme source which contained 0.25-0.4 mg proteimmg, depending on the extraction procedures. For all of the experiments in this figure, the lipid fraction obtained by the double extraction of lyophilized microsomes with chloroform followed by a single extraction with acetone was added, except for the experiment with chloroform-methanol microsomes which were assayed with the addition of chloroformmethanol-extracted total microsomal lipid. x Untreated microsomes; A acetone-extracted microsomes; 0 chloroform (single)-extracted microsomes; 0 chloroform (double) and acetone (single)-extracted microsomes; W chloroform-methanol-extracted microsomes. The amounts of lipid required for the optimal restoration of enzymatic activity roughly corresponded to those that have been extracted from the respective preparations. Although not shown, the isobutanol-extracted microsomes could not be restored in the hydrolase activity with the addition of lipids.
574
CHOI AND SUZUKI TABLE
I
RATBRAIN MICROSOMALLIPIDAND CHOLESTEROLESTERHYDROLASE BEFORE ANDAFTER DOUBLE EXTRACTIONWITH~ILOROFORM FOLLOWEDBYA~INGLE EXTRACTION WITHACETONE~ Lipid content (mg/mg protein) Enzyme source Untreated CHCl,-extracted
Enzymatic activity (nmolihimg protein)
Cholesterol
Phospholipid
Without lipid
With additional lipid
0.424 f 0.034 (6) 0.036 + 0.008 (4)
0.915 + 0.075 (6) 0.268 2 0.030 (6)
20.3 + 2.0 (5) 1.01 2 0.64 (6)
28.6 + 3.7 (7) 31.2 k 6.3 (9)
a Values are expressed as averages *SD with the number of separate experiments in parentheses. The lipid contents of the untreated and chloroform-extracted microsomes were determined as described in the text. The cholesterol ester hydrolase activity was determined either with or without the optimal amounts of the chloroform-extracted microsomal lipid mixture. The values of protein determined by the dye-binding assay were used for the calculation.
an excellent system for further study-total loss of the enzymatic activity after extraction and the complete restoration of the activity by the addition of the extracted lipid-the nature of the chloroform-extracted and remaining lipids was examined. The double extraction with cold chloroform removed lipids constituting 33 & 2% of the dry weight of microsomes. The extracted lipid fraction had the following composition; ethanolamine phospholipid (33%), cholesterol (25%), phosphatidylserine (20%), phosphatidylcholine (14%), and others (each less than 6%, including galactosylceramide and sphingomyelin). Semiquantitative thin-layer chromatographic comparison of lipid fractions from intact microsomes, the chloroform extract, and the chloroform-extracted microsomes indicated that chloroform extracted almost all cholesterol, most of ethanolamine phospholipids, perhaps 80% of phosphatidylcholine, and about a half of the phosphatidylserine (Fig. 3). Quantitative determinations indicated that more than 90% of cholesterol and approximately 70% of total phospholipid were extracted by the standard chloroform extraction procedure (Table 1). Restoration of the Activity Lipids
with Individual
Capacities of pure individual lipids and their combinations to restore the cholesterol ester hydrolase activity in the chloroformextracted microsomes were examined (Fig. 4). Phospholipids were generally more
effective than cholestrol, except for sphingomyelin. Galactosylceramide was poor in restoring the enzymatic activity. However, none of the individual pure lipids alone were as effective as the chloroformextracted lipid mixture (Fig. 4A). Since the chloroform-extracted lipid contained approximately 25% cholesterol and 65% phospholipids, mixtures of cholesterol and individual phospholipids were tested. When the amount of cholesterol was kept constant at 100 pg/tube and that of phospholipid was varied, phosphatidylserine, phosphatidylethanolamine, and phosphatidylcholine were all capable of restoring the hydrolase activity to the level of the system to which the optimal amount of the chloroformextracted lipid mixture was added (Fig. 4B). Again, sphingomyelin showed essentially no effect. The amount of individual glycerophospholipids necessary to achieve the optimal reactivation in combination with 100 pg cholesterol varied, with phosphatidylserine being the most effective followed by phosphatidylethanolamine and then by phosphatidylcholine. It was also clear that the reactivating effects of cholesterol and the glycerophospholipids were not additive. Effects of Exogenous Lipids on Solubilixed Microsomal Cholesterol Ester Hydrolase
The microsomal cholesterol ester hydrolase was solubilized from the chloroform-extracted microsomes as described under Methods.
MICROSOMAL
CHOLESTEROL
PE
ESTER HYDROLASE
575
individual lipids required for the optimal restoration of the activity were generally 20-25% of those required for the chloroformextracted microsome-bound enzyme. Finally, cholesterol, which, when added alone, could reactivate the hydrolase in the chloroform-extracted microsomes only to 20% of the optimal activity, activated the solubilized enzyme up to 80% of the optimal
PC
SM
PS
I
II
III
FIG. 3. Thin-layer chromatography of rat brain microsomal lipid. The total lipid fractions of untreated microsomes, chloroform-extracted microsomes, and the combined chloroform extracts were run on a silica gel G thin-layer plate, 250 pm thick, in the solvent system of chloroform-methanol-water (65:25:4, by volume), and were visualized by sulfuric acid spray and heating. I: Residual lipid of chloroform-extracted microsome; II: chloroform-extracted lipid; III: lipid fraction of untreated microsomes. Chol: cholesterol; PE: ethanolamine phospholipid; PC: phosphatidylcholine; SM: sphingomyelin; PS: phosphatidylserine. The spot above PS is an artifact (sucrose). Lipid fractions from comparable amounts of microsomes are chromatographed. Therefore, the sum of I and II should be approximately III.
The solubilized preparations were then tested for the hydrolase activity with or without additional exogenous lipids (Fig. 5). Although the results were generally similar to those obtained with the chloroformextracted microsomes as the enzyme source, there were several points of quantitative differences that appeared significant. The solubilized preparations exhibited considerably higher activities of the cholesterol ester hydrolase than the starting chloroform-extracted microsomes in the absence of additional lipids. The amounts of
FIG. 4. Effect of individual lipids on the cholesterol ester hydrolase activity of chloroform-extracted rat brain microsomes. Lyophilized microsomes were extracted twice with cold chloroform followed by a single extraction with acetone and the cholesterol esterase activity was determined with the addition of varying amounts of different lipids. Each tube contained 1 mg of the enzyme source (0.4 mg protein). For both A and B, the broken lines represent the results obtained with addition of the chloroformextracted lipid mixture. Other results in A were obtained when a single lipid was added: 0 phosphatidylserine; 0 ethanolamine phospholipid; 0 cholesterol. In B, 106 pg of cholesterol was also added in addition to the respective test lipids: 0 phosphatidylserine; 0 ethanolamine phospholipid; m phosphatidylcholine; q sphingomyelin.
