Planta
Planta (1984)161:249 254
9 Springer-Verlag 1984
Oleate metabolism in microsomes from developing leaves of Pisum sativum L.* Denis J. Murphy 1 **, Kumar D. Mukherjee 2 and Erwin Latzko 1 1 Botanisches Institut der Universit/it Mfinster, SchloBgarten 3, D-4400 Mfinster, and 2 Bundesanstalt fiir Fettforschung, Institut ffir Biochemie und Technologie, H.P. Kaufmann-Institut, Piusallee 68, D-4400 Mfinster, Federal Republic of Germany
Abstract. Microsomes from young leaves of pea, Pisum sativum L., metabolized oleate principally by the reactions mediated by oleoyl-CoA synthetase, oleoyl-CoA thioesterase, oleoyl-CoA: phosphatidylcholine acyltransferase and oleoyl phosphatidylcholine desaturase. Hydrogen peroxide specifically inhibited oleate desaturation and the evidence presented argues for a specific inhibition of the terminal enzyme of the desaturase system, i.e. oleoyl phosphatidylcholine desaturase. Catalase, ascorbic acid, or ascorbate peroxidase, in conjunction with ascorbic acid, stimulated oleate desaturation, possibly by the removal of hydrogen peroxide. Lysophosphatidylcholine was found to be the preferred acceptor for acyl transfer from oleoyl-CoA, which indicates that the transfer of oleoyl moieties was catalyzed predominantly by oleoyl-CoA: lysophosphatidylcholine acyltransferase. Acyl exchange between oleoyl-CoA and phosphatidylcholine, with a possible involvement of phospholipases, was also detected but at much lower rates than acyl transfer. When intact or broken chloroplasts were added to microsomes, which had been preincubated with oleoyl-CoA, some stimulation of the reactions catalyzed by oleoylCoA: phosphatidylcholine acyltransferase and oleoyl phosphatidylcholine desaturase was observed. However, only minor amounts of microsomal linoleoyl phosphatidylcholine were converted to galactolipids containing linolenoyl moieties. Key words: Acyltransferase - Desaturase - Microsome (oleate metabolism) - Oleate metabolism Pisum (oleate metabolism) - Thioesterase. * Dedicated to Professor Helmut K. Mangold, Bundesanstalt ffir Fettforschung, Miinster, on his 60th birthday ** P e r m a n e n t address: Robert Hill Laboratory of the A.R.C. Research Group on Photosynthesis, Department of Botany, University of Sheffield, Sheffield $10 2TN, U K Abbreviations: FA = unesterified fatty acid (s); PC - phosphatidylcholines; 18 : 1 = oleoyl moieties; 18 : 2 = linoleoyl moieties
Introduction In photosynthetic tissues of plants, oleic acid, the central metabolite of lipid biosynthesis, is formed mainly, if not exclusively, within the chloroplast on soluble enzymes of the stroma (Stumpf et al. 1980). Subsequent metabolism of oleic acid to linoleoyl moieties of chloroplast-membrane lipids seems to occur on membrane-bound cytosolic enzymes that are generally recovered in the microsomal fraction (Roughan and Slack 1982). Studies using microsomes from both photosynthetic (Slack et al. 1976) and nonphotosynthetic (Slack etal. 1979; Stymne and Appelqvist 1978; Stymne and Glad 1981) plant tissues have revealed that desaturation of oleate occurs on oleoyl moieties of phosphatidylcholines (PC) that are directly converted to linoleoyl moieties of this lipid class. The 18:1-PC desaturase has an absolute requirement for molecular oxygen and N A D H (Slack et al. 1976, 1979; Stymne and Appelqvist 1978), and it is inhibited by cyanide (Stymne and Appelqvist 1978). Recently (Murphy et al. 1983a), we have detected in microsomes from young pea leaves, activities of all the enzymes that are needed to convert oleic acid of chloroplastic origin to 18:1-COA, 18:1-PC and 18:2-PC according to the following scheme (FA, unesterified fatty acids): 18" 1-FA ~ 18" 1-CoA ~ 18" I-PC ~ 18 : 2-PC Moreover, we detected a thioesterase activity that readily hydrolyzes 18 : I-CoA. Apparently, the conversion of oleate to linoleate on the microsome, catalyzed by 18:1-PC desaturase, is intimately linked with the activities of 18:1-COA synthetase, 18: 1-CoA thioesterase and 18 : I-CoA: PC acyltransferase. Some of these enzymes were partially solubilized from the microsomal membranes (Murphy et al. 1983b). We report here on the effects of addition and removal of hydrogen peroxide on
250
the reactions catalyzed by these enzymes. We also investigated the various reaction mechanisms of acyl transfer. The results are discussed in terms of a possible regulation of oleate metabolism in vivo. Material and methods Materials. Seedlings of dwarf pea (Pisum sativum L. cv. Kleine Rheinl/inderin; Bruno Nebelung, Miinster, FRG) were grown on vermiculite at 15 ~ C and 60% relative humidity maintaining a 12-h photoperiod under cool-white fluorescent light (80 gE
m-2s-l). The substrate, [l-14C]oleoyl-CoA (2.072 GBq mmol-1), was purchased from Amersham Buchler, Braunschweig, FRG. The cofactors, ATP, CoA, N A D H , catalase and bovine serum albumin (fatty acid-free), were obtained from Sigma Chemie, Mfinchen, FRG. Ascorbate peroxidase was isolated from pea leaves (Kelly and Latzko 1979). Analytical grade reagents and adsorbents for thinqayer chromatography were from E. Merck AG., Darmstadt, FRG. Distilled solvents were used throughout. Lipid standards and column packing for gas-liquid chromatography were purchased from Applied Science Laboratories, Inc., State College, Pa., USA.
Isolation of organellefi'actions. Microsomes were isolated from the leaves of 8-d-old pea seedlings using the procedures described elsewhere (Diesperger et al. 1974; Murphy et al. 1983 a). Chloroplasts were isolated from the leaves of 8-d-old pea seedlings by the procedure of Nakatani and Barber (1977) following the modifications described elsewhere (Murphy and Walker 1982). The preparations contained 90-95% intact chloroplasts as judged by the ferricyanide method (Heber and Santarius 1970). Broken chloroplasts were obtained from the intact chloroplast preparations by osmotic shock following their addition to resuspension buffer (Nakatani and Barber 1977) from which sorbitot had been omitted.
Incubations, lipid extraction and analytical procedures. The incubation medium routinely consisted of 0.1 M potassium-phosphate buffer, pH7.2, 1% bovine serum albumin, 1 0 m M MgC12, 1 mM N A D H , 0.3 mM CoA and 4 m M ATP. To some of the incubations were added catalase (2000 units) and - or various amounts of H202, ascorbic acid, ascorbate peroxidase, or liposomes of oleoyl lysophosphatidylcholine or those of dioleoyl phosphatidylcholine. Liposomes were prepared by adding solutions of phospholipids in chloroform to the assay tubes, removing chloroform under a stream of N 2, and sonicating using a probe tip for 5 s in incubation medium. Incubations were carried out in loosely fitted screw-cap tubes by gently shaking in a water bath at 22 ~ C under light from fluorescent tubes. The incubation mixture (total volume 1 ml) consisted of incubation medium, about 1 mg microsomal protein and 1.5-8 KBq of [1-14C]18:1-CoA. In several experiments, the pea-leaf microsomes were preincubated with [l-~4C]18:1-CoA, as described above, for defined periods; subsequently, known amounts of intact or broken chloroplasts, freshly isolated from the pea leaves, were added and the incubations continued. Incubations were terminated by adding 18 ml chloroformmethanol (2: 1, v/v). The chloroform-soluble lipids and the fraction containing acyl-CoAs were recovered as described earlier (Murphy et al. 1983 a). Protein was determined according to Lowry et al. (1951) and chlorophyll by the method of Arnon (1949). The methods used for fractionation and analysis of lipids were identical to those described earlier (Murphy et al. 1983 a).
D.J. Murphy et al. : Oleate metabolism in microsomes
Enzyme assays. The methods described for the assay of N A D H : ferricyanide reductase (Jolliot et al. 1978) and N A D H : cytochrome b s reductase (Strittmatter 1967) were used.
