Coenzyme A-dependent, ATP-independent acylation of 2-acyl lysophosphatidylinositol in rat liver microsomes

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Bh;tq#mica et Biophysk'u Ac:a. 1084 ( It)91 ) 292-299 1991 Elsevier Science Publishers B.V. 1101)5-27fi0/91/S03.50 A DONIS 11{11~5276119111[]2I6S

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Coenzyme A-dependent, ATP-independent acylation of 2-acyl lysophosphatidylinositol in rat liver microsomes Jennifer Cordes Darnell and Alan R. Saltiel I.abm'at~W of Moh'cular Om'ology, The R.ckefi'ller Uniw'rsity, New York, I~T (U.S..4.) (Received 19 February 1991]

Key w~rds: 2-Acyl lysoPI; Phospholip.d remodelling; ATP-independenl acylalion

Phosphatidylinositol (PI) is synthesized from cytidine.diphosphodiacylglyceroi (CDP-DAG) and inositol by the enzyme P! synthase. CDP-DAG is itself synthesized from phosphatidic acid and CTP. The observation that PI differs in fatty acid composition from i~s precursors CDP-DAG and phosphatidic acid led to the proposal that following its synthesis the fatty acids of P! are removed and replaced by others in a process called fatty acid remodelling. Previously, we used rat liver microsomes to study the molecular mechanisms of PI remodelling. Following its synthesis, PI is rapidly deacylated to form lysoPl which is teac:,'lated to form. new P! species. PI remodelling occurs predominantly at the I-position. We demonstrate here that lysoP! can be aeylated in the l-position in an ATP-independent manner. The acylation of 2-acyl lysoPI by the ¢oenzyme A-dependent, ATP-independent mechanism was examined. The acylation exhibits a pH optimum of 7.5, does not require a divalent cation, and is not inhibited by Ca 2+ or Mg 2+, although Zn '+ is a potent inhibitor. The apparent K m values for coenzyme A and 2-acyl lysoPl are 14 ~M and 30 /zM, respectively. The acylation of 2-acyl lysoPl incorporates primarily stearic acid into the l-position of PI, as would be expected based on the fatty acid composition of steady-state PI in rat hepatocytes.

Introduction Phosphatidylinositol (PI) that is synthesized in the cndoplasmic reticulum has a fatty acid composition that is similar to that of its precursors, cytidine diphosphodiacylglycerol (CDP-DAG) and phosphatidic acid (PA). This newly synthesized PI has a saturated fatty acid in the 1-position and a mono- or diunsaturated fatty acid in the 2-position [1-3]. Interestingly, the composition of P! extracted from mammalian liver, brain, or platelets has a different fatty acid composition [1,4-7]. Approx. 811% of this 'mature' Pl exhibits a 1-stearoyl, 2-arachidonoyl structure. ] h e molecular mechanisms involved in the generation of these te-

Abbreviations: I't. phosphatidylil~mil,~l; CDP-DAG, cytidinediphosphodiglyccridc: PA. p h m p h a t i d i ¢ acid; TL('. thin-layer cimmlatograph.v; PI,A ,_. phospholipase A ,. -

('orrcsptmdcncc: A.R. Satticl Parke-Davis Pharmacculical Division. Warner Lamltcrl. 2801) Plymo,.th Rd.. Ann Arbor, MI 48105, U.S.A.

traenoic PI molecules, and the regulatory processes involved are not yet defined. Work by Holub [I,2,8] and others [3,9,10] has defined the processes involved in the fatty acid remodelling of PI. The fatty acids present in newly synthesized PI are apparently removed by phospholipase(s) A [ll,12] and subsequently replaced by acyltransferases or transacylases [13-16]. The rapidity of this process was suggested by the appearance of remodelled forms of Pl in rat liver rapidly following whole animal labelling with ~-'P or [3H]glycerol [l]. Moreover, the inability to detect the accumulation of lysoP! suggested that the deacylation and reacylation reactions are coupled. In previous studies we developed an in vitro system for evaluating Pl remodelling in rat liver microsomes [17.18]. In this system newly synthesized P! undergoes a cycle of deacytation/reacylation in the l-position, resulting in the incorporation of primarily stearate and to a lesser extent oleate. Interestingly, the remodelling of the l-position fatty acid occurs in the absence of added cofactors. In this report we have examined the mechanisms involved in the reacylation of 2-aeyl lysoPI. Evidence