576
CHOI AND SUZUKI
activity. The most likely explanation for these quantitative differences is that the residual lipid, relatively rich in phosphatidylserine, is not available to the enzyme which is bound to the chloroform-extracted microsomes but becomes available during the enzyme solubilization procedure in the presence of the detergent. Effects of Lipids on Other Enz ynw Properties
Heat stability of the cholesterol ester hydrolase was tested by heating water suspensions of untreated microsomes, chloroform-extracted microsomes with or without adding back the chloroform extract, and the solubilized prepartion (Fig. 6). The enzyme in the untreated microsomes was relatively heat stable, tolerating 50°C heating for 15 min without inactivation. It ,was inactivated only by 20% after 60 min at 50°C. The solubilized preparation was very heat labile and 50% inactivation occurred after 5 min at 45°C. When heated without added lipid, the enzyme in the chloroformextracted microsomes showed intermediate heat stability. When the chloroform-extracted microsomes were heated in the presence of
FIG. 5. Effect of lipids on solubilized rat brain microsomal cholesterol ester hydrolase. The enzyme was solubilized from the chloroform-extracted microsomes as described in the text. The activity was then assayed in the assay mixture optimized for the solubilized enzyme with added lipids as indicated. A Chloroform-extracted microsomal lipid mixture; 0 phosphatidylserine; 0 cholesterol; 0 phosphatidylcholine. Each assay tube contained 0.42 mg of solubilized protein.
FIG. 6. Heat stability of rat brain microsomal cholesterol ester hydrolase. Water suspensions of untreated microsomes, chloroform-extracted microsomes with or without the chloroform-extracted lipid, and the solubilized preparation were heated at 50°C for the indicated periods of time, except for the solubilized enzyme which was heated at 45°C. The solubilized enzyme was too heat labile for reliable results when heated at 50°C. At the end of heating, the enzyme was assayed with the standard procedure with the optimal amount of lipids. 0 Untreated microsomes; 0 chloroform-extracted microsomes heated in the absence of the additional lipid; A chloroform-extracted microsomes heated in the presence of the optimal amount of the chloroform-extracted lipid; 0 solubilized enzyme preparation.
the extracted lipid, it appeared to show slight protective effects against heat inactivation but the heat stability of the untreated microsomes could not be restored. Some membrane-bound enzymes show characteristic discontinuities in the Arrhenius plots, which are usually interpreted as reflecting phase transition of the lipid phase around the enzyme proteins (13). The cholesterol ester hydrolase in the untreated and chloroform-extracted microsomes was assayed at different temperatures (Fig. 7). The Arrhenius plot of the untreated microsomes appeared to be linear throughout the temperature range studied. On the other hand, the chloroform-extracted microsomes assayed with the addition of the optimal amount of the extracted lipid gave a sharp discontinuity in the Arrhenius plot at 16.9”C. The discontinuity point shifted to 21.5”C when the chloroform-extracted microsomes were assayed in the presence of a mixture of phospholipids free of cholesterol.
MICROSOMAL
CHOLESTEROL
FIG. 7. The Arrhenius plots of rat brain microsomal cholesterol ester hydrolase. 0 Untreated microsomes; 0 chloroform-extracted microsomes assayed in the presence of the optimal amount of the chloroformextracted lipid fraction; 0 chloroform-extracted microsomes assayed in the presence of the chloroformextracted lipid from which cholesterol had been removed by the silicic acid column chromatography.
These findings suggested perturbation of the lipid phase of the microsomes during the extraction-reactivation procedure. The effects of the substrate concentration were tested with the untreated microsomes and the chloroform-extracted microsomes (Fig. 8). The latter was assayed with three different concentrations of the chloroformextracted lipids. Untreated microsomes were assayed without exogenous lipids. Since the Lineweaver-Burk plots were not linear, the kinetic parameters, V and K,, were estimated directly from the graph of V vs [S]. While the V varied as expected, the K, remained quite constant, indicating chat the lipid altered the reaction rate without affecting the affinity of the enzyme to the substrate. DISCUSSION
The present series of studies was initiated when we observed during our attempt to solubilize the rat brain microsomal cholesterol ester hydrolase that delipidation of lyophilized microsomes preliminary to solubilization of
ESTER HYDROLASE
577
the enzyme resulted in apparent loss of the enzymatic activity. It was possible to correlate the apparent loss of the enzymatic activity of the partially delipidated microsomes with the amount of lipid removed, and the activity could be restored by the addition of the extracted lipids. The optimal restoration of the activity could be achieved when the original lipid content of the microsomes was restored. These findings are consistent with the criteria of lipid dependency of an enzyme (6). For the reconstitution of the catalytic activity, we employed only the direct addition of the extracted lipid to the assay mixture without preassociation. Under these conditions, the restored activity showed some properties that were different from those of the native enzyme in the untreated microsomes, although the general kinetic properties were similar to those of the untreated enzyme. The maximum reaction rate was slightly but consistently greater in the restored enzyme; the heat stability of the untreated enzyme could not be restored; and there were distinct alterations in the Arrhenius plots. When microsomes were partially delipidated with dry, cold chloroform, almost all cholesterol and substantial portions of ethanolamine and choline phospholipids were extracted, leaving a mixture of lipids relatively enriched with phosphatidylserine. Such preparations were essentially inactive in catalyzing hydrolysis of cholesteryl oleate unless appropriate types and amounts of lipids were added to the assay mixture. Restoring cholesterol alone showed little effect. Similarly, any of the glycerophospholipids alone could not restore the original activity. When combined with cholesterol, each of the glycerophospholipids could restore the original activity, and with respect to the amount required for the optimal restoration, phosphatidylserine was the most effective. These observations indicated that it is not merely the restoration of the original total microsomal lipid composition but a combination of specific lipids-cholesterol and phosphatidylserinethat is required for restoration of the enzymatic activity. The finding on the solubilized enzyme appeared to be consistent with this. When the enzyme was solubilized
578
CHOI AND SUZUKI
l/[Sl FIG. 8. Effect of the substrate concentration on the rat brain microsomal cholesterol ester hydrolase. The hydrolytic activities were determined with the standard assay procedure except that the amount of the substrate and the amount of exogenous lipids were varied as indicated. (A) Activity vs substrate concentration, (B) Lineweaver-Burk plots. 0 Untreated microsomes assayed without exogenous lipid; 0 chloroform-extracted microsomes assayed with the optimal amount of the chloroformextracted lipid; x chloroform-extracted microsomes assayed with twice the optimal amount of the chloroform-extracted lipid; 0 chloroform-extracted microsomes assayed with one-fourth of the optimal amount of chloroform-extracted lipid. The Lineweaver-Burk plots were not linear. The K, estimated from A was relatively constant at 7-8 X 10m5M (+30 pg/O.6 ml).