Results
In accordance with earlier observations (Murphy et al. 1983a; Slack et al. 1976), microsomes from young pea leaves were found to metabolize [1-14C]18 : I-CoA mainly to labeled 18 : 1-FA and PC containing labeled 18:1 and 18:2 moieties. Other minor labeled products formed from [1-14C]18:1-COA include monogalactosyldiacylglycerols, phosphatidylethanolamines, diacylglycerols and triacylglycerols. Time-course experiments (data not shown) revealed that 18 : 1-PC was rapidly formed from 18 : 1-CoA until a steady state was reached after about 12 min. The level of 14C-18:2 moieties progressively increased in PC before they were detected in other lipid classes, including acylCoAs. Table 1. Effect of hydrogen peroxide on metabolism of oleoylCoA by pea-leaf microsomes. Pea-leaf microsomes containing 0.8 mg protein were incubated for 1 h with [1-~4C]18:1-CoA (t.665 KBq; 0.8 nmol) in the presence of various amounts of H 2 0 2 according to procedures described under Material and methods. Each incubation mixture contained N A D H (1 mM), ATP (4 mM) and CoA (0.3 mM)
H2O 2
Distribution (%) of radioactivity in products
(mM)
0 0.5 2.5 10.0
18:1FA
18:2FA
18:1PC
18:2PC
18:1CoA
18:2CoA
Others
10.0 12.5 10.5 25.0
0.1 0.2 0.1 0.0
18.2 19.1 22.0 26.0
10.1 8.1 4.9 0.0
56.1 50.2 57.3 42.8
1.9 2.1 0.6 0.0
3.6 7.8 4.6 6.2
Table 2. Effect of catalase on desaturation of oleate by pea-leaf microsomes. Different preparations of pea-leaf microsomes containing 1.3 mg protein (experiment I), 0.8 mg protein (experiment II) and 1.2 mg protein (experiment III) were incubated for i h with [1-14C]oleoyl-CoA (7.4 KBq; 3.57 nmol) with or without added catalase (2000 units ml -~) to the medium for isolation of microsomes and the incubation mixture. Isolation of microsomes and incubations were carried out as described under Material and methods. Each incubation mixture contained N A D H (1 raM), ATP (4 raM) and CoA (0.3 mM) Expt,
Additions
I~C-18 : 2 in total lipids (%)
I
None Catalase
27.0 34.0
II
None Catalase Catalase+H202 (0.1 mM) None Catalase+ H202 (1.0 raM) Catalase + H20 a (5.0 raM) H20 2 (5.0 raM)
17.0 23.0 24.0 19.8 19.6 19.0 4.8
III
D.J. Murphy et ak : Oteate metabolism in microsomes
251
Table 3. Effect of ascorbic acid and ascorbate peroxidase on metabolism of oleoyl-CoA by pea-leaf microsomes. Different preparations of pea-leaf microsomes containing 0.9 mg protein (experiment I) and 1.0 mg protein (experiment II) were incubated with [1-14C]oleoyl-CoA (7.4 KBq; 3.57 nmol). Isolation of microsomes and incubations were carried out as described under Material and methods. Each incubation mixture contained N A D H (1 mM), ATP (4 mM) and CoA (0.3 mM) Expt.
Additions
14C-18:2 in total lipids
Ascorbic acid (raM)
Ascorbate peroxidase (units per assay)
I
0.05 1.0
-
31.4 35.3
II
1.0 1.0 1.0
60 60 60
29.7 32.2 36.0 0.0 30.2
EIzO 2
(raM)
10.0 10.0
Distribution (%) of radioactivity in products FA
PC
Aeyl-CoA
Others
16.2 15.5 21.1 14.5 10.9
26.5 28.5 32.3 50.4 18.9
46.0 50.0 34.0 27.0 69.0
11.3 6.0 12.6 8.1 1.2
(%)
The effects of following factors on oleate metabolism in pea-leaf microsomes were investigated.
Effect of H202 and H202 scavenging agents. When pea-leaf microsomes were incubated with [1-14C]18 : 1-CoA in the presence of increasing concentrations of H202, the proportion of 14C-18:2 moieties in the major lipid classes progressively decreased (Table 1). Most striking was the reduction in the level of 14C-18:2-PC. At a concentration of 10 m M H 2 0 2 in the incubation mixture, ~4C18:2 moieties were no longer detectable, either in PC or in the other lipid classes. Although H202 inhibited the formation of 18:2, it barely affected the distribution of label in the individual lipid classes. Upon incubation of pea-leaf microsomes with [1-14C]18:1-CoA, a stimulation in the formation o f 1 4 C - 1 8 : 2 moieties occurred when catalase was added both to the medium for isolation of the microsomes and to the incubation mixture (Table 2, experiments I and II). Moreover, the inhibitory effect of H202 on the formation of 18:2 moieties was completely overcome by the use of catalase (Table 2, experiment III). In order to test the hypothesis that catalase was stimulating oleate desaturation by acting as a n H 2 0 2 scavenger, the effect of an alternative H202-removing system was investigated. Accordingly, pea-leaf microsomes were incubated with [1-~4C]18: 1-CoA in the presence of ascorbic acid and a semi-purified form of ascorbate peroxidase that was also isolated from pea leaves. Some increase in the level of ~4C-18:2 moieties in the lipids was observed with increasing concentration of ascorbic acid in the incubation mixture (Table 3, experiments I and II). Inclusion of ascorbate peroxidase with ascorbic acid in the incubation mixture resulted in a further increase in the level of
Table 4. Effect of phospholipids on metabolism of oleoyl-CoA by pea-leaf microsomes. Pea-leaf microsomes containing 1.0 mg protein were incubated for l h with [1-x4C]18:l-CoA (3.33 KBq; 1.61 nmol) in the presence of lyso-PC or PC, that had been added as liposomes. Procedures for isolation of microsomes and incubation were as given under Material and methods. Each incubation mixture contained N A D H (1 raM), ATP (4 mM) and CoA (0.3 mM) Phospho- Concn. lipid (~tM)
None Lyso-PC PC
68 136 68 170
Distribution (%) of radioactivity in products FA
18:1PC
18:2PC
AcylCoA
Others
10.1 18.2 16.3 16.1 19.5
28.9 37.6 42.6 34.4 34.6
7.2 11.8 11.1 8.6 8.2
51.5 27.7 24.3 32.7 29.7
2.3 4.6 5.7 8.3 7.9
14C-18:2 moieties in the total lipids (Table 3, experiment II), in addition to completely relieving the inhibition of oleate desaturation caused by exogenous H 2 0 2.
Effect of exogenous lipids. When pea-leaf microcomes were incubated with [1-14C] 18: 1-CoA in the presence of unlabeled lyso-PC or PC, added as liposomes at two different concentrations, the results given in Table 4 were obtained. They show that higher proportions of labeled 18:1-PC and 18:2-PC were formed when unlabeled lyso-PC, rather than unlabeled PC, was added to the incubation mixture.
Effect of intact and broken chloroplasts. Two different preparations of pea-leaf microsomes were incubated with [1-14C]18:1-CoA for 1 h after which the corresponding preparations of intact or broken chloroplasts were added and incubations continued for times specified in Table 5. The distribution of radioactivity in chloroform-soluble reaction
252
D.J. Murphy et al. : Oleate metabolism in microsomes
Table 5. Effect of intact chloroplasts (IC) and broken chloroplasts (BC) on metabolism of oleoyl-CoA by pea-leaf microsomes.
Different preparations of pea-leaf microsomes containing 0.9 mg protein (experiment I) and 1.0 mg protein (experiment II) were incubated for specified times with [1-14C]18:1-COA (5.18 KBq; 2.5 nmol). The corresponding chloroplast preparations containing final concentrations of 136 gg chlorophyll m1-1 (experiment I) and 263 gg chlorophyll m1-1 (experiment II) were added and the incubations were continued for specified periods. Procedures for the isolation of microsomes and chloroplast preparations and for the incubations were as described under Material and methods. Each incubation mixture contained NADH (1 mM), ATP (4 raM), CoA (0.3 raM), catalase (2000 units), uridine 5'-diphosphate-galactose (1 raM) and glyeerol-3-phosphate (1 mM); MGDG = monogalactosyldiacylglycerols,PE = phosphatidylethanolamines Expt.
