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ACYLTRANSFERASES
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Concluding Remarks Current evidence suggests that the activities of 1-alkylglycerophosphocholine and 1-alkenylglycerophosphocholine acyltransferases are located only in selected tissues. Since small amounts of 1-alkyl-2-acylGPC and 1-alkenyl-2-acyl-GPC are always found in major mammalian organs, and these phospholipids undergo acyl chain remodeling, the enzymes for such remodeling should also be present. It is likely that the acyltransferases are present in these tissues, but their low activities have eluded detection.
[10] D i h y d r o x y a c e t o n e P h o s p h a t e A c y l t r a n s f e r a s e
By KEITH O. WEBBER and AMIYA K. HA~RA Introduction Dihydroxyacetone phosphate acyltransferase (DHAPAT; glycerone3-phosphate acyltransferase, EC 2.3.1.42) catalyzes the transfer of the fatty acid moiety from long-chain acyl-coenzyme A (acyl-CoA) to the free hydroxyl group of dihydroxyacetone phosphate: Dihydroxyacetone phosphate + acyl-CoA--~ acyldihydroxyacetone phosphate + CoASH
This reaction initiates the synthesis of the ether-linked glycerolipids as well as the more common glycerol ester lipids. 1,2 DHAPAT is an integral membrane-bound protein located on the luminal side of animal cell peroxisomes. 3-5 This acyltransferase has been solubilized and partially purified from guinea pig liver. 6'7 The present method is based on that described for the preparation of highly purified enzyme from guinea pig liver peroxi-
somes.7
1 A. 2 R. 3 A. 4 A. 5 C. 6 C. 7 K.
K. Hajra, Biochem. Soc. Trans. 5, 34 (1977). Manning and D. N. Brindley, Biochem. J. 130, 1003 (1972). K. Hajra, C. L. Burke, and C. L. Jones, J. Biol. Chem. 244, 8289 (1989). K. Hajra and J. E. Bishop, Ann. N.Y. Acad. Sci. 386, 170 (1982). L. Jones and A. K. Hajra, J. Biol. Chem. 255, 8289 (1980). L. Jones and A. K. Hajra, Arch. Biochem. Biophys. 226, 155 (1983). O. Webber, Ph.D. Dissertation, University of Michigan, Ann Arbor (1988).
METHODSIN ENZYMOLOGY,VOL. 209
Copyright© 1992by AcademicPress, Inc. All rightsof reproductionin any formreserved.
[10]
DIHYDROXYACETONE PHOSPHATE ACYLTRANFERASE
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Assay Method
Principle. Enzyme activity is measured as the amount of lipophilic (at low pH) radioactivity formed from [32p]DHAP in the presence of palmitoylCoA and e n z y m e ) Reagents 5 mM Dihydroxyacetone [32p]phosphate: [32p]DHAP is prepared by enzymatic phosphorylation of dihydroxyacetone with [y-32p]ATP. 8,9The radioactive DHAP is diluted with nonradioactive DHAP (Li + salt) to the desired specific activity [5000-10,000 counts/min (cpm)/nmol] 1 mM Palmitoyl-CoA, lithium salt 0.3 M Morpholinoethanesulfonic acid (MES), pH 5.7 0.3 M Tris-HCl buffer, pH 7.5 0.1 M Sodium fluoride 0.1 M Magnesium chloride 20 mg/ml Bovine serum albumin (BSA), fatty acid poor, fraction V Asolectin suspension (liposomes) in 10 mM Tris-HC1, I mM EDTA, pH 7.5: Suspend 2 g asolectin (Associated Concentrates, Woodside, NY) in 100 ml of 10 mM Tris-HCl (pH 7.5)- 1 mM EDTA by sonicating with an ultrasonic probe and then centrifuge the suspension at 35,000 g for 30 min. Use the supernatant (15-20/xmol lipid phosphate/ml) for the assay. When stored at 4° under N2, this suspension is stable for 1 month. 2 M potassium chloride in 0.2 M phosphoric acid Chloroform Chloroform-methanol (1 : 2, v/v) Chloroform-methanol-0.5 N aqueous H3PO 4 (1 : 12 : 12, v/v) Procedure. Add the following reagents to 16 × 125 mm screw-topped tubes: buffer 1° (0.3 M Tris-HC1 (or 0.3 M MES) 0.15 ml, BSA 0.05 ml, palmitoyl-CoA 0.05 ml, NaF 0.1 ml, MgC12 0.05 ml, asolectin suspension 0.1 ml, 1~ enzyme sample, and water to make the final volume 0.6 ml. Incubate the mixture at 37° for 15 min in a reciprocating shaker water bath. Stop the reaction by adding 2.25 ml chloroform-methanol (1 : 2, v-v) and mix (vortex). Add 0.75 ml methanol and 0.75 ml KCI-H3PO4 (2-0.2 M), vortex, and centrifuge for 5 rain at 1000 g. Aspirate off the upper layers s A. K. Hajra, J. Biol. Chem. 2,43, 3458 (1968). 9 A. K. Hajra and C. L. Burke, J. Neurochem. 31, 125 (1978). 10 U s e M E S buffer for the m e m b r a n e - b o u n d e n z y m e and Tris buffer for the solubilized enzyme. 11 Addition of asolectin is not n e c e s s a r y for the a s s a y of m e m b r a n e - b o u n d e n z y m e .