from the chloroform-extracted microsomes, substantial reactivation could be obtained by the addition of cholesterol alone, although cholesterol alone had little effect on the chloroform-extracted microsomes. One plausible interpretation is that, during the enzyme solubilization procedure which required a high concentration of Triton X-100, the remaining phosphatidylserine became available for lipid-enzyme interactions, thus requiring only the addition of cholesterol for activity. This finding appears analogous to Na+, K+-ATP’ase, which is a phospholipid-requiring enzyme (14). However, when the enzyme source was partially delipidated by organic solvents under controlled conditions, the addition of cholesterol alone could restore the ATP’ase activity, presumably because the required phospholipids were still present in the enzyme source (15). Several pieces of observations are suggestive of the added cholesterol primarily interacting with the phospholipid rather than with the enzyme. Since cholesterol is an end product of the enzymatic reaction, it is unlikely to interact with the enzyme and activate it. Perhaps more importantly,
the point of discontinuity in the Arrhenius plots was shifted depending on the presence or absence of exogenous cholesterol in the assay mixture. Since the discontinuity is likely to reflect the phase transition of the lipid phase associated with the enzyme (13), this suggests interactions of cholesterol with phospholipids, thereby altering the physical characteristics of the lipid phase. Cholesterol is known to affect the physical properties of phospholipid monolayers (16) or vesicles (17). The mechanism with which the lipid exerts its effect on the activity of the rat brain microsomal cholesterol ester hydrolase is difficult to assess precisely, because of the residual lipid in the delipidated microsomes and of the presence of Triton X-100 in the assay system. We have not been able to prepare completely lipid-free active enzyme source or to eliminate the unnatural detergent from the reaction mixture. There is a possibility, for example, that the different lipid effects may be due to differential competition of different lipids with Triton X-100. Although this possibility cannot be excluded completely, we think it is unlikely because our preliminary results
MICROSOMAL
CHOLESTEROL
indicate that the ratio of detergent to the enzyme source is more critical than the ratio of detergent to lipid for the activity of the solubilized enzyme (Choi and Suzuki, unpublished). Similarly, the inhibition observed at high concentrations of lipids cannot be explained rigorously at the present time except for speculative interpretations, such as substrate or detergent dilution by the excess lipids. However, even under these conditions, our findings strongly suggested the importance of the interactions between the normally membrane-bound cholesterol ester hydrolase and its lipid environment within the membrane for the enzymatic activity. The appearance of the discontinuity in the Arrhenius plots when the delipidated microsomes were used as the enzyme source indicated that although the catalytic activity could be restored, the restored lipid environment of the enzyme was not identical with that in the untreated microsomes which showed no discontinuous point. This interpretation is also consistent with the finding that the heat stability of the enzyme in the untreated microsomes could not be restored by the simple addition of lipid to the chloroform-extracted microsomes or to the solubilized enzyme. When varying amounts of lipid mixture were added to the delipidated microsomes, the maximum reaction velocity varied but the K, for the substrate remained constant. Therefore, the lipid appeared to act as a heterotropic effector primarily affecting the reaction turnover rate, rather than altering the affinity of the enzyme to the substrate. The series of studies presented in this report was necessitated because of the practical need of our ongoing attempts at solubilization and purification of the rat brain microsomal cholesterol ester hydrolase. These pieces of information would be essential for monitoring the recovery and the specific activity of the enzyme in various purification steps. These data, however, provided more fundamental information than of merely pragmatic value. The activity of the rat brain microsomal cholesterol ester hydrolase is dependent on the lipid environment and there are considerable degrees of specificity with respect
ESTER
HYDROLASE
579
to different lipids in their capacity as the effector of the enzyme. Our previous studies showed that the myelin-specific cholesterol ester hydrolase was also dependent on exogenously added lipid, particularly on phosphatidylserine, for its activity (18, 19). The acidic cholesterol ester hydrolase is specifically activated by bovine brain ethanolamine phospholipid (Ogino and Suzuki, unpublished). Therefore, activities of all of the three known cholesterol ester hydrolases in rat brain are affected by specific lipids added to the assay mixtures. REFERENCES 1. ETO, Y., AND SUZUKI, K. (1971) Biochim. Biophys. Acta 239, 293-311. 2. ETO, Y., AND SUZUKI, K. (1973) J. Biol. Chem. 248, 1986-1991. 3. ETO, Y., AND SUZUKI, K. (1973) J. Neurochem. 20, 1475- 1477. 4. IGARASHI, M., AND SUZUKI, K. (1974) Esp. Neurol. 45, 549-553. 5. SINGER, S. J. (1974) Annu. Rev. Biochem. 43, 805-833. 6. FLEISCHER, S., BRIERLEY, G., KLOUWEN, H., AND SLAUTTERBACK, D. B. (1962) J. Biol. Chem. 237, 3264-3272. 7. FOLCH-PI, J., LEES, M. B., AND SLOANESTANLEY, G. H. (1957) d. Biol. Chem. 226, 497-509. 8. NORTON, W. T., AND AUTILIO, L. A. (1965) Ann. N. Y. Acad. Sci. 122, 77-85. 9. CHEN, P. S., TORIBARA, T. Y., AND WARNER, H. (1956) Anal. Chem. 28, 1756-1758. 10. SEARCY, R. L., AND BERGQUIST, L. M. (1960) Clin. Chim. Acta 5, 192-199. 11. BRADFORD, M. M. (1976) Anal. Biochem. 72, 248-254. 12. LOWRY, 0. H., ROSEBROUGH, N. J., FARR, A. L., AND RANDALL, R. J. (1951) J. Biol. Chem. 193, 265-275. 13. WALKER, J. A., AND WHEELER, K. P. (1975) Biochem. J. 151, 439-442. 14. DAHL, J. L., AND HOKIN, L. E. (1974) Annu. Rev. Biochem. 43, 327-356. 15. NOGUCHI, T., AND FREED, S. (1971) Nature New Biol. 230, 148-150. 16. DEMEL, R. A., VAN DEENEN, L. L. M., AND PETHICA, B. A. (1967) Bioehim. Biophys. Acta 135, 11-19. 17. HUNT, G. R., AND TIPPING, L. R. H. (1978) Biochim. Biophys. Actu 507, 242-261. 18. IGARASHI, M., AND SUZUKI, K. (1976) J. Neurothem. 27, 859-866. 19. IGARASHI, M., AND SUZUKI, K. (1977) J. Neurothem. 28, 729-738.