II
Incubation time (min)
Distribution (%) of radioactivity in chloroform-soluble lipids
Microsomes _+ Chloroplasts
FA
18:I-PC
18:2-PC
PE
MGDG
Others ~
(180) (60) (60)
none IC (120) BC (120)
62 47 56
10 14 16
4 13 9
4 5 2
1 3 3
19 18 14
(180) (60) (60)
none IC ( 6 0 ) IC (120)
64 42 52
7 19 16
4 13 12
5 2 2
3 3 4
17 21 14
a Including lyso-PC, diacylglycerols and triacylglycerols
products shows, for both experiments, an increase in the labeling of 18:1-PC and 18:2-PC by the addition of chloroplast preparations to microsomes, yet, only a minor labeling of galactolipids occurred and only traces of lipids containing 14Clinolenoyl moieties were detected. Discussion
Earlier findings (Murphy et al. 1983 a; Slack et al. 1976) taken together with the time-course studies mentioned before corroborate the pathways by which 18:1-FA is activated to 18:1-COA in the presence of ATP and CoA, then transferred to PC and finally desaturated to 18 : 2-PC. Each of these steps is catalyzed by membrane-bound enzymes in pea-leaf microsomes. Contamination by chloroplast-envelope-derived activities was ruled out on the basis of subcellular distribution and enzymemarker studies (Murphy et al. 1983 b). Hydrogen peroxide has been found to inhibit stearoyl-CoA desaturase in rat-liver microsomes (Baker et al. 1976) and stearoyl-acyl carrier protein desaturase in maturing safflower seeds (McKeon and Stumpf 1982), although little is known about the possible source of H 2 0 2 o r its mechanism of action on desaturases. We found that H 2 0 2 a t low concentrations hardly affects 18:1-PC desaturation, but with increasing concentration of H202 (2.5 to 10 raM) 18!1-PC desaturation is progressively inhibited (Table 1). The distribution of radioactivity in the products of incubation reveals that H202 specifically inhibits 18: 1-PC desaturation but has hardly any effect on the reactions catalyzed by 18:I-CoA:PC acyltransferase or 18 : 1-CoA thioesterase (Table 1). In agreement with earlier findings with stearoyl-CoA desaturase (Baker et al. 1976), stearoyl-
acyl carrier protein desaturase (McKeon and Stumpf 1982) and "linoleate desaturase" (Browse and Slack 1981), catalase was found to stimulate oleate desaturation and overcome the inhibitory effect of H20 2 on 18: I-PC desaturation in pea-leaf microsomes (Table 2). Stimulation of the desaturase by catalase was likely caused by removal of H 2 0 2 , although a specific action of catalase on the desaturation reaction itself cannot be ruled out. We have shown earlier (Murphy et al. 1983a) that the inhibition of 18:1-PC desaturation by H202 is neither the consequence of~oxidative damage to the lipids of microsomal membranes nor the results of oxidation of NADH which is one of the cofactors required by the desaturase system (Slack et al. 1976; Styrene and Appelqvist 1978). Oleate desaturation in plants is inhibited by cyanide (Stymne and Appelqvist 1978; Vijay and Stumpf 1972), but not by carbon monoxide (Vijay and Stumpf 1972), as is the case with stearate desaturation in animal systems (Pugh and Kates 1979). It is, therefore, likely that the plant 18 : 1-PC desaturase is terminally linked with proteins similar to NADH : cytochrome b s reductase and cytochrome b s for electron transfer from NADH, as is the case in desaturase systems from animals, such as stearoyl-CoA desaturase (Pugh and Kates 1979). We detected NADH: ferricyanide reductase and NADH: cytochrome c reductase activities in our preparations of pea leaf-microsomes using the spectrophotometric techniques (Jolliot et al. 1978; Strittmatter 1967) which were routinely used to assay for cytochrome b s reductase and cytochrome b s. In order to assess how these two likely components of the oleate desaturase system are affected b y H202, we incubated the pea-leaf microsomes with up to 10 mM H20 2 in the standard incubation medium and followed the changes in the activ-
D.J. Murphyet al. : Oleatemetabolismin microsomes ity of NADH: ferricyanide reductase and NADH: cytochrome c reductase. In each case it was possible to rule out a specific inhibition of electrontransport activity by H20 2 at either of these sites, since H20 2 at up to 10 mM affected neither activity. All these findings strongly indicate that HzO 2 specifically inhibits the terminal 18:1-PC desaturase itself, rather than any of the components of its associated electron-transport chain. Ascorbic acid has been found to stimulate the acyl-CoA AS-desaturase in developing meadowfoam seeds (Moreau etal. 1981). We observed some stimulation of oleate desaturation in pea-leaf microsomes by the addition of ascorbic acid (Table 3). Addition of ascorbic acid with ascorbate peroxidase resulted in further stimulation of oleate desaturation (Table 3). It is likely that the stimulation of oleate desaturation by ascorbate peroxidase is caused by removal of H20 2 since the latter is a cosubstrate for this enzyme, which converts ascorbic acid to dehydroascorbic acid (Kelly and Latzko 1979). The specific effect of H20 2 upon the oleate desaturase system was seen only at relatively high H202 concentrations (>0.5 raM). While the cytosolic HzO 2 concentration is probably less than 0.5 mM under physiological conditions, it is possible that local H20 z concentrations may considerably exceed this value. Leaf cells from a variety of plants have been found to contain an extremely effective HzOz-scavenging system - ascorbate peroxidase (Groden and Beck 1979; Kelly and Latzko 1980). One of the physiological roles of this and other H2Oz-scavenging systems may be to protect susceptible proteins, such as the terminal enzyme of the oleate-desaturase system, from oxidative damage. As found earlier (Murphy et al. 1983a; Slack etal. 1976), the transfer of 18:1 moieties from 18:1-COA to PC is a prerequisite for desaturation of oleate in pea-leaf microsomes. In microsomes from developing safflower seeds, the transfer of 18:1 moieties from 18:1-COA to PC has been shown to be catalyzed predominantly by 18:lCoA:lysophospholipid acyltransferase and lysoPC seems to be the major acceptor in this reaction (Moreau and Stumpf 1982). On the other hand, in microsomes from developing cotyledons of soya bean and safflower, the transfer of 18:1 moieties from 18:1-COA to PC seems to occur mainly by acyl exchange (Styrene and Glad 1981; Stymne et al. 1983). When pea-leaf microsomes were incubated with radioactive 18:1-COA in the presence of exogenous lyso-PC or PC at two different concentrations, a distinctly higher labeling of both total PC and 18:2-PC was observed by the inclusion of lyso-PC rather than PC in the incubation mix-
253 ture (Table 4). These findings are in agreement with lyso-PC being the preferred acceptor for acyl transfer from 18:1-COA. On the other hand, we consistently detected small proportions of labeled 18:2-CoA in the reaction products (Table 1) which indicates that a small amount of acyl exchange between acyl-CoA and PC occurred as well. Moreover, we detected in the reaction products, minor proportions of labeled lyso-PC (5-10% of toal labeled PC; data not shown) which indicates that deacylation of PC by phospholipases and reacylation of the resulting lyso-PC by acyl-CoA might also be involved in the transfer of acyl moieties to PC. However, the contribution of the latter two reactions to the net transfer of oleoyl moieties to PC was very minor as compared with the direct transfer from oleoyl-CoA to form PC. It is likely, however, that in vivo the deacylation-reacylation reactions play an important role in sustaining a pool of lyso-PC, to which the oleoyl moieties are transferred from 18 : I-CoA. It has been proposed that co-operation between chloroplasts and endoplasmic reticulum (enriched in the microsomal fraction) is involved in the biosynthesis and assembly of chloroplast-membrane lipids (Roughan etal. 1980; Tr~moli&es etal. 1980). Such a co-operation is believed to result in the transport and conversion of 18:2-PC, synthesized on the microsomal membranes, to galactolipids containing 18 : 2 and 18 : 3 moieties in the chloroplast. These conclusions were drawn from the pattern of labeling of lipids when isolated chloroplasts were incubated with radioactive acetate with or without added microsomes in the presence of uridine 5'-diphosphate (UDP)-galactose and glycerol-3-phosphate. In order to detect such a cooperation, we preincubated the pea-leaf microsomes with radioactive 18: I-CoA in the presence of UDP-galactose and glycerol-3-phosphate. Subsequently, intact or broken pea-leaf chloroplasts were added to the incubation mixture and the transfer of label to various lipid classes was followed (Table 5). We found that the addition of either intact or broken chloroplasts to the microsomes resulted in some stimulation in the formation of both 18:1-PC and 18:2-PC. But the galactolipids were labeled only to a small extent and the labeled linolenoyl moieties formed barely exceeded 1-2% of the labeled acyl moieties in the total lipids. Even the incubation of [1-agC]oleoyl CoA with freshly prepared whole homogenate from pea leaves did not result in more than a small (1-2%) labeling of galactolipids or linolenate (data not shown). It has previously been shown that chloroplasts, liposomes and microsomes will readily fuse or clump together even in low-salt media (Murphy and Kuhn 1981). Indeed it has recently
254
been reported that spontaneous phospholipid transfer or exchange readily occurs between phospholipid vesicles of different composition (De Cuyper etal. 1983). Therefore, labeled 18:2-PC was presumably transferred from microsomes to chloroplast membranes but was not significantly metabolized thereafter in either whole-tissue homogenates or cell preparations of pea leaves. The observed stimulation in 18:2-PC formation that was found following the addition of chloroplasts to actively desaturating microsomes may be caused by the removal of 18:2-PC from the microsomes to the chloroplasts and the consequent relief of a product inhibition of the 18 : 1-PC desaturase system. A part of this work was supported by a grant from Deutsche Forschungsgemeinschaft (DFG).