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and add 2.5 ml of chloroform-methanol-0.5 N aqueous H3PO 4 (1 : 12: 12, v/v), mix, and centrifuge. Aspirate off the upper layers and wash the lower layers again with chloroform-methanol-aqueous H3PO4 as described above. Transfer aliquots of the washed lower layers (generally 1 ml, i.e., two-thirds of the total) to counting vials, evaporate off the solvents with a stream of air or N2, add scintillation solvent mixture, and determine the radioactivity in a liquid scintillation counter. Units. One unit (U) of enzyme activity is defined as 1 nmol of product formed per minute at 37°. The specific activity is expressed as nanomoles per minute per milligram protein. Purification of Enzyme The purification of DHAPAT can be divided into three processes: (1) isolation ofperoxisomes, (2) solubilization of the peroxisomal membranes, and (3) chromatographic purification of DHAPAT. Purification o f Peroxisomes from Guinea Pig Liver Subcellular Fractionation. Livers are homogenized according to the protocol of deDuve et al. 12in a buffer containing 0.25 M sucrose, 10 mM N-tris(hydroxymethyl)methyl-2-aminoethanesulfonicacid (TES; pH 7.5), 1 mM EDTA, 0.1% ethanol (v/v), 0.2 mM phenylmethylsulfonyl fluoride (PMSF), and 1/xM leupeptin. Fractionate the liver by differential centrifugation to produce the "light mitochondrial" fraction (L-fraction, sedimenting between 33,000 and 250,000 g.min), 12which is enriched in peroxisomes. The method used in our laboratory for subcellular fractionation of guinea pig liver is essentially that described by deDuve et al. 12with minor modifications. 3 Suspend the L-fraction in the homogenization buffer to a volume of 0.25 ml/g of original liver weight. Density Gradient Centrifugation. The peroxisomes are isolated from the L-fraction by centrifugation through 30% (w/v) Nycodenz as described in Ref. 13 with some modifications. 7
1. Into 25-ml polycarbonate centrifuge bottles (Beckman, Fullerton, CA, #340382), place 15 ml of a solution containing 30% Nycodenz (w/v), 10 mM TES, pH 7.5, and 1 mM EDTA. 2. Overlay the 15 ml of Nycodenz solution with 2 ml of L-fraction. 3. Centrifuge at 75,000 g for 45 min in a Ti-55 fixed-angle rotor (BeckJ2 C. deDuve, B. C. Pressman, R. Gianetto, R. Wattiaux, and F. Appelmans, Biochem. J. 60, 604 (1955). 13 M. K. Ghosh and A. K. Hajra, Anal. Biochem. 159, 169 (1986).
[10]
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95
man). Peroxisomes will sediment through the Nycodenz solution while mitochondria, microsomes, and lysosomes will not. Carefully aspirate away the entire supernatant. 5. Resuspend the remaining pellet in homogenization buffer to a volume equivalent to 20% of the original liver weight. Store at - 20°. .
