Glycerolipid biosynthesis by microsomal fractions from olive fruits

Phytochemistry, Vol. 31, No. 1. pp. 129-134, 1992 Printedin Great Britam.

GLYCEROLIPID

0031.9422/92 $5.00+0.00 G 1991 PergamonPress plc

BIOSYNTHESIS BY MICROSOMAL OLIVE FRUITS

JUAN SANCHEZ, M. TERESA DEL CUVILLO

and JOHN L.

FRACTIONS

FROM

HARWOOD*

Instituto de la Grasa, C.S.I.C.,Av. P. Garcia Tejero 4,41012 Sevilla, Spain; *Department of Biochemistry, University of Wales, P.O. Box 903, Cardiff CFl IST, U.K. (Received Key Word Index-Oh

europea;

4 April 1991)

Oleacea; olive fruits; biosynthesis; glycerolipids; triacylglycerols.

Abstract-Microsomal fractions prepared from maturing olive (Olea europea cv Picual) fruits incorporated label from both [‘4C]palmitoyl-CoA and [i4C]oleoyl-CoA into several glycerolipids, including triacylglycerols. The preparations contained high acyl-CoA thioesterase activity which was inhibited by Mg2’ and bovine serum albumin. Incorporation into glycerolipids was strongly stimulated by Mg2+, whereas addition of ATP was inhibitory; a low stimulation by glycerol-3-phosphate was observed. Under all conditions used oleoyl-CoA was a better substrate for glycerolipid synthesis than palmitoyl-CoA, whereas the latter seemed to be a better substrate for the acyl-CoA thioesterase.

INTRODUCTION

Much work has been done on the formation of glycerolipids in oilseeds [ 11. As a result it is well documented that synthesis of storage triacylglycerols (TAG) in such oleaginous organs takes place according to the Kennedy pathway, glycerol-3-phosphate is acylated at positions sn-1 and sn-2 in a stepwise manner by acyl-CoAs, yielding phosphatidic acid (PA). This is in turn dephosphorylated to render diacylglycerol (DAG), which is eventually acylated at the sn-3 position to form TAG. In contrast to the wealth of available information regarding oilseeds, very little work has been carried out with oleaginous fruits, such as avocado, palm and olive, despite the fact that these crops have a high commercial value. The reason for this seems to reside in the fact that fruits in the right stage of development are not as readily available as seeds, because they grow on trees and mature more slowly than seeds. In addition, the presence of high amounts of polyphenols and tannins in the mesocarp of some of these fruits, particularly olives, makes it difficult to prepare active cell free extracts. Olives are the source of the most important edible vegetable oil of many Mediterranean countries and, therefore, a crop of paramount importance for them. Knowledge of enzymatic processes responsible for the formation of TAG in olives should be the basis for future programme designed to improve oil quality and yield. We describe the preparation of microsomes from maturing olive fruits capable of incorporating radiolabelled acylCoAs into glycerolipids, among them TAG. RESULTS

Previous attempts to prepare active subcellular fractions from maturing olives, using grinding media of different compositions, resulted in a rapid browning of the extract and a concomitant enzyme inactivation. As the browning process was likely to be elicited by the

oxidation of polyphenols present in the fruits, several polyphenol oxidase inhibitors (KC& sodium metabisulphite), polyphenol scavengers (PVPP), antioxidants (ascorbate) and thiol reagents (DTE, /I-mercaptoethanol) have been tested in the grinding buffer. Although the presence of these ingredients in the medium proved to be effective in preventing the browning of the resulting extract, the subcellular fractions obtained displayed very little or no activity when assayed for glycerolipid synthesis from [i4C]-acyl-CoA. However, addition of glycerol, together with other protectants, allowed the preservation of enzyme activity in the resulting microsomal fraction. Olive microsomes were capable of rapid metabolism of the [14C]acyl-CoA precursors (Fig. 1). The distribution of label among acyl classes differed according to the nature of the radiolabelled substrate used, which is likely to reflect the acyl specificity of enzymes involved in the biosynthesis of glycerolipids. Thus, label from [i’C]palmitoyl-CoA appeared mostly as non-esterified fatty acid (NEFA), whereas that from [‘4C]oleoyl-CoA was more actively incorporated into glycerolipids. This may reflect either a high acyl-CoA thioesterase activity against palmitoyl-CoA or a comparatively higher activity of the acyltransferases for oleoyl-CoA. Effect