References Arnon, D.I. (1949) Copper enzymes in isolated chloroplasts. Polyphenoloxidase in Beta vulgaris. Plant Physiol. 24, 1-14 Baker, R.C., Wykle, R.L., Lockmiller, J.S., Snyder, F. (1976) Identification of a soluble protein stimulator of plasmalogen biosynthesis and stearoyl-coenzyme A desaturase. Arch. Biochem. Biophys. 177, 29%306 Browse, J.A., Slack, C.R. (1981) Catalase stimulates linoleate desaturase activity in microsomes from developing linseed cotyledons. FEBS Lett. 131, 111-114 De Cuyper, M., Joniau, M., Dangreau, H. (1983) Intravesicular phospholipid transfer. A free-flow electrophoresis study. Biochemistry 22, 415-420 Diesperger, H., Miiller, C.R., Sandermann, H. (1974) Rapid isolation of a plant microsomal fraction by magnesium (2 +) precipitation. FEBS Lett. 43, 155-158 Groden, D., Beck, E. (lp79) HzO 2 destruction by ascorbatedependent systems f~om chloroplasts. Biochim. Biophys. Acta 546, 426-435 [ Heber, U., Santarius, KtA. (1970) Direct and indirect transfer of ATP and ADP acr6ss the chloroplast envelope. Z. Naturforsch. Teil B 25, 718-728 Jolliot, A., Demandre, C., Mazliak, P. (1978) Role of lipids in electron transport chain function in potato microsomes. Biochimie 60, 767-775 Kelly, G.J., Latzko, E. (1979) Soluble ascorbate peroxidase. Detection in plants and use in vitamin C estimation. Naturwissenschaften 66, 617-618 Kelly, G.J., Latzko, E. (1980) Prospect of a specific enzymatic assay for ascorbic acid (vitamin C). J. Agric. Food Chem. 28, 132(~1321 Lowry, O.H., Rosebrough, N.J., Farr, A.L., Randall, R.S. (1951) Protein measurement with the folin phenol reagent. J. Biol. Chem. 193, 265-275 McKeon, T.A., Stumpf, P.K. (1982) Purification and characterization of the stearoyl-acyl carrier protein desaturase and the acyl-acyl carrier protein thioesterase from maturing seeds of safflower. J. Biol. Chem. 257, 12141-12147 Moreau, R.A., Pollard, M.R., Stumpf, P.K. (1981) Properties of a A5-fatty acyl-CoA desaturase in the cotyledons of developing Limnanthes alba. Arch. Biochem. Biophys. 209, 376-384
D.J. Murphy et al. : Oleate metabolism in microsomes Moreau, R.A., Stumpf, P.K. (1982) Solubilization and characterization of an acyl-coenzyme A: O-lysophospholipid acyltransferase from the microsomes of developing safflower seeds. Plant Physiol. 69, 1293-1297 Murphy, D.J., Kuhn, D.N. (1981) The lack of a phospholipidexchange-protein activity in soluble fractions of Spinacia oleracea leaves. Biochem. J. 194, 257-264 Murphy, D.J., Mukherjee, K.D., Latzko, E. (1983a) Lipid metabolism in microsomal fraction from photosynthetic tissue: effects of catalase and hydrogen peroxide on oleate desaturation. Biochem. J. 213, 2149-252 Murphy, D.J., Woodrow, I.E., Latzko, E., Mukherjee, K.D. (1983b) The oleate desaturase system of pea leaf microsomes: solubilisation of oleoyl-CoA thioesterase, oleoylCoA: phosphatidylcholine acyltransferase and oleoyl-phosphatidylcholine desaturase. FEBS Lett. 162, 442-446 Murphy, D.J., Walker, D.A. (1982) Acetyl coenzyme A biosynthesis in the chloroplast. What is the physiological precursor? Planta 156, 84-88 Nakatani, H.V., Barber, J. (1977) An improved method for isolating chloroplast retaining their outer membranes. Biochim. Biophys. Acta 461, 510-512 Pugh, E.L., Kates, M. (1979) Membrane-bound phospholipid desaturases. Lipids 14, 159-165 Roughan, P.G., Holland, R., Slack, C.R. (1980) The role of chloroplasts and microsomal fractions in polar lipid synthesis from [1-1~C]acetate by cell-free preparations from spinach (Spinacia oleracea) leaves. Biochem. J. 188, 1224 Roughan, P.G., Slack, C.R. (1982) Cellular organization of gtycerolipid metabolism. Annu. Rev. Plant Physiol. 33, 97-132 Slack, C.R., Roughan, P.G., Browse, J. (1979) Evidence for an oleoyl phosphatidylcholine desaturase in microsomal preparations from cotyledons of safflower (Carthamus tinctorius) seed. Biochem. J. 179, 649-656 Slack, C.R., Roughan, P.G., Terpstra, J. (1976) Some properties of a microsomal oleate desaturase from leaves. Biochem. J. 155, 71-80 Strittmatter, P. (1967) NADH-cytochrome b 5 reductase. Methods Enzymol. i0, 561-565 Stumpf, P.K., Kuhn, D.N., Murphy, D.J., Pollard, M.R., McKeon, T., MacCarthy, J. (1980) Oleic acid, the central substrate. In: Biogenesis and function of plant lipids, pp. 310, Mazliak, P., Benveniste, P., Costes, C., Douce, R., eds. Elsevier/North-Holland Biomedical Press, Amsterdam Stymne, S., Appelqvist, L.-A. (1978) The biosynthesis of linoleate from oleoyl-CoA via oleoyl-phosphatidylcholinein microsomes of developing safflower seeds. Eur. J. Biochem. 90, 223-229 Stymne, S., Glad, G. (1981) Acyl exchange between oleoyl-CoA and phosphatidylcholine in microsomes of developing soya bean cotyledons and its role in fatty acid desaturation. Lipids 16, 298-305 Stymne, S., Stobart, A.K., Glad, G. (1983) The role of the acyl-CoA pool in the synthesis of polyunsaturated 18-carbon fatty acids and triacylglycerol production in the microsomes of developing safflower seeds. Biochim. Biophys. Acta 752, 198-208 Tr6moli+res, A., Dubacq, J.P., Drapier, D., Mfiller, M., Mazliak, P. (1980) In vitro cooperation between plastids and microsomes in the biosynthesis of leaf lipids. FEBS Lett. 114, 135-138 Vijay, I.K., Stumpf, P.K. (1972) Properties of oleoyl-coenzyme A desaturase of Carthamus tinctorius. J. Biol. Chem. 247, 36~366 Received 14 November 1983; accepted 27 January 1984
Planta (1984)161:249 254
9 Springer-Verlag 1984
Oleate metabolism in microsomes from developing leaves of Pisum sativum L.* Denis J. Murphy 1 **, Kumar D. Mukherjee 2 and Erwin Latzko 1 1 Botanisches Institut der Universit/it Mfinster, SchloBgarten 3, D-4400 Mfinster, and 2 Bundesanstalt fiir Fettforschung, Institut ffir Biochemie und Technologie, H.P. Kaufmann-Institut, Piusallee 68, D-4400 Mfinster, Federal Republic of Germany