Solubilization of Dihydroxyacetone Phosphate Membrane Preparation. Initial purification of DHAPAT entails separation of the peroxisomal membrane from the soluble, matrix proteins. The peroxisomes are osmotically ruptured by 10-fold dilution with 10 mM sodium pyrophosphate, pH 9.0, containing 1 tzM leupeptin, 1 /zM pepstatin, 0.2 mM PMSF, and 1 mM EDTA. Stir the mixture for 1 hr on ice. Centrifuge at 100,000 g for 30 min to separate membranes (pellet) from the soluble matrix proteins (supernatant). Resuspend the pellet in 25 mM TES (pH 7) to a protein concentration of approximately 10 mg/ml. This membrane suspension may be stored at - 2 0 °. Solubilization. Mix equal volumes of peroxisomal membrane suspension and a solution containing 300 mM NaC1, 30 mM 3-[(3-cholamidopropyl)dimethylammonio]-l-propanesulfonate (CHAPS), 20 mM MES (pH 6.5), 2 mM dithiothreitol (DTT), 2/.LM leupeptin, 2/xM pepstatin, 2 mM EDTA, and 0.4 mM PMSF. Stir this mixture gently for 15 min on ice. Centrifuge the mixture at I00,000 g for 60 min. The supernatant (a clear brown liquid) contains the solubilized DHAPAT with a specific activity of approximately 80-90 U/rag protein. Column Chromatography The solubilized DHAPAT is purified to near homogeneity by a multistep regimen of both low-pressure and high-pressure column chromatography. Low-Pressure Size-Exclusion Chromatography. Pass the solubilized peroxisomal membrane proteins over a column of Sephacryl S-200 (1.6 x 95 cm; Pharmacia, Piscataway, NJ) at 4°. The mobile phase is 150 mM NaCl, 5 mg/ml CHAPS, 10 mM MES (pH 6.5), 1 mM DTT, 0.02% NaN3 flowing at 6.0 ml/hr. Collect 3-ml fractions. Pool the fractions (fractions 12-15) comprising the peak DHAPAT activity. Cation-Exchange Chromatography. Cation-exchange chromatography is performed on a 0.5 × 5 cm Mono S column (Pharmacia) attached to a suitable high-performance liquid chromatography (HPLC) system allowing binary gradient elution and monitoring of effluent absorbance at 280 nm. Equilibrate the column with 25 mM MES, pH 6.5, 5 mg/ml CHAPS, 1 mM DTT (buffer A) and then load the sample onto the column
96
ACYLTRANSFERASES
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TABLE 1 PURIFICATION OF DIHYDROXYACETONE PHOSPHATE ACYLTRANSFERASE
Fraction
Protein (rag)
Total activity (U)
Specific activity (U/mg)
Enrichment (-fold)
Yield (%)
Liver homogenate Peroxisomes Solubilized membranes Sephacryl S-200 Mono S Hydroxylapatite TSK-3000
15,150 276 94 31 1.0 0. | 8 0.08
7900 2820 6020 3640 1400 450 260
0.52 10.2 64 118 1400 2400 3350
1 19.6 123 227 2700 4600 6440
100 36 76 46 18 6 3
at 0.5 ml/min. Wash the unbound proteins off the column with buffer A until the 280 nm absorbance of the effluent returns to baseline. While collecting 1.0-ml fractions, elute the adherent proteins from the column with a linear salt gradient from 0 to 400 mM NaCI in buffer A at a flow rate of 0.5 ml/min (total gradient volume 40 ml). Pool the fractions (fractions 11-15) comprising the peak DHAPAT activity. Hydroxylapatite Chromatography. Equilibrate a high-pressure hydroxylapatite column (0.5 x 4.8 cm; Bio-Rad Laboratories, Richmond, CA) with 10 mM potassium phosphate, pH 6.8, 0.3 mM CaCI2, 5 mg/ml CHAPS, 50 mM NaCI, I mM DTT, 0.05% NAN3. Load the pooled DHAPAT-containing fractions from the cation-exchange column at 0.5 ml/min. Wash the unbound proteins from the column with equilibration buffer until the absorbance of the effluent at 280 nm returns to baseline. Elute the bound proteins with a 25-ml linear phosphate gradient starting with equilibration buffer and ending with 0.3 M potassium phosphate, pH 6.8, 10/zM CaCI2, 5 mg/ml CHAPS, I mM DTT, 0.05% NAN3. Collect 1-ml fractions. High-Pressure Size-Exclusion Chromatography. Pool the DHAPATcontaining fractions (fractions 12-14) eluted from the hydroxylapatite column and concentrate the combination to approximately 1.0 ml in a centrifugal microconcentrator (Bio-Rad). Inject the concentrated enzyme solution onto the HPLC gel-filtration column (TSK-3000; Toyo-Soda, Japan) and elute with 150 mM NaC1, 5 mg/ml CHAPS, 10 mM Bis-Tris-propane, pH 7.5, 1 mM DTT, 0.02% NaN 3 at 0.5 ml/min. Collect 1.0-ml fractions while monitoring the absorbance of the effluent at 280 nm. The enzyme generally elutes in fractions 4 to 7. Combine the fractions, concentrate the enzyme by ultrafiltration (centrifugal microconcentrator), and store at 4 °. The enzyme could be purified further by chromatofocusing but at such
[10]
DIHYDROXYACETONE PHOSPHATE ACYLTRANFERASE
97
a low yield that the specific activity could not be accurately determined. 7 A typical purification procedure is summarized in Table I. Properties
Physical Characteristics. DHAPAT activity copurifies with a protein which has an apparent Mr of 69,000 as determined by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate (SDS-PAGE). Gel filtration of trace amounts of solubilized DHAPAT gives a M r determination of approximately 90,000, indicating that the active enzyme is probably not multimeric. Thermostability. Membrane-bound peroxisomal DHAP acyltransferase is remarkably heat stable. In fact, heating membranes to 50° prior to assay increases the measurable enzyme activity. The solubilized enzyme does not possess this thermostability and loses significant activity at temperatures as low as 40° . Kinetics. The guinea pig liver enzyme is most active at pH 5.5 in the membrane-bound state; however, on solubilization with either ionic or nonionic detergents, the pH optimum shifts to pH 7.4 and becomes somewhat less stringent. At pH 7.4, the membrane-bound enzyme gives biphasic kinetics with Km (DHAP) values of 40 and 200 ~M which increase to 100 and 500 ~M on solubilization. When the solubilized enzyme is assayed in the presence of asolectin (soybean lipids) the kinetics are monophasic with a Km (DHAP) of 100/zM. After extensive purification (as above), the Km (DHAP) was determined to be about 60/zM; however, at subsaturating levels of palmitoyl-CoA the value drops to 35 ~M. Activators and Inhibitors. The crude membrane-bound enzyme activity is stimulated by Mg 2+ and F - ; however, after solubilization these ions have no effects on the enzyme activity. The solubilized enzyme is stimulated by phospholipid vesicles, such as asolectin or phosphatidylcholine. 6 Palmitoyl-CoA inhibits the enzyme activity, and the presence of BSA in the reaction mixture prevents this inhibition. Both the membrane-bound and solubilized enzymes are relatively unaffected by thiol-modifying agents such as N-ethylmaleimide and iodoacetamide; however, bulkier, charged sulfhydryl-reactive compounds such as p-chloromercuriphenylsulfonic acid or 5,5'-dithiobis(2-nitrobenzoic acid) produce moderate levels of inhibition. Diagnostic Use of Enzyme A number of genetic diseases involving peroxisomal disorders are prenatally or postnatally diagnosed by the decreased activity of DHAPAT
98
ACYLTRANSFERASES
[ 1 1]
in aminocytes, chorionic villi, leukocytes, or in cultured skin fibroblasts obtained from the patients. ~4-~6 i4 N. S. Datta, G. N. Wilson, and A. K. Hajra, N. Engl. J. Med. 311, 1080 (1984). 15A. K. Hajra, N. S. Datta, G. L. Jackson, A. B. Moser, H. W. Moser, J. W. Larsen, and J. Powers, N. Engl. J. Med. 312, 455 (1985). 16 R. B. Schutgens, H. S. Heyman, R. J. Wanders, H. van den Bosch, and J. M. Tager, Eur. J. Pediatr. 144, 430 (1986).
[11] D i a c y l g l y c e r o l A c y l t r a n s f e r a s e a n d M o n o a c y l g l y c e r o l Acyltransferase from Liver and Intestine B y ROSALIND A . COLEMAN
Introduction The acylation of diacylglycerol catalyzed by the diacylglycerol acyltransferase is the only enzyme reaction unique to triacylglycerol synthesis. This reaction lies at the diacylglycerol branch point of phosphatidylcholine, phosphatidylethanolamine, and triacylglycerol synthesis) In most tissues the major pathway for the biosynthesis of the diacylglycerol substrate proceeds from the acylation of glycerol 3-phosphate. In intestine and liver, however, the monoacylglycerol pathway provides an alternate route for diacylglycerol synthesis. 2-Monoacylglycerol, produced by the action of gastric and pancreatic lipases on dietary triacylglycerol, enters the intestinal mucosa where monoacylglycerol acyltransferase plays a major role in resynthesizing triacylglycerol. In the liver of certain species, monoacylglycerol acyltransferase activity is high during early development; however, neither its specific role in liver nor the source of the monoacylglycerol substrate is known. 2-4 Both diacylglycerol acyltransferase and monoacylglycerol acyltransferase are intrinsic membrane proteins whose active sites face the cytosolic surface of the endoplasmic reticulum. 5 Neither activity has been substantially purified. i R. M. Bell and R. A. Coleman, in "The Enzymes" (P. D. Boyer, ed.), 3rd Ed., Vol. 16, p. 87. Academic Press, New York, 1983. 2 R. A. Coleman and E. B. Haynes, J. Biol. Chem. 259, 8934 (1984). 3 K. Sansbury, D. S. Millington, and R. A. Coleman, J. Lipid Res. 30, 1251 (1989). 4 R. A. Coleman, E. B. Haynes, and C. D. Coates, J. Lipid Res. 28, 320 (1987). 5 R. A. Coleman and R. M. Bell, in "The Enzymes" (P. D. Boyer, ed.), 3rd Ed., Vol. 16, p. 605. Academic Press, New York, 1983.
METHODS IN ENZYMOLOGY, VOL. 209
Copyright © 1992 by Academic Press, Inc. All rights of reproduction in any form reserved.