Microsomal Cholesterol Ester Hydrolase of Rat Brain: Lipids as Effector of Enzymatic Activity’ MYUNG-UN The
CHOI AND KUNIHIKO
Saul R. Korey Department of Neurology, Department of Neuroscience, for Research in Mental Retardation and Human Development, Albert Bronx,
New
York
SUZUKI and the Rose F. Kennedy Center College of Medicine,
Einstein
10461
Received February 26, 1979; revised May 15, 1979 Evidence is presented that lipid plays an important role in the function of the microsomal cholesterol ester hydrolase of rat brain. The catalytic activity was almost completely lost when most of cholesterol and up to 70% of phospholipids were removed from lyophilized microsomes by extraction with chloroform at -20°C. The activity was completely restored when the chloroform-extracted lipid was added back to the assay mixture in the amount equal to the original concentration. Cholesterol or individual phospholipid alone was not effective in reconstituting the lost enzymatic activity. Effective restoration of the activity required addition of cholesterol and a phospholipid. Among the phospholipids tested, phosphatidylserine was the most effective, followed by ethanolamine phospholipids and phosphatidylcholine. The apparent V was dependent on the amount of the lipid added, while the K, for the substrate, cholesteryl oleate, remained relatively constant, indicating that the effect of the added lipid was primarily on the reaction rate and not on the affinity of the enzyme to the substrate. The similar lipid dependence was observed with the Triton X-lOOsolubilized enzyme preparation. When the lipid phase of the microsomal membrane was perturbed, the enzyme became unstable when heated at 50°C and its activity showed a discontinuity in the Arrhenius plots. Therefore, not only the concentration of the added lipid but also the physical state of the lipid phase around the enzyme appeared to be important for the activity of the rat brain microsomal cholesterol ester hydrolase.
ately by sodium taurocholate (2). Exogenous lipids added to the assay system did not activate this microsomal cholesterol ester hydrolase as greatly as they did other two enzymes (4). However, during the course of our attempts at solubilization and purification of the rat brain microsomal cholesterol ester hydrolase, the enzyme exhibited the characteristic properties of an integral membrane protein (5). High concentrations of detergents were required for solubilization, and once solubilized, it appeared to associate with lipids during experimental manipulations, such as gel filtration. Therefore, by utilizing the commonly employed criteria to demonstrate lipid dependence of membranebound enzymes (6), we partially delipidated rat brain microsomes with organic solvents and examined the possible functional importance of lipids on the activity of the cholesterol ester hydrolase. The results,
At least three distinct cholesterol ester hydrolases (cholesterol esterase, EC 3.1.1.13) are present in rat brain (l-3). They are localized in the crude mitochondria, microsomes, and in the myelin sheath, respectively, and have different properties with respect to the pH optimum, requirements for detergents and/or exogenous lipids, and changes during brain development. One of the three, localized primarily in microsomes, has the pH optimum of 6.0, and is highly activated by Triton X-100 but only moder1 This investigation was supported by Research Grants NS-13578, NS-03356, and HD-01799 from the United States Public Health Service. A part of the content of this article was presented at the 10th annual meeting of the American Society for Neurochemistry, March 12- 16,1979, in Charleston, S. C. and was published in the form of an abstract (M. Choi and K. Suzuki, 1979, Trans. Amer. Sot. Neurochem. 10, 69). 0003-9861/79/120570$02.00/0 Copyright 8 1979 by Academic Press, Inc. All rights of reproduction in any form reserved.
570
MICROSOMAL
CHOLESTEROL
taken collectively, indicated strongly that lipids, particularly cholesterol and phosphatidylserine, act as effecters for the activity of the enzyme, when brain microsomes were delipidated by cold chloroform and acetone. EXPERIMENTAL
PROCEDURE
Materials [4-‘%]Cholesteryl oleate (54.0 mCi/mmol) and Econofluor were purchased from New England Nuclear Corporation (Boston, Mass.). The labeled cholesteryl oleate was diluted IOO-fold with unlabeled cholesteryl oleate (Sigma Chemical Co., St. Louis, MO.). Its radiopurity was examined by thin-layer chromatography, and if necessary, it was purified by silicic acid (Unisil, Clarkson Chemical Co., Williamsport, Pa.) column chromatography (1). Cholesterol and Triton X-100 were from Sigma Chemical Company. Phosphatidylserine, sphingomyelin, and galactosylceramide (all from bovine brain) were products of Koch-Light Laboratories, purchased through Research Products International (Elk Grove Village, Ill.). Synthetic distearoyl phosphatidylcholine was from Applied Science Laboratories (State College, Pa.) and precoated silica gel G thin-layer plates were from Analtech (Newark, Del.). Digitonin and all of the organic solvents used were supplied by Fisher Scientific Company (Fairlawn, N. J.). The Bio-Rad dye-binding protein assay kit was from Bio-Rad Laboratories (Richmond, Calif.). All commercial lipids were tested by thin-layer chromatography. When 100 pg of the material was spotted, all preparations were without detectable impurities, except for phosphatidylserine, which contained detectable impurity that cochromatographed with cholesterol in two solvent systems, chloroform-methanol-coned ammonia (‘70:30:5, by volume) and chloroform-methanol (9:1, v/v).
Methods Preparation of rat brain microsomes. Young adult Sprague-Dawley rats were used throughout. The animals were decapitated and the whole brains were removed and homogenized in 9 vol of ice-cold 0.32 M sucrose solution in a hand-operated Dounce homogenizer (Kontes Glass Co., Vineland, N. J.). The homogenate was centrifuged at 11,OOOg x 20 min. The pellet was resuspended in 0.32 M sucrose and centrifuged once more. The combined supernatants were then centrifuged at 100,OOOg x 60 min to obtain the microsomal fraction. The microsomal pellet was washed once with ice-cold distilled water by suspension and recentrifugation. The final pellet was suspended in water and lyophilized. Lyophilization did not significantly affect the enzymatic activity and the lyophilized microsomes could be
ESTER
HYDROLASE
571
stored at -20°C in a desiccator for several months without loss of the activity. Extraction with organic solvents. Lipid-depleted microsomes were prepared by extractions with organic solvents. Lyophilized microsomes, 100 mg, were placed in a glass centrifuge tube, and 10 ml of the extracting solvent, chilled to -20°C was added. This ratio of the microsomes to the solvent was maintained for all routine extractions. After 5 min with gentle shaking, the mixture was centrifuged at -20°C and the organic solvent phase was transferred. When chloroform was the extracting solvent, special care was required so as not to collect floating microsomes. The extraction was repeated once more with the same solvent. Finally, the residue was extracted with 10 ml of acetone at -20°C. All of the solvent phases including the final acetone extract were combined and used as the source of the extracted lipids for reactivation studies. The final acetone extraction was not used when microsomes were extracted with other organic solvents other than chloroform. The lipid-depleted microsomes were dried gently under a stream of nitrogen and kept at -20°C in a desiccator. For studies of the effect of the extraction conditions, variations were introduced to the above standard procedure, such as different numbers of extractions. Characterization of extracted microsomal lipid. The extracted lipid in the combined organic