Abstract. Microsomes from young leaves of pea, Pisum sativum L., metabolized oleate principally by the reactions mediated by oleoyl-CoA synthetase, oleoyl-CoA thioesterase, oleoyl-CoA: phosphatidylcholine acyltransferase and oleoyl phosphatidylcholine desaturase. Hydrogen peroxide specifically inhibited oleate desaturation and the evidence presented argues for a specific inhibition of the terminal enzyme of the desaturase system, i.e. oleoyl phosphatidylcholine desaturase. Catalase, ascorbic acid, or ascorbate peroxidase, in conjunction with ascorbic acid, stimulated oleate desaturation, possibly by the removal of hydrogen peroxide. Lysophosphatidylcholine was found to be the preferred acceptor for acyl transfer from oleoyl-CoA, which indicates that the transfer of oleoyl moieties was catalyzed predominantly by oleoyl-CoA: lysophosphatidylcholine acyltransferase. Acyl exchange between oleoyl-CoA and phosphatidylcholine, with a possible involvement of phospholipases, was also detected but at much lower rates than acyl transfer. When intact or broken chloroplasts were added to microsomes, which had been preincubated with oleoyl-CoA, some stimulation of the reactions catalyzed by oleoylCoA: phosphatidylcholine acyltransferase and oleoyl phosphatidylcholine desaturase was observed. However, only minor amounts of microsomal linoleoyl phosphatidylcholine were converted to galactolipids containing linolenoyl moieties. Key words: Acyltransferase - Desaturase - Microsome (oleate metabolism) - Oleate metabolism Pisum (oleate metabolism) - Thioesterase. * Dedicated to Professor Helmut K. Mangold, Bundesanstalt ffir Fettforschung, Miinster, on his 60th birthday ** P e r m a n e n t address: Robert Hill Laboratory of the A.R.C. Research Group on Photosynthesis, Department of Botany, University of Sheffield, Sheffield $10 2TN, U K Abbreviations: FA = unesterified fatty acid (s); PC - phosphatidylcholines; 18 : 1 = oleoyl moieties; 18 : 2 = linoleoyl moieties
Introduction In photosynthetic tissues of plants, oleic acid, the central metabolite of lipid biosynthesis, is formed mainly, if not exclusively, within the chloroplast on soluble enzymes of the stroma (Stumpf et al. 1980). Subsequent metabolism of oleic acid to linoleoyl moieties of chloroplast-membrane lipids seems to occur on membrane-bound cytosolic enzymes that are generally recovered in the microsomal fraction (Roughan and Slack 1982). Studies using microsomes from both photosynthetic (Slack et al. 1976) and nonphotosynthetic (Slack etal. 1979; Stymne and Appelqvist 1978; Stymne and Glad 1981) plant tissues have revealed that desaturation of oleate occurs on oleoyl moieties of phosphatidylcholines (PC) that are directly converted to linoleoyl moieties of this lipid class. The 18:1-PC desaturase has an absolute requirement for molecular oxygen and N A D H (Slack et al. 1976, 1979; Stymne and Appelqvist 1978), and it is inhibited by cyanide (Stymne and Appelqvist 1978). Recently (Murphy et al. 1983a), we have detected in microsomes from young pea leaves, activities of all the enzymes that are needed to convert oleic acid of chloroplastic origin to 18:1-COA, 18:1-PC and 18:2-PC according to the following scheme (FA, unesterified fatty acids): 18" 1-FA ~ 18" 1-CoA ~ 18" I-PC ~ 18 : 2-PC Moreover, we detected a thioesterase activity that readily hydrolyzes 18 : I-CoA. Apparently, the conversion of oleate to linoleate on the microsome, catalyzed by 18:1-PC desaturase, is intimately linked with the activities of 18:1-COA synthetase, 18: 1-CoA thioesterase and 18 : I-CoA: PC acyltransferase. Some of these enzymes were partially solubilized from the microsomal membranes (Murphy et al. 1983b). We report here on the effects of addition and removal of hydrogen peroxide on
250
the reactions catalyzed by these enzymes. We also investigated the various reaction mechanisms of acyl transfer. The results are discussed in terms of a possible regulation of oleate metabolism in vivo. Material and methods Materials. Seedlings of dwarf pea (Pisum sativum L. cv. Kleine Rheinl/inderin; Bruno Nebelung, Miinster, FRG) were grown on vermiculite at 15 ~ C and 60% relative humidity maintaining a 12-h photoperiod under cool-white fluorescent light (80 gE
m-2s-l). The substrate, [l-14C]oleoyl-CoA (2.072 GBq mmol-1), was purchased from Amersham Buchler, Braunschweig, FRG. The cofactors, ATP, CoA, N A D H , catalase and bovine serum albumin (fatty acid-free), were obtained from Sigma Chemie, Mfinchen, FRG. Ascorbate peroxidase was isolated from pea leaves (Kelly and Latzko 1979). Analytical grade reagents and adsorbents for thinqayer chromatography were from E. Merck AG., Darmstadt, FRG. Distilled solvents were used throughout. Lipid standards and column packing for gas-liquid chromatography were purchased from Applied Science Laboratories, Inc., State College, Pa., USA.
Isolation of organellefi'actions. Microsomes were isolated from the leaves of 8-d-old pea seedlings using the procedures described elsewhere (Diesperger et al. 1974; Murphy et al. 1983 a). Chloroplasts were isolated from the leaves of 8-d-old pea seedlings by the procedure of Nakatani and Barber (1977) following the modifications described elsewhere (Murphy and Walker 1982). The preparations contained 90-95% intact chloroplasts as judged by the ferricyanide method (Heber and Santarius 1970). Broken chloroplasts were obtained from the intact chloroplast preparations by osmotic shock following their addition to resuspension buffer (Nakatani and Barber 1977) from which sorbitot had been omitted.
Incubations, lipid extraction and analytical procedures. The incubation medium routinely consisted of 0.1 M potassium-phosphate buffer, pH7.2, 1% bovine serum albumin, 1 0 m M MgC12, 1 mM N A D H , 0.3 mM CoA and 4 m M ATP. To some of the incubations were added catalase (2000 units) and - or various amounts of H202, ascorbic acid, ascorbate peroxidase, or liposomes of oleoyl lysophosphatidylcholine or those of dioleoyl phosphatidylcholine. Liposomes were prepared by adding solutions of phospholipids in chloroform to the assay tubes, removing chloroform under a stream of N 2, and sonicating using a probe tip for 5 s in incubation medium. Incubations were carried out in loosely fitted screw-cap tubes by gently shaking in a water bath at 22 ~ C under light from fluorescent tubes. The incubation mixture (total volume 1 ml) consisted of incubation medium, about 1 mg microsomal protein and 1.5-8 KBq of [1-14C]18:1-CoA. In several experiments, the pea-leaf microsomes were preincubated with [l-~4C]18:1-CoA, as described above, for defined periods; subsequently, known amounts of intact or broken chloroplasts, freshly isolated from the pea leaves, were added and the incubations continued. Incubations were terminated by adding 18 ml chloroformmethanol (2: 1, v/v). The chloroform-soluble lipids and the fraction containing acyl-CoAs were recovered as described earlier (Murphy et al. 1983 a). Protein was determined according to Lowry et al. (1951) and chlorophyll by the method of Arnon (1949). The methods used for fractionation and analysis of lipids were identical to those described earlier (Murphy et al. 1983 a).
D.J. Murphy et al. : Oleate metabolism in microsomes
Enzyme assays. The methods described for the assay of N A D H : ferricyanide reductase (Jolliot et al. 1978) and N A D H : cytochrome b s reductase (Strittmatter 1967) were used.