ACYLTRANSFERASES
[10]
Concluding Remarks Current evidence suggests that the activities of 1-alkylglycerophosphocholine and 1-alkenylglycerophosphocholine acyltransferases are located only in selected tissues. Since small amounts of 1-alkyl-2-acylGPC and 1-alkenyl-2-acyl-GPC are always found in major mammalian organs, and these phospholipids undergo acyl chain remodeling, the enzymes for such remodeling should also be present. It is likely that the acyltransferases are present in these tissues, but their low activities have eluded detection.
[10] D i h y d r o x y a c e t o n e P h o s p h a t e A c y l t r a n s f e r a s e
By KEITH O. WEBBER and AMIYA K. HA~RA Introduction Dihydroxyacetone phosphate acyltransferase (DHAPAT; glycerone3-phosphate acyltransferase, EC 2.3.1.42) catalyzes the transfer of the fatty acid moiety from long-chain acyl-coenzyme A (acyl-CoA) to the free hydroxyl group of dihydroxyacetone phosphate: Dihydroxyacetone phosphate + acyl-CoA--~ acyldihydroxyacetone phosphate + CoASH
This reaction initiates the synthesis of the ether-linked glycerolipids as well as the more common glycerol ester lipids. 1,2 DHAPAT is an integral membrane-bound protein located on the luminal side of animal cell peroxisomes. 3-5 This acyltransferase has been solubilized and partially purified from guinea pig liver. 6'7 The present method is based on that described for the preparation of highly purified enzyme from guinea pig liver peroxi-
somes.7
1 A. 2 R. 3 A. 4 A. 5 C. 6 C. 7 K.
K. Hajra, Biochem. Soc. Trans. 5, 34 (1977). Manning and D. N. Brindley, Biochem. J. 130, 1003 (1972). K. Hajra, C. L. Burke, and C. L. Jones, J. Biol. Chem. 244, 8289 (1989). K. Hajra and J. E. Bishop, Ann. N.Y. Acad. Sci. 386, 170 (1982). L. Jones and A. K. Hajra, J. Biol. Chem. 255, 8289 (1980). L. Jones and A. K. Hajra, Arch. Biochem. Biophys. 226, 155 (1983). O. Webber, Ph.D. Dissertation, University of Michigan, Ann Arbor (1988).
METHODSIN ENZYMOLOGY,VOL. 209
Copyright© 1992by AcademicPress, Inc. All rightsof reproductionin any formreserved.
[10]
DIHYDROXYACETONE PHOSPHATE ACYLTRANFERASE
93
Assay Method
Principle. Enzyme activity is measured as the amount of lipophilic (at low pH) radioactivity formed from [32p]DHAP in the presence of palmitoylCoA and e n z y m e ) Reagents 5 mM Dihydroxyacetone [32p]phosphate: [32p]DHAP is prepared by enzymatic phosphorylation of dihydroxyacetone with [y-32p]ATP. 8,9The radioactive DHAP is diluted with nonradioactive DHAP (Li + salt) to the desired specific activity [5000-10,000 counts/min (cpm)/nmol] 1 mM Palmitoyl-CoA, lithium salt 0.3 M Morpholinoethanesulfonic acid (MES), pH 5.7 0.3 M Tris-HCl buffer, pH 7.5 0.1 M Sodium fluoride 0.1 M Magnesium chloride 20 mg/ml Bovine serum albumin (BSA), fatty acid poor, fraction V Asolectin suspension (liposomes) in 10 mM Tris-HC1, I mM EDTA, pH 7.5: Suspend 2 g asolectin (Associated Concentrates, Woodside, NY) in 100 ml of 10 mM Tris-HCl (pH 7.5)- 1 mM EDTA by sonicating with an ultrasonic probe and then centrifuge the suspension at 35,000 g for 30 min. Use the supernatant (15-20/xmol lipid phosphate/ml) for the assay. When stored at 4° under N2, this suspension is stable for 1 month. 2 M potassium chloride in 0.2 M phosphoric acid Chloroform Chloroform-methanol (1 : 2, v/v) Chloroform-methanol-0.5 N aqueous H3PO 4 (1 : 12 : 12, v/v) Procedure. Add the following reagents to 16 × 125 mm screw-topped tubes: buffer 1° (0.3 M Tris-HC1 (or 0.3 M MES) 0.15 ml, BSA 0.05 ml, palmitoyl-CoA 0.05 ml, NaF 0.1 ml, MgC12 0.05 ml, asolectin suspension 0.1 ml, 1~ enzyme sample, and water to make the final volume 0.6 ml. Incubate the mixture at 37° for 15 min in a reciprocating shaker water bath. Stop the reaction by adding 2.25 ml chloroform-methanol (1 : 2, v-v) and mix (vortex). Add 0.75 ml methanol and 0.75 ml KCI-H3PO4 (2-0.2 M), vortex, and centrifuge for 5 rain at 1000 g. Aspirate off the upper layers s A. K. Hajra, J. Biol. Chem. 2,43, 3458 (1968). 9 A. K. Hajra and C. L. Burke, J. Neurochem. 31, 125 (1978). 10 U s e M E S buffer for the m e m b r a n e - b o u n d e n z y m e and Tris buffer for the solubilized enzyme. 11 Addition of asolectin is not n e c e s s a r y for the a s s a y of m e m b r a n e - b o u n d e n z y m e .
