Expression and properties of acyl-CoA binding protein from Brassica napus

Plant

Physiol.

Biochem.,

1998,

36 (9), 629-635

Expression and properties of acyl-CoA binding protein from Brassica napus Adrian P. Brown’, Philip Johnson, Stephen Rawsthorne, Matthew

J. Hills*

John Innes Centre, Norwich Research Park, Colney, Norwich, NR4 7UH, UK. *Author to whom correspondence should be addressed (fax +44-1603 259882; e-mail [email protected]) (Received March 24,1998;

accepted June 6,1998)

Abstract - Expression and characteristics of an acyl-CoA binding protein (ACBP) from Brussica napus L. were examined. A cDNA encoding an ACBP from rape was over-expressed in E. coli and the resulting protein (rACBP) was purified by ammonium sulphate precipitation followed by gel filtration chromatography. SDS-PAGE showed the protein to be greater than 99 % pure and to have a molecular weight of approximately 10 kDa. Lipidex-1000 competition assays showed that the rACBP purified by this method is fully functional, binding both oleoyl- and palmitoyl-CoA in the ratio 1 mol protein:1 mol acyl-CoA. Native isoelectric focusing revealed the presence of two isoforms of rACBP in ammonium sulphate and gel-filtration purified preparations. These isoforms were separated by chromatofocusing and shown to differ in mass by 131 Da, the mass of one methionine residue. Both of these isoforms bound palmitoyl-CoA with similar affinities. Antibodies raised to purified rACBP were used to study tissue specific and developmental expression of ACBP. Western blots revealed the presence of ACBP in all tissues examined and the level of expression was similar in many of those tissues. The amount of ACBP was not, however, strongly correlated with rates of lipid biosynthesis during embryo development nor with lipid degradation during seedling germination. The addition of rACBP stimulated microsomal glycerol-3-phosphate acyl-transferase (GPAT, EC 2.3.1.15) activity in in vitro assays, but concentrations in excess of a 1: 1 ratio of ACBP:oleoyl-CoA caused decreases in GPAT activity. 0 Elsevier, Paris Acyl-CoA

I acyl-CoA

binding

protein

(r)ACBP, (recombinant)acyl-CoA dodecyl sulphate-polyacrylamide

I acyltransferase

/ lipids I Brassica

binding protein / GPAT, glycerol-3-phosphate acyltransferase gel electrophoresis / HPLC, high pressure liquid chromatography

1. INTRODUCTION

Acyl-CoA binding proteins (ACBPs) are small (ca. 10 kDa) proteins which bind long-chain acyl-CoAs but not fatty acids or CoA. They are structurally quite different from the plant lipid transfer proteins which are involved in transport of a wide range of lipidic substrates [2]. ACBP was first purified from bovine liver by virtue of its ability to stimulate goat fatty acid synthetase in in vitro assays [ 121 and is likely to be ubiquitous in eukaryotes. It has since been identified in, and the genes encoding it cloned from, a number of plant sources including Brussica napus [9] and Arubidopsis thaliana [5]. Where ACBPs have been localized, all were soluble cytosolic proteins. Long chain acyl-CoA and ACBP have been suggested to play a role in regulation of both enzyme activity and gene expression. In vitro studies have shown that long chain acyl-CoAs regulate amongst others acetyl-CoA ’ Present

address:

Plant Physiol.

Department

Biochem.,

napus

of Biological

Sciences,

098 I -9428/98/09/O

Elsevier,

University

Paris

of Durham,

/ SDS-PAGE,

sodium

carboxylase (ACCase), the AMP kinase kinase and the FadR protein in E. coli [6], all of which are enzymes and proteins involved in the regulation of lipid biosynthesis. Although these enzymes have not been shown to be inhibited by long chain acyl-CoA in vivo, it is possible that long chain acyl-CoA regulates its own synthesis by inhibition of these or other proteins. In both these cases, ACBP could relieve inhibition of enzyme activity by binding long chain acylCoA. In addition, ACBP can protect long chain acylCoA from long chain acyl-hydrolases in vitro [5]. ACBP has been shown to donate long chain acyl-CoA to microsomal membranes for glycerolipid synthesis or for g-oxidation in mitochondria derived from rat liver [15]. These observations suggest that ACBP is involved in the formation of a stable long chain acylCoA pool in the cytosol. In rat liver, the concentration of long chain acyl-CoAs corresponds closely with that of ACBP which implies that the concentration of free Durham,

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3LE,

UK.

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long chain acyl-CoA is likely to be quite low [6]. On the other hand. experiments in yeast show that the acyl-CoA pool was actually increased in a strain in which the ACBP gene had been ablated [19]. Long chain acyl-CoA occupies a central point in lipid metabolism and its possible importance as a general regulator of metabolism has recently been discussed [6]. Little information on the role of ACBP in plant tissues during lipid biosynthesis is available. In plants, fatty acids are synthesized by the plastid but little is known on the mechanism of acyl-chain export from the plastid to the cytosol and their conversion to long chain acyl-CoAs. During storage lipid or phospholipid synthesis, long chain acyl-CoAs are used to sequentially acylate a glycerol-3-phosphate backbone via the Kennedy pathway [ 131. In order to investigate roles of long chain acyl-CoA and ACBP in plant metabolism, we have over-expressed in E. coli a cDNA encoding ACBP cloned from B. napus embryos [9]. Recombinant, purified protein was used to raise antibodies to determine expression of ACBP present in different tissues and developmental stages. In vitro studies of the effect of ACBP on glycerol-3-phosphate acyltransferase activities (the first enzyme of the Kennedy pathway of glycerolipid synthesis) are also presented. 2. RESULTS AND DISCUSSION 2.1. Expression

of Brassica ACBP in E. coli

The rACBP from cleared E. coli lysate was purified by ammonium sulphate precipitation and gel filtration chromatography. The majority of rACBP precipitated from solution in the 80-100 % ammonium sulphate fraction. This fraction was approximately 95 % pure rACBP, as estimated by SDS-PAGE, and was carried over for further purification by gel filtration to obtain rACBP which was over 99 % pure. The concentration of rACBP protein was estimated by hydrolysis of the purified protein followed by HPLC analysis of the amino acids according to standard protocols. This concentration was compared with that estimated by dye binding assay [3] using thyroglobulin as a standard. The protein estimation using the dye binding assay was only 62 % of that determined by the amino acid analysis. This was not unexpected since protein estimations using the Bradford assay vary considerably depending on the protein being determined. Since some of the experiments required an accurate determination of the absolute amount of protein, estimations of rACBP protein concentration made using the Plant Physiol.

Biochem

dye binding assay were adjusted accordingly. The yield of rACBP following purification was typically 1O-15 mg.L-’ culture depending on the preparation. 2.2. Characterization

of the rACBP

The purified rACBP was analysed by IEF under denaturing conditions followed by western blotting @gure 1, lane 1). As expected, this revealed the presence of one major isoform of rACBP with a p1 of 5.2. This is close to the estimated p1 of 5.3 derived from the ACBP sequence [9]. Unexpectedly, however, IEF of recombinant protein under native conditions revealed two isoforms with p1 values of 4.9 and 5.15 @gure I, lanes 2-4). Both forms were present in approximately equal amounts but it was not clear whether the difference was due to differences in folding of the same polypeptide or whether an error in translation of processing had occurred. Electrospray mass spectroscopy (EMS) of the purified rACBP showed the presence of 2 proteins with masses of 10 038.6 Da and 10 170.3 Da (data not shown). The difference is equivalent to the mass of one methionine residue raising the possibility that a proportion of the over-expressed protein was not being processed during protein synthesis in E. co/i. To confirm this, the two proteins were separated by chromatofocusing on a Mono-P column with a pH gradient from 5.5-4.5 (data not shown). This yielded one peak of rACBP with a mass, estimated by EMS, of 10 038.6 Da and the second with a mass of 10 170.3 Da. The pl values for the two proteins were found to be 4.9 and 5.15, respectively. The two proteins were subjected to N-terminal sequencing and the protein with a mass of 10 170 Da

1

Figure 1. Western from B. napus. Z-4, native IEF focusing purified focusing purified

2 3 4

blot of isoelectric focusing gels of purified rACBP 1, denaturing IEF of gel filtration purified rACBP: gel of gel filtration purified rACBP(2), chromatorACBP with terminal methionine (3) and chromatorACBP lacking terminal methionine (4).