293 suggests that 2-acyl lysoPl can be acylated in a coenzyme A (CoA~-dependent, ATP-independent manner in rat liver microsomes.

standard ( R v = 0.4) was located by iodine staining and the region corresponding to tysoPI was scraped and exhaustively eluted with solvent B. This lysoPl was used in assays for l-acyl lysoPl acylation.

Materials and Methods

Mater;als [3H]lnositol (15 C i / m m o l ) w a s from American Radiolabelled Chemicals. [~H]Arachidonic acid (94.5 Ci/mmol) :rod EN~HANCE were from New England Nuclear. CDP-DAG was obtained from Serdary Lipids. C-8 rever~ed-pha~c TLC plate~ and silica gel 6(1 plate,,, were from EM Science, Silica Gel G plates were from Analtech. Ready Safe was f-om Beckman and protcir~ assay kit was from Bio-Rad. Triacylglycerol lipase (EC 3.1.1.3) type Xl from Rhizopus arrhizus, phospholipa.~e A , (EC 3.1.1.41 from porcine pancreas and all other biocitcmieais were purchased from Sigma.

Preparation of licer m&rosomes Rat liver microsomes were prepared from male Sprague-Dawley rats (1811-250 g) (Charles RiverJ ag previously described [17]. Following ultracentrifugation the microsomal pellets were resuspcnded using a Teflon-glass homogenizer to a final protein concentration of 15-20 m g / m l in cold 101) mM phosphate buf,.(pH 7.4). Microsomes were either used immediately or stored at - 7 0 ° C for up to 6 months with no s;gnificant decrease in P! synthase, deacyiase or reacylasc activities. Protein concentration was determined by Bradford using the Bio-Rad protein assay kit.

Preparation of microsomal Pi labelled by head group e~'chatlge Rat liver microsomes (I m g / m l protein) were incubated with Buffer A and 25 #.Ci/ml [~H]inositol (10 p.M) in the [:resence of 10 mM MnCI.,. Following incubation at 37°C for 90-120 min, the reaction was terminated with solvent B. The tipid~ that separated with the lower organic phase were dried under nitrogem rcsuspcnded in 1011/.tl of methanol and streaked over 4-5 cm at the origin of a silica gel 6{1 plate. The plate was developed to 17 cm from the origin in chloroform/' methanol/acetic acid/water {56: 30:8:4, v/v~. A PI standard (Rv = 0.7) was visualized with iodine arid the edge of the labelled lipids was scraped in 1 cm ["....,.is and counted. The band corresponding to labeled PI was scraped from the plate and exhaustively eluted with sequential 2 ml aliquots of chloroform/methanol (2: t. v/v) and dried under nitrogen. These lipid.,, wcrc resuspendcd by son)cation in 50 t,tl of methanol and streaked across 2-3 cm o~. a '~ilica gel 6t1 plate. The plate was developed in c h l o r o f o r m / m e t . h a n o i / acetone/acetic acid/water (5(1: 10:20 : 10 : 5. v/v) to 17 cm. On this system Pi typically ran with an RI.. of 0.25. PI was scraped, elated and evaporated to dryness as above.

Lipase digestions Preparation of I "H]P! and [~H]lysoPl from dipabnitoyl CDP-DAG These substrates were prepared by iv, vitro synthesis in rat liver microsomes in a total volume of 1 mi. The reaction contained 50 mM Hepes buffer (pH 7.5), with 3 mM MgCI.,, I mM EDTA, 0.1 mM EGTA (Buffer A) and 25 ~Ci of [~H]inositol (10 ~,M). A stock solution (10 m g / m l ) of dipalmitoyl CDP-DAG in CHCIff MeOH (2: 1, v/v) was prepