- Email: [email protected]
[75] Monoacylglycerol lipase from rat adipose tissue
646
HYDROLASES
[75]
incubation for 30 min at 37° with 0.25 mM emulsified tri[aH]oleoylglycerol as substrate in a final volume of 200 ~l (as described above). Acknowledgments This work has been supported by grants from Phhlssons Foundation, Segerfalks Foundation, Novo Insulin Foundation, the Swedish Diabetes Foundation, the Medical Faculty of the University of Lund, and the Swedish Medical Research Council (Grant No. 3362).
[75] M o n o a c y l g l y c e r o l L i p a s e f r o m R a t A d i p o s e T i s s u e EC 3.1.1.23 Glycerol-monoester acylhydrolase By HANS TORNQVIST and PER BELFRAGE
Monoacylglycerol [1(3)- or 2-isomer] + 1-120 --->glycerol + fatty acid Rat adipose tissue contains very active long-chain monoacylglycerol hydrolase activity. Most of this is due to monoacylglycerol lipase ~ an enzyme first described by Vaughan et al. 2 The enzyme has been extensively purified and partially characterized) The physiological role of the enzyme is not clear; its activity is not influenced by the nutritional state of the animal or by hormones. ~It has been suggested that monoacylglycerol lipase catalyzes the hydrolysis of monoacylglycerols derived both from the hormone-sensitive lipase-catalyzed intraceUular lipolysis and from the lipoprotein lipase-catalyzed hydrolysis of chylomicron and very lowdensity lipoprotein lipids.~ Thus, the enzyme may account for the observation that monoacylglycerols do not accumulate in adipose tissue.
Assay Method
Principle. Free [3H]glycerol produced by hydrolysis of l(3)-monooleoyl[3H]glycerol in mixed micelles with nonionic detergent is isolated with a simple liquid-liquidpartition system, and the radioactivity is determined by liquid scintillationcounting. 4 Hormone-sensitive lipase1"5and I H. 2 M. H. 4 H. 5 G.
Tornqvist, P. Nilsson-Ehle, and P. Belfrage. Biochim. Biophys. Acta 530, 474 (1978). Vaughan, J. E. Berger, and D. Steinberg. J. Biol. Chem. 239, 401 (1964). Tornqvist and P. Belfrage. J. Biol. Chem. 251, 813 (1976). Tornqvist, L. Krabisch, and P. Belfi'age. J. Lipid Res. 15, 291 (1974). Fredrikson, P. Strfilfors, N. 0 . Nilsson, and P. Belfrage, this volume [74].
METHODS IN ENZYMOLOGY.VOL. 71
Copyright© 1981by AcademicPress,Inc. All rightsof reproductionin any formreserved. ISBN 0-12-181971-X
[75]
RAT ADIPOSE TISSUE MONOACYLGLYCEROLLIPASE
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lipoprotein lipase I are also active against long-chain monoacylglycerols, but contribute relatively little to this enzyme activity in crude adipose tissue extracts. The activity of these enzymes is strongly inhibited by the concentration of nonionic detergent used in the assay, rendering it highly specific for monoacylglycerol lipase. We prefer to use the assay described here because of its high sensitivity compared to assays with unlabeled substrate and because the reaction products can be rapidly determined in a simple way. Monooleoylglycerol is preferred as substrate over other monoacylglycerols for several technical reasons.4 Synthesis of Substrate. Monooleoyl[3H]glycerol is synthesized as follows4: Sodium mono[3H]glycerate is obtained by allowing [l(3)-3H]glycerol (10 mCi, 1 mmol) to react with NaOH at 150°. The glycerol is dissolved in 1 ml of methanol, 1 ml of 1 M NaOH is added, and the solvents are evaporated with dry nitrogen at 150°; the reaction is allowed to continue for another 30 min at 150°. The sodium mono[3H]glycerate is dried overnight in a desiccator. Ethyl acetate (20 ml), which has been dried with molecular sieve type 4A to remove water and trace amounts of alcohols, is thoroughly mixed with the compound in a closed vial, and the glycerate is subsequently acylated at room temperature for 2.5 hr with 400 /xl of oleoyl chloride. The reaction is interrupted by addition of water, and the lipids are extracted with diethyl ether, which is evaporated after drying over Na2SO4. Unlabeled monooleoylglycerol (0.7 g) is added and the monooleoyl[aH]glycerol is purified by silicic acid column chromatography. The compound is applied to the column (30 mg of lipid per gram of silicic acid) in hexane. Tri- and dioleoylglycerol and oleic acid are eluted with up to 25% diethyl ether in hexane and the monooleoylglycerol is subsequently eluted with 50% diethyl ether in hexane. Fractions containing more than 99.5% pure monooleoylglycerol are pooled, the solvent is evaporated, and the lipid is dissolved in dry redistilled benzene at 4° to minimize hydrolysis during storage. Purity is regularly checked by thinlayer chromatography on silicic acid. The monooleoyl[3H]glycerol so obtained should have a specific activity of about 2/~Ci//.Lmol. It is sufficient for at least 5000 enzyme determinations.