Results
In accordance with earlier observations (Murphy et al. 1983a; Slack et al. 1976), microsomes from young pea leaves were found to metabolize [1-14C]18 : I-CoA mainly to labeled 18 : 1-FA and PC containing labeled 18:1 and 18:2 moieties. Other minor labeled products formed from [1-14C]18:1-COA include monogalactosyldiacylglycerols, phosphatidylethanolamines, diacylglycerols and triacylglycerols. Time-course experiments (data not shown) revealed that 18 : 1-PC was rapidly formed from 18 : 1-CoA until a steady state was reached after about 12 min. The level of 14C-18:2 moieties progressively increased in PC before they were detected in other lipid classes, including acylCoAs. Table 1. Effect of hydrogen peroxide on metabolism of oleoylCoA by pea-leaf microsomes. Pea-leaf microsomes containing 0.8 mg protein were incubated for 1 h with [1-~4C]18:1-CoA (t.665 KBq; 0.8 nmol) in the presence of various amounts of H 2 0 2 according to procedures described under Material and methods. Each incubation mixture contained N A D H (1 mM), ATP (4 mM) and CoA (0.3 mM)
H2O 2
Distribution (%) of radioactivity in products
(mM)
0 0.5 2.5 10.0
18:1FA
18:2FA
18:1PC
18:2PC
18:1CoA
18:2CoA
Others
10.0 12.5 10.5 25.0
0.1 0.2 0.1 0.0
18.2 19.1 22.0 26.0
10.1 8.1 4.9 0.0
56.1 50.2 57.3 42.8
1.9 2.1 0.6 0.0
3.6 7.8 4.6 6.2
Table 2. Effect of catalase on desaturation of oleate by pea-leaf microsomes. Different preparations of pea-leaf microsomes containing 1.3 mg protein (experiment I), 0.8 mg protein (experiment II) and 1.2 mg protein (experiment III) were incubated for i h with [1-14C]oleoyl-CoA (7.4 KBq; 3.57 nmol) with or without added catalase (2000 units ml -~) to the medium for isolation of microsomes and the incubation mixture. Isolation of microsomes and incubations were carried out as described under Material and methods. Each incubation mixture contained N A D H (1 raM), ATP (4 raM) and CoA (0.3 mM) Expt,
Additions
I~C-18 : 2 in total lipids (%)
I
None Catalase
27.0 34.0
II
None Catalase Catalase+H202 (0.1 mM) None Catalase+ H202 (1.0 raM) Catalase + H20 a (5.0 raM) H20 2 (5.0 raM)
17.0 23.0 24.0 19.8 19.6 19.0 4.8
III
D.J. Murphy et ak : Oteate metabolism in microsomes
251
Table 3. Effect of ascorbic acid and ascorbate peroxidase on metabolism of oleoyl-CoA by pea-leaf microsomes. Different preparations of pea-leaf microsomes containing 0.9 mg protein (experiment I) and 1.0 mg protein (experiment II) were incubated with [1-14C]oleoyl-CoA (7.4 KBq; 3.57 nmol). Isolation of microsomes and incubations were carried out as described under Material and methods. Each incubation mixture contained N A D H (1 mM), ATP (4 mM) and CoA (0.3 mM) Expt.
Additions
14C-18:2 in total lipids
Ascorbic acid (raM)
Ascorbate peroxidase (units per assay)
I
0.05 1.0
-
31.4 35.3
II
1.0 1.0 1.0
60 60 60
29.7 32.2 36.0 0.0 30.2
EIzO 2
(raM)
10.0 10.0
Distribution (%) of radioactivity in products FA
PC
Aeyl-CoA
Others
16.2 15.5 21.1 14.5 10.9
26.5 28.5 32.3 50.4 18.9
46.0 50.0 34.0 27.0 69.0
11.3 6.0 12.6 8.1 1.2
(%)
The effects of following factors on oleate metabolism in pea-leaf microsomes were investigated.
Effect of H202 and H202 scavenging agents. When pea-leaf microsomes were incubated with [1-14C]18 : 1-CoA in the presence of increasing concentrations of H202, the proportion of 14C-18:2 moieties in the major lipid classes progressively decreased (Table 1). Most striking was the reduction in the level of 14C-18:2-PC. At a concentration of 10 m M H 2 0 2 in the incubation mixture, ~4C18:2 moieties were no longer detectable, either in PC or in the other lipid classes. Although H202 inhibited the formation of 18:2, it barely affected the distribution of label in the individual lipid classes. Upon incubation of pea-leaf microsomes with [1-14C]18:1-CoA, a stimulation in the formation o f 1 4 C - 1 8 : 2 moieties occurred when catalase was added both to the medium for isolation of the microsomes and to the incubation mixture (Table 2, experiments I and II). Moreover, the inhibitory effect of H202 on the formation of 18:2 moieties was completely overcome by the use of catalase (Table 2, experiment III). In order to test the hypothesis that catalase was stimulating oleate desaturation by acting as a n H 2 0 2 scavenger, the effect of an alternative H202-removing system was investigated. Accordingly, pea-leaf microsomes were incubated with [1-~4C]18: 1-CoA in the presence of ascorbic acid and a semi-purified form of ascorbate peroxidase that was also isolated from pea leaves. Some increase in the level of ~4C-18:2 moieties in the lipids was observed with increasing concentration of ascorbic acid in the incubation mixture (Table 3, experiments I and II). Inclusion of ascorbate peroxidase with ascorbic acid in the incubation mixture resulted in a further increase in the level of
Table 4. Effect of phospholipids on metabolism of oleoyl-CoA by pea-leaf microsomes. Pea-leaf microsomes containing 1.0 mg protein were incubated for l h with [1-x4C]18:l-CoA (3.33 KBq; 1.61 nmol) in the presence of lyso-PC or PC, that had been added as liposomes. Procedures for isolation of microsomes and incubation were as given under Material and methods. Each incubation mixture contained N A D H (1 raM), ATP (4 mM) and CoA (0.3 mM) Phospho- Concn. lipid (~tM)
None Lyso-PC PC
68 136 68 170
Distribution (%) of radioactivity in products FA
18:1PC
18:2PC
AcylCoA
Others
10.1 18.2 16.3 16.1 19.5
28.9 37.6 42.6 34.4 34.6
7.2 11.8 11.1 8.6 8.2
51.5 27.7 24.3 32.7 29.7
2.3 4.6 5.7 8.3 7.9
14C-18:2 moieties in the total lipids (Table 3, experiment II), in addition to completely relieving the inhibition of oleate desaturation caused by exogenous H 2 0 2.
Effect of exogenous lipids. When pea-leaf microcomes were incubated with [1-14C] 18: 1-CoA in the presence of unlabeled lyso-PC or PC, added as liposomes at two different concentrations, the results given in Table 4 were obtained. They show that higher proportions of labeled 18:1-PC and 18:2-PC were formed when unlabeled lyso-PC, rather than unlabeled PC, was added to the incubation mixture.
Effect of intact and broken chloroplasts. Two different preparations of pea-leaf microsomes were incubated with [1-14C]18:1-CoA for 1 h after which the corresponding preparations of intact or broken chloroplasts were added and incubations continued for times specified in Table 5. The distribution of radioactivity in chloroform-soluble reaction
252
D.J. Murphy et al. : Oleate metabolism in microsomes
Table 5. Effect of intact chloroplasts (IC) and broken chloroplasts (BC) on metabolism of oleoyl-CoA by pea-leaf microsomes.
Different preparations of pea-leaf microsomes containing 0.9 mg protein (experiment I) and 1.0 mg protein (experiment II) were incubated for specified times with [1-14C]18:1-COA (5.18 KBq; 2.5 nmol). The corresponding chloroplast preparations containing final concentrations of 136 gg chlorophyll m1-1 (experiment I) and 263 gg chlorophyll m1-1 (experiment II) were added and the incubations were continued for specified periods. Procedures for the isolation of microsomes and chloroplast preparations and for the incubations were as described under Material and methods. Each incubation mixture contained NADH (1 mM), ATP (4 raM), CoA (0.3 raM), catalase (2000 units), uridine 5'-diphosphate-galactose (1 raM) and glyeerol-3-phosphate (1 mM); MGDG = monogalactosyldiacylglycerols,PE = phosphatidylethanolamines Expt.