of cofactors

The effect of the presence of different cofactors in the incubation mixture is shown in Table 1. In the absence of cofactors the acyl-CoA substrates are mostly hydrolysed to NEFA, with the apparent activity being higher for palmitoyl-CoA. Even under these conditions, however, a significant part of the label from both palmitoyl- and oleoyl-CoA was incorporated into glycerolipids, indicating the existence of endogenous acceptors in the microsomal preparation. Addition of Mg2+ resulted in a severe reduction in the proportion of label in NEFA and a significant increase of 129

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cially in the case of oleoyl-CoA. The action does not appear simply to be due to chelation of Mg2+. Contrary to what might have been expected, addition of DL-glycerol-3-phosphate (G3P) only elicited a small stimulation in the formation of glycerolipids and optimal conditions for glycerolipid synthesis were met when Mg2+ and G3P were present together in the incubation medium. In this context it is noteworthy to point out that experiments carried out under the same conditions but using 14C-G3P as the labelled precursor, gave rather low incorporation rates into glycerolipids (data not shown). Bovine serum albumin (BSA) has been routinely used in assays of acyltransferase activity to prevent the detergent effect of acyl-CoAs as well as to protect these substrates against thioesterase activity. We have observed that in the absence of any other cofactor, BSA certainly protects acyl-CoAs against hydrolase activity (as judged by the low percentage of label in NEFA) but the incorporation into glycerolipids was also severely impaired when compared to controls. This adverse effect on acyltransferase activity by BSA is well known [2] and is probably due to the tight binding of acyl-CoAs to albumin [3]. Substrate concentration curve

L

Fig. 1. Incorporation of [14Clpalmitoyl-CoA (A) and [‘~oleoyl-CoA (B) into dilkent acyl lipids. The incubation mixture contained 2.5 mM ATP, 0.5 mM CoA, 5 mM MgCl,, 2.5 mM DL-glycerol-3-phosphate, 44 nM ‘T-acyl-CoA and microsomes equivalent to 0.5 mg protein ml-‘. Incubation was carried out at 30” for 7.5 min. Under these conditions 33% of the label added as palmitoyl-CoA and 44% of that from oleoyl-CoA were recovered in the organic extract, which was then chromatographed on silica gel with petrol-Et,O-HCO,H (66:33:1). Chromatograms were analysed using a radio TLC-linear analyser.

the radioactivity associated with water-soluble products (acyl-CoAs). This effect of Mg2+ may be through an inhibition of acyl-CoA thioesterase activity directly or because they lead to changes in membrane structure and aggregation. On the other hand, the presence of Mg2+ in the medium stimulated the incorporation of both precursors into glycerohpids, an effect which was especially noticeable in the TAG fraction. When ATP was added at the same concentration as Mg2+, both in the absence and in the presence of CoA, then a severe inhibition in the incorporation of acyl-CoAs into glycerolipids was observed, with most of the label remaining in the aqueous fraction. This suggests either inhibition of acyl-CoA thioesterase activity, or resynthesis of acyl-CoA from the released fatty acids catalysed by acyl-CoA synthetase, stimulated by the presence of ATP and CoA. It seems likely that ATP exerts its major effect through stimulation of acyl-CoA resynthesis, espe-

Variations in the concentration of acyl-CoA substrates resulted in sign&ant changes in the pattern of distribution of label among acyl classes. The results (Fig. 2) show that increasing concentrations of acyl-CoA in the incubation medium mainly affect the accumulation of label in NEFA, that is, thioesterase activity. As incubations were carried out in the presence of added ATP and CoA, rather small proportions of the labelled substrates were incorporated into TAG (l-2%). Similar curves were obtained in another experiment performed in the absence of added cofactors, where higher percentages of incorporation into TAG were observed (results not shown). In both experiments (Fig. 2) the proportion of label incorporated into glycerolipids was reduced by increasing concentrations of acyl-CoAs, which might be due to the detergent effect of these acylthioesters. Time course The variations in the distribution pattern of label with time were analysed in a time-course experiment (Fig. 3). Under the conditions used, over 50% of both radiolabelled precursors were metabolised in 15 min and a plateau was reached after 30 min. As indicated above the distribution of label among different products depended on the nature of the precursor. Thus, label from [ “C]palmitoylCoA is mainly channelled to NEFA, which probably reflects thioesterase activity, whereas that from [ 14C] oleoyl-CoA is mostly incorporated into glycerolipids, indicating that this acylthioester is a better substrate for the acyltransferases. This agrees with data for other oil tissues where, for example, transfer of acyl groups into phosphatidylcholine (PC) is very active for oleate [l]. Efect of microsomal concentration Increasing amounts of microsomal preparation in the incubation mixture resulted in an increase in the incorporation of radioactivity into glycerolipids with a concomitant decrease in the label associated with water-soluble

Glycerolipid biosynthesis

131

Table 1. Effect of cofactors

Precursor

Addition

Palmitoyl-CoA

None Mgs+ Mg2+ +ATP Mgs+ +ATP+CoA G3P Mgs+ +G3P Mg2+ +G3P+ATP+CoA BSA None Mg2+ Mg2+ +ATP Mg2 + + ATP + CoA G3P Mg2+ +G3P Mg2+ +G3P+ATP+CoA BSA

Oleoyl-CoA

Aqu*

Distribution of label (nmol hr-‘mgg’) PC DAG NEFA TAG

60 169 233 237 50 159 191 280 52 98 232 240 43 102 164 248

20 31 tr tr 21 34 tr 9 52 84 5 5 56 85 8 29

2

2 3 2 tr 5 5 3 tr 5 4 6 1

195 71 62 59 199 82 100 12 131 54 31 30 126 44 74 3

0th

2 4

14 11

2 6 tr 1 4 9 .4 10 tr tr

15 15 tr 2 18 22 3 tr 30 27 15 2

-

Incubations were carried out at 30” for 1 hr in the presence of microsomes equivalent to 0.25 mg proteinml-‘, and where stated 5 mM MgCI,, 5 mM ATP, 1 mM CoA, 5 mM DL-glycerol-3-phosphate (G3P) and 6 mg/ml bovine serum albumin (BSA). The concentration of acyl-CoA precursors was 75 PM. *Abbreviations: Aqu, watersoluble products; PC, phosphatidyl choline; DAG, diacylglycerol; NEFA, non-ester&d fatty acid, TAG, triacylglycerol; Oth, other lipids in organic extract.

products (acyl-CoAs) and NEFA (Table 2). These incubations were carried out for one hr so that the patterns

represent the final balance of products (Fig. 3). Thus, it appears that high rates of incorporation into glycerolipids require high protein concentrations, whereas thioesterase activity is somewhat favoured by lower amounts of protein. C PE IO

Eflect

ofpH

The formation of TAG from either [r4C]palmitoylCoA or [14C]oleoyl-CoA showed a strong pH dependence (Table 3), with the highest activity at pH ca 8, whereas incorporation into the other acyl fractions was not so much affected by pH.

lo

E DAG

DISCUSSION

fecyl -CoAl

pM

Effect of concentration of acyl-CoA substrates on incorporation into different acyl fractions. The reaction mixture contained 2.5 mM ATP, 0.5 mM CoA, 2.5 mM MgCl,, 2.5 mM DLglycerol-3-phosphate and microsomes equivalent to 0.54 mg protein ml-‘. Incubations were carried out at 30” for 1 hr. Open circles correspond to [t4C]palmitoyl-CoA and full circles to [‘~]oleoyl-CoA. Notice the difference between scales of the ordinate axes.

The results presented here show that microsomes prepared from maturing olive fruits are active in the formation of different glycerolipids, including TAG, from labelled acyl-CoAs. For the first time we have been able to define reaction conditions which permit good rates of labelling of glycerolipids in such preparations. Microsomal preparations displayed a high acyl-CoA thioesterase activity, which is in agreement with the observations made in preparations from other oleaginous fruits, such as avocado ([4], Eccleston, V. S. and Harwood, J. L., unpublished results) and palm [S]. This activity has been found to be located in the chloroplast envelope of spinach [6] and pea [7] and in both cases it was reported to be highly stable. Our results suggest that this thioesterase activity in olives is higher for palmitoylCoA than for oleoyl-CoA, which agrees with the results of

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both Sambanthamurthi and 00 for palm [S] and Joyard and Stumpf for spinach [6], and is apparently inhibited by Mg2+ ions, which is in agreement with the results of Andrews and Keegstra [7] for pea leaves. Increasing concentrations of acyl-CoA substrates, on the other hand, enhanced the accumulation of label in NEFA in a way which seems to reflect a stimulatory effect on the acylCoA thioesterase (Fig. 2A); this effect might be due to the

60

GL

Fig. 3. Time-course of incorporation of [‘4C]pahnitoyl-CoA (A) and [‘4C]oleoyl-CoA (B) into non-esterified fatty acids (NEFA) and giycerolipids (GL). Aqu represents radioactivity in water-soluble products. The incubation mixture contained 2.5mM ATP, 0.5mM CoA, 2.5mM MgCl,, 2.5mM DLglycerol-3-phosphate, 45 PM [‘4C]acyl-CoA and microsomes equivalent to 0.52 mg proteinml-‘.

solubilisation of the enzyme induced by the detergent-like properties of acyl-CoAs. In this regard it has been reported that acyl-CoA thioesterase can be readily solubilized by detergent from pea microsomes, a treatment that resulted in a large increase of activity [S]. There are many, and sometimes contradictory, reports on the effect of Mg ‘+ ions on the formation of storage TAG by oil seeds. Glycerophosphate acyltransferase from castor bean depended on the presence on divalent cations [9], but Mg ‘+ showed little effect on the same activity from safflower [lo]. Mg2+ was reported to stimulate lysophosphatidate acyltransferase from palm and maize, but was found to inhibit that from rapeseed [ 111. Diacylglycerol acyltransferase from safflower was inhibited by Mg2 ’ [ 121, but it was found to activate that of spinach leaves [13]. Finally phosphatidate phosphohydrolase from safflower has been reported to be highly dependent on Mgz+ [14-161. Our results (Table 1) show a clear increase in the incorporation of label from both [‘4C]palmitoyl-CoA and [‘“Cl oleoyl-CoA into glycerolipids, which was most obvious in the case of TAG. However, it is not possible to identify the specific enzyme steps affected by Mg ‘+ in our experiments. There was no accumulation of any Kennedy pathway intermediates so that the total flux of carbon through the pathway did not seem to be affected by a particular stimulation of the glycerophosphate acyltransferases. However, DAG acyltransferase and/or phosphatidate phosphohydrolase may have been increased, thus allowing the observable rise in TAG labelling. Addition of ATP, which has been reported to stimulate glycerophosphate acyltransferase of Escherichia coli [17], resulted in severe inhibition of the incorporation of label into glycerolipids under the conditions used. The formation of glycerolipids from acyl-CoAs in olive microsomes seems to a large extent to be independent of

Table 2. Effect of microsomal concentration

Precursor 16:0-CoA

18: I-COA

Microsomes (pg prot)

Aqu*

PC

34 68 136 310 34 68 136 310

15 12 10 9 29 22 14 10

11 12 21 32 14 30 41 56

Distribution of label (%) NEFA DAG TAG 71 70 61 47 53 40 34 20

0th

tr 1 2 3 1 2 3 4

Incubations were carried in 250 4 containing 2.5 mM ATP, 0.5 mM CoA, 2.5 mM MgCl, and 2.5 mM glycerol-3-phosphate. Radiolabelled precursors were added at a concentration of 40 PM. *Abbreviations as in Table 1.

133

Glycerolipid biosynthesis Table 3. Effect of pH. Distribution of label (nmol hr - ’ mg- ’ ) Aqu* PC DAG NEFA

Precursor

PH

Palmitoyl-CoA

5.8 6.5 7.2 7.9 8.6

39 21 34 22 31

24 30 21 34 21

1 1 2 4 3

Oleoyl-CoA

5.8 6.5 7.2 7.9 8.6

63 55 36 41 40

71 65 61 85 92

4 3 5 9 10

TAG

0th

93 90 68 61 73

1 2 5 13 9

5 11 24 18 16

54 60 52 36 40

3 4 13 16 12

13 21 35 22 17

Incubation mixtures contained 2.5 mM ATP, 0.5 mM CoA, 2.5 mM MgCl,, 5 mM DLglycerol-3-phosphate and microsomes equivalent to 0.3 mg proteinml-i. The concentration of labelled precursors were 50 PM for palmitoyl-CoA and 65 PM for oleoyl-CoA. *Abbreviations as in Table 1.

the presence of added G3P (Table 1) and the precursor [14C]G3P was poorly incorporated into glycerolipids (results not shown). This suggests that acyl-CoAs might also be channelled to TAG via a route other than the Kennedy pathway. One possibility is the incorporation of acyl-CoA into phosphatidylcholine via acylCoA: lysophosphatidylcholine acyltransferase, followed by conversion of the phosphatidylcholine thus formed into DAG, catalysed by cholinephosphotransferase. The first activity, which is well documented in plants [18], would account for the high incorporation of label into PC observed in our experiments, and is consistent with the enhancement in the incorporation into PC induced by increasing amounts of microsomes (Table 2), which would provide the acceptor for acylation. To summarise, high rates of TAG synthesis in olive microsomes require the presence of MgZ+, G3P and high concentrations of microsomal protein; the optimal pH for the reaction is close to 8 and the concentration of acylCoA substrates should be adjusted to the amount of microsomes in the reaction medium, in order to avoid adverse detergent effects. The successful development of this in vitro system should be of great use in further studies on the regulation of TAG synthesis in this important edible oil crop. EXPERIMENTAL PIant material. OIea europea cv. Picual fruits used in expts were taken from 20-yr-old trees, growing in a grove near Seville, which had been provided with drop irrigation before blossoming Expts described here were performed in the period from September to November, when fruits were 22-30 weeks after flowering. Harvesting of fruits was routinely done early in the morning and the olives were utilized for expts within 1 hr. Preparation ofmicrosomes. Fr. fruits were thoroughly rinsed with H,O and then wiped with paper tissue. Fruits (50 g) were ground in 200 ml of a buffer containing 25 mM Hepes, pH 7.2, 330 mM sorbitol, 1 mM EDTA, 10 mM KCl, 5 mM Na metabisulphite, 1 mM DTE, 12 mM b-mercaptoethanol, 0.1% BSA, 0.1% ascorbic acid, 10% glycerol and 1% PVPP. Grinding was done in a precooled glass domestic blender for 30 set and the

homogenate filtered through two layers of Miracloth. The filtrate was centrifuged at 4300 g for 10 min. The floating layer formed was carefully removed by filtration through a pad of glass wool and the resulting filtrate was centrifuged at 27 Ooo g for 20 min. The supernatant from the last centrifugation was subsequently centrifuged at 100000 t_~for 80 min. The microsomal pellet so obtained was resuspended in a small vol (l-2ml) of 50 mM Hepes, pH 7.2, 330 mM sorbitol, 1 mM DTE using a glass homogeniser. Assays. Incubations were carried out in buffered mixts consisting of 35 mM Hepes, pH 7.2, 300 mM sorbitol and 0.5 mM DTE, as well as the additions stated in the legends of Tables and Figs. Reactions were allowed to proceed for 1 hr at 30” with constant shaking. The [Y]acyl-CoA substrates used in the assays were adjusted to a sp act of 12 000 dpm nmol-i by mixing [i4CJpalmitoyl-CoA (59 Cimol-i) or [WJoleoyl-CoA (54 Ci mol-‘) (Amersham) with suitable amounts of the corresponding non-labelled acyl-CoA (Sigma). Analyses. Incubations were stopped by addition of 2Ovols (usually 5 ml) of petrol-iso-PrGH (3:2) to extract lipids according to ref. [19]. After sepn of phases, aliquots were taken from both the aq and organic layers to assay for radioactivity. The lipid extract, after evapn of solvent under a stream of N,, was then submitted to TLC on precoated silica gel plates using two solvent systems run successively in the same direction. First the plate was developed with petrol-Et&-HCO,H (20:80: 1) up to 2 cm below the top edge; then the plate was thoroughly dried with N, and the left edge, where a DAG standard had been run, was briefly exposed to I, vapour to visualize the position of the DAG standard. The plate was subsequently developed wtih CHCl,-MeOH-Hz0 (65:25:4), up to 1 cm below the DAG mark. This system allowed the sepn of all major neutral and polar lipid classes from olives. Further characterization of neutral lipids was achieved by using petrol-Et,O-HOAc (67: 33: 1). Bands were visualized with I, vapour and identified by comparison with stds. Radioactive products were located first using a radio TLC-linear analyser and then by autoradiography. Radioactivity in each product was determined by liquid scintillation counting with quench correction. Protein was determined by a modification of the Lowry’s method involving detergent solubilisation and subsequent pptn of microsomal proteins [20].

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Acknowfedyements--This work is part of a research project funded by the Comisibn Interministerial de Ciencia y Tecnologia (CICYT), Spain, and the European Ecomonic Community through the ECLAIR program.

REFERENCES

Stymne, S. and Stobart, A. K. (1987) in The Biochemistry of Plants Vol. 9 (Stumpf, P. K. and Conn, E. E., eds), pp. 175-214. Academic Press. New York. 2. Woldegiorgis, G., Bremer, J. and Shrago, E. (1985) Biochim. 1.

Biophys. Acta 837, 135. 3. Richards, E. W., Humm, M. W., Fletcher, J. E. and Otto, D. A. (1990) Biochim. Biophys. Acta 1044,361. 4. Ohlrogge, J. B., Shine, W. E. and Stumpf, P. K. (1978) Arch. Biochem. Biophys. 189,382. 5. Sambanthamurthi, R., 00, K. C. (1990) in Plant Lipid Biochemistry, Structure and Utilization (Quinn, P. J. and

Harwood, J. L., eds), pp. 166168. Portland Press. London. 6. Joyard, J. and Stumpf, P. K. (1980) Plant Physiol. 65, 1039. 7. Andrews, J. and Keegstra, K. (1983) Plant Physiol. 72, 735.

8. Murphy, D. J., Woodrow, I. E., Latzko, E. and Mukherjee, K. D. (1983) FEBS Letters 162, 442. 9. Vick, B. and Beevers, H. (1977) Plant Physiol. 59,459. 10. Ichihara, K. (1984) Arch. Biochem. Biophys. 232, 685. 11. 00, K. C. and Huang, A. H. C. (1989) Plant Physiol. 91,1288. 12. Ichihara, K. and Noda, M. (1982) Phytochemistry 21, 1895. 13. Martin, B. A. and Wilson, R. F. (1983) Lipids 18, 1. 14. Griffiths, G., Stobart, A. K. and Stymne, S. (1985) Biochem. J. 230, 376. 15. Ichihara, K., Norikura, S. and Fujii, S. (1989) Plant Physiol.

90,413. 16. Harwood, J. L. and Price-Jones, M. J. (1988) in Phosphatidate Phasphohydrolase, Vol. 2 (Brindley, D. N., ed.), pp. l-37. CRC Press, Boca Raton. 17. Rock, C. O., Goelz, S. E. and Cronan, J. E., Jr (1981) Arch. Biochem. Biophys. 211, 113. 18. Moreau, R. A. and Stumpf, P. K. (1982) Plant Physiol. 69, 1293. 19. Hara, A. and Radin, N. S. (1978) Anal. Biochem. 90,420. 20. Kresze, G. B. (1983) in Methods of EnzymaticAnalysis 3rd

Edn, Vol. 2 (Bergmeyer, H. U., ed.), pp. 84-99. Verlag Chemie, Weinheim