solvent phase was recovered by vacuum evaporation of the solvent. The resulting residue was dissolved in a mixture of chloroform-methanol (21, v/v). The insoluble material was eliminated by centrifugation and transfer of the soluble material to a tared tube. The amount of the extracted lipid was determined after drying under a stream of nitrogen. It was then redissolved in a known volume of chloroformmethanol (21, v/v) for further analysis. For comparison, the total microsomal lipid fraction was prepared by extractingintact microsomes with chloroform-methanol (21, v/v), essentially according to Folch et al. (7). The lipid remaining after chloroform extraction was also extracted similarly from the “lipid-depleted” microsomes with chloroform-methanol (2:1, v/v). The extracted lipids were fractionated into seven groups by silicic acid column chromatography according to the elution scheme of Norton and Autilo (8), which involved application of the lipid onto the column in chloroform and stepwise elution with increasing proportion of methanol in chloroform. Each fraction was semiquantitatively examined by thin-layer chromatography and was dried for gravimetric determination of the amount. Semiquantitative comparison of various lipid fractions was carried out by thin-layer chromatography in the solvent system of chloroform-methanolwater (65254, by volume), followed by sulfuric acid spray and charring at 135°C. Several lipid standards were chromatographed simultaneously to aid identification of the spots,
572
CHOI AND SUZUKI
Solubilization of the enzyme. Rat brain microsomal cholesterol ester hydrolase could be solubilized with reasonable recovery and stability by a medium consisting of 0.75% Triton X-100, 120 mM KCl, 40 mM Tris-HCl buffer, pH 7.6, and 10% glycerol. This formula was arrived at after extensive examinations of various solubilizing conditions (Choi and Suzuki, unpublished). The lipid-depleted microsomes, extracted twice with chloroform and once with acetone as described above (chloroform-extracted microsome), was suspended in this solubilizing medium at the ratio of 10 mg dry chloroform-extracted microsome to 1 ml medium in a glass homogenizer and was kept in ice for 30 min with occasional brief homogenization. After centrifugation at 100,OOOgfor 60 min, the clear supernatant was dialyzed overnight against 10 mM Tris-maleate buffer, pH 6.2, containing 0.01% Triton X-100, 1 mM EDTA, and 20% glycerol. The turbidity that developed during dialysis was eliminated by another centrifugation at 100,OOOg x 60 min. The final solubilized enzyme was stable for at least several days at 4°C. Enzyme assay. The assay procedure for the rat brain microsomal cholesterol ester hydrolase was essentially as described previously (2, 4). For assays of microsome-bound activity, 125 pg of [4-W]cholesteryl oleate (specific activity, 0.54 mCi/mmol), 1.25 mg of Triton X-100, and when included, appropriate amounts of lipid, were dried together in a 13 x loo-mm screw-capped test tube under a stream of nitrogen. The mixture was dispersed by the addition of 25 ~1 of ethanol and sonication of a water-bath type sonicator, followed by further addition of 0.5 ml of 0.1 M sodium phosphate buffer, pH 6.0 and sonication. When solubilized enzyme was assayed, the amount of Triton X-100 was reduced to 0.12 mg/tube. The reaction was initiated by addition of 0.1 ml of microsomal suspension in water (10 mg dry wt/ml) (0.25-0.4 mg protein), or 0.1 ml of the solubilized enzyme source (0.4-0.45 mg protein). The incubation was at 37°C for 60 min. All assays were carried out at least in duplicate, and blank tubes containing boiled enzyme source were included. The reaction was terminated by the addition of 5 ml of acetone -ethanol (1: 1, v/v) containing 100 pg of unlabeled cholesterol as carrier. The radioactivity of the cholesterol liberated by the enzymatic reaction was determined by the digitonin precipitation procedure (4). The results of duplicate assays always agreed within 10%. Analytical methods. Phospholipid was determined by its phosphorus content by the method of Chen et al. (9). The average molecular weight of phospholipids was assumed to be 775. The amount of cholesterol was determined by the method of Searcy and Bergquist (10). The Bio-Rad dye-binding assay system was used for most protein determination. This is a procedure developed commercially by Bio-Rad Laboratories based on the method of Bradford (11). Bovine serum albumin was used as the standard.
The values for membrane-bound protein obtained with the dye-binding assay gave approximately 55% of the values obtained by the more conventional Lowry method (12). Comparison of the results obtained by the two different protein assays could be made with satisfactory accuracy using the above conversion factor. The dye-binding assay was relatively insensitive to interference from Triton X-100 but equivalent amounts of Triton X-100 were added to the standard bovine serum albumin when the sample contained Triton X-100. RESULTS
Effect of Organic Solvent Extraction on the Microsowu~l Cholesterol Ester Hydrolase When the lyophilized microsomes were extracted with varieties of organic solvents, the activities of the microsomal cholesterol ester hydrolase decreased in varying degrees. The residual activities of organic solvent extracted microsomes correlated well with the amount of lipid left unextracted in these preparations (Fig. 1). When only a small fraction of the total lipid was extracted, such as single extraction with acetone or diethylether, the reduction of the cholesterol esterase activity was minor, while the microsomes from which 75% of the lipid was extracted with chloroform completely lost the hydrolytic activity. Extraction with chloroform-methanol (2:1, v/v) resulted essentially in complete loss of both lipid and the cholesterol esterase activity. Unlike in other extraction conditions, however, the chloroform-methanol extraction resulted in partial but irreversible inactivation of the enzyme, as described below. Restoration of the Enzyme Activities Extraction with Organic Solvents
After
With the exceptions of extractions with chloroform-methanol (2:1, v/v) and with iso-butanol, the cholesterol ester hydrolase activities of the organic solvent-extracted microsomes could be restored to the original level by adding back the extracted lipid fraction to the assay mixture (Fig. 2). When varying amounts of the lipid mixture obtained by cold chloroform extraction of dry microsomes were added to the assay system, the cholesterol ester hydrolase in the native intact microsomes was activated by 30% at low concentration of the added
MICROSOMAL
CHOLESTEROL
FIG. 1. Effect of partial delipidation of microsomes. The lyophilized rat brain microsomal fraction was extracted with organic solvents and then assayed for activity of the cholesterol ester hydrolase with the standard procedure in the absence of exogenous lipids. The amounts of cholesterol and phospholipids remaining in the microsomes were determined as described under Methods. The vertical and horizontal bars at “a” represent the means and the standard deviations of the lipid content and hydrolase activity of untreated microsomes (n = 5). The bars at “b” give the same data for the microsomes extracted twice with cold chloroform (n = 6) followed by a single extraction with acetone. 0 Single extraction with acetone; 0 single extraction with diethylether; n double extraction with acetone; 13 double extraction diethylether; 0 double extraction with iso-butanol; 0 double extraction with 2-propanol; A single extraction with chloroform followed by single extraction with acetone; A single extraction with chloroformmethanol (2:1, v/v). Except for the chloroformmethanol extraction, there was a good correlation between the amount of the residual lipid and the enzymatic activity.