II
Incubation time (min)
Distribution (%) of radioactivity in chloroform-soluble lipids
Microsomes _+ Chloroplasts
FA
18:I-PC
18:2-PC
PE
MGDG
Others ~
(180) (60) (60)
none IC (120) BC (120)
62 47 56
10 14 16
4 13 9
4 5 2
1 3 3
19 18 14
(180) (60) (60)
none IC ( 6 0 ) IC (120)
64 42 52
7 19 16
4 13 12
5 2 2
3 3 4
17 21 14
a Including lyso-PC, diacylglycerols and triacylglycerols
products shows, for both experiments, an increase in the labeling of 18:1-PC and 18:2-PC by the addition of chloroplast preparations to microsomes, yet, only a minor labeling of galactolipids occurred and only traces of lipids containing 14Clinolenoyl moieties were detected. Discussion
Earlier findings (Murphy et al. 1983 a; Slack et al. 1976) taken together with the time-course studies mentioned before corroborate the pathways by which 18:1-FA is activated to 18:1-COA in the presence of ATP and CoA, then transferred to PC and finally desaturated to 18 : 2-PC. Each of these steps is catalyzed by membrane-bound enzymes in pea-leaf microsomes. Contamination by chloroplast-envelope-derived activities was ruled out on the basis of subcellular distribution and enzymemarker studies (Murphy et al. 1983 b). Hydrogen peroxide has been found to inhibit stearoyl-CoA desaturase in rat-liver microsomes (Baker et al. 1976) and stearoyl-acyl carrier protein desaturase in maturing safflower seeds (McKeon and Stumpf 1982), although little is known about the possible source of H 2 0 2 o r its mechanism of action on desaturases. We found that H 2 0 2 a t low concentrations hardly affects 18:1-PC desaturation, but with increasing concentration of H202 (2.5 to 10 raM) 18!1-PC desaturation is progressively inhibited (Table 1). The distribution of radioactivity in the products of incubation reveals that H202 specifically inhibits 18: 1-PC desaturation but has hardly any effect on the reactions catalyzed by 18:I-CoA:PC acyltransferase or 18 : 1-CoA thioesterase (Table 1). In agreement with earlier findings with stearoyl-CoA desaturase (Baker et al. 1976), stearoyl-
acyl carrier protein desaturase (McKeon and Stumpf 1982) and "linoleate desaturase" (Browse and Slack 1981), catalase was found to stimulate oleate desaturation and overcome the inhibitory effect of H20 2 on 18: I-PC desaturation in pea-leaf microsomes (Table 2). Stimulation of the desaturase by catalase was likely caused by removal of H 2 0 2 , although a specific action of catalase on the desaturation reaction itself cannot be ruled out. We have shown earlier (Murphy et al. 1983a) that the inhibition of 18:1-PC desaturation by H202 is neither the consequence of~oxidative damage to the lipids of microsomal membranes nor the results of oxidation of NADH which is one of the cofactors required by the desaturase system (Slack et al. 1976; Styrene and Appelqvist 1978). Oleate desaturation in plants is inhibited by cyanide (Stymne and Appelqvist 1978; Vijay and Stumpf 1972), but not by carbon monoxide (Vijay and Stumpf 1972), as is the case with stearate desaturation in animal systems (Pugh and Kates 1979). It is, therefore, likely that the plant 18 : 1-PC desaturase is terminally linked with proteins similar to NADH : cytochrome b s reductase and cytochrome b s for electron transfer from NADH, as is the case in desaturase systems from animals, such as stearoyl-CoA desaturase (Pugh and Kates 1979). We detected NADH: ferricyanide reductase and NADH: cytochrome c reductase activities in our preparations of pea leaf-microsomes using the spectrophotometric techniques (Jolliot et al. 1978; Strittmatter 1967) which were routinely used to assay for cytochrome b s reductase and cytochrome b s. In order to assess how these two likely components of the oleate desaturase system are affected b y H202, we incubated the pea-leaf microsomes with up to 10 mM H20 2 in the standard incubation medium and followed the changes in the activ-
D.J. Murphyet al. : Oleatemetabolismin microsomes ity of NADH: ferricyanide reductase and NADH: cytochrome c reductase. In each case it was possible to rule out a specific inhibition of electrontransport activity by H20 2 at either of these sites, since H20 2 at up to 10 mM affected neither activity. All these findings strongly indicate that HzO 2 specifically inhibits the terminal 18:1-PC desaturase itself, rather than any of the components of its associated electron-transport chain. Ascorbic acid has been found to stimulate the acyl-CoA AS-desaturase in developing meadowfoam seeds (Moreau etal. 1981). We observed some stimulation of oleate desaturation in pea-leaf microsomes by the addition of ascorbic acid (Table 3). Addition of ascorbic acid with ascorbate peroxidase resulted in further stimulation of oleate desaturation (Table 3). It is likely that the stimulation of oleate desaturation by ascorbate peroxidase is caused by removal of H20 2 since the latter is a cosubstrate for this enzyme, which converts ascorbic acid to dehydroascorbic acid (Kelly and Latzko 1979). The specific effect of H20 2 upon the oleate desaturase system was seen only at relatively high H202 concentrations (>0.5 raM). While the cytosolic HzO 2 concentration is probably less than 0.5 mM under physiological conditions, it is possible that local H20 z concentrations may considerably exceed this value. Leaf cells from a variety of plants have been found to contain an extremely effective HzOz-scavenging system - ascorbate peroxidase (Groden and Beck 1979; Kelly and Latzko 1980). One of the physiological roles of this and other H2Oz-scavenging systems may be to protect susceptible proteins, such as the terminal enzyme of the oleate-desaturase system, from oxidative damage. As found earlier (Murphy et al. 1983a; Slack etal. 1976), the transfer of 18:1 moieties from 18:1-COA to PC is a prerequisite for desaturation of oleate in pea-leaf microsomes. In microsomes from developing safflower seeds, the transfer of 18:1 moieties from 18:1-COA to PC has been shown to be catalyzed predominantly by 18:lCoA:lysophospholipid acyltransferase and lysoPC seems to be the major acceptor in this reaction (Moreau and Stumpf 1982). On the other hand, in microsomes from developing cotyledons of soya bean and safflower, the transfer of 18:1 moieties from 18:1-COA to PC seems to occur mainly by acyl exchange (Styrene and Glad 1981; Stymne et al. 1983). When pea-leaf microsomes were incubated with radioactive 18:1-COA in the presence of exogenous lyso-PC or PC at two different concentrations, a distinctly higher labeling of both total PC and 18:2-PC was observed by the inclusion of lyso-PC rather than PC in the incubation mix-
253 ture (Table 4). These findings are in agreement with lyso-PC being the preferred acceptor for acyl transfer from 18:1-COA. On the other hand, we consistently detected small proportions of labeled 18:2-CoA in the reaction products (Table 1) which indicates that a small amount of acyl exchange between acyl-CoA and PC occurred as well. Moreover, we detected in the reaction products, minor proportions of labeled lyso-PC (5-10% of toal labeled PC; data not shown) which indicates that deacylation of PC by phospholipases and reacylation of the resulting lyso-PC by acyl-CoA might also be involved in the transfer of acyl moieties to PC. However, the contribution of the latter two reactions to the net transfer of oleoyl moieties to PC was very minor as compared with the direct transfer from oleoyl-CoA to form PC. It is likely, however, that in vivo the deacylation-reacylation reactions play an important role in sustaining a pool of lyso-PC, to which the oleoyl moieties are transferred from 18 : I-CoA. It has been proposed that co-operation between chloroplasts and endoplasmic reticulum (enriched in the microsomal fraction) is involved in the biosynthesis and assembly of chloroplast-membrane lipids (Roughan etal. 1980; Tr~moli&es etal. 1980). Such a co-operation is believed to result in the transport and conversion of 18:2-PC, synthesized on the microsomal membranes, to galactolipids containing 18 : 2 and 18 : 3 moieties in the chloroplast. These conclusions were drawn from the pattern of labeling of lipids when isolated chloroplasts were incubated with radioactive acetate with or without added microsomes in the presence of uridine 5'-diphosphate (UDP)-galactose and glycerol-3-phosphate. In order to detect such a cooperation, we preincubated the pea-leaf microsomes with radioactive 18: I-CoA in the presence of UDP-galactose and glycerol-3-phosphate. Subsequently, intact or broken pea-leaf chloroplasts were added to the incubation mixture and the transfer of label to various lipid classes was followed (Table 5). We found that the addition of either intact or broken chloroplasts to the microsomes resulted in some stimulation in the formation of both 18:1-PC and 18:2-PC. But the galactolipids were labeled only to a small extent and the labeled linolenoyl moieties formed barely exceeded 1-2% of the labeled acyl moieties in the total lipids. Even the incubation of [1-agC]oleoyl CoA with freshly prepared whole homogenate from pea leaves did not result in more than a small (1-2%) labeling of galactolipids or linolenate (data not shown). It has previously been shown that chloroplasts, liposomes and microsomes will readily fuse or clump together even in low-salt media (Murphy and Kuhn 1981). Indeed it has recently
254
been reported that spontaneous phospholipid transfer or exchange readily occurs between phospholipid vesicles of different composition (De Cuyper etal. 1983). Therefore, labeled 18:2-PC was presumably transferred from microsomes to chloroplast membranes but was not significantly metabolized thereafter in either whole-tissue homogenates or cell preparations of pea leaves. The observed stimulation in 18:2-PC formation that was found following the addition of chloroplasts to actively desaturating microsomes may be caused by the removal of 18:2-PC from the microsomes to the chloroplasts and the consequent relief of a product inhibition of the 18 : 1-PC desaturase system. A part of this work was supported by a grant from Deutsche Forschungsgemeinschaft (DFG).