Reagents Monooleoyl[aH]glycerol, 5 mg/ml in benzene, stored at 4° Monooleoylglycerol, 20 mg/ml in heptane, - 2 0 ° Tris-HC1, 0.2 M, pH 8.0, with C13E12nonionic detergent, e 0.8% (w/v), 4o
6CtaE12is a polydispersepreparation of alkylpolyoxyethyleneswiththe indicatedaverage c o m p o s i t i o n ; C = a l k y l c a r b o n s , E = o x y e t h y l e n e units. Detergentsof this type are available from AB Berolkemi,Stenungsund,Sweden.Othercommerciallyavailabledetergents
648
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M e t h a n o l - c h l o r o f o r m - h e p t a n e , 1.41 : 1.25:1 (v/v/v) NaC1, 2%, w/v InstagelT-toluene 1 : 1 (v/v)
Preparation of Monooleoyl[aH]glycerol Substrate. Monooleoyl[all]glycerol (approximately 4 × 106 cpm) is mixed with monooleoylglycerol to a combined amount of 14.2 mg (40/zmol) in a small glass vial, and the solvents are evaporated with dry N~. The substrate is then dispersed by sonication on ice in 4.0 ml o f the Tris-HCl buffer with detergent, until a water-clear solution is obtained. Conditions for sonication are not critical. If not used the same day, the substrate solution is frozen at - 2 0 ° and resonicated before use. Procedure. E n z y m e and buffer, in a total volume of 100/zl, is incubated with 100/.d of the above substrate solution in a disposable test tube (10 × 140 mm) for 5-60 min at 21 ° or 37°. The amount of e n z y m e and incubation time are chosen such that total hydrolysis does not exceed 10%. The reaction is terminated by adding 3.25 ml o f the m e t h a n o l - c h l o r o f o r m heptane solution and 1.05 ml of the sodium chloride solution. The tubes are shaken vigorously in a vortex mixer, and two phases are separated by centrifugation. Without delay, a 0.5 ml sample o f the upper m e t h a n o l water phase (2.5 ml) containing all the [all]glycerol produced is transferred to a counting vial with 5 ml o f Instagel-toluene, and the radioactivity is determined. Units. One unit o f enzyme activity corresponds to the release of 1 ~mol of glycerol per minute at 21 ° unless otherwise indicated. Purification Procedure a An overall purification of the enzyme o f about 2500-fold is obtained by the purification procedure described below, s The table shows a s u m m a r y o f a purification. Unless otherwise indicated all steps were carried out at of similar composition, e.g., those of the Lubrol or Brij series, or alkylphenoxy derivatives, e.g. Triton X-100, can also be used in the assay below. Optimal concentration may be different. Detergents containing aromatic groups, however, have strong UV absorbance and are thus not suitable for use during protein fractionation. 7 Scintillator solution is commercially available from Packard Instruments Inc. s Monoacylglycerollipase can also be prepared starting from a fraction obtained during the purification of hormone-sensitive lipase from rat adipose tissue,5 using a slightly different procedure. During gradient sievorptive chromatography on QAE-Sephadex of the latter enzyme, monoacylglycerollipase is eluted with the unretained proteins. After dialysis and concentration by pressure ultrafiltration, the pooled enzyme is subjected to isoelectric focusing at pH 6-8 (step 6), but in the LKB 440 ml electrofocusingcolumn. The enzyme is then, after concentration by pressure ultrafiltration, taken through the Sephadex G-150gel filtration step (step 5) and finally again electrofocused at pH 8-6 (step 7).
