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[6]
[6] G l y c e r o p h o s p h a t e A c y l t r a n s f e r a s e f r o m L i v e r
By DIPAK HALDAR and ALES VANCURA Introduction Glycerol 3-phosphate acyltransferase (acyl-CoA : sn-glycerol 3-phosphate O-acyltransferase, EC 2.3.1.15) catalyzes the following reaction: sn-Glycerol3-phosphate + acyl-CoA--->1-acyl-sn-glycerol3-phosphate + CoA The enzyme converts water-soluble glycerophosphate to a lipid product and catalyzes the committed step in the biosynthesis of phosphoglycerides and triacylglycerols from glycerophosphate. The enzyme can divert fatty acids from/3-oxidation to esterification. Thus, the reaction repcesents a probable regulatory site in fatty acid metabolism. In mammalian organs, glycerophosphate acyltranferase is present in both the endoplasmic reticulum (microsomes) and mitochondrial outer membrane. 1'2 In liver the glycerophosphate acyltransferase activity is nearly equal in these two organelles, whereas in other organs the microsomal acyltransferase is approximately 10 times more active than the mitochondrial enzyme. 3 Assay
Principle. The enzyme activity can be measured by following the convcrsion of sn-[2-3H]glycerol 3-phosphate to l-butanol-extractable lipid.2,3 Reagents 0.20 M MES-TES-glycylglycine buffer, pH 7.5 [4-morpho-lineethanesulfonic acid, N-tris(hydroxymethyl)methyl-2-aminoethanesulfonic acid, and glycylglycine] 0.10 M MgCIz 0.72 mM Palmitoyl-CoA 0.10 M KCN 50 mg/ml Bovine serum albumin (BSA; fatty acid free) 20 mg/ml Asolectin (sonicated suspension) J L. N. W. Daae and J. Bremer, Biochim. Biophys. Acta 210, 92 (1970). 2 G. Monroy, F. H. Rola, and M. E. Pullman, J. Biol. Chem. 247, 6884 (1972). 3 D. Haldar, W.-W. Tso, and M. E. Pullman, J. Biol. Chem. 254, 4502 (1979).
METHODS IN ENZYMOLOGY,VOL. 209
Copyright © 1992by Academic Press, Inc. All fights of reproduction in any form reserved.
[6]
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7.5 mM sn-[2-3H]Glycerol 3-phosphate [~10,000 counts/min (cpm)/ nmol] Scintillation cocktail MES-TES-glycylglycine buffer 2 is prepared by dissolving appropriate amounts of each compound in water to a concentration of 0.4 M, adjusting the pH with KOH, and finally diluting the solution to 0.2 M. The concentration of palmitoyl-CoA is determined from the optical density of the solution at 232 and 260 nm using the millimolar extinction coefficients of 8.7 and 16.8, 2'4 respectively. An unsaturated fatty acyl-CoA can also be used as an acyl group donor in the glycerophosphate acyltransferase assay, but it should be freshly prepared to avoid oxidation. KCN is used to prevent oxidation of acyl-CoAs by mitochondria and should also be freshly prepared. It has no effect on glycerophosphate acyltransferase. Asolectin, a soybean phospholipid preparation, is suspended in 10 mM Tris-HCl, pH 7.5, and the mixture is exposed at 0° to sonic irradiation until a translucent suspension is obtained. Fifteen-second exposures with 15-sec intervals between exposures for a total exposure of 2 to 3 min with a Megason Ultrasonic Disintegrator (Ultrasonic Instruments International, Farmingdale, NY) at nine-tenths of its maximum output is adequate. The suspension is then centrifuged at 105,000 g for 10 min and the supernatant taken. The suspension can be stored at 4 ° for a period not exceeding 2 weeks, after which the phospholipid starts sedimenting. Radioactive glycerophosphate is commercially available but can also be prepared enzymatically from [2-3H]glycerol by phosphorylation with ATP in the presence of glycerokinase 5followed by purification.6 The specific activity of the radioactive glycerophosphate is expressed in cpm/nmol. Although the assay is carried out with 1.5 mM glycerol 3-phosphate for economic reasons, maximal activity of the acyltransferase is achieved at higher concentrations (7.5 to 15 mM glycerol 3-phosphate for mitochondria). Unless mentioned otherwise, all of the above reagents can be stored at - 20°.
Preparation of Subcellular Fractions Rat liver mitochondrial and microsomal fractions can be prepared following any standard procedure such as the one previously described. 2,7 An 8000 to 12,000 g intermediate fraction is discarded. The washed mitochondrial and microsomal fractions are suspended in 0.25 M sucrose (5-I0 mg subcellular protein/ml) and are either immediately used or divided into 0.5- to 1.0-ml aliquots and stored at - 70°. Storage under these conditions 4 E. 5 E. 6 E. 7 E.
Stadtman, this series, Vol. 3, p. 931. P. Kennedy, this series, Vol. 5, p. 476. E. Hill, D. R. Husbands, and W. E. M. Lands, J. Biol. Chem. 243, 4440 (1968). C. Weinbach, Anal. Biochem. 2, 335 (1961).
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results in only a little loss of activity over the course of 6 months. Protein is estimated by the method of Lowry et al. s using BSA as a standard. P r o c e d u r e . Reaction mixtures contain the following in a final volume of 0.5 ml: 40 mM MES-TES-glycylglycine buffer; 2 mM MgCI 2 ; an optimal concentration (20-100 /~M) of palmitoyl-CoA; 2 mM KCN; an optimal concentration (1-4 mg) of BSA; 0.2 mg asolectin; 1.5 mM sn-[2-3H]glycerol 3-phosphate; and 0.05 to 0.2 mg subcellular protein. The reaction mixture is preincubated for 3 min at 37° before the subceUular fraction is added, and the incubation is continued for another 3 min. The reaction is stopped by adding 0.5 ml of 1-butanol and vortexing the mixture. One milliliter of water is added, mixed well, and the mixture centrifuged for 5 rain at room temperature in a tabletop centrifuge. The bottom water layer is removed and may be stored and used later for recycling of the radioactive glycerophosphate. The butanol layer (upper) is washed with 1.5 ml of butanolsaturated water. To an aliquot (usually 0.1-0.2 ml) of the butanol is added a scintillation cocktail, and the mixture is counted. The specific activity of the enzyme is calculated on the basis of nanomoles of glycerophosphate acylated per minute of incubation in the presence of the enzyme per milligram of protein (nmol/min/mg). An endogenous control, incubated without palmitoyl-CoA, is subtracted from the experimental value. Typical values for freshly prepared rat liver mitochondria and microsomes, using palmitoyl-CoA as the acyl donor, are 3-5 and 4-6 nmol/min/mg, respectively. The reaction rate is rectilinear with time (up to 9 min) and subcellular protein (up to 2 mg/ml). The optimal concentration of palmitoyl-CoA presumably depends on the amount of total protein--subcellular fraction and BSA--and is lower for the mitochondria than for microsomes. The optimal concentration of BSA varied in our hands according to the source of the protein. Asolectin, although stimulatory to the glycerophosphate acyltransferase, is not essential for the assay and is usually omitted from the assay because of the complexities involved in the preparation and stability of the suspension of this soybean phospholipid mixture. Variations of the assay include the use of palmitoylcarnitine and CoA to generate palmitoyl-CoA.1 This assay works, but when an activator or inhibitor is used, one is not sure if the effect is on glycerophosphate acyltransferase or palmitoylcarnitine acyltransferase. In another variation, 9 chloroform-methanol (2: 1) is used instead of l-butanol to extract the lipids. However, chloroform-methanol shows some bias against lysophosphatidic acid, 2 which is the main product of mitochondrial incubation s O. H. Lowry, N. J. Rosebrough, A. L. Farr, and R. J. Randall, J. Biol. Chem. 193, 265 (1951). 9 H. Eibl, E. F. Hill, and W. E. M. Lands, Eur. J. Biochem. 9, 250 (1969).
[6]
GLYCEROPHOSPHATE ACYLTRANSFERASE FROM LIVER
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under the above conditions. 2'3 Also, the butanol extraction procedure is somewhat more efficient. 1,2 The products of acylation of glycerophosphate under the above conditions are mainly lysophosphatidic acid and phosphatidic acid along with a small amount, if any, of mono- and diglycerides. The lipids can be separated by thin-layer chromatography on silica gel plates with a solvent system containing chloroform, methanol, acetic acid, and water (65 : 25 : I : 4). 2'1° An input of approximately 2000 cpm is adequate. The radioactivity of the lipids is determined by scraping 0.5 × 2 cm sections of each lane into mini-scintillation vials. The scrapings are soaked for 15 min in 0.2-ml portions of 10 mM EGTA, pH 4.6. Three milliliters of scintillation fluid is added and the mixture counted. Recovery of radioactivity from the plates is between 80 and 110%. Properties
pH Optimum and K m . Both the microsomal n and mitochondrial glycerophosphate acyltransferase exhibit a broad pH optimum between 6.6 and 9.0. The Km for sn-glycerol 3-phosphate for the microsomal enzyme is lower (0.1-0.2 mM) than that for the mitochondrial acyltransferase (~1 rnM). 2'11 Substrate and Position Specificity. In the glycerophosphate acyltransferase assay, the microsomes from liver as well as from other mammalian organs can use both saturated and unsaturated fatty acyl-CoAs with almost equal efficiency. Mitochondria, on the other hand, prefer saturated acylCoA thioesters.2 However, regardless of whether saturated or unsaturated acyl-CoA is used as acyl donor, 1-acyl-sn-glycerol 3-phosphate is the major if not the sole product of sn-glycerol 3-phosphate acylation. During the processing of the samples for determining positional specificity, approximately 6% isomerization of 1- to 2-monoacylglycerol and 20% conversion of the 2- to the 1-isomer have occurred. 3 The above properties of the mitochondrial glycerophosphate acyltransferase provide an excellent mechanism for the observed preferential positioning of saturated fatty acids in the sn-1 position and unsaturated fatty acids in the sn-2 position in naturally occurring acylglycerols. 2 This possibility is further supported by the facts that (1) in Ehflich cells, which exhibit an abnormally high proportion of unsaturated fatty acids at the sn-1 position of some phosphoglycerides, a mitochondrial glycerophosphate acyltransferase is undectable 3 and (2) in cell cultures, there is an agel0 V. P. Skipski and M. Barclay, this series, Vol. 14, p. 530. i~ S. Yamashita and S. Numa, this series, Vol. 71, p. 550.
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16]
dependent decline of mitochondrial glycerophosphate acyltransferase activity and a parallel increase in the amount of unsaturated fatty acids at the sn-1 position in choline phosphoglycerides. 12 Recently, experimental support of the above hypothesis has come from the observation that in the presence of BSA, mitochondrially produced monoacylglycerophosphate can exit the organelles, be translocated to the microsomes, and converted to diacylglycerophosphate. 13 Also, a monoacylglycerophosphate-binding liver cytosolic protein has been purified, which may be involved in the transport of mitochondrially made monoacylglycerophosphate to the endoplasmic reticulum.14 Activators and Inhibitors. Some divalent cations, such as Mg 2+ and Ca 2+, stimulate both the mitochondria115 and microsoma111 glycerophosphate acyltranferase, especially when the enzyme is partially purified (see later). Acetone 3 and polymyxin B 16 stimulate the mitochondrial but inhibit the microsomal acyltranferase. Sulfhydryl group reagents, such as N-ethylmaleimide and iodoacetamide, strongly inhibit the microsomal acyltransferase; the mitochondrial activity is unaffected by these reagents. 2'3 This distinguishing property of mitochondrial and microsomal glycerophosphate acyltransferase has been used to determine the microsomal contamination of the mitochondrial fraction 3'17or to assay the mitochondrial enzyme present in a whole cell homogenate. 12'18 Phospholipids modulate liver mitochondrial and microsomal glycerophosphate acyltransferase. Phosphatidylserine, asolectin, and phosphatidylcholine stimulate, whereas mono- and diacylglycerophosphate and cardiolipin inhibit the mitochondrial activity. 15Microsomes are stimulated by phosphatidylcholine and phosphatidylethanolamine but are inhibited by phosphatidylserine and phosphatidylinositol. H These results have been obtained from experiments performed with partially purified subcellular glycerophosphate acyltransferase. Among other lipids, monoacylglycerols and some of their analogs inhibit both the mitochondrial and microsomal glycerophosphate acyltransferase. 28 The liver microsomal glycerophosphate acyltransferase is inhibited by 12 W. Stern and M. E. Pullman, J. Biol. Chem. 253, 8047 (1978). 13 D. Haldar and L. Lipfert, J. Biol. Chem. 265, 11014 (1990). 14 A. Vancura, M. A. Carroll, and D. Haldar, Biochem. Biophys. Res. Commun. 175, 339 (1991). 15 G. Monroy, H. C. Kelker, and M. E. Pullman, J. Biol. Chem. 248, 2845 (1973). 16 M. A. Carroll, P. E. Morris, C. D. Grosjean, T. Anzalone, and D. Haldar, Arch. Biochem. Biophys. 214, 17 (1982). J7 R. J. Pavlica, C. B. Hesler, L. Lipfert, I. N. Hirshfield, and D. Haldar, Biochim. Biophys. Acta 1022, 115 (1990). I8 R. A. Coleman, Biochim. Biophys. Acta 963, 367 (1988).
[6]
GLYCEROPHOSPHATE ACYLTRANSFERASE FROM LIVER
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all proteases. ~9'2° The mitochondrial enzyme, on the other hand, is not inhibited by trypsin and chymotrypsin. Non-site-specific proteases, such as proteinase K and subtilisin, inhibit the mitochondrial enzyme. The degree of inhibition increases if the ionic strength of the incubation medium is lowered. The protease sensitivity of the microsomal enzyme does not respond to any such changes in the ionic strength of the incubation medium. Exposure of trypsin- or chymotrypsin-sensitive domains of glycerophosphate acyltransferase on the inner surface of the mitochondrial outer membrane can be directly demonstrated by incubating trypsin-loaded outer membrane vesicles. 2° These results, taken together, suggest that the mitochondrial glycerophosphate acyltransferase spans the transverse plane of the outer membrane.
Purification Both microsomal and mitochondrial glycerophosphate acyltransferase have been only partially purified. In contrast, the Escherichia coli glycerophosphate acyltranferase has been completely purified, its properties studled, 21 and the gene cloned and sequenced. EEThe successful purification of the prokaryotic enzyme has been facilitated by massive overproduction of the enzyme owing to molecular c l o n i n g . E1
Microsomal Glycerophosphate Acyltransferase All operations are conducted at 0 ° to 5°. Rat liver microsomes are treated for 2 hr with 6 mM Triton X- 100 at pH 8.6 at a protein concentration of 10 mg/ml and then passed through a Sepharose 2B column. The first few fractions of the eluate which contain protein and the enzyme activity are combined and centrifuged through a sucrose gradient (0.5 to 1.5 M layered on 2 M). This treatment resolves the two enzyme activities: glycerophosphate acyltransferase and 1-acylglycerophosphate acyltransferase. However, the enrichment of glycerophosphate acyltransferase is only 3.6 times, with approximately 90% loss of activity. For a detailed account of this method, the reader is referred to an earlier volume in this series. H It is interesting to note that, unlike the microsomes, the partially purified enzyme prefers saturated over unsaturated fatty acyl-CoA thioesters as substrates. i9 R. A. Coleman and R. M. Bell, J. Cell Biol. 76, 245 (1978). 2o C. B. Hesler, M. A. Carroll, and D. Haldar, J. Biol. Chem. 260, 7452 (1985). 21 M. A. Scheideler and R. M. Bell, J. Biol. Chem. 264, 12455 (1989). 22 p. R. Green, T. C. Vanaman, P. Mordich, and R. M. Bell, J. Biol. Chem. 258, 10862 (1983).
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Mitochondrial Glycerophosphate Acyltransferase There are two methods available for the partial purification of the mitochondrial acyltransferase. All operations are carried out at 0 ° to 4 °. In the first method, 15 submitochondrial particles are prepared by sonic irradiation of an aqueous suspension of mitochondria (I0 mg/ml) for 10 min with a Branson Sonifier operating at maximum output. The particles are centrifuged down and finally suspended (10 mg/ml) for 10 rain in a mixture containing 0.25 M sucrose, 10 mM Tris-HCl, pH 8.0, 0.5% potassium cholate, pH 7.8, and 1.0 M KC1. The mixture is centrifuged at 150,000 g, and to the supernatant is added 0.35% asolectin suspension followed by the addition of a saturated solution of ammonium sulfate to a final concentration of 15%. After 10 min the mixture is centrifuged at 105,000 g for 15 min. The supernatant fluid is adjusted to 45% saturation with ammonium sulfate and centrifuged. The precipitate is dissolved in Tris-HCl, pH 8.0, containing 0.35% asolectin and dialyzed thoroughly against the same solution. This preparation represents 6-fold purification with no monoacylglycerophosphate acyltransferase activity. In the other method, 23 submitochondrial particles are prepared by suspending the mitochondria ( 10 mg/ml) in 20 mM Tris-HCl buffer, pH 8.4, containing I mM phenylmethylsulfonyl fluoride. The mixture is exposed to sonic irradiation for 4 min (30-sec exposures with 15-sec intervals) using a Megason Ultrasonic Disintegrator working at nine-tenths of its maximum output. The mixture is then centrifuged at 170,000 g for 90 min. The sediment containing the submitochondrial particles is suspended in a solution containing 0.25 M sucrose, 1 M KCI, 20 mM Tris-HCl buffer, pH 8.4, 1 mM phenylmethylsulfonyl fluoride, and 0.5% (ultrapure) Lubrol PX. The detergent-to-protein ratio is maintained between 2 and 4. After 30 min, the mixture is centrifuged at 170,000 g for 90 min. The supernatant fraction, containing most of the enzyme activity, is desalted on a Pharmacia (Piscataway, N J) PD-10 column equilibrated with 20 mM Tris-HC1, pH 8.4. The mixture is then loaded on Sepharose Q column (I .5 x 30 cm), equilibrated with 20 mM Tris-HC1, pH 8.4, containing 0.5% Lubrol PX and 10% glycerol. The column is eluted with the same buffer, and fractions containing the glycerophosphate acyltransferase activity are combined and loaded on a BioGel HT (Bio-Rad, Richmond, CA) column (1 x I0 cm) equilibrated with 20 mM Tris-HC1, pH 8.4, containing 0.5% Lubrol PX and 10% glycerol. The column is washed with 100 ml of the same buffer. Delipidated glycerophosphate acyltransferase is eluted from the column with 50 ml of a gradient of 20 to 500 mM potassium phosphate 2t A. Vancura and D. Haldar, manuscript in preparation.
[6]
71
GLYCEROPHOSPHATE ACYLTRANSFERASE FROM LIVER 0.5"
"6
-5
2
0.4 1
~.
i
A x
0.3"
-3
~
'1
~
.~
0.2
0.1
0.0
.
.
.
.
.
.
.
.
.
T
.
10
.
.
.
.
.
.
20 Fraction No.
30
40
FIG. 1. Purification of mitochondrial glycerophosphate acyltransferase on a BioGel HT column. The column was eluted at 14 ml/hr with a linear gradient of phosphate buffer (20-500 mM) (line 3), and 1.3-ml fractions were collected for determining absorbance at 280 nm (line 1) and glycerophosphate acyltransferase activity (line 2).
buffer, pH 7.4, containing 0.2% Lubrol PX and 10% glycerol (Fig. 1). From this step, determination of the glycerophosphate acyltransferase activity is completely dependent on reconstitution of the enzyme with asolectin suspension. Fractions exhibiting glycerophosphate acyltransferase activity are combined, supplemented with NaC1 to 0.5 M concentration, and loaded on an octyl-Sepharose CL-4B column (I x 5 cm), equilibrated with 20 mM Tris-HC1 buffer, pH 7.4, containing 0.2% Lubrol PX, 10% glycerol, and 0.5 M NaCI. The column is washed with 10 ml of the same buffer, and then the enzyme is eluted with 20 mM Tris-HC1, pH 8.4, containing 0.5% Lubrol PX and 10% glycerol. Results of a typical purification procedure are summarized in Table I. TABLE I PURIFICATION OF MITOCHONDRIAL GLYCEROPHOSPHATE ACYLTRANSFERASE
Step
Protein (rag)
Total activity (nmol/min)
Specific activity (nmol/min/mg)
Purification (-fold)
Yield (%)
Submitochondrial particles Lubrol PX extract Sepharose Q fast flow BioGel HT Octyl-Sepharose CL-4B
119.2 84.9 12.1 2.2 1.5
122.1 73.1 41.3 33.8 31.7
1.03 0.86 3.42 15.36 21.20
1.00 0.83 3.32 14.91 20.58
100 60 34 28 26
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ACYLTRANSFERASES
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Additional improvement in purification of mitochondrial glycerophosphate acyltranferase was achieved using affinity chromatography on palmityl-CoA-agarose or glycerol 3-phosphate-agarose. When a glycerol 3-phosphate agarose column was used instead of octyl-Sepharose CL-4B column, the overall purification of glycerophosphate acyltransferase was over 40-fold. The molecular weight of the native glycerophosphate acyltransferase was 60-85 kDa as determined by gel filtration on Sephacryl S-300 HR in 0.2% CHAPS. Comparison of this result with electrophoretic data strongly suggests the mitochondrial glycerophosphate acyltransferase to be a monomeric enzyme. SDS-PAGE of the purified glycerophosphate acyltransferase followed by Coomassie blue staining exhibited a single band with a molecular weight of 80-85 kDa. Acknowledgment This work was supported by a grant from the National Science Foundation (DCB8801535).
[7] C o e n z y m e A - I n d e p e n d e n t A c y l t r a n s f e r a s e
By TAKAYUKI SUGIURA and KEIZO WAKU Introduction The coenzyme A (CoA)-independent transacylation system was first described by Kramer and Deykin 1'2 for human platelets. Similar enzyme activity was also found for several mammalian tissues and cells 36 including rabbit alveolar macrophages. 7-9 This system catalyzes the transfer of fatty I R. M. Kramer and D. Deykin, J. Biol. Chem. 258, 13806 (1983). 2 R. M. Kramer, G. M. Patton, C. R. Pritzker, and D. Deykin, J. Biol. Chem. 259, 13316 (1984). P. V. Reddy and H. H. O. Schmid, Biochem. Biophys. Res. Commun. 129, 381 (1985). 4 y . Masuzawa, S. Okano, Y. Nakagawa, A. Ojima, and K. Waku, Biochim. Biophys. Acta 876, 80 (1986). 5 A. Ojima, Y. Nakagawa, T. Sugiura, Y. Masuzawa, and K. Waku, J. Neurochem. 48, 1403 (1987). 6 0 . V. Reddy and H. H. O. Schmid, Biochim. Biophys. Acta 879, 369 (1986). 7 T. Sugiura and K. Waku, Biochem. Biophys. Res. Commun. 127, 384 (1985). 8 M. Robinson, M. L. Blank, and F. Snyder, J. Biol. Chem. 260, 7889 (1985). 9 T. Sugiura, Y. Masuzawa, Y. Nakagawa, and K. Waku, J. Biol. Chem. 262, 1199 (1987).
METHODS IN ENZYMOLOGY,VOL. 209
Copyright© 1992by AcademicPress, Inc. All rightsof reproductionin any formreserved.
ACYLTRANSFERASES
[6]
[6] G l y c e r o p h o s p h a t e A c y l t r a n s f e r a s e f r o m L i v e r
By DIPAK HALDAR and ALES VANCURA Introduction Glycerol 3-phosphate acyltransferase (acyl-CoA : sn-glycerol 3-phosphate O-acyltransferase, EC 2.3.1.15) catalyzes the following reaction: sn-Glycerol3-phosphate + acyl-CoA--->1-acyl-sn-glycerol3-phosphate + CoA The enzyme converts water-soluble glycerophosphate to a lipid product and catalyzes the committed step in the biosynthesis of phosphoglycerides and triacylglycerols from glycerophosphate. The enzyme can divert fatty acids from/3-oxidation to esterification. Thus, the reaction repcesents a probable regulatory site in fatty acid metabolism. In mammalian organs, glycerophosphate acyltranferase is present in both the endoplasmic reticulum (microsomes) and mitochondrial outer membrane. 1'2 In liver the glycerophosphate acyltransferase activity is nearly equal in these two organelles, whereas in other organs the microsomal acyltransferase is approximately 10 times more active than the mitochondrial enzyme. 3 Assay
Principle. The enzyme activity can be measured by following the convcrsion of sn-[2-3H]glycerol 3-phosphate to l-butanol-extractable lipid.2,3 Reagents 0.20 M MES-TES-glycylglycine buffer, pH 7.5 [4-morpho-lineethanesulfonic acid, N-tris(hydroxymethyl)methyl-2-aminoethanesulfonic acid, and glycylglycine] 0.10 M MgCIz 0.72 mM Palmitoyl-CoA 0.10 M KCN 50 mg/ml Bovine serum albumin (BSA; fatty acid free) 20 mg/ml Asolectin (sonicated suspension) J L. N. W. Daae and J. Bremer, Biochim. Biophys. Acta 210, 92 (1970). 2 G. Monroy, F. H. Rola, and M. E. Pullman, J. Biol. Chem. 247, 6884 (1972). 3 D. Haldar, W.-W. Tso, and M. E. Pullman, J. Biol. Chem. 254, 4502 (1979).
METHODS IN ENZYMOLOGY,VOL. 209
Copyright © 1992by Academic Press, Inc. All fights of reproduction in any form reserved.
[6]
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7.5 mM sn-[2-3H]Glycerol 3-phosphate [~10,000 counts/min (cpm)/ nmol] Scintillation cocktail MES-TES-glycylglycine buffer 2 is prepared by dissolving appropriate amounts of each compound in water to a concentration of 0.4 M, adjusting the pH with KOH, and finally diluting the solution to 0.2 M. The concentration of palmitoyl-CoA is determined from the optical density of the solution at 232 and 260 nm using the millimolar extinction coefficients of 8.7 and 16.8, 2'4 respectively. An unsaturated fatty acyl-CoA can also be used as an acyl group donor in the glycerophosphate acyltransferase assay, but it should be freshly prepared to avoid oxidation. KCN is used to prevent oxidation of acyl-CoAs by mitochondria and should also be freshly prepared. It has no effect on glycerophosphate acyltransferase. Asolectin, a soybean phospholipid preparation, is suspended in 10 mM Tris-HCl, pH 7.5, and the mixture is exposed at 0° to sonic irradiation until a translucent suspension is obtained. Fifteen-second exposures with 15-sec intervals between exposures for a total exposure of 2 to 3 min with a Megason Ultrasonic Disintegrator (Ultrasonic Instruments International, Farmingdale, NY) at nine-tenths of its maximum output is adequate. The suspension is then centrifuged at 105,000 g for 10 min and the supernatant taken. The suspension can be stored at 4 ° for a period not exceeding 2 weeks, after which the phospholipid starts sedimenting. Radioactive glycerophosphate is commercially available but can also be prepared enzymatically from [2-3H]glycerol by phosphorylation with ATP in the presence of glycerokinase 5followed by purification.6 The specific activity of the radioactive glycerophosphate is expressed in cpm/nmol. Although the assay is carried out with 1.5 mM glycerol 3-phosphate for economic reasons, maximal activity of the acyltransferase is achieved at higher concentrations (7.5 to 15 mM glycerol 3-phosphate for mitochondria). Unless mentioned otherwise, all of the above reagents can be stored at - 20°.
Preparation of Subcellular Fractions Rat liver mitochondrial and microsomal fractions can be prepared following any standard procedure such as the one previously described. 2,7 An 8000 to 12,000 g intermediate fraction is discarded. The washed mitochondrial and microsomal fractions are suspended in 0.25 M sucrose (5-I0 mg subcellular protein/ml) and are either immediately used or divided into 0.5- to 1.0-ml aliquots and stored at - 70°. Storage under these conditions 4 E. 5 E. 6 E. 7 E.
Stadtman, this series, Vol. 3, p. 931. P. Kennedy, this series, Vol. 5, p. 476. E. Hill, D. R. Husbands, and W. E. M. Lands, J. Biol. Chem. 243, 4440 (1968). C. Weinbach, Anal. Biochem. 2, 335 (1961).
66
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results in only a little loss of activity over the course of 6 months. Protein is estimated by the method of Lowry et al. s using BSA as a standard. P r o c e d u r e . Reaction mixtures contain the following in a final volume of 0.5 ml: 40 mM MES-TES-glycylglycine buffer; 2 mM MgCI 2 ; an optimal concentration (20-100 /~M) of palmitoyl-CoA; 2 mM KCN; an optimal concentration (1-4 mg) of BSA; 0.2 mg asolectin; 1.5 mM sn-[2-3H]glycerol 3-phosphate; and 0.05 to 0.2 mg subcellular protein. The reaction mixture is preincubated for 3 min at 37° before the subceUular fraction is added, and the incubation is continued for another 3 min. The reaction is stopped by adding 0.5 ml of 1-butanol and vortexing the mixture. One milliliter of water is added, mixed well, and the mixture centrifuged for 5 rain at room temperature in a tabletop centrifuge. The bottom water layer is removed and may be stored and used later for recycling of the radioactive glycerophosphate. The butanol layer (upper) is washed with 1.5 ml of butanolsaturated water. To an aliquot (usually 0.1-0.2 ml) of the butanol is added a scintillation cocktail, and the mixture is counted. The specific activity of the enzyme is calculated on the basis of nanomoles of glycerophosphate acylated per minute of incubation in the presence of the enzyme per milligram of protein (nmol/min/mg). An endogenous control, incubated without palmitoyl-CoA, is subtracted from the experimental value. Typical values for freshly prepared rat liver mitochondria and microsomes, using palmitoyl-CoA as the acyl donor, are 3-5 and 4-6 nmol/min/mg, respectively. The reaction rate is rectilinear with time (up to 9 min) and subcellular protein (up to 2 mg/ml). The optimal concentration of palmitoyl-CoA presumably depends on the amount of total protein--subcellular fraction and BSA--and is lower for the mitochondria than for microsomes. The optimal concentration of BSA varied in our hands according to the source of the protein. Asolectin, although stimulatory to the glycerophosphate acyltransferase, is not essential for the assay and is usually omitted from the assay because of the complexities involved in the preparation and stability of the suspension of this soybean phospholipid mixture. Variations of the assay include the use of palmitoylcarnitine and CoA to generate palmitoyl-CoA.1 This assay works, but when an activator or inhibitor is used, one is not sure if the effect is on glycerophosphate acyltransferase or palmitoylcarnitine acyltransferase. In another variation, 9 chloroform-methanol (2: 1) is used instead of l-butanol to extract the lipids. However, chloroform-methanol shows some bias against lysophosphatidic acid, 2 which is the main product of mitochondrial incubation s O. H. Lowry, N. J. Rosebrough, A. L. Farr, and R. J. Randall, J. Biol. Chem. 193, 265 (1951). 9 H. Eibl, E. F. Hill, and W. E. M. Lands, Eur. J. Biochem. 9, 250 (1969).
[6]
GLYCEROPHOSPHATE ACYLTRANSFERASE FROM LIVER
67
under the above conditions. 2'3 Also, the butanol extraction procedure is somewhat more efficient. 1,2 The products of acylation of glycerophosphate under the above conditions are mainly lysophosphatidic acid and phosphatidic acid along with a small amount, if any, of mono- and diglycerides. The lipids can be separated by thin-layer chromatography on silica gel plates with a solvent system containing chloroform, methanol, acetic acid, and water (65 : 25 : I : 4). 2'1° An input of approximately 2000 cpm is adequate. The radioactivity of the lipids is determined by scraping 0.5 × 2 cm sections of each lane into mini-scintillation vials. The scrapings are soaked for 15 min in 0.2-ml portions of 10 mM EGTA, pH 4.6. Three milliliters of scintillation fluid is added and the mixture counted. Recovery of radioactivity from the plates is between 80 and 110%. Properties
pH Optimum and K m . Both the microsomal n and mitochondrial glycerophosphate acyltransferase exhibit a broad pH optimum between 6.6 and 9.0. The Km for sn-glycerol 3-phosphate for the microsomal enzyme is lower (0.1-0.2 mM) than that for the mitochondrial acyltransferase (~1 rnM). 2'11 Substrate and Position Specificity. In the glycerophosphate acyltransferase assay, the microsomes from liver as well as from other mammalian organs can use both saturated and unsaturated fatty acyl-CoAs with almost equal efficiency. Mitochondria, on the other hand, prefer saturated acylCoA thioesters.2 However, regardless of whether saturated or unsaturated acyl-CoA is used as acyl donor, 1-acyl-sn-glycerol 3-phosphate is the major if not the sole product of sn-glycerol 3-phosphate acylation. During the processing of the samples for determining positional specificity, approximately 6% isomerization of 1- to 2-monoacylglycerol and 20% conversion of the 2- to the 1-isomer have occurred. 3 The above properties of the mitochondrial glycerophosphate acyltransferase provide an excellent mechanism for the observed preferential positioning of saturated fatty acids in the sn-1 position and unsaturated fatty acids in the sn-2 position in naturally occurring acylglycerols. 2 This possibility is further supported by the facts that (1) in Ehflich cells, which exhibit an abnormally high proportion of unsaturated fatty acids at the sn-1 position of some phosphoglycerides, a mitochondrial glycerophosphate acyltransferase is undectable 3 and (2) in cell cultures, there is an agel0 V. P. Skipski and M. Barclay, this series, Vol. 14, p. 530. i~ S. Yamashita and S. Numa, this series, Vol. 71, p. 550.
68
ACYLTRANSFERASES
16]
dependent decline of mitochondrial glycerophosphate acyltransferase activity and a parallel increase in the amount of unsaturated fatty acids at the sn-1 position in choline phosphoglycerides. 12 Recently, experimental support of the above hypothesis has come from the observation that in the presence of BSA, mitochondrially produced monoacylglycerophosphate can exit the organelles, be translocated to the microsomes, and converted to diacylglycerophosphate. 13 Also, a monoacylglycerophosphate-binding liver cytosolic protein has been purified, which may be involved in the transport of mitochondrially made monoacylglycerophosphate to the endoplasmic reticulum.14 Activators and Inhibitors. Some divalent cations, such as Mg 2+ and Ca 2+, stimulate both the mitochondria115 and microsoma111 glycerophosphate acyltranferase, especially when the enzyme is partially purified (see later). Acetone 3 and polymyxin B 16 stimulate the mitochondrial but inhibit the microsomal acyltranferase. Sulfhydryl group reagents, such as N-ethylmaleimide and iodoacetamide, strongly inhibit the microsomal acyltransferase; the mitochondrial activity is unaffected by these reagents. 2'3 This distinguishing property of mitochondrial and microsomal glycerophosphate acyltransferase has been used to determine the microsomal contamination of the mitochondrial fraction 3'17or to assay the mitochondrial enzyme present in a whole cell homogenate. 12'18 Phospholipids modulate liver mitochondrial and microsomal glycerophosphate acyltransferase. Phosphatidylserine, asolectin, and phosphatidylcholine stimulate, whereas mono- and diacylglycerophosphate and cardiolipin inhibit the mitochondrial activity. 15Microsomes are stimulated by phosphatidylcholine and phosphatidylethanolamine but are inhibited by phosphatidylserine and phosphatidylinositol. H These results have been obtained from experiments performed with partially purified subcellular glycerophosphate acyltransferase. Among other lipids, monoacylglycerols and some of their analogs inhibit both the mitochondrial and microsomal glycerophosphate acyltransferase. 28 The liver microsomal glycerophosphate acyltransferase is inhibited by 12 W. Stern and M. E. Pullman, J. Biol. Chem. 253, 8047 (1978). 13 D. Haldar and L. Lipfert, J. Biol. Chem. 265, 11014 (1990). 14 A. Vancura, M. A. Carroll, and D. Haldar, Biochem. Biophys. Res. Commun. 175, 339 (1991). 15 G. Monroy, H. C. Kelker, and M. E. Pullman, J. Biol. Chem. 248, 2845 (1973). 16 M. A. Carroll, P. E. Morris, C. D. Grosjean, T. Anzalone, and D. Haldar, Arch. Biochem. Biophys. 214, 17 (1982). J7 R. J. Pavlica, C. B. Hesler, L. Lipfert, I. N. Hirshfield, and D. Haldar, Biochim. Biophys. Acta 1022, 115 (1990). I8 R. A. Coleman, Biochim. Biophys. Acta 963, 367 (1988).
[6]
GLYCEROPHOSPHATE ACYLTRANSFERASE FROM LIVER
69
all proteases. ~9'2° The mitochondrial enzyme, on the other hand, is not inhibited by trypsin and chymotrypsin. Non-site-specific proteases, such as proteinase K and subtilisin, inhibit the mitochondrial enzyme. The degree of inhibition increases if the ionic strength of the incubation medium is lowered. The protease sensitivity of the microsomal enzyme does not respond to any such changes in the ionic strength of the incubation medium. Exposure of trypsin- or chymotrypsin-sensitive domains of glycerophosphate acyltransferase on the inner surface of the mitochondrial outer membrane can be directly demonstrated by incubating trypsin-loaded outer membrane vesicles. 2° These results, taken together, suggest that the mitochondrial glycerophosphate acyltransferase spans the transverse plane of the outer membrane.
Purification Both microsomal and mitochondrial glycerophosphate acyltransferase have been only partially purified. In contrast, the Escherichia coli glycerophosphate acyltranferase has been completely purified, its properties studled, 21 and the gene cloned and sequenced. EEThe successful purification of the prokaryotic enzyme has been facilitated by massive overproduction of the enzyme owing to molecular c l o n i n g . E1
Microsomal Glycerophosphate Acyltransferase All operations are conducted at 0 ° to 5°. Rat liver microsomes are treated for 2 hr with 6 mM Triton X- 100 at pH 8.6 at a protein concentration of 10 mg/ml and then passed through a Sepharose 2B column. The first few fractions of the eluate which contain protein and the enzyme activity are combined and centrifuged through a sucrose gradient (0.5 to 1.5 M layered on 2 M). This treatment resolves the two enzyme activities: glycerophosphate acyltransferase and 1-acylglycerophosphate acyltransferase. However, the enrichment of glycerophosphate acyltransferase is only 3.6 times, with approximately 90% loss of activity. For a detailed account of this method, the reader is referred to an earlier volume in this series. H It is interesting to note that, unlike the microsomes, the partially purified enzyme prefers saturated over unsaturated fatty acyl-CoA thioesters as substrates. i9 R. A. Coleman and R. M. Bell, J. Cell Biol. 76, 245 (1978). 2o C. B. Hesler, M. A. Carroll, and D. Haldar, J. Biol. Chem. 260, 7452 (1985). 21 M. A. Scheideler and R. M. Bell, J. Biol. Chem. 264, 12455 (1989). 22 p. R. Green, T. C. Vanaman, P. Mordich, and R. M. Bell, J. Biol. Chem. 258, 10862 (1983).
70
ACYLTRANSFERASES
[6]
Mitochondrial Glycerophosphate Acyltransferase There are two methods available for the partial purification of the mitochondrial acyltransferase. All operations are carried out at 0 ° to 4 °. In the first method, 15 submitochondrial particles are prepared by sonic irradiation of an aqueous suspension of mitochondria (I0 mg/ml) for 10 min with a Branson Sonifier operating at maximum output. The particles are centrifuged down and finally suspended (10 mg/ml) for 10 rain in a mixture containing 0.25 M sucrose, 10 mM Tris-HCl, pH 8.0, 0.5% potassium cholate, pH 7.8, and 1.0 M KC1. The mixture is centrifuged at 150,000 g, and to the supernatant is added 0.35% asolectin suspension followed by the addition of a saturated solution of ammonium sulfate to a final concentration of 15%. After 10 min the mixture is centrifuged at 105,000 g for 15 min. The supernatant fluid is adjusted to 45% saturation with ammonium sulfate and centrifuged. The precipitate is dissolved in Tris-HCl, pH 8.0, containing 0.35% asolectin and dialyzed thoroughly against the same solution. This preparation represents 6-fold purification with no monoacylglycerophosphate acyltransferase activity. In the other method, 23 submitochondrial particles are prepared by suspending the mitochondria ( 10 mg/ml) in 20 mM Tris-HCl buffer, pH 8.4, containing I mM phenylmethylsulfonyl fluoride. The mixture is exposed to sonic irradiation for 4 min (30-sec exposures with 15-sec intervals) using a Megason Ultrasonic Disintegrator working at nine-tenths of its maximum output. The mixture is then centrifuged at 170,000 g for 90 min. The sediment containing the submitochondrial particles is suspended in a solution containing 0.25 M sucrose, 1 M KCI, 20 mM Tris-HCl buffer, pH 8.4, 1 mM phenylmethylsulfonyl fluoride, and 0.5% (ultrapure) Lubrol PX. The detergent-to-protein ratio is maintained between 2 and 4. After 30 min, the mixture is centrifuged at 170,000 g for 90 min. The supernatant fraction, containing most of the enzyme activity, is desalted on a Pharmacia (Piscataway, N J) PD-10 column equilibrated with 20 mM Tris-HC1, pH 8.4. The mixture is then loaded on Sepharose Q column (I .5 x 30 cm), equilibrated with 20 mM Tris-HC1, pH 8.4, containing 0.5% Lubrol PX and 10% glycerol. The column is eluted with the same buffer, and fractions containing the glycerophosphate acyltransferase activity are combined and loaded on a BioGel HT (Bio-Rad, Richmond, CA) column (1 x I0 cm) equilibrated with 20 mM Tris-HC1, pH 8.4, containing 0.5% Lubrol PX and 10% glycerol. The column is washed with 100 ml of the same buffer. Delipidated glycerophosphate acyltransferase is eluted from the column with 50 ml of a gradient of 20 to 500 mM potassium phosphate 2t A. Vancura and D. Haldar, manuscript in preparation.
[6]
71
GLYCEROPHOSPHATE ACYLTRANSFERASE FROM LIVER 0.5"
"6
-5
2
0.4 1
~.
i
A x
0.3"
-3
~
'1
~
.~
0.2
0.1
0.0
.
.
.
.
.
.
.
.
.
T
.
10
.
.
.
.
.
.
20 Fraction No.
30
40
FIG. 1. Purification of mitochondrial glycerophosphate acyltransferase on a BioGel HT column. The column was eluted at 14 ml/hr with a linear gradient of phosphate buffer (20-500 mM) (line 3), and 1.3-ml fractions were collected for determining absorbance at 280 nm (line 1) and glycerophosphate acyltransferase activity (line 2).
buffer, pH 7.4, containing 0.2% Lubrol PX and 10% glycerol (Fig. 1). From this step, determination of the glycerophosphate acyltransferase activity is completely dependent on reconstitution of the enzyme with asolectin suspension. Fractions exhibiting glycerophosphate acyltransferase activity are combined, supplemented with NaC1 to 0.5 M concentration, and loaded on an octyl-Sepharose CL-4B column (I x 5 cm), equilibrated with 20 mM Tris-HC1 buffer, pH 7.4, containing 0.2% Lubrol PX, 10% glycerol, and 0.5 M NaCI. The column is washed with 10 ml of the same buffer, and then the enzyme is eluted with 20 mM Tris-HC1, pH 8.4, containing 0.5% Lubrol PX and 10% glycerol. Results of a typical purification procedure are summarized in Table I. TABLE I PURIFICATION OF MITOCHONDRIAL GLYCEROPHOSPHATE ACYLTRANSFERASE
Step
Protein (rag)
Total activity (nmol/min)
Specific activity (nmol/min/mg)
Purification (-fold)
Yield (%)
Submitochondrial particles Lubrol PX extract Sepharose Q fast flow BioGel HT Octyl-Sepharose CL-4B
119.2 84.9 12.1 2.2 1.5
122.1 73.1 41.3 33.8 31.7
1.03 0.86 3.42 15.36 21.20
1.00 0.83 3.32 14.91 20.58
100 60 34 28 26
72
ACYLTRANSFERASES
[7]
Additional improvement in purification of mitochondrial glycerophosphate acyltranferase was achieved using affinity chromatography on palmityl-CoA-agarose or glycerol 3-phosphate-agarose. When a glycerol 3-phosphate agarose column was used instead of octyl-Sepharose CL-4B column, the overall purification of glycerophosphate acyltransferase was over 40-fold. The molecular weight of the native glycerophosphate acyltransferase was 60-85 kDa as determined by gel filtration on Sephacryl S-300 HR in 0.2% CHAPS. Comparison of this result with electrophoretic data strongly suggests the mitochondrial glycerophosphate acyltransferase to be a monomeric enzyme. SDS-PAGE of the purified glycerophosphate acyltransferase followed by Coomassie blue staining exhibited a single band with a molecular weight of 80-85 kDa. Acknowledgment This work was supported by a grant from the National Science Foundation (DCB8801535).
[7] C o e n z y m e A - I n d e p e n d e n t A c y l t r a n s f e r a s e
By TAKAYUKI SUGIURA and KEIZO WAKU Introduction The coenzyme A (CoA)-independent transacylation system was first described by Kramer and Deykin 1'2 for human platelets. Similar enzyme activity was also found for several mammalian tissues and cells 36 including rabbit alveolar macrophages. 7-9 This system catalyzes the transfer of fatty I R. M. Kramer and D. Deykin, J. Biol. Chem. 258, 13806 (1983). 2 R. M. Kramer, G. M. Patton, C. R. Pritzker, and D. Deykin, J. Biol. Chem. 259, 13316 (1984). P. V. Reddy and H. H. O. Schmid, Biochem. Biophys. Res. Commun. 129, 381 (1985). 4 y . Masuzawa, S. Okano, Y. Nakagawa, A. Ojima, and K. Waku, Biochim. Biophys. Acta 876, 80 (1986). 5 A. Ojima, Y. Nakagawa, T. Sugiura, Y. Masuzawa, and K. Waku, J. Neurochem. 48, 1403 (1987). 6 0 . V. Reddy and H. H. O. Schmid, Biochim. Biophys. Acta 879, 369 (1986). 7 T. Sugiura and K. Waku, Biochem. Biophys. Res. Commun. 127, 384 (1985). 8 M. Robinson, M. L. Blank, and F. Snyder, J. Biol. Chem. 260, 7889 (1985). 9 T. Sugiura, Y. Masuzawa, Y. Nakagawa, and K. Waku, J. Biol. Chem. 262, 1199 (1987).
METHODS IN ENZYMOLOGY,VOL. 209
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