lipid. The acetone- or chloroform-extracted microsomes required larger amounts of lipid for optimal activation. Despite the almost complete loss of the activity after the extraction, the cholesterol ester hydrolase activity in the chloroform-extracted microsomes could be restored completely (Fig. 2 and Table I). The amounts of the chloroform-extracted lipids for the optimal restoration of the hydrolase activity differed, depending on how the microsomes had been depleted of lipid. However, in each instance, the optimal amount of the lipid to be added to the assay system was approximately equal to the amount of lipid that had been removed by the solvent extraction. The cholesterol ester hydrolase activity in the chloroform-extracted microsome, restored
ESTER HYDROLASE
573
with the optimal amount of added lipid, showed general characteristics similar to those in the untreated microsomes with respect to the linearity against the incubation time and the enzyme source, the pH optimum, and the effect of Triton X-100 (2). The hydrolase activity in the chloroform-methanol-extracted microsomes could not be restored to more than 25% of the original activity, suggesting that substantial hydrolase activity was irreversibly inactivated by the chloroform-methanol extraction (Fig. 2). Nature of Chloroform-Extracted Microsomal Lipid Since the cold chloroform extraction of lyophilized microsomes appeared to provide
FIG. 2. Effect of added lipid mixture on the cholesterol ester hydrolase. Untreated and variously delipidated preparations of rat brain microsomes were assayed with or without additional lipid in the assay mixture. Each assay tube contained 1 mg of delipidated dry enzyme source which contained 0.25-0.4 mg proteimmg, depending on the extraction procedures. For all of the experiments in this figure, the lipid fraction obtained by the double extraction of lyophilized microsomes with chloroform followed by a single extraction with acetone was added, except for the experiment with chloroform-methanol microsomes which were assayed with the addition of chloroformmethanol-extracted total microsomal lipid. x Untreated microsomes; A acetone-extracted microsomes; 0 chloroform (single)-extracted microsomes; 0 chloroform (double) and acetone (single)-extracted microsomes; W chloroform-methanol-extracted microsomes. The amounts of lipid required for the optimal restoration of enzymatic activity roughly corresponded to those that have been extracted from the respective preparations. Although not shown, the isobutanol-extracted microsomes could not be restored in the hydrolase activity with the addition of lipids.
574
CHOI AND SUZUKI TABLE
I
RATBRAIN MICROSOMALLIPIDAND CHOLESTEROLESTERHYDROLASE BEFORE ANDAFTER DOUBLE EXTRACTIONWITH~ILOROFORM FOLLOWEDBYA~INGLE EXTRACTION WITHACETONE~ Lipid content (mg/mg protein) Enzyme source Untreated CHCl,-extracted
Enzymatic activity (nmolihimg protein)
Cholesterol
Phospholipid
Without lipid
With additional lipid
0.424 f 0.034 (6) 0.036 + 0.008 (4)
0.915 + 0.075 (6) 0.268 2 0.030 (6)
20.3 + 2.0 (5) 1.01 2 0.64 (6)
28.6 + 3.7 (7) 31.2 k 6.3 (9)
a Values are expressed as averages *SD with the number of separate experiments in parentheses. The lipid contents of the untreated and chloroform-extracted microsomes were determined as described in the text. The cholesterol ester hydrolase activity was determined either with or without the optimal amounts of the chloroform-extracted microsomal lipid mixture. The values of protein determined by the dye-binding assay were used for the calculation.
an excellent system for further study-total loss of the enzymatic activity after extraction and the complete restoration of the activity by the addition of the extracted lipid-the nature of the chloroform-extracted and remaining lipids was examined. The double extraction with cold chloroform removed lipids constituting 33 & 2% of the dry weight of microsomes. The extracted lipid fraction had the following composition; ethanolamine phospholipid (33%), cholesterol (25%), phosphatidylserine (20%), phosphatidylcholine (14%), and others (each less than 6%, including galactosylceramide and sphingomyelin). Semiquantitative thin-layer chromatographic comparison of lipid fractions from intact microsomes, the chloroform extract, and the chloroform-extracted microsomes indicated that chloroform extracted almost all cholesterol, most of ethanolamine phospholipids, perhaps 80% of phosphatidylcholine, and about a half of the phosphatidylserine (Fig. 3). Quantitative determinations indicated that more than 90% of cholesterol and approximately 70% of total phospholipid were extracted by the standard chloroform extraction procedure (Table 1). Restoration of the Activity Lipids
with Individual
Capacities of pure individual lipids and their combinations to restore the cholesterol ester hydrolase activity in the chloroformextracted microsomes were examined (Fig. 4). Phospholipids were generally more
effective than cholestrol, except for sphingomyelin. Galactosylceramide was poor in restoring the enzymatic activity. However, none of the individual pure lipids alone were as effective as the chloroformextracted lipid mixture (Fig. 4A). Since the chloroform-extracted lipid contained approximately 25% cholesterol and 65% phospholipids, mixtures of cholesterol and individual phospholipids were tested. When the amount of cholesterol was kept constant at 100 pg/tube and that of phospholipid was varied, phosphatidylserine, phosphatidylethanolamine, and phosphatidylcholine were all capable of restoring the hydrolase activity to the level of the system to which the optimal amount of the chloroformextracted lipid mixture was added (Fig. 4B). Again, sphingomyelin showed essentially no effect. The amount of individual glycerophospholipids necessary to achieve the optimal reactivation in combination with 100 pg cholesterol varied, with phosphatidylserine being the most effective followed by phosphatidylethanolamine and then by phosphatidylcholine. It was also clear that the reactivating effects of cholesterol and the glycerophospholipids were not additive. Effects of Exogenous Lipids on Solubilixed Microsomal Cholesterol Ester Hydrolase
The microsomal cholesterol ester hydrolase was solubilized from the chloroform-extracted microsomes as described under Methods.
MICROSOMAL
CHOLESTEROL
PE
ESTER HYDROLASE
575
individual lipids required for the optimal restoration of the activity were generally 20-25% of those required for the chloroformextracted microsome-bound enzyme. Finally, cholesterol, which, when added alone, could reactivate the hydrolase in the chloroform-extracted microsomes only to 20% of the optimal activity, activated the solubilized enzyme up to 80% of the optimal
PC
SM
PS
I
II
III
FIG. 3. Thin-layer chromatography of rat brain microsomal lipid. The total lipid fractions of untreated microsomes, chloroform-extracted microsomes, and the combined chloroform extracts were run on a silica gel G thin-layer plate, 250 pm thick, in the solvent system of chloroform-methanol-water (65:25:4, by volume), and were visualized by sulfuric acid spray and heating. I: Residual lipid of chloroform-extracted microsome; II: chloroform-extracted lipid; III: lipid fraction of untreated microsomes. Chol: cholesterol; PE: ethanolamine phospholipid; PC: phosphatidylcholine; SM: sphingomyelin; PS: phosphatidylserine. The spot above PS is an artifact (sucrose). Lipid fractions from comparable amounts of microsomes are chromatographed. Therefore, the sum of I and II should be approximately III.
The solubilized preparations were then tested for the hydrolase activity with or without additional exogenous lipids (Fig. 5). Although the results were generally similar to those obtained with the chloroformextracted microsomes as the enzyme source, there were several points of quantitative differences that appeared significant. The solubilized preparations exhibited considerably higher activities of the cholesterol ester hydrolase than the starting chloroform-extracted microsomes in the absence of additional lipids. The amounts of
FIG. 4. Effect of individual lipids on the cholesterol ester hydrolase activity of chloroform-extracted rat brain microsomes. Lyophilized microsomes were extracted twice with cold chloroform followed by a single extraction with acetone and the cholesterol esterase activity was determined with the addition of varying amounts of different lipids. Each tube contained 1 mg of the enzyme source (0.4 mg protein). For both A and B, the broken lines represent the results obtained with addition of the chloroformextracted lipid mixture. Other results in A were obtained when a single lipid was added: 0 phosphatidylserine; 0 ethanolamine phospholipid; 0 cholesterol. In B, 106 pg of cholesterol was also added in addition to the respective test lipids: 0 phosphatidylserine; 0 ethanolamine phospholipid; m phosphatidylcholine; q sphingomyelin.
576
CHOI AND SUZUKI
activity. The most likely explanation for these quantitative differences is that the residual lipid, relatively rich in phosphatidylserine, is not available to the enzyme which is bound to the chloroform-extracted microsomes but becomes available during the enzyme solubilization procedure in the presence of the detergent. Effects of Lipids on Other Enz ynw Properties
Heat stability of the cholesterol ester hydrolase was tested by heating water suspensions of untreated microsomes, chloroform-extracted microsomes with or without adding back the chloroform extract, and the solubilized prepartion (Fig. 6). The enzyme in the untreated microsomes was relatively heat stable, tolerating 50°C heating for 15 min without inactivation. It ,was inactivated only by 20% after 60 min at 50°C. The solubilized preparation was very heat labile and 50% inactivation occurred after 5 min at 45°C. When heated without added lipid, the enzyme in the chloroformextracted microsomes showed intermediate heat stability. When the chloroform-extracted microsomes were heated in the presence of
FIG. 5. Effect of lipids on solubilized rat brain microsomal cholesterol ester hydrolase. The enzyme was solubilized from the chloroform-extracted microsomes as described in the text. The activity was then assayed in the assay mixture optimized for the solubilized enzyme with added lipids as indicated. A Chloroform-extracted microsomal lipid mixture; 0 phosphatidylserine; 0 cholesterol; 0 phosphatidylcholine. Each assay tube contained 0.42 mg of solubilized protein.
FIG. 6. Heat stability of rat brain microsomal cholesterol ester hydrolase. Water suspensions of untreated microsomes, chloroform-extracted microsomes with or without the chloroform-extracted lipid, and the solubilized preparation were heated at 50°C for the indicated periods of time, except for the solubilized enzyme which was heated at 45°C. The solubilized enzyme was too heat labile for reliable results when heated at 50°C. At the end of heating, the enzyme was assayed with the standard procedure with the optimal amount of lipids. 0 Untreated microsomes; 0 chloroform-extracted microsomes heated in the absence of the additional lipid; A chloroform-extracted microsomes heated in the presence of the optimal amount of the chloroform-extracted lipid; 0 solubilized enzyme preparation.
the extracted lipid, it appeared to show slight protective effects against heat inactivation but the heat stability of the untreated microsomes could not be restored. Some membrane-bound enzymes show characteristic discontinuities in the Arrhenius plots, which are usually interpreted as reflecting phase transition of the lipid phase around the enzyme proteins (13). The cholesterol ester hydrolase in the untreated and chloroform-extracted microsomes was assayed at different temperatures (Fig. 7). The Arrhenius plot of the untreated microsomes appeared to be linear throughout the temperature range studied. On the other hand, the chloroform-extracted microsomes assayed with the addition of the optimal amount of the extracted lipid gave a sharp discontinuity in the Arrhenius plot at 16.9”C. The discontinuity point shifted to 21.5”C when the chloroform-extracted microsomes were assayed in the presence of a mixture of phospholipids free of cholesterol.
MICROSOMAL
CHOLESTEROL
FIG. 7. The Arrhenius plots of rat brain microsomal cholesterol ester hydrolase. 0 Untreated microsomes; 0 chloroform-extracted microsomes assayed in the presence of the optimal amount of the chloroformextracted lipid fraction; 0 chloroform-extracted microsomes assayed in the presence of the chloroformextracted lipid from which cholesterol had been removed by the silicic acid column chromatography.
These findings suggested perturbation of the lipid phase of the microsomes during the extraction-reactivation procedure. The effects of the substrate concentration were tested with the untreated microsomes and the chloroform-extracted microsomes (Fig. 8). The latter was assayed with three different concentrations of the chloroformextracted lipids. Untreated microsomes were assayed without exogenous lipids. Since the Lineweaver-Burk plots were not linear, the kinetic parameters, V and K,, were estimated directly from the graph of V vs [S]. While the V varied as expected, the K, remained quite constant, indicating chat the lipid altered the reaction rate without affecting the affinity of the enzyme to the substrate. DISCUSSION
The present series of studies was initiated when we observed during our attempt to solubilize the rat brain microsomal cholesterol ester hydrolase that delipidation of lyophilized microsomes preliminary to solubilization of
ESTER HYDROLASE
577
the enzyme resulted in apparent loss of the enzymatic activity. It was possible to correlate the apparent loss of the enzymatic activity of the partially delipidated microsomes with the amount of lipid removed, and the activity could be restored by the addition of the extracted lipids. The optimal restoration of the activity could be achieved when the original lipid content of the microsomes was restored. These findings are consistent with the criteria of lipid dependency of an enzyme (6). For the reconstitution of the catalytic activity, we employed only the direct addition of the extracted lipid to the assay mixture without preassociation. Under these conditions, the restored activity showed some properties that were different from those of the native enzyme in the untreated microsomes, although the general kinetic properties were similar to those of the untreated enzyme. The maximum reaction rate was slightly but consistently greater in the restored enzyme; the heat stability of the untreated enzyme could not be restored; and there were distinct alterations in the Arrhenius plots. When microsomes were partially delipidated with dry, cold chloroform, almost all cholesterol and substantial portions of ethanolamine and choline phospholipids were extracted, leaving a mixture of lipids relatively enriched with phosphatidylserine. Such preparations were essentially inactive in catalyzing hydrolysis of cholesteryl oleate unless appropriate types and amounts of lipids were added to the assay mixture. Restoring cholesterol alone showed little effect. Similarly, any of the glycerophospholipids alone could not restore the original activity. When combined with cholesterol, each of the glycerophospholipids could restore the original activity, and with respect to the amount required for the optimal restoration, phosphatidylserine was the most effective. These observations indicated that it is not merely the restoration of the original total microsomal lipid composition but a combination of specific lipids-cholesterol and phosphatidylserinethat is required for restoration of the enzymatic activity. The finding on the solubilized enzyme appeared to be consistent with this. When the enzyme was solubilized
578
CHOI AND SUZUKI
l/[Sl FIG. 8. Effect of the substrate concentration on the rat brain microsomal cholesterol ester hydrolase. The hydrolytic activities were determined with the standard assay procedure except that the amount of the substrate and the amount of exogenous lipids were varied as indicated. (A) Activity vs substrate concentration, (B) Lineweaver-Burk plots. 0 Untreated microsomes assayed without exogenous lipid; 0 chloroform-extracted microsomes assayed with the optimal amount of the chloroformextracted lipid; x chloroform-extracted microsomes assayed with twice the optimal amount of the chloroform-extracted lipid; 0 chloroform-extracted microsomes assayed with one-fourth of the optimal amount of chloroform-extracted lipid. The Lineweaver-Burk plots were not linear. The K, estimated from A was relatively constant at 7-8 X 10m5M (+30 pg/O.6 ml).