[75]
RAT ADIPOSE TISSUE MONOACYLGLYCEROL LIPASE
649
PURIFICATION OF MONOACYLGLYCEROL LIPASE FROM RAT ADIPOSE TISSUEa
Steps 1. 110,000 g supernatant 2. pH 5.2 precipitate 3. Solubilized pH 5.2 precipitate 4. TEAE chromatography 5. Ultrafiltration, PM-30 gel filtration (Sephadex G-150) 6. First isoelectric focusing, pH 6-8 7. Second isoelectric focusing, pH 8-6 Gel filtration of peak fractions (Sephadex G-50)
Volume (ml) 822 85 87 338 72
Total protein (mg) 720 220 220 47 4.7
6.5
0.11
6.8
--
4.0
0.04
Specific activity (units/mg protein) 0.14 0.43 0.42 1.5 11
Yield (%)
Purification (fold)
100 93 91
l 3.1 3.0
70 52
11 79
270
29
1900
--
16
--
350
13
2500
a Epididymal fat pads, 120 g, from 100 rats were used as enzyme source. Activity units are micromoles per minute at 21°. Data are from Tornqvist and Belfrage.a
4° . Reagents, buffers, and water of highest available purity were used because the enzyme contains functionally important free SH groups. All solutions contained 1 mM EDTA and 1 mM dithiothreitol and, from step 3 on, the nonionic detergent ClaE126(0.2% w/v), unless otherwise stated. All pH values were measured at 21°. Steps I and 2. Crude Fractionation of Adipose Tissue Homogenate. Epididymal fat pads from 10 Sprague-Dawley rats (200-250 g) were homogenized in l0 ml of 0.25 M sucrose, pH 7.4, per gram of tissue, with a glass homogenizer with a rotating Teflon pestle for at least l0 rain and further with a ground-glass grinder for another l0 min. The homogenate was then centrifuged in a swing-out rotor at 110,000 g for 45 rain. The floating fat cake was removed by cutting the tubes, and the clear supernatant solution containing more than 80% of total enzymatic activity was decanted. (A more concentrated homogenate, e.g., 1 g of adipose tissue per two volumes of sucrose solution, can be prepared, but a slightly lower yield of enzyme in the 110,000 g supernatant is obtained.) The combined supernatants were precipitated at pH 5.2 by addition of 0.2 M acetic acid. After 30 min, the precipitate formed was collected by centrifugation and suspended in 20 mM Tris-HCl, pH 7.4, 1.0 ml/g adipose tissue. Suspensions of pH 5.2 precipitate from 100 rats were prepared and stored at - 7 0 ° until further use.
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Step 3. Detergent Solubilization. ClaE12 nonionic detergent was added to the pH 5.2 precipitate suspension to a concentration of 0.2% (w/v); the mixture was sonicated on ice with a Branson Model B 12 sonifier at setting 3 for 2 min (in 30-sec intervals to avoid excessive heating) until the suspension became visually clear. Step 4. TEAE Column Chromatography. TEAE-cellulose was prepared for chromatography according to the manufacturer's instructions. It was then washed with distilled water several times, and fines were removed by sedimentation. The TEAE-cellulose was packed in a column (4 × 16 cm) and equilibrated by running 10 volumes of 20 mM Tris-HC1, pH 7.4, through the column. The sample, approximately 90 ml of step 3 material was adjusted to pH 7.4 and applied to the column. The enzyme was eluted (180 ml/hr) with about 800 ml of starting buffer, and the main part of the contaminating proteins were retained on the column. Fractions containing more than 0.1 unit of enzyme activity per milliliter were pooled. Step 5. Sephadex G-150 Gel Filtration. Dry Sephadex G-150 superfine gel beads were fractionated to get more uniform size allowing better separation at higher elution rates. 9 A column (5.0 x 70 cm) was packed and equilibrated in 20 mM Tris-HC1, pH 7.0. The pooled enzyme peak fractions from the TEAE-cellulose column were concentrated by pressure ultrafiltration through a PM-30 Diaflo membrane (cutoff limit: MW -~ 30,000) to 20 ml and applied to the gel filtration column. This was eluted at 10-15 ml/hr, and 10-ml fractions were collected. The enzyme was obtained at an elution volume of approximately 550 ml. Fractions containing more than 0.2 unit of enzyme activity per milliliter were pooled. Step 6. First Isoelectric Focusing. An LKB 110 ml electrofocusing column was filled as follows: (a) anode solution (lower end): sucrose, 57% (w/v), phosphoric acid, 1% (v/v); (b) cathode solution: ethanolamine, 1% (v/v); (c) 100 ml of a linear sucrose gradient made up from 50 ml of sucrose, 46% (w/v), containing Ampholine, 1° pH 6-8, 1.6% (w/v), and 50 ml Ampholine solution, 0.4% (w/v). The pooled enzyme from step 5 was included in the sucrose gradient, if necessary after concentration by pressure ultrafiltration, as described above. The isoelectric focusing was performed at a constant effect of 5 W with a maximum of 1400 V or 10 mA for 80 hr. The column was emptied from the bottom with a peristaltic pump (50 ml/hr) and 1-ml fractions were collected. Fractions containing approximately 80% of total enzyme activity were combined. Step 7. Second lsoelectric Focusing. A second isoelectric focusing colR. Ekman, B. G. Johansson, and U. Ravnskov, Anal. Biochem. 70, 628 (1976). 10Ampholine ampholytes, LKB, Stockholm, Sweden.
