P l a n t a 9 Springer-Verlag1991
Substrate specificities of the membrane-bound and partially purified microsomal acyl-CoA: 1-acylglycerol-3-phosphate acyltransferase from etiolated shoots of Pisum satirum (L.) Woifgang Hares and Margrit Frentzen* Institut ffir Allgemeine Botanik, Universitfit Hamburg, Ohnhorststrasse 18, W-2000 Hamburg 52, Federal Republic of Germany Received 21 February; accepted 15 April 1991
Abstract. Membrane fractions enriched in rough endoplasmic reticulum and not contaminated with plastidial membranes were isolated from etiolated shoots of Pi su m sativum (L.). F r o m these fractions the acyl-CoA:l-acylsn-glycerol-3-phosphate acyltransferase (EC 220.127.116.11) was solubilized by extracting the membranes with the zwitterionic detergent 3-[(3-cholamidopropyl)-dimethylammonio]-l-propanesulfonate at high ionic strength. The subsequent separation of the solubilized fractions on a M o n o Q column resulted in a tenfold enriched enzymic activity, which could be stabilized by polyethyleneglycol precipitation. A comparison of the substrate specificities and selectivities of the solubilized, enriched 1-acylglycerol-3-phosphate acyltransferase and the corresponding membrane-bound activity revealed no appreciable difference. Both enzymic forms specifically utilized acylCoA thioesters as acyl donors whereas the corresponding acyl-acyl carrier protein thioesters were not used. Furthermore, the membrane-bound as well as the solubilized enriched form showed not only higher activities with 1-oleoyl- than with 1-palmitoylglycerol-3-phosphate but also pronounced specificities and selectivities for unsaturated C18-CoA thioesters. Hence, the extraplastidial l-acylglycerol-3-phosphate acyltransferase which catalyses the formation of phosphatidic acid with an eukaryotic fatty-acid pattern was partially purified. Key words: Acyl-CoA:l-acylglycerol-3-phosphate acyltransferase - Endoplasmic reticulum - Eukaryotic fatty acid pattern - Membrane protein purification - Pisum Introduction Glycerol-3-phosphate and l-acylglycerol-3-phosphate acyltransferase (LPA-AT), which catalyse the stepwise * To whom correspondence should be addressed Abbreviations." ACP = acyl carrier protein; CHAPS = 3-[(3-cholamidopropyl)-dimethylammonio]-l-propanesulfonate; LPA-AT = acyl-CoA:l-acylglycerol-3-phosphate acyltransferase; PEG = polyethyleneglycol
acylation of glycerol-3-phosphate in the course of denovo biosynthesis of glycerolipids, are located in different subcellular compartments of plant cells, namely in plastids, mitochondria, and microsomes, mainly in the E R (for a review, see Frentzen 1986). In all compartments the L P A - A T is firmly bound to the membranes (Joyard and Douce 1977; Vick and Beevers 1977; Sparace and Moore 1979; Block et al. 1983; Andrews et al. 1985; Sauer and Robinson 1985; Hares and Frentzen 1987; Ichihara et al. 1987; Alban et al. 1989; Frentzen et al. 1990). However, distinct differences between the isofunctional enzymes have been observed with respect to their fatty-acid specificities and selectivities (Frentzen et al. 1983, 1990; Griffith et al. 1985; Hares and Frentzen 1987; Ichihara et al. 1987). These experiments strongly indicate that the LPA-ATs of the different compartments play a crucial role in determining whether glycerolipids with prokaryotic or eukaryotic fatty-acid patterns are formed (Frentzen 1986; Frentzen et al. 1990). So far, the properties of the L P A - A T s of the different compartments have been investigated by using subcellular fractions as enzyme source. Furthermore, in the case of the microsomal L P A - A T the plastidial envelope membranes present in microsomal fractions can interfere with the determination of its substrate specificities (Hares and Frentzen 1987). Hence, in order to elucidate the substrate dependencies of the microsomal L P A - A T devoid of critical contaminations, we have started to purify this enzymic activity from microsomal fractions of etiolated pea shoots and separated it from the interfering plastidial L P A - A T . The solubilization and partial purification of this L P A - A T as well as a comparison of certain properties of the solubilized enriched and the membrane-bound enzymic form are presented.
Materials and methods Chemicals. [1-14C]Palmitoyl-CoA(1.92 GBq "mmol-1), [l_14C]stea_ royl-CoA (2.04 GBq. mmol-1), sn-[U-14C]glycerol-3-phosphate (6.29 GBq. mmol-1), [l-14C]palmitic acid (2.15 GBq" mmol-1),
W. Hares and M. Frentzen: Eukaryotic acyltransferase [l-14C]oleic acid (2.07 G B q . m m o l - l ) , UDP-o[U-14C]galac tose (11.8 GBq - mmol - 1) and UDP-D[U-t4C]glucose (8.62 GBq-mmo1-1) were purchased from Amersham Buchler (Braunschweig, FRG). [l-14C]Oleoyl-CoA (1.99 GBq" mmo1-1) and [1-x4C]linoleoyl-CoA (1.82 GBq- mmol- t) came from DuPont (Bad Homburg, FRG). 1-Oleoyl-sn-[U-14C]glycerol-3-phosphate was synthesized from oleoyl-CoA (Sigma, Deisenhofen, FRG) and [U-t4C]glycerol-3 phosphate with purified glycerol-3-phosphate acyltransferase, from pea chloroplasts according to Bertrams and Heinz (1981). [1-14C]Palmitoyl- and [1-14C]oleoyl-acyl carrier protein (ACP) were synthesized from ACP (Sigma) and the corresponding labelled fatty acid by acyl-ACP synthetase (Sigma), and subsequently purified as described by Rock and Garwin (1979). Fine chemicals and enzymes came from Sigma if not stated otherwise.
Plants. Pea seeds (Pisum sativum L. ev. Kleine Rheinl~inderin; Zwaan Pannevis, Kleve, FRG) were soaked overnight in running tap water, sown in vermiculite and grown for 6 d in the dark at 20~ C.
Membrane preparation. Six-day-old etiolated pea shoots were ground in a precooled mortar with homogenization buffer (1 ml 9g FW -~) consisting of 0.1 M Tris-HCl pH 7.7, 0.3 M sucrose, 3 mM MgCIz and 1 mM dithiothreitol. The homogenate was filtered through a nylon mesh (200 ~tm pore size) and spun for 15 min at 10 000 9g. The resulting supernatant was layered on top of a cushion of 60% (w/w) sucrose and centrifuged for 1 h at 100 000 9g in an SW-28 rotor (Beckmann, Miinchen, FRG). The membranes concentrated at the sucrose interphase were collected, layered on top of a linear sucrose gradient (2245% (w/w) sucrose in homogenization buffer) and centrifuged for 3 h at 100 000 99 (SW-28 rotor). For marker-enzyme measurements the gradients were fractionated into 1-ml portions with an Auto Densiflow (Haake Buchler, Saddle Brook, N.J., USA) connected to a peristaltic pump. For the solubilization and purification of the microsomal LPA-AT, the membranes which zoned as a distinct band at a density of about 1.17 g 9cm-3 were collected, diluted with 50 mM Tris-HC1 pH 7.7 and sedimented by high-speed centrifugation (100 000 .g, 1 h). The resulting pellets were resuspended in 50 mM Tris-HC! pH 7.7 containing 50% glycerol and 10mM dithiothreitol and stored at -- 20~ C. Protein was assayed as described by Bradford (1976). The analysis of the lipid composition of the membrane fractions was carried out as described by Haschke et al. (1990).
Solubilization. For standard solubilizations, four volumes of membrane fraction were mixed with one volume of a stock solution of 2% (w/v) 3-[(3-cholamidopropyl)-dimethylammonio]-l-propanesulfonate (CHAPS) in 2.5 M NaCI to give a final detergent-toprotein ratio of 1.5 (w/w). After a 15-min incubation at 20~ C an aliquot was taken as a control and solubilization mixtures were centrifuged for 1 h at 130 000 - g. The resulting supernatant fractions, termed detergent extracts, were carefully removed and used for further experiments. Column chromatography. The detergent extract was desalted on prepacked Sephadex G-25 columns (PD-10; Pharmacia, Freiburg, FRG), which had been equilibrated with elution buffer (0.3 % (w/v) CHAPS, 25 mM Tris-HC1 pH 7.7, 10 mM dithiothreitol, 20% (w/v) glycerol, 0.02% (w/v) NAN3). The desalted extracts were applied to a Mono Q HR 5/5 analytical column (Pharmacia). After rinsing the column with elution buffer to remove the unbound material, it was eluted with a linear gradient mixed from 7.5 ml of elution buffer and 7.5 ml of 0.7 M NaC1 in elution buffer (flow rate 1 ml 9min-~), and l-ml fractions were collected. Peak fractions of LPA-AT activity were combined and concentrated by polyethyleneglycol (PEG) precipitation. To this end the fractions were adjusted to 20% (w/v) PEG 6000 (Serva, Heidel-
125 berg, FRG) stirred for 15 min on ice and centrifuged for 45 min at 200 000"g. The pellets were resuspended in 0.3% CHAPS, 40% glycerol, 50mM Tris-HC1 pH 7.7, 0.02% NaN3 and stored at - 20~ C.
Enzyme assays. Acyl-CoA:l-acylglycerol-3-phosphate acyltransferase was assayed as described before (Hares and Frentzen 1987). For the determination of the substrate specificities and selectivities of the LPA-AT, assays consisted of 30 mM 3-(cyclohexylamino)-1propanesulfonic acid-NaOH pH 10, up to 5 lag protein and substrate concentrations as indicated in the figures in a final volume of 80 lal. After 2-min incubations at 25 ~ C the reaction was terminated and lipophilic products were extracted as described by Hajra (1974). The analysis of the reaction products and their fatty-acid composition was carried out as described before (Bertrams and Heinz 1981). Glucan synthase II (EC 18.104.22.168) was assayed as described by Depta et al. (1987). The incorporation of UDP-glucose into lipophilic products was also analysed by extracting the ethanol layer of glucan-synthase-I assays with CHCI3 and 0.48 % NaC1 and separating the concentrated organic layer on precoated silica gel G plates (Merck, Darmstadt, FRG) in chloroform/methanol (80/20; v/v). This solvent system was also used to analyse the reaction products from UDP-galactose incubations which were carried out as described by Douce (1974). NADPH:cytochrome-c oxidoreductase (EC 22.214.171.124) was measured according to Douce et al. (1973). Results and discussion
Isolation o f microsomalfractions not contaminated with plastidial membranes. The m i c r o s o m a l L P A - A T is p r e d o m i n a n t l y located in the E R (Hares a n d F r e n t z e n 1987) whereas the c o r r e s p o n d i n g plastidial activity is firmly b o u n d to the envelope m e m b r a n e s ( J o y a r d a n d D o u c e 1977). Hence, in o r d e r to characterize the mic r o s o m a l activity a n d to separate it f r o m the interfering plastidial one, a t t e m p t s were u n d e r t a k e n to o b t a i n m e m b r a n e fractions enriched in E R a n d n o t c o n t a m i n a t e d with plastidial m e m b r a n e s . T o this end, crude m i c r o s o m a l fractions f r o m etiolated pea shoots were separated o n linear sucrose gradients in the presence o f MgC12. After c e n t r i f u g a t i o n , three m e m b r a n e b a n d s were detectable in the g r a d i e n t (Fig. 1B) while a l m o s t all o f the L P A - A T activity was recovered in the b a n d which z o n e d at a d e n s i t y o f 1.17 g" c m -3 a n d which displayed highest N A D P H : c y t o c h r o m e - c o x i d o r e d u c t a s e activity, too (Fig. 1A). I n this b a n d , g l u c a n synthetase II a n d sterol glycosyltransferase activities, k n o w n e n z y m i c activities o f p l a s m a m e m b r a n e s ( H a r t m a n n - B o u i l l o n et al. 1979; D e p t a et al. 1987), were also detectable. H o w e v e r , this b a n d displayed n o activity o f the diacylglycerol g a l a c t o s y l t r a n s ferase (Fig. 1B), a m a r k e r e n z y m e o f plastidial envelope m e m b r a n e s ( J o y a r d a n d D o u c e 1987). E v e n after conc e n t r a t i n g the m e m b r a n e s o f this b a n d by high-speed centrifugation, an incorporation of UDP-galactose into galactolipids was n o t observed, These results indicate that the m e m b r a n e fractions o f the sucrose g r a d i e n t c o n t a i n e d E R a n d p l a s m a m e m b r a n e - d e r i v e d vesicles b u t n o plastidial envelopes. This was c o n f i r m e d b y the analysis o f the lipid comp o s i t i o n o f this f r a c t i o n which, as depicted in Fig. 2, consisted o f p h o s p h o l i p i d s , free sterols, a n d the glycolipids characteristic o f p l a s m a m e m b r a n e s a n d t o n g -
W. Hares and M. Frentzen: Eukaryotic acyltransferase
J _;/ !
r q _ . ~ . Azl
. m 1
Fig. 1A, B. Distribution of various enzymic activities along the linear sucrose gradient (22-45% (w/w)) used for the separation of crude microsomal fractions of etiolated pea shoots. A LPA-AT (e), NADPH :cytochrome-c oxidoreductase (), B glycan synthetase II (v), sterol glycosyltransferase (*), diacylglycerol galactosyltransferase (9 protein concentration (...) t,0-
SteHGO DGO AS6 Cer $8
PG PE PA PS
Fig. 2. Lipid composition of the microsomal fraction purified on the sucrose gradient shown in Fig. l (Ste, free sterols; MGD, monogalactosyldiacylglycerol; DGD, digalactosyldiacylglycerol; ASG, acylated steryl glycoside; Cer, cerebroside; SG, steryl glycoside; PC, phosphatidylcholine; PI, phosphatidylinositol; PG, phosphatidylglycerol; PA, phosphatidic acid; PS, phosphatidylserine)
plasts (Verhoek et al. 1983; Yoshida and Uemura 1986; Lynch and Steponkus 1987; Rochester et al. 1987; Haschke et al. 1990), namely cerebroside, steryl glycoside and acylated steryl glycoside. However, galactolipids, the predominant polar lipids o f plastids, were not found. In summary, the marker-enzyme measurements as well as the analysis o f the membrane lipids clearly re-
vealed that the microsomal fractions isolated from etiolated pea shoots were not contaminated by plastidial membranes. Thus, a separation of the microsomal L P A - A T from the interfering plastidial activity was achieved. In these fractions most of the L P A - A T activity was localized in the E R membranes since the activity peak of this enzyme was shifted to a lower density in the sucrose gradient when MgC12 was omitted throughout the separation procedure as described before (Hares and Frentzen 1987). These fractions were used on the one hand as an enzyme source to determine the properties of the membrane bound L P A - A T and on the other hand as a starting material for the enzyme purification. Solubilization of the L P A - A T . In order to purify the microsomal L P A - A T , sotubilization experiments were carried out by extracting the membrane fractions with various detergents and salts. Best results were obtained with the zwitterionic bile-salt derivative CHAPS at high ionic strength. As shown in Fig. 3A, without the addition of NaC1, CHAPS extracted less than 10% of the L P A - A T activity from the membranes whereas the solubilization yields were substantially increased when the extractions were carried out with CHAPS at high ionic strength. In the presence of 0.5 M NaC1, a CHAPS concentration of about 0.4% (w/v) (Fig. 3A) and a detergent/ protein ratio of about 1.5 (Fig. 3B) were found to be optimal. Under these conditions about 60% of the microsomal L P A - A T activity and about 70% of the microsomat protein could usually be recovered in the detergent extract, so that it showed slightly lower specific activities than the microsomal fraction (Table 1). Higher detergent concentrations or higher detergent/protein ratios, however, resulted in an irreversible inhibition of the L P A - A T activity and, therefore, in lower activity yields in the detergent extracts although the percentage of the solubilized protein was further increased up to 80 % of the microsomal protein (Fig. 3). In the solubilized fractions the L P A - A T activity was stable for at least 1 d when stored at 4 ~ C. Purification o f the L P A - A T. The detergent extracts were subsequently used for column-chromatography experiments. Of the various types of column materials tested, anion-exchange chromatography on a Mono Q column was found to be most efficient for the initial purification step. As shown in Fig. 4, the L P A - A T activity eluted in two peaks from this column. One part (peak I) of the activity was eluted at about 0.3 M NaC1 along with most of the applied protein whereas the other part (peak II) was released from the Mono Q column at a distinctly higher ionic strength and behind the main protein peak. As a result of this elution pattern, the peak-I fraction displayed twofold lower, but the peak-II fraction tenfold higher specific activities of the L P A - A T than the desalted detergent extract (Table 1). The rechromatography of each of the peak fractions on the Mono Q column revealed that the L P A - A T activity of the peak-II fraction was eluted in only one peak at high ionic
W. Hares and M. Frentzen: Eukaryotic acyltransferase