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ACYLTRANSFERASES
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and add 2.5 ml of chloroform-methanol-0.5 N aqueous H3PO 4 (1 : 12: 12, v/v), mix, and centrifuge. Aspirate off the upper layers and wash the lower layers again with chloroform-methanol-aqueous H3PO4 as described above. Transfer aliquots of the washed lower layers (generally 1 ml, i.e., two-thirds of the total) to counting vials, evaporate off the solvents with a stream of air or N2, add scintillation solvent mixture, and determine the radioactivity in a liquid scintillation counter. Units. One unit (U) of enzyme activity is defined as 1 nmol of product formed per minute at 37°. The specific activity is expressed as nanomoles per minute per milligram protein. Purification of Enzyme The purification of DHAPAT can be divided into three processes: (1) isolation ofperoxisomes, (2) solubilization of the peroxisomal membranes, and (3) chromatographic purification of DHAPAT. Purification o f Peroxisomes from Guinea Pig Liver Subcellular Fractionation. Livers are homogenized according to the protocol of deDuve et al. 12in a buffer containing 0.25 M sucrose, 10 mM N-tris(hydroxymethyl)methyl-2-aminoethanesulfonicacid (TES; pH 7.5), 1 mM EDTA, 0.1% ethanol (v/v), 0.2 mM phenylmethylsulfonyl fluoride (PMSF), and 1/xM leupeptin. Fractionate the liver by differential centrifugation to produce the "light mitochondrial" fraction (L-fraction, sedimenting between 33,000 and 250,000 g.min), 12which is enriched in peroxisomes. The method used in our laboratory for subcellular fractionation of guinea pig liver is essentially that described by deDuve et al. 12with minor modifications. 3 Suspend the L-fraction in the homogenization buffer to a volume of 0.25 ml/g of original liver weight. Density Gradient Centrifugation. The peroxisomes are isolated from the L-fraction by centrifugation through 30% (w/v) Nycodenz as described in Ref. 13 with some modifications. 7
1. Into 25-ml polycarbonate centrifuge bottles (Beckman, Fullerton, CA, #340382), place 15 ml of a solution containing 30% Nycodenz (w/v), 10 mM TES, pH 7.5, and 1 mM EDTA. 2. Overlay the 15 ml of Nycodenz solution with 2 ml of L-fraction. 3. Centrifuge at 75,000 g for 45 min in a Ti-55 fixed-angle rotor (BeckJ2 C. deDuve, B. C. Pressman, R. Gianetto, R. Wattiaux, and F. Appelmans, Biochem. J. 60, 604 (1955). 13 M. K. Ghosh and A. K. Hajra, Anal. Biochem. 159, 169 (1986).
[10]
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95
man). Peroxisomes will sediment through the Nycodenz solution while mitochondria, microsomes, and lysosomes will not. Carefully aspirate away the entire supernatant. 5. Resuspend the remaining pellet in homogenization buffer to a volume equivalent to 20% of the original liver weight. Store at - 20°. .
Solubilization of Dihydroxyacetone Phosphate Membrane Preparation. Initial purification of DHAPAT entails separation of the peroxisomal membrane from the soluble, matrix proteins. The peroxisomes are osmotically ruptured by 10-fold dilution with 10 mM sodium pyrophosphate, pH 9.0, containing 1 tzM leupeptin, 1 /zM pepstatin, 0.2 mM PMSF, and 1 mM EDTA. Stir the mixture for 1 hr on ice. Centrifuge at 100,000 g for 30 min to separate membranes (pellet) from the soluble matrix proteins (supernatant). Resuspend the pellet in 25 mM TES (pH 7) to a protein concentration of approximately 10 mg/ml. This membrane suspension may be stored at - 2 0 °. Solubilization. Mix equal volumes of peroxisomal membrane suspension and a solution containing 300 mM NaC1, 30 mM 3-[(3-cholamidopropyl)dimethylammonio]-l-propanesulfonate (CHAPS), 20 mM MES (pH 6.5), 2 mM dithiothreitol (DTT), 2/.LM leupeptin, 2/xM pepstatin, 2 mM EDTA, and 0.4 mM PMSF. Stir this mixture gently for 15 min on ice. Centrifuge the mixture at I00,000 g for 60 min. The supernatant (a clear brown liquid) contains the solubilized DHAPAT with a specific activity of approximately 80-90 U/rag protein. Column Chromatography The solubilized DHAPAT is purified to near homogeneity by a multistep regimen of both low-pressure and high-pressure column chromatography. Low-Pressure Size-Exclusion Chromatography. Pass the solubilized peroxisomal membrane proteins over a column of Sephacryl S-200 (1.6 x 95 cm; Pharmacia, Piscataway, NJ) at 4°. The mobile phase is 150 mM NaCl, 5 mg/ml CHAPS, 10 mM MES (pH 6.5), 1 mM DTT, 0.02% NaN3 flowing at 6.0 ml/hr. Collect 3-ml fractions. Pool the fractions (fractions 12-15) comprising the peak DHAPAT activity. Cation-Exchange Chromatography. Cation-exchange chromatography is performed on a 0.5 × 5 cm Mono S column (Pharmacia) attached to a suitable high-performance liquid chromatography (HPLC) system allowing binary gradient elution and monitoring of effluent absorbance at 280 nm. Equilibrate the column with 25 mM MES, pH 6.5, 5 mg/ml CHAPS, 1 mM DTT (buffer A) and then load the sample onto the column
96
ACYLTRANSFERASES
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TABLE 1 PURIFICATION OF DIHYDROXYACETONE PHOSPHATE ACYLTRANSFERASE
Fraction
Protein (rag)
Total activity (U)
Specific activity (U/mg)
Enrichment (-fold)
Yield (%)
Liver homogenate Peroxisomes Solubilized membranes Sephacryl S-200 Mono S Hydroxylapatite TSK-3000
15,150 276 94 31 1.0 0. | 8 0.08
7900 2820 6020 3640 1400 450 260
0.52 10.2 64 118 1400 2400 3350
1 19.6 123 227 2700 4600 6440
100 36 76 46 18 6 3
at 0.5 ml/min. Wash the unbound proteins off the column with buffer A until the 280 nm absorbance of the effluent returns to baseline. While collecting 1.0-ml fractions, elute the adherent proteins from the column with a linear salt gradient from 0 to 400 mM NaCI in buffer A at a flow rate of 0.5 ml/min (total gradient volume 40 ml). Pool the fractions (fractions 11-15) comprising the peak DHAPAT activity. Hydroxylapatite Chromatography. Equilibrate a high-pressure hydroxylapatite column (0.5 x 4.8 cm; Bio-Rad Laboratories, Richmond, CA) with 10 mM potassium phosphate, pH 6.8, 0.3 mM CaCI2, 5 mg/ml CHAPS, 50 mM NaCI, I mM DTT, 0.05% NAN3. Load the pooled DHAPAT-containing fractions from the cation-exchange column at 0.5 ml/min. Wash the unbound proteins from the column with equilibration buffer until the absorbance of the effluent at 280 nm returns to baseline. Elute the bound proteins with a 25-ml linear phosphate gradient starting with equilibration buffer and ending with 0.3 M potassium phosphate, pH 6.8, 10/zM CaCI2, 5 mg/ml CHAPS, I mM DTT, 0.05% NAN3. Collect 1-ml fractions. High-Pressure Size-Exclusion Chromatography. Pool the DHAPATcontaining fractions (fractions 12-14) eluted from the hydroxylapatite column and concentrate the combination to approximately 1.0 ml in a centrifugal microconcentrator (Bio-Rad). Inject the concentrated enzyme solution onto the HPLC gel-filtration column (TSK-3000; Toyo-Soda, Japan) and elute with 150 mM NaC1, 5 mg/ml CHAPS, 10 mM Bis-Tris-propane, pH 7.5, 1 mM DTT, 0.02% NaN 3 at 0.5 ml/min. Collect 1.0-ml fractions while monitoring the absorbance of the effluent at 280 nm. The enzyme generally elutes in fractions 4 to 7. Combine the fractions, concentrate the enzyme by ultrafiltration (centrifugal microconcentrator), and store at 4 °. The enzyme could be purified further by chromatofocusing but at such
[10]
DIHYDROXYACETONE PHOSPHATE ACYLTRANFERASE
97
a low yield that the specific activity could not be accurately determined. 7 A typical purification procedure is summarized in Table I. Properties
Physical Characteristics. DHAPAT activity copurifies with a protein which has an apparent Mr of 69,000 as determined by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate (SDS-PAGE). Gel filtration of trace amounts of solubilized DHAPAT gives a M r determination of approximately 90,000, indicating that the active enzyme is probably not multimeric. Thermostability. Membrane-bound peroxisomal DHAP acyltransferase is remarkably heat stable. In fact, heating membranes to 50° prior to assay increases the measurable enzyme activity. The solubilized enzyme does not possess this thermostability and loses significant activity at temperatures as low as 40° . Kinetics. The guinea pig liver enzyme is most active at pH 5.5 in the membrane-bound state; however, on solubilization with either ionic or nonionic detergents, the pH optimum shifts to pH 7.4 and becomes somewhat less stringent. At pH 7.4, the membrane-bound enzyme gives biphasic kinetics with Km (DHAP) values of 40 and 200 ~M which increase to 100 and 500 ~M on solubilization. When the solubilized enzyme is assayed in the presence of asolectin (soybean lipids) the kinetics are monophasic with a Km (DHAP) of 100/zM. After extensive purification (as above), the Km (DHAP) was determined to be about 60/zM; however, at subsaturating levels of palmitoyl-CoA the value drops to 35 ~M. Activators and Inhibitors. The crude membrane-bound enzyme activity is stimulated by Mg 2+ and F - ; however, after solubilization these ions have no effects on the enzyme activity. The solubilized enzyme is stimulated by phospholipid vesicles, such as asolectin or phosphatidylcholine. 6 Palmitoyl-CoA inhibits the enzyme activity, and the presence of BSA in the reaction mixture prevents this inhibition. Both the membrane-bound and solubilized enzymes are relatively unaffected by thiol-modifying agents such as N-ethylmaleimide and iodoacetamide; however, bulkier, charged sulfhydryl-reactive compounds such as p-chloromercuriphenylsulfonic acid or 5,5'-dithiobis(2-nitrobenzoic acid) produce moderate levels of inhibition. Diagnostic Use of Enzyme A number of genetic diseases involving peroxisomal disorders are prenatally or postnatally diagnosed by the decreased activity of DHAPAT
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ACYLTRANSFERASES
[ 1 1]
in aminocytes, chorionic villi, leukocytes, or in cultured skin fibroblasts obtained from the patients. ~4-~6 i4 N. S. Datta, G. N. Wilson, and A. K. Hajra, N. Engl. J. Med. 311, 1080 (1984). 15A. K. Hajra, N. S. Datta, G. L. Jackson, A. B. Moser, H. W. Moser, J. W. Larsen, and J. Powers, N. Engl. J. Med. 312, 455 (1985). 16 R. B. Schutgens, H. S. Heyman, R. J. Wanders, H. van den Bosch, and J. M. Tager, Eur. J. Pediatr. 144, 430 (1986).
[11] D i a c y l g l y c e r o l A c y l t r a n s f e r a s e a n d M o n o a c y l g l y c e r o l Acyltransferase from Liver and Intestine B y ROSALIND A . COLEMAN
Introduction The acylation of diacylglycerol catalyzed by the diacylglycerol acyltransferase is the only enzyme reaction unique to triacylglycerol synthesis. This reaction lies at the diacylglycerol branch point of phosphatidylcholine, phosphatidylethanolamine, and triacylglycerol synthesis) In most tissues the major pathway for the biosynthesis of the diacylglycerol substrate proceeds from the acylation of glycerol 3-phosphate. In intestine and liver, however, the monoacylglycerol pathway provides an alternate route for diacylglycerol synthesis. 2-Monoacylglycerol, produced by the action of gastric and pancreatic lipases on dietary triacylglycerol, enters the intestinal mucosa where monoacylglycerol acyltransferase plays a major role in resynthesizing triacylglycerol. In the liver of certain species, monoacylglycerol acyltransferase activity is high during early development; however, neither its specific role in liver nor the source of the monoacylglycerol substrate is known. 2-4 Both diacylglycerol acyltransferase and monoacylglycerol acyltransferase are intrinsic membrane proteins whose active sites face the cytosolic surface of the endoplasmic reticulum. 5 Neither activity has been substantially purified. i R. M. Bell and R. A. Coleman, in "The Enzymes" (P. D. Boyer, ed.), 3rd Ed., Vol. 16, p. 87. Academic Press, New York, 1983. 2 R. A. Coleman and E. B. Haynes, J. Biol. Chem. 259, 8934 (1984). 3 K. Sansbury, D. S. Millington, and R. A. Coleman, J. Lipid Res. 30, 1251 (1989). 4 R. A. Coleman, E. B. Haynes, and C. D. Coates, J. Lipid Res. 28, 320 (1987). 5 R. A. Coleman and R. M. Bell, in "The Enzymes" (P. D. Boyer, ed.), 3rd Ed., Vol. 16, p. 605. Academic Press, New York, 1983.
METHODS IN ENZYMOLOGY, VOL. 209
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