[75]
RAT ADIPOSE TISSUE MONOACYLGLYCEROLLIPASE
651
umn was prepared exactly as described above, except that electrodes were reversed [anode solution: phosphoric acid, 1% (v/v); cathode solution: ethanolamine, 1% (v/v) and sucrose, 57% (v/v)]. Fractions containing approximately 80% of total enzyme activity were pooled. Most of the contaminating proteins had slightly higher pI values than monoacylglycerol lipase; care should be taken not to include these with the pooled enzyme. The purified enzyme was then run through a Sephadex G-50 Superfine column (1.6 × 40 cm) in 20 mM phosphate buffer, pH 7.0, to remove sucrose and Ampholine, and the pooled enzyme was stored at - 7 0 °. Properties 3
Purity. The enzyme prepared by the above procedure accounts for more than 85% of the stainable protein after SDS-polyacrylamide gel electrophoresis. Molecular Properties. Owing to the small amount of enzyme protein obtained, no extensive molecular characterization has been performed. The SDS-polyacrylamide gel electrophoresis indicated a minimum molecular weight of 32,900. The purified enzyme has a pI of 7.2 (4°) and a Stokes' radius of 39/~. It should be observed that the latter value presumably is a measure of an enzyme protein-detergent complex. Stability. The enzyme is stable for several months at - 7 0 ° in 20 mM Tris-HC1 or phosphate buffer, pH 7.0, containing 0.2% ClaE12, 1 mM dithiothreitol, and l mM EDTA. Under these conditions half-lives were approximately 9 hr at 37°, 36 hr at +21 °, and 9 days at +4 °. Removal of the detergent, H or dithiothreitol, drastically reduces enzyme stability. The enzyme rapidly loses activity if heated above +44 °. Inhibitors. The enzyme is strongly inhibited by SH reagents such as mercuric chloride and p-chloromercuribenzoate. Diisopropylfluorophosphate (10/zM) totally inhibits its activity, indicating a functional serine residue. Substrate Specificity. The enzyme catalyzes the hydrolysis of 1(3)- and 2-monooleoylglycerol at equal rates (apparent Km at 21 ° = 0.2 mM). It has no activity against long-chain di- and triacylglycerols, cholesterol ester, or lysophosphatidylcholine. The enzyme catalyzes the hydrolysis of a variety of medium- and long-chain monoacylglycerols.4 Using aqueous dispersions of monodecanoyl- and monododecanoylglycerol it has been found that the enzyme requires an organized substrate structure, a minimal substrate interphase, to be brought into proper alignment for optimal H p. Strhlfors, H. Tornqvist, B. Jergil, and P. Belfrage,Biochim. Biophys. Acta 533, 90 (1978).
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catalytic action. 12,13Because of this characteristic "activation" by lipidwater interphases, monoacylglycerol lipase should be classified as a " t r u e " lipase, like phospholipase A2 and pancreatic lipase.14 Acknowledgments This w o r k was su pported by grants from A. P~hissons Foundation, Maim6; P. H~.kanssons
Foundation, Esl6v; SegerfalksFoundation, Helsingborg;The Medical Faculty, University of Lund, Swedenand the Swedish MedicalResearch Council(project No. 3362). lz H. Tornqvist and P. Belfrage,unpublishedobservation. 13H. Tornqvist. Thesis, Universityof Lund, Sweden, 1975. 14R. Vergerand G. H. de Haas, Annu. Rev. Biophys. Bioeng. 5, 77 (1976).
[76]
Cutinases from Fungi and Pollen
By P. E. KOLATTUKUDY,R. E. PURDY, and I. B. MAITI Cutinase is an enzyme that catalyzes hydrolysis of an insoluble biopolyester, cutin, the structural component of plant cuticle. This polyester is composed of the following fatty acids: o-hydroxyfatty acids, dihydroxypalmitic acid, saturated and A12-monounsaturated 18-hydroxy9,10-epoxy Cls acids, and saturated and AlZ-monounsaturated 9,10,18trihydroxy Cls acids. 1 The actual composition of cutin is dependent on the species; but, in general, fast growing plants appear to contain predominantly the C1o acids, particularly dihydroxypalmitate, whereas in slower growing plants a mixture of C~0 and C~s acids is found (Fig. 1). Cutinase hydrolytically releases all types of monomers from the polymer. Preparation of the Polymer Mature Golden delicious apple fruits are cut into quarters, and as much internal tissue as possible is removed from the slices. The peel is boiled for a few hours 'in water to remove as much internal tissue as possible and collected by filtration through cheesecloth. To remove the remaining internal tissue from the cuticular layer the peel is boiled for 4 hr in an aqueous solution containing 4 g of oxalic acid and 16 g of ammonium oxalate per liter. The cuticular layers collected by filtration through cheesecloth are thoroughly washed with water and mixed with an excess of a 2 : 1 mixture of chloroform and methanol (20 ml/g wet weight). The solid material collected by filtration is reextracted twice with the i p. E. K o l a t t u k u d y , Recent Adv. Phytochem. 11, 185 (1972).