References Arnon, D.I. (1949) Copper enzymes in isolated chloroplasts. Polyphenoloxidase in Beta vulgaris. Plant Physiol. 24, 1-14 Baker, R.C., Wykle, R.L., Lockmiller, J.S., Snyder, F. (1976) Identification of a soluble protein stimulator of plasmalogen biosynthesis and stearoyl-coenzyme A desaturase. Arch. Biochem. Biophys. 177, 29%306 Browse, J.A., Slack, C.R. (1981) Catalase stimulates linoleate desaturase activity in microsomes from developing linseed cotyledons. FEBS Lett. 131, 111-114 De Cuyper, M., Joniau, M., Dangreau, H. (1983) Intravesicular phospholipid transfer. A free-flow electrophoresis study. Biochemistry 22, 415-420 Diesperger, H., Miiller, C.R., Sandermann, H. (1974) Rapid isolation of a plant microsomal fraction by magnesium (2 +) precipitation. FEBS Lett. 43, 155-158 Groden, D., Beck, E. (lp79) HzO 2 destruction by ascorbatedependent systems f~om chloroplasts. Biochim. Biophys. Acta 546, 426-435 [ Heber, U., Santarius, KtA. (1970) Direct and indirect transfer of ATP and ADP acr6ss the chloroplast envelope. Z. Naturforsch. Teil B 25, 718-728 Jolliot, A., Demandre, C., Mazliak, P. (1978) Role of lipids in electron transport chain function in potato microsomes. Biochimie 60, 767-775 Kelly, G.J., Latzko, E. (1979) Soluble ascorbate peroxidase. Detection in plants and use in vitamin C estimation. Naturwissenschaften 66, 617-618 Kelly, G.J., Latzko, E. (1980) Prospect of a specific enzymatic assay for ascorbic acid (vitamin C). J. Agric. Food Chem. 28, 132(~1321 Lowry, O.H., Rosebrough, N.J., Farr, A.L., Randall, R.S. (1951) Protein measurement with the folin phenol reagent. J. Biol. Chem. 193, 265-275 McKeon, T.A., Stumpf, P.K. (1982) Purification and characterization of the stearoyl-acyl carrier protein desaturase and the acyl-acyl carrier protein thioesterase from maturing seeds of safflower. J. Biol. Chem. 257, 12141-12147 Moreau, R.A., Pollard, M.R., Stumpf, P.K. (1981) Properties of a A5-fatty acyl-CoA desaturase in the cotyledons of developing Limnanthes alba. Arch. Biochem. Biophys. 209, 376-384
D.J. Murphy et al. : Oleate metabolism in microsomes Moreau, R.A., Stumpf, P.K. (1982) Solubilization and characterization of an acyl-coenzyme A: O-lysophospholipid acyltransferase from the microsomes of developing safflower seeds. Plant Physiol. 69, 1293-1297 Murphy, D.J., Kuhn, D.N. (1981) The lack of a phospholipidexchange-protein activity in soluble fractions of Spinacia oleracea leaves. Biochem. J. 194, 257-264 Murphy, D.J., Mukherjee, K.D., Latzko, E. (1983a) Lipid metabolism in microsomal fraction from photosynthetic tissue: effects of catalase and hydrogen peroxide on oleate desaturation. Biochem. J. 213, 2149-252 Murphy, D.J., Woodrow, I.E., Latzko, E., Mukherjee, K.D. (1983b) The oleate desaturase system of pea leaf microsomes: solubilisation of oleoyl-CoA thioesterase, oleoylCoA: phosphatidylcholine acyltransferase and oleoyl-phosphatidylcholine desaturase. FEBS Lett. 162, 442-446 Murphy, D.J., Walker, D.A. (1982) Acetyl coenzyme A biosynthesis in the chloroplast. What is the physiological precursor? Planta 156, 84-88 Nakatani, H.V., Barber, J. (1977) An improved method for isolating chloroplast retaining their outer membranes. Biochim. Biophys. Acta 461, 510-512 Pugh, E.L., Kates, M. (1979) Membrane-bound phospholipid desaturases. Lipids 14, 159-165 Roughan, P.G., Holland, R., Slack, C.R. (1980) The role of chloroplasts and microsomal fractions in polar lipid synthesis from [1-1~C]acetate by cell-free preparations from spinach (Spinacia oleracea) leaves. Biochem. J. 188, 1224 Roughan, P.G., Slack, C.R. (1982) Cellular organization of gtycerolipid metabolism. Annu. Rev. Plant Physiol. 33, 97-132 Slack, C.R., Roughan, P.G., Browse, J. (1979) Evidence for an oleoyl phosphatidylcholine desaturase in microsomal preparations from cotyledons of safflower (Carthamus tinctorius) seed. Biochem. J. 179, 649-656 Slack, C.R., Roughan, P.G., Terpstra, J. (1976) Some properties of a microsomal oleate desaturase from leaves. Biochem. J. 155, 71-80 Strittmatter, P. (1967) NADH-cytochrome b 5 reductase. Methods Enzymol. i0, 561-565 Stumpf, P.K., Kuhn, D.N., Murphy, D.J., Pollard, M.R., McKeon, T., MacCarthy, J. (1980) Oleic acid, the central substrate. In: Biogenesis and function of plant lipids, pp. 310, Mazliak, P., Benveniste, P., Costes, C., Douce, R., eds. Elsevier/North-Holland Biomedical Press, Amsterdam Stymne, S., Appelqvist, L.-A. (1978) The biosynthesis of linoleate from oleoyl-CoA via oleoyl-phosphatidylcholinein microsomes of developing safflower seeds. Eur. J. Biochem. 90, 223-229 Stymne, S., Glad, G. (1981) Acyl exchange between oleoyl-CoA and phosphatidylcholine in microsomes of developing soya bean cotyledons and its role in fatty acid desaturation. Lipids 16, 298-305 Stymne, S., Stobart, A.K., Glad, G. (1983) The role of the acyl-CoA pool in the synthesis of polyunsaturated 18-carbon fatty acids and triacylglycerol production in the microsomes of developing safflower seeds. Biochim. Biophys. Acta 752, 198-208 Tr6moli+res, A., Dubacq, J.P., Drapier, D., Mfiller, M., Mazliak, P. (1980) In vitro cooperation between plastids and microsomes in the biosynthesis of leaf lipids. FEBS Lett. 114, 135-138 Vijay, I.K., Stumpf, P.K. (1972) Properties of oleoyl-coenzyme A desaturase of Carthamus tinctorius. J. Biol. Chem. 247, 36~366 Received 14 November 1983; accepted 27 January 1984