from the chloroform-extracted microsomes, substantial reactivation could be obtained by the addition of cholesterol alone, although cholesterol alone had little effect on the chloroform-extracted microsomes. One plausible interpretation is that, during the enzyme solubilization procedure which required a high concentration of Triton X-100, the remaining phosphatidylserine became available for lipid-enzyme interactions, thus requiring only the addition of cholesterol for activity. This finding appears analogous to Na+, K+-ATP’ase, which is a phospholipid-requiring enzyme (14). However, when the enzyme source was partially delipidated by organic solvents under controlled conditions, the addition of cholesterol alone could restore the ATP’ase activity, presumably because the required phospholipids were still present in the enzyme source (15). Several pieces of observations are suggestive of the added cholesterol primarily interacting with the phospholipid rather than with the enzyme. Since cholesterol is an end product of the enzymatic reaction, it is unlikely to interact with the enzyme and activate it. Perhaps more importantly,
the point of discontinuity in the Arrhenius plots was shifted depending on the presence or absence of exogenous cholesterol in the assay mixture. Since the discontinuity is likely to reflect the phase transition of the lipid phase associated with the enzyme (13), this suggests interactions of cholesterol with phospholipids, thereby altering the physical characteristics of the lipid phase. Cholesterol is known to affect the physical properties of phospholipid monolayers (16) or vesicles (17). The mechanism with which the lipid exerts its effect on the activity of the rat brain microsomal cholesterol ester hydrolase is difficult to assess precisely, because of the residual lipid in the delipidated microsomes and of the presence of Triton X-100 in the assay system. We have not been able to prepare completely lipid-free active enzyme source or to eliminate the unnatural detergent from the reaction mixture. There is a possibility, for example, that the different lipid effects may be due to differential competition of different lipids with Triton X-100. Although this possibility cannot be excluded completely, we think it is unlikely because our preliminary results
MICROSOMAL
CHOLESTEROL
indicate that the ratio of detergent to the enzyme source is more critical than the ratio of detergent to lipid for the activity of the solubilized enzyme (Choi and Suzuki, unpublished). Similarly, the inhibition observed at high concentrations of lipids cannot be explained rigorously at the present time except for speculative interpretations, such as substrate or detergent dilution by the excess lipids. However, even under these conditions, our findings strongly suggested the importance of the interactions between the normally membrane-bound cholesterol ester hydrolase and its lipid environment within the membrane for the enzymatic activity. The appearance of the discontinuity in the Arrhenius plots when the delipidated microsomes were used as the enzyme source indicated that although the catalytic activity could be restored, the restored lipid environment of the enzyme was not identical with that in the untreated microsomes which showed no discontinuous point. This interpretation is also consistent with the finding that the heat stability of the enzyme in the untreated microsomes could not be restored by the simple addition of lipid to the chloroform-extracted microsomes or to the solubilized enzyme. When varying amounts of lipid mixture were added to the delipidated microsomes, the maximum reaction velocity varied but the K, for the substrate remained constant. Therefore, the lipid appeared to act as a heterotropic effector primarily affecting the reaction turnover rate, rather than altering the affinity of the enzyme to the substrate. The series of studies presented in this report was necessitated because of the practical need of our ongoing attempts at solubilization and purification of the rat brain microsomal cholesterol ester hydrolase. These pieces of information would be essential for monitoring the recovery and the specific activity of the enzyme in various purification steps. These data, however, provided more fundamental information than of merely pragmatic value. The activity of the rat brain microsomal cholesterol ester hydrolase is dependent on the lipid environment and there are considerable degrees of specificity with respect
ESTER
HYDROLASE
579
to different lipids in their capacity as the effector of the enzyme. Our previous studies showed that the myelin-specific cholesterol ester hydrolase was also dependent on exogenously added lipid, particularly on phosphatidylserine, for its activity (18, 19). The acidic cholesterol ester hydrolase is specifically activated by bovine brain ethanolamine phospholipid (Ogino and Suzuki, unpublished). Therefore, activities of all of the three known cholesterol ester hydrolases in rat brain are affected by specific lipids added to the assay mixtures. REFERENCES 1. ETO, Y., AND SUZUKI, K. (1971) Biochim. Biophys. Acta 239, 293-311. 2. ETO, Y., AND SUZUKI, K. (1973) J. Biol. Chem. 248, 1986-1991. 3. ETO, Y., AND SUZUKI, K. (1973) J. Neurochem. 20, 1475- 1477. 4. IGARASHI, M., AND SUZUKI, K. (1974) Esp. Neurol. 45, 549-553. 5. SINGER, S. J. (1974) Annu. Rev. Biochem. 43, 805-833. 6. FLEISCHER, S., BRIERLEY, G., KLOUWEN, H., AND SLAUTTERBACK, D. B. (1962) J. Biol. Chem. 237, 3264-3272. 7. FOLCH-PI, J., LEES, M. B., AND SLOANESTANLEY, G. H. (1957) d. Biol. Chem. 226, 497-509. 8. NORTON, W. T., AND AUTILIO, L. A. (1965) Ann. N. Y. Acad. Sci. 122, 77-85. 9. CHEN, P. S., TORIBARA, T. Y., AND WARNER, H. (1956) Anal. Chem. 28, 1756-1758. 10. SEARCY, R. L., AND BERGQUIST, L. M. (1960) Clin. Chim. Acta 5, 192-199. 11. BRADFORD, M. M. (1976) Anal. Biochem. 72, 248-254. 12. LOWRY, 0. H., ROSEBROUGH, N. J., FARR, A. L., AND RANDALL, R. J. (1951) J. Biol. Chem. 193, 265-275. 13. WALKER, J. A., AND WHEELER, K. P. (1975) Biochem. J. 151, 439-442. 14. DAHL, J. L., AND HOKIN, L. E. (1974) Annu. Rev. Biochem. 43, 327-356. 15. NOGUCHI, T., AND FREED, S. (1971) Nature New Biol. 230, 148-150. 16. DEMEL, R. A., VAN DEENEN, L. L. M., AND PETHICA, B. A. (1967) Bioehim. Biophys. Acta 135, 11-19. 17. HUNT, G. R., AND TIPPING, L. R. H. (1978) Biochim. Biophys. Actu 507, 242-261. 18. IGARASHI, M., AND SUZUKI, K. (1976) J. Neurothem. 27, 859-866. 19. IGARASHI, M., AND SUZUKI, K. (1977) J. Neurothem. 28, 729-738.