METHODS IN ENZYMOLOGY, VOL. 71
Copyright © 1981by AcademicPress, Inc. All rights of reproductionin any form reserved. ISBN 0-12-181971-X
HYDROLASES
[75]
incubation for 30 min at 37° with 0.25 mM emulsified tri[aH]oleoylglycerol as substrate in a final volume of 200 ~l (as described above). Acknowledgments This work has been supported by grants from Phhlssons Foundation, Segerfalks Foundation, Novo Insulin Foundation, the Swedish Diabetes Foundation, the Medical Faculty of the University of Lund, and the Swedish Medical Research Council (Grant No. 3362).
[75] M o n o a c y l g l y c e r o l L i p a s e f r o m R a t A d i p o s e T i s s u e EC 3.1.1.23 Glycerol-monoester acylhydrolase By HANS TORNQVIST and PER BELFRAGE
Monoacylglycerol [1(3)- or 2-isomer] + 1-120 --->glycerol + fatty acid Rat adipose tissue contains very active long-chain monoacylglycerol hydrolase activity. Most of this is due to monoacylglycerol lipase ~ an enzyme first described by Vaughan et al. 2 The enzyme has been extensively purified and partially characterized) The physiological role of the enzyme is not clear; its activity is not influenced by the nutritional state of the animal or by hormones. ~It has been suggested that monoacylglycerol lipase catalyzes the hydrolysis of monoacylglycerols derived both from the hormone-sensitive lipase-catalyzed intraceUular lipolysis and from the lipoprotein lipase-catalyzed hydrolysis of chylomicron and very lowdensity lipoprotein lipids.~ Thus, the enzyme may account for the observation that monoacylglycerols do not accumulate in adipose tissue.
Assay Method
Principle. Free [3H]glycerol produced by hydrolysis of l(3)-monooleoyl[3H]glycerol in mixed micelles with nonionic detergent is isolated with a simple liquid-liquidpartition system, and the radioactivity is determined by liquid scintillationcounting. 4 Hormone-sensitive lipase1"5and I H. 2 M. H. 4 H. 5 G.
Tornqvist, P. Nilsson-Ehle, and P. Belfrage. Biochim. Biophys. Acta 530, 474 (1978). Vaughan, J. E. Berger, and D. Steinberg. J. Biol. Chem. 239, 401 (1964). Tornqvist and P. Belfrage. J. Biol. Chem. 251, 813 (1976). Tornqvist, L. Krabisch, and P. Belfi'age. J. Lipid Res. 15, 291 (1974). Fredrikson, P. Strfilfors, N. 0 . Nilsson, and P. Belfrage, this volume [74].
METHODS IN ENZYMOLOGY.VOL. 71
Copyright© 1981by AcademicPress,Inc. All rightsof reproductionin any formreserved. ISBN 0-12-181971-X
[75]
RAT ADIPOSE TISSUE MONOACYLGLYCEROLLIPASE
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lipoprotein lipase I are also active against long-chain monoacylglycerols, but contribute relatively little to this enzyme activity in crude adipose tissue extracts. The activity of these enzymes is strongly inhibited by the concentration of nonionic detergent used in the assay, rendering it highly specific for monoacylglycerol lipase. We prefer to use the assay described here because of its high sensitivity compared to assays with unlabeled substrate and because the reaction products can be rapidly determined in a simple way. Monooleoylglycerol is preferred as substrate over other monoacylglycerols for several technical reasons.4 Synthesis of Substrate. Monooleoyl[3H]glycerol is synthesized as follows4: Sodium mono[3H]glycerate is obtained by allowing [l(3)-3H]glycerol (10 mCi, 1 mmol) to react with NaOH at 150°. The glycerol is dissolved in 1 ml of methanol, 1 ml of 1 M NaOH is added, and the solvents are evaporated with dry nitrogen at 150°; the reaction is allowed to continue for another 30 min at 150°. The sodium mono[3H]glycerate is dried overnight in a desiccator. Ethyl acetate (20 ml), which has been dried with molecular sieve type 4A to remove water and trace amounts of alcohols, is thoroughly mixed with the compound in a closed vial, and the glycerate is subsequently acylated at room temperature for 2.5 hr with 400 /xl of oleoyl chloride. The reaction is interrupted by addition of water, and the lipids are extracted with diethyl ether, which is evaporated after drying over Na2SO4. Unlabeled monooleoylglycerol (0.7 g) is added and the monooleoyl[aH]glycerol is purified by silicic acid column chromatography. The compound is applied to the column (30 mg of lipid per gram of silicic acid) in hexane. Tri- and dioleoylglycerol and oleic acid are eluted with up to 25% diethyl ether in hexane and the monooleoylglycerol is subsequently eluted with 50% diethyl ether in hexane. Fractions containing more than 99.5% pure monooleoylglycerol are pooled, the solvent is evaporated, and the lipid is dissolved in dry redistilled benzene at 4° to minimize hydrolysis during storage. Purity is regularly checked by thinlayer chromatography on silicic acid. The monooleoyl[3H]glycerol so obtained should have a specific activity of about 2/~Ci//.Lmol. It is sufficient for at least 5000 enzyme determinations.