Acyl-CoA binding protein from Brassica napus

was found to begin with MGLK whereas the other with a mass of 10039 Da began with GLKD. This confirmed that the rACBP was incompletely processed during its synthesis. Since methionine residues do not carry a charge, it could be speculated that the change in p1 caused by the presence of an N terminal methionine is due to masking of a charged residue or, less likely, a conformational change in the protein structure. 2.3. Binding

of long chain acyl-CoA by ACBP

It was previously shown that rACBP from Arubidupsis bound long chain acyl-CoA, but the binding shown in that study was relatively weak [5]. Binding of radiolabeled oleoyl- and palmitoyl-CoA by rACBP from rape was determined using the Lipidex binding assay as described previously [14]. In this case, binding of long chain acyl-CoA saturated at concentrations of long chain acyl-CoA close to the ratio of 1 mol protein: 1 mol acyl-CoA at saturating acyl-CoA concentrations (figure 2). The binding constants of the rACBP for oleoyl-CoA and palmitoyl-CoA were calculated to be 6.9 x 10m6 and 1.5 x 10e5, respectively, by least squares regression analysis of Scatchard plots of the data. These results are consistent with binding constants obtained for rat ACBP [14] using the Lipidex method. It should be noted, however, that the Lipidex competition assay does not give absolute binding constants but binding relative to the affinity of Lipidex1000 [ 141. The binding of palmitoyl-CoA to both rACBP isoforms which had been purified by chromatofocusing was very similar and confirmed that the presence of the N-terminal methionine did not detect-

0.2 0

1 [Acyl-CoAl

Figure 2. Binding of palmitoyl-CoA rACBP from B. napus determined Methods.

2

3

@MI

(0) and oleoyl-CoA (0) by Lipidex method detailed

by in

ably affect the affinity (data not shown).

631

of the protein for its ligand

2.4. Expression of ACBP and isoform composition in different tissues of Brassica napus The amount of ACBP in a range of tissues of Brassica napus was assessed by western blots made with SDS-PAGE of 50 yg of proteins in cleared extracts @gut-e 3 B). For comparison, a serial dilution of rACBP (ng protein) is given in fisure 3 A. The blot shows that the antibody was monospecific for the ACBP in all of the tissues examined. ACBP was detected in all of the tissues examined and the level of ACBP varied between tissues over about a four-fold range on a protein basis. The amount of ACBP in each band was estimated by densitometry since the standards showed a linear response with respect to colour development on the blot. The amount of ACBP was similar in most tissues including those which are known to synthesize lipids at high rates, such as developing seeds, petals and expanding leaves. It varied between 1.3 pg.mg-’ protein in buds and 2.3 pg.mg-’ protein in young leaves. ACBP concentrations in cotyledons of seedlings and anthers were a little lower at 1.0 and 0.7 yg.mg-’ protein, respectively. These data are broadly similar to those reported recently for a more limited range of B. napus tissues [5]. Having studied nine tissues, it is clear that ACBP is likely to be expressed constitutively in plants, though the absolute amount of protein varies to a relatively small degree between tissues.

Figure 3. Western blot showing ACBP in 50 ,ug of cytosolic protein isolated from a range of tissues of B. nopus. A, Dilution series of rACBP in ng. B, Tissues: E, embryo; C, cotyledon from 4-d seedlings; B, 4 mm long buds; P, petals; A, anthers; yL, young leaves; mL, mature leaf; S, shoot from flowering region; R, root. The entire gel is presented with prestained molecular mass standards shown on the left from 8-206 kDa.

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123456789

EC

B

P

A

yL mL

S

R

Figure 4. The isoform composition of ACBP from a range of tissues was determined by isoelectric focusing followed by western blotting as described in Methods. A pH gradient from 4 to 6 was used and the blot is oriented with the alkaline side at the top. E, embryo; C. cotyledon from 4-d seedlings; B, 4 mm long buds; P, petals; A, anthers; yL, young leaves; mL, mature leaf; S, shoot from flowering region; R, root.

It had been shown previously that B. napus contains 6 ACBP genes [9] so it was possible that one or more of these genes was expressed differentially in different tissues. The ACBP isoform composition was assessed using isoelectric focusing under denaturing conditions (figure 4). One isoform predominated in all of the tissues (except for mature leaves) but there were two relatively minor and more alkaline isoforms present in developing seeds and cotyledons of 4-d seedlings. These alkaline forms were also present in some other tissues but at much lower levels. Both young and mature leaves lacked the alkaline forms but contained two more acidic isoforms which were faintly detectable in buds and stems. The ACBP isoform which predominated in most tissues was almost undetectable in mature leaves, where only the acid isoforms were present. 2.5. Changes in ACBP during and germination

seed development

The amount of ACBP in extracts of embryos taken at nine stages of development from torpedo stage to desiccated seeds was assessed on western blots (figure 5). The amount of ACBP increased from the torpedo stage onwards and was highest during the mid to late cotyledonary stages when lipids are synthesized at the highest rates. The amount of ACBP detected during these latter stages was similar to that reported by Engeseth et al. [5]. However, in the present study, the amount of ACBP detected at early stages of embryo development was very much higher than that reported previously [5] and overall, we observed much smaller changes in ACBP during embryo developPlant

Ph ysiol.

Biochrm.

Figure 5. ACBP content during seed development. Fifty pg of cytosolic protein isolated from developing seeds of B. napus isolated at a number of stages following anthesis were separated by SDS-PAGE, blotted and probed with I:4 000 dilution of antisera raised against rACBP. I, torpedo stage; 2, late torpedo; 3, early cotyledon; 4, mid cotyledon (2.1 mg fresh weight): 5, mid cotyledon (2.9 mg); 6. late cotyledon; 7 and 8, stages during seed desiccation: 9, dry seed.

ment. At later stages of seed development during the desiccation phase, the amount of ACBP per embryo fell to almost undetectable levels. Since oil synthesis has ceased by this stage [lo], the loss of ACBP is not surprising. Following seed germination storage lipid is broken down quite rapidly and after three days, the amount of lipid in the cotyledons is only about 50 % that of the dry seed (figure 6). However, although flux of lipid through catabolism is at its most rapid over this period, the amount of ACBP is low and it only becomes expressed at high levels when lipid oxidation is almost complete. Although it is correlative evidence, it would appear that ACBP may not play an important role in lipid oxidation. This may not be unexpected since ACBP is a cytosolic protein and fatty acid oxidation occurs in the glyoxysome. 2.6. Effect of ACBP on microsomal glycerol-3-phosphate acyltransferase

activity

Although concentrations of long chain acyl-CoA and ACBP in B. napus have not yet been determined, it is likely that the situation will be similar to that found in animals or yeast where the concentrations of both tend to be closely matched [6]. In order to assess the possible effect of ACBP on acyl-CoA metabolism, microsomal glycerol-3-phosphate acyltransferase (GPAT) was assayed in reactions containing a range of ratios of [‘4C]-oleoyl-CoA to ACBP @gure 7). At each of the [‘4C]-oleoyl-CoA concentrations used, the addition of ACBP stimulated acyltransferase activity up to a point but at an ACBP concentration above that of [‘4C]-oleoyl-CoA, the GPAT activity was inhibited. These results differ to an extent from those found for acyltransferases in rat liver where even the presence of low concentrations of ACBP in the reaction mixture caused inhibition of acyltransferase activity [ 161. This inhibition of the rape microsomal GPAT was by as much as 90 % when the ratio free ACBP:oleoyl-CoA bound

Acyl-CoA binding protein from Brassica napus

1

0.0 1 0

2

0

1

4

2

8

6

3

4

6

10

8

12

11

Figure 6. ACBP and lipid content of cotyledons following seed germination. A, Total lipid was extracted from cotyledon pairs in duplicate samples and quantified as described in Methods. B, Total protein from the equivalent of 1 cotyledon pair excised at various days after germination was separated by SDS-PAGE, blotted and probed with 1:4 000 dilution of antisera raised against rACBP. The lane numbers refer to the days after germination.

633

CoAs so strongly at an ACBP concentration much above equimolar with that of long chain acyl-CoA, the free long chain acyl-CoA concentration will be very low. In this circumstance, the acyltransferase is most likely to receive the acyl-CoA substrate directly from the ACBl? It can therefore be speculated that free ACBP at higher concentrations will compete with the acyl-CoA-ACBP thereby reducing the activity of the acyltransferase. The role of ACBPs in plants (and indeed other organisms) is not entirely clear. Studies in yeast have shown that knocking out the ACBP gene makes the cells much less competitive when co-cultured with wild type, but otherwise they grow well on their own [ 191. Interestingly, it appears that the pool of acyl-CoA actually increased by about 2-fold in the knockout yeast and expression of the stearoyl-CoA desaturase gene was also affected. Whether or not ACBP is required for growth and development of plants, it is certainly present at relatively high levels in plant tissues (0.14.2 % of cell protein). It should be noted that an Arabidopsis gene encoding a membrane bound protein with a domain with high homology to ACBP has been reported [4]. In addition, an Arabidopsis Expressed Sequence Tag (GenBank accession T04081) encoding a protein with almost 50 % amino acid sequence similarity to ACBP (including conservative substitutions) shows that there may well be a number of soluble and membrane bound acyl-CoA binding proteins in plants. Knockout or antisense experiments in the future may help us to understand the role of ACBP(s) in plants. 3. CONCLUSION

0

20

40

60 IACBPI

Figure 7. Glycerol-3-phosphate formed as described in Methods. 10 uM (A), 50 pM (0) and 100 pM assays were done in triplicate and dard error.

80

100

120

(jiMI

acyltransferase assays were per[‘4C]-oleoyl-CoA was included at (m) and ACBP up to 120 pM. The results are shown as mean + stan-

ACBP was 12: 1. It is not immediately clear why increasing the ACBP concentration causes progressive inhibition of acyltransferase activity. An explanation may arise from the high binding affinity of the ACBP and its possible role in delivery of long chain acylCoAs to enzymes. Since ACBP binds long-chain acyl-

Acyl-CoA binding proteins are present in a wide range of plant tissues and at similar concentrations on a protein basis. They bind acyl-CoAs very strongly and with affinities similar to the animal and yeast homologs. The amount of ACBP did not correlate strongly with the rate of lipid metabolism in developing embryos or cotyledons of seedlings, but was almost undetectable in desiccating seeds. The exact role of ACBP in plants is not yet clear and may be manifold. It was previously shown to protect acylCoAs from the action of thioesterase [5] and in this study, it was shown that the ACBP is likely to deliver acyl-CoAs to glycerol-3-phosphate acyltransferase. 4. METHODS 4.1. Plant material. were

grown

Plants in the glasshouse

of

Brussica at day

and

napus night

L. cv Topas temperatures

vol. 36 (9) 1998

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A.P. Brown et al.

of 18 “C and 12 “C, respectively; 16 h of supplementary lighting was provided from October to March. Siliques were harvested and kept on ice whilst the developing embryos were excised from the testae using a razor blade.

4.2. Expression of ACBP in E. coli. B. napus ACBP was over-expressed in E. coli using the vector pET15b from Novagen (Abingdon, UK). The open reading frame from plasmid Bn411 [9] was amplified by PCR using the following primers. N-terminal primer: 5’-CTACGAATTEATGGGTTTGAAGG-3’ (NcoI site underlined), C-terminal primer: 5’.TACGGACATGGATCCGCACTAATAGATCACC-3’ (BamHI site underlined). The PCR conditions used were 20 cycles at 94 “C for 40 s, 42 “C for 1 min and 72 “C for 2 min. The 321 bp product was digested with Ncol and BamHI and the resulting fragment ligated into suitably prepared pET15b. Expression of ACBP was done in E. coli strain BL21(DE3) according to the suppliers’ instructions. Cultures were grown in 2YT medium [17] at 37 “C to an OD,, of approximately 0.5. After addition of 0.5 mM IPTG, they were incubated for a further 3 h before harvesting and lysis of the bacteria following Novagen instructions. 4.3. Purification

of recombinant

ACBP. Cultures

were chilled on ice for 20 min before harvesting and all further steps were carried out at 4 “C or on ice. Bacteria were collected by centrifugation at 4 000 x g for 10 min and washed in 100 mM NaCl, 10 mM Tris-HCl pH 8.0, 1 mM EDTA. After recentrifugation, the pellet was resuspended in lysis buffer containing 50 mM Tris. HCI pH 8.0, 1 mM EDTA, 1 mM DTT (50 mL per 2 L bacterial culture) containing Complete Protease Inhibitor Cocktail tablets from Boehringer Mannheim (GmbH Germany). Bacteria were lysed by sonication for three periods of 45 s (7 mW peak to peak amplitude) with 30 s gaps in between to allow cooling and the lysate was cleared by two centrifugations at 12 000 x g for 10 min. The initial stage of purification of the recombinant ACBP (rACBP) was by (NH&SO, precipitation in steps of 20 % up to 100 % saturation. Protein precipitates were collected by centrifugation at 10 000 x g for 10 min after each addition of (NH&SO,. The precipitate from the SO-100 % saturation step was resuspended in buffer A (100 mM NaCl, 20 mM Tris-HCl pH 8.0). ACBP was further purified by gel filtration using a HiLoad 16/60 column (bed volume 120 mL) containing Superdex 75 and using an FPLC machine from Pharmacia (St Albans, UK). The sample volume was 5 mL and column was eluted at 1 mL.min-’ with buffer A. Fractions containing ACBP (analysed by Coomassie blue staining of protein gels) were pooled and frozen in 0.5 mL aliquots before storage at -80 “C. A portion of the ACBP purified by gel filtration was further resolved by chromatofocusing. The ACBP sample was exchanged from buffer A to buffer B (25 mM Bis-Tris-HCl pH 6.3) using a PD- 10 column (Pharmacia) following instructions from the manufacturer. Samples containing up to 3 mg ACBP were loaded onto a Mono P HR 5/20 column from Pharmacia equilibrated in buffer B and protein eluted with 80 mL buffer

Plant

Physiol.

Biochem.

C (5 mL Polybuffer 74 diluted to 100 mL pH 4.0, HCI) at a flow rate of 0.5 mL.min-’

4.4. Analysis of ACBP. Proteins were dialysed into 1 mM Tris-HCl pH 6.8 and diluted to a concentration of 0.5 mg.mL-’ in this buffer before analysis by electrospray mass spectrometry. Three samples of 20 pL were injected into a Fisons Micromass VG platform, Quadrupole mass spectrometer as previously described. The amino acid content of ACBP was determined by first dialysing the sample against pure water and then dilution to a concentration of 1 mg.mL-’ as estimated by Bradford assay [3] using thyroglobulin as a standard. Samples of 50 pL from the same stock ACBP solution were then hydrolysed in 6 N HCl, under nitrogen, for 24 h before analysis using an LKB 4 15 1 amino acid analyser according to the manufacturers’ instructions. 4.5. Raising antibodies against recombinant ACBP. Antibodies were raised standard procedures neous injection of adjuvant followed Freunds incomplete

in rabbits against the rACBP following [7]. The rabbit was given 1 sub-cuta0.5 mg of rACBP in Freunds complete by 3 injections of 0.5 mg rACBP in adjuvant at monthly intervals.

4.6. SDS-PAGE, isoelectric focusing and immunobiotting. Proteins were separated by SDS-PAGE using a Mini-Protean dual slab cell (Bio-Rad, Hemel Hempstead, UK) and the buffer system described by Schagger and von Jagow 118). Electrophoresis was performed with 1.5 mm slab gels containing 16 % polyacrylamide (w/v) according to manufacturers instructions. Proteins were transferred from the gel to nitrocellulose (Hybond C, 0.45 pm, Amersham, UK.) using Mini-Trans-blot electrophoretic transfer cell (Bio-Rad, UK) according to the manufacturers’ instructions. The blot was probed with a 1:8 000 dilution of rabbit serum raised against the E. coli expressed rACBP following the procedure of Lacey and Hills [ 1 I] and binding was revealed using alkaline phosphatase conjugated goat anti-rabbit IgG (Sigma, Poole, UK). Isoelectric focusing gels were run according to the method described by Bollag and Edelstein I l] with a pH gradient from 4 to 6. The gels were western blotted and the blots treated as described above. 4.7. Lipid Extraction. Lipids were extracted from cotyledon pairs isolated (in duplicate) from seedlings at a number of days following seed imbibition by the method previously described [8]. Total lipid was quantified gravimetrically following removal of solvent from the sample.

4.8. Determination of binding constant. Binding of [ 1-14CJpalmitoyl-CoA and [ l-‘4C]-oleoyl-CoA to recombinant ACBP was determined by the method of Rasmussen et al. [ 141. The rACBP (0.2 FM) and varying concentrations of [ l-‘4C]-acyl-CoA were incubated at 37 “C for 30 min in binding buffer (10 pM KH,PO,, pH 7.4) in a final volume of 200 pL. Samples were then chilled on ice for 10 min and mixed with 400 pL of ice cold Lipidex 1000 (Packard Inc., Meridian CT, USA) in binding buffer (50 % v/v). After 10 min incubation on ice, samples were centrifuged at 12 000 x g for 5 min at 0 “C. Radiolabeled acyl-CoA bound

Acyl-CoA

to ACBP was measured by scintillation counting of 200 pL of the resulting supernatant. For each acyl-CoA concentration the results indicate the mean and standard error of 6 individual experiments. Blank assays containing no ACBP and varying concentrations of [ 1-‘4C]-acyl-CoA were performed to confirm that all acyl-CoA remained bound to Lipidex in the absence of ACBP. 4.9. Glycerol-3-phosphate acyltransferase assay. Microsomal membranes were prepared from about 200 embryos by grinding in 10 mM KCl, 5 mM EDTA, 2 mM DTT, 100 mM Hepes/NaOH pH 7.5 and 0.24 M sucrose. After centrifugation at I.5 000 x R for 15 min, the supernatant was spun at 300 000 x g for 30 min and the pellet resusupended in a small volume of grinding buffer (at about 10 mg.mL-’ protein). The GPAT assay buffer contained 0.1 mM 18: l-CoA, 0.5 mM [‘“Cl-G3l? 8 mM MgCl,, 60 mM KH*PO,/NaOH pH 7.5 and varying amounts of ACBP. The label was added at I .85 kBq per 200 pL assay/time point. Samples were incubated IO min at 30 “C. Reactions were stopped by addition of 0.7 ml CHCl,/MeOH ( 1: I, v/v) and stored on ice till addition of I mL KCI/H,PO, (0.7 %/0.2 M) and separation of the phases. The chloroform phase was transferred to a scintillation vial, dried (nitrogen stream) and chloroform-soluble radiolabeled lipids measured after addition of scintillant. Acknowledgements. We thank Doug Hobbs and Denis Murphy for their helpful comments on the manuscript. We thank Ian Moss at the Advanced Biotechnology Centre, Charing Cross and Westminster Medical School, for amino acid determination and Rick Evans-Gowing (Department of Chemistry, University of East Anglia) for help with electrospray mass spectrometry. DNA sequencing was done by Julia Bartley at the University of Durham. This work was supported by the competitive strategic grant to the JIC from the Biotechnology and Biological Sciences Research Council, UK and by a John Innes Foundation Studentship to P.J.

REFERENCES [l] Bollag D.M., Edelstein S.J., Protein Liss, New York, 199 1.

Methods,

Wiley-

[2] Bourgis F., Kader J.-C., Lipid-transfer proteins: tools for manipulating membrane lipids, Physiol. Plant. 100 ( 1997) 78-84. ]3] Bradford M.M., A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding, Anal. Biothem. 72 (1976) 248-254. [4] Chye M.-L., Cheung K.-Y., Membrane associated acyl Co-enzyme A binding protein from Arubidopsis thaliuna, ISPMB 5th International Congress, Singapore, 1997, Abstract 699. [5] Engeseth N.J., Pacovsky R.S., Newman T., Ohlrogge J., Characterization of an Acyl-CoA-Binding Protein from

binding

protein

from Brassica napus

635

Arabidopsis thaliana, Arch. Biochem. Biophys. 33 1 (1996) 5.5-62. [6] Faergemann N.J., Knudsen J., Role of long-chain fatty acyl-CoA esters in the regulation of metabolism and in cell signalling, Biochem. J. 323 (I 997) I -I 2. [7] Harlow E., Lane D., Antibodies: A Laboratory Manual. Cold Spring Harbor Laboratory Press. Cold Spring Harbor, NY. 1988. [S] Hills M.J., Mukherjee K.D., Triacylglycerol lipase from rape (Brussica napus) suitable for biotechnological purposes, Appl. Biochem. Biotechnol. 26 (I 990) I -I 0. [9] Hills M.J., Dann R., Lydiate D., Sharpe A., Molecular cloning of a cDNA from Brassica nupus L. for a homologue of acyl-CoA-binding protein, Plant Mol. Biol. 25 ( 1994) 9 17-920. [IO] Kang F., Ridout C.J., Morgan CL., Rawsthorne S., The activity of acetyl-CoA carboxylase is not correlated with the rate of lipid synthesis during development of oilseed rape (Brassica nupus L.) embryos, Planta 193 ( 1994) 320-325. [I l] Lacey D.J., Hills M.J., Heterogeneity of the endoplasmic reticulum with respect to lipid synthesis in developing seeds of Brassica napus L., Planta 199 (1996) 545-55 1 [ 121 Mogensen LB., Schulenberg H., Hansen H.O., Spener F.. Knudsen J., A novel acyl-CoA binding protein from bovine liver, Biochem. J. 241 (1997) 189-192. [ 131 Ohlrogge J., Browse J., Lipid Biosynthesis, Plant Cell 7 (1995) 957-970. [ 141 Rasmussen J.T., Boerchers T., Knudsen J., Comparison of the binding activities of acyl-CoA-binding protein and fatty-acid-binding protein for long chain acyl-CoA esters, Biochem. J. 265 (I 990) 849-855. 1151 Rasmussen J.T.. Faergemann N.J., Kristiansen K.. Knudsen J., Acyl-CoA-binding protein (ACBP) can mediate intermembrane acyl-CoA transport and donate acyl-CoA for R-oxidation and glycerolipid synthesis, Biochem. J. 299 (1994) 165-170. [ 161 Rasmussen J.T., Rosendal J., Knudsen J., Interaction of acyl-CoA binding protein on processes for which acylCoA is a substrate, product or inhibitor. Biochem. J. 292 (1993) 907- 913. [I71 Sambrook J., Fritsch E.F., Maniatis T., Molecular Cloning: A Laboratory Manual, 2nd ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY. 1989. [I81 Schagger H., von Jagow G., Tricine-sodium dodecyl sulphate polyacrylamide gel electrophoresis for the separation of proteins in the range I to 100 kDa, Anal. Biochem. I66 (1987) 368-379. [19] Schjerling C.J., Hummel R., Hansen J.K., Borsting C., Mikkelsen J.M., Kristiansen K., Knudsen J., Disruption of the gene encoding the acyl-CoA-binding protein (ACB 1) perturbs acyl-CoA metabolism in Succharomyccs crrevisiue, J. Biol. Chem. 271 (1996) 225 1422521.

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