Reagents Monooleoyl[aH]glycerol, 5 mg/ml in benzene, stored at 4° Monooleoylglycerol, 20 mg/ml in heptane, - 2 0 ° Tris-HC1, 0.2 M, pH 8.0, with C13E12nonionic detergent, e 0.8% (w/v), 4o
6CtaE12is a polydispersepreparation of alkylpolyoxyethyleneswiththe indicatedaverage c o m p o s i t i o n ; C = a l k y l c a r b o n s , E = o x y e t h y l e n e units. Detergentsof this type are available from AB Berolkemi,Stenungsund,Sweden.Othercommerciallyavailabledetergents
648
HYDROLASES
[75]
M e t h a n o l - c h l o r o f o r m - h e p t a n e , 1.41 : 1.25:1 (v/v/v) NaC1, 2%, w/v InstagelT-toluene 1 : 1 (v/v)
Preparation of Monooleoyl[aH]glycerol Substrate. Monooleoyl[all]glycerol (approximately 4 × 106 cpm) is mixed with monooleoylglycerol to a combined amount of 14.2 mg (40/zmol) in a small glass vial, and the solvents are evaporated with dry N~. The substrate is then dispersed by sonication on ice in 4.0 ml o f the Tris-HCl buffer with detergent, until a water-clear solution is obtained. Conditions for sonication are not critical. If not used the same day, the substrate solution is frozen at - 2 0 ° and resonicated before use. Procedure. E n z y m e and buffer, in a total volume of 100/zl, is incubated with 100/.d of the above substrate solution in a disposable test tube (10 × 140 mm) for 5-60 min at 21 ° or 37°. The amount of e n z y m e and incubation time are chosen such that total hydrolysis does not exceed 10%. The reaction is terminated by adding 3.25 ml o f the m e t h a n o l - c h l o r o f o r m heptane solution and 1.05 ml of the sodium chloride solution. The tubes are shaken vigorously in a vortex mixer, and two phases are separated by centrifugation. Without delay, a 0.5 ml sample o f the upper m e t h a n o l water phase (2.5 ml) containing all the [all]glycerol produced is transferred to a counting vial with 5 ml o f Instagel-toluene, and the radioactivity is determined. Units. One unit o f enzyme activity corresponds to the release of 1 ~mol of glycerol per minute at 21 ° unless otherwise indicated. Purification Procedure a An overall purification of the enzyme o f about 2500-fold is obtained by the purification procedure described below, s The table shows a s u m m a r y o f a purification. Unless otherwise indicated all steps were carried out at of similar composition, e.g., those of the Lubrol or Brij series, or alkylphenoxy derivatives, e.g. Triton X-100, can also be used in the assay below. Optimal concentration may be different. Detergents containing aromatic groups, however, have strong UV absorbance and are thus not suitable for use during protein fractionation. 7 Scintillator solution is commercially available from Packard Instruments Inc. s Monoacylglycerollipase can also be prepared starting from a fraction obtained during the purification of hormone-sensitive lipase from rat adipose tissue,5 using a slightly different procedure. During gradient sievorptive chromatography on QAE-Sephadex of the latter enzyme, monoacylglycerollipase is eluted with the unretained proteins. After dialysis and concentration by pressure ultrafiltration, the pooled enzyme is subjected to isoelectric focusing at pH 6-8 (step 6), but in the LKB 440 ml electrofocusingcolumn. The enzyme is then, after concentration by pressure ultrafiltration, taken through the Sephadex G-150gel filtration step (step 5) and finally again electrofocused at pH 8-6 (step 7).
[75]
RAT ADIPOSE TISSUE MONOACYLGLYCEROL LIPASE
649
PURIFICATION OF MONOACYLGLYCEROL LIPASE FROM RAT ADIPOSE TISSUEa
Steps 1. 110,000 g supernatant 2. pH 5.2 precipitate 3. Solubilized pH 5.2 precipitate 4. TEAE chromatography 5. Ultrafiltration, PM-30 gel filtration (Sephadex G-150) 6. First isoelectric focusing, pH 6-8 7. Second isoelectric focusing, pH 8-6 Gel filtration of peak fractions (Sephadex G-50)
Volume (ml) 822 85 87 338 72
Total protein (mg) 720 220 220 47 4.7
6.5
0.11
6.8
--
4.0
0.04
Specific activity (units/mg protein) 0.14 0.43 0.42 1.5 11
Yield (%)
Purification (fold)
100 93 91
l 3.1 3.0
70 52
11 79
270
29
1900
--
16
--
350
13
2500
a Epididymal fat pads, 120 g, from 100 rats were used as enzyme source. Activity units are micromoles per minute at 21°. Data are from Tornqvist and Belfrage.a
4° . Reagents, buffers, and water of highest available purity were used because the enzyme contains functionally important free SH groups. All solutions contained 1 mM EDTA and 1 mM dithiothreitol and, from step 3 on, the nonionic detergent ClaE126(0.2% w/v), unless otherwise stated. All pH values were measured at 21°. Steps I and 2. Crude Fractionation of Adipose Tissue Homogenate. Epididymal fat pads from 10 Sprague-Dawley rats (200-250 g) were homogenized in l0 ml of 0.25 M sucrose, pH 7.4, per gram of tissue, with a glass homogenizer with a rotating Teflon pestle for at least l0 rain and further with a ground-glass grinder for another l0 min. The homogenate was then centrifuged in a swing-out rotor at 110,000 g for 45 rain. The floating fat cake was removed by cutting the tubes, and the clear supernatant solution containing more than 80% of total enzymatic activity was decanted. (A more concentrated homogenate, e.g., 1 g of adipose tissue per two volumes of sucrose solution, can be prepared, but a slightly lower yield of enzyme in the 110,000 g supernatant is obtained.) The combined supernatants were precipitated at pH 5.2 by addition of 0.2 M acetic acid. After 30 min, the precipitate formed was collected by centrifugation and suspended in 20 mM Tris-HCl, pH 7.4, 1.0 ml/g adipose tissue. Suspensions of pH 5.2 precipitate from 100 rats were prepared and stored at - 7 0 ° until further use.
650
HYDROLASES
[75]
Step 3. Detergent Solubilization. ClaE12 nonionic detergent was added to the pH 5.2 precipitate suspension to a concentration of 0.2% (w/v); the mixture was sonicated on ice with a Branson Model B 12 sonifier at setting 3 for 2 min (in 30-sec intervals to avoid excessive heating) until the suspension became visually clear. Step 4. TEAE Column Chromatography. TEAE-cellulose was prepared for chromatography according to the manufacturer's instructions. It was then washed with distilled water several times, and fines were removed by sedimentation. The TEAE-cellulose was packed in a column (4 × 16 cm) and equilibrated by running 10 volumes of 20 mM Tris-HC1, pH 7.4, through the column. The sample, approximately 90 ml of step 3 material was adjusted to pH 7.4 and applied to the column. The enzyme was eluted (180 ml/hr) with about 800 ml of starting buffer, and the main part of the contaminating proteins were retained on the column. Fractions containing more than 0.1 unit of enzyme activity per milliliter were pooled. Step 5. Sephadex G-150 Gel Filtration. Dry Sephadex G-150 superfine gel beads were fractionated to get more uniform size allowing better separation at higher elution rates. 9 A column (5.0 x 70 cm) was packed and equilibrated in 20 mM Tris-HC1, pH 7.0. The pooled enzyme peak fractions from the TEAE-cellulose column were concentrated by pressure ultrafiltration through a PM-30 Diaflo membrane (cutoff limit: MW -~ 30,000) to 20 ml and applied to the gel filtration column. This was eluted at 10-15 ml/hr, and 10-ml fractions were collected. The enzyme was obtained at an elution volume of approximately 550 ml. Fractions containing more than 0.2 unit of enzyme activity per milliliter were pooled. Step 6. First Isoelectric Focusing. An LKB 110 ml electrofocusing column was filled as follows: (a) anode solution (lower end): sucrose, 57% (w/v), phosphoric acid, 1% (v/v); (b) cathode solution: ethanolamine, 1% (v/v); (c) 100 ml of a linear sucrose gradient made up from 50 ml of sucrose, 46% (w/v), containing Ampholine, 1° pH 6-8, 1.6% (w/v), and 50 ml Ampholine solution, 0.4% (w/v). The pooled enzyme from step 5 was included in the sucrose gradient, if necessary after concentration by pressure ultrafiltration, as described above. The isoelectric focusing was performed at a constant effect of 5 W with a maximum of 1400 V or 10 mA for 80 hr. The column was emptied from the bottom with a peristaltic pump (50 ml/hr) and 1-ml fractions were collected. Fractions containing approximately 80% of total enzyme activity were combined. Step 7. Second lsoelectric Focusing. A second isoelectric focusing colR. Ekman, B. G. Johansson, and U. Ravnskov, Anal. Biochem. 70, 628 (1976). 10Ampholine ampholytes, LKB, Stockholm, Sweden.
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RAT ADIPOSE TISSUE MONOACYLGLYCEROLLIPASE
651
umn was prepared exactly as described above, except that electrodes were reversed [anode solution: phosphoric acid, 1% (v/v); cathode solution: ethanolamine, 1% (v/v) and sucrose, 57% (v/v)]. Fractions containing approximately 80% of total enzyme activity were pooled. Most of the contaminating proteins had slightly higher pI values than monoacylglycerol lipase; care should be taken not to include these with the pooled enzyme. The purified enzyme was then run through a Sephadex G-50 Superfine column (1.6 × 40 cm) in 20 mM phosphate buffer, pH 7.0, to remove sucrose and Ampholine, and the pooled enzyme was stored at - 7 0 °. Properties 3
Purity. The enzyme prepared by the above procedure accounts for more than 85% of the stainable protein after SDS-polyacrylamide gel electrophoresis. Molecular Properties. Owing to the small amount of enzyme protein obtained, no extensive molecular characterization has been performed. The SDS-polyacrylamide gel electrophoresis indicated a minimum molecular weight of 32,900. The purified enzyme has a pI of 7.2 (4°) and a Stokes' radius of 39/~. It should be observed that the latter value presumably is a measure of an enzyme protein-detergent complex. Stability. The enzyme is stable for several months at - 7 0 ° in 20 mM Tris-HC1 or phosphate buffer, pH 7.0, containing 0.2% ClaE12, 1 mM dithiothreitol, and l mM EDTA. Under these conditions half-lives were approximately 9 hr at 37°, 36 hr at +21 °, and 9 days at +4 °. Removal of the detergent, H or dithiothreitol, drastically reduces enzyme stability. The enzyme rapidly loses activity if heated above +44 °. Inhibitors. The enzyme is strongly inhibited by SH reagents such as mercuric chloride and p-chloromercuribenzoate. Diisopropylfluorophosphate (10/zM) totally inhibits its activity, indicating a functional serine residue. Substrate Specificity. The enzyme catalyzes the hydrolysis of 1(3)- and 2-monooleoylglycerol at equal rates (apparent Km at 21 ° = 0.2 mM). It has no activity against long-chain di- and triacylglycerols, cholesterol ester, or lysophosphatidylcholine. The enzyme catalyzes the hydrolysis of a variety of medium- and long-chain monoacylglycerols.4 Using aqueous dispersions of monodecanoyl- and monododecanoylglycerol it has been found that the enzyme requires an organized substrate structure, a minimal substrate interphase, to be brought into proper alignment for optimal H p. Strhlfors, H. Tornqvist, B. Jergil, and P. Belfrage,Biochim. Biophys. Acta 533, 90 (1978).
652
HYDROLASES
[76]
catalytic action. 12,13Because of this characteristic "activation" by lipidwater interphases, monoacylglycerol lipase should be classified as a " t r u e " lipase, like phospholipase A2 and pancreatic lipase.14 Acknowledgments This w o r k was su pported by grants from A. P~hissons Foundation, Maim6; P. H~.kanssons
Foundation, Esl6v; SegerfalksFoundation, Helsingborg;The Medical Faculty, University of Lund, Swedenand the Swedish MedicalResearch Council(project No. 3362). lz H. Tornqvist and P. Belfrage,unpublishedobservation. 13H. Tornqvist. Thesis, Universityof Lund, Sweden, 1975. 14R. Vergerand G. H. de Haas, Annu. Rev. Biophys. Bioeng. 5, 77 (1976).
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Cutinases from Fungi and Pollen
By P. E. KOLATTUKUDY,R. E. PURDY, and I. B. MAITI Cutinase is an enzyme that catalyzes hydrolysis of an insoluble biopolyester, cutin, the structural component of plant cuticle. This polyester is composed of the following fatty acids: o-hydroxyfatty acids, dihydroxypalmitic acid, saturated and A12-monounsaturated 18-hydroxy9,10-epoxy Cls acids, and saturated and AlZ-monounsaturated 9,10,18trihydroxy Cls acids. 1 The actual composition of cutin is dependent on the species; but, in general, fast growing plants appear to contain predominantly the C1o acids, particularly dihydroxypalmitate, whereas in slower growing plants a mixture of C~0 and C~s acids is found (Fig. 1). Cutinase hydrolytically releases all types of monomers from the polymer. Preparation of the Polymer Mature Golden delicious apple fruits are cut into quarters, and as much internal tissue as possible is removed from the slices. The peel is boiled for a few hours 'in water to remove as much internal tissue as possible and collected by filtration through cheesecloth. To remove the remaining internal tissue from the cuticular layer the peel is boiled for 4 hr in an aqueous solution containing 4 g of oxalic acid and 16 g of ammonium oxalate per liter. The cuticular layers collected by filtration through cheesecloth are thoroughly washed with water and mixed with an excess of a 2 : 1 mixture of chloroform and methanol (20 ml/g wet weight). The solid material collected by filtration is reextracted twice with the i p. E. K o l a t t u k u d y , Recent Adv. Phytochem. 11, 185 (1972).
METHODS IN ENZYMOLOGY, VOL. 71
Copyright © 1981by AcademicPress, Inc. All rights of reproductionin any form reserved. ISBN 0-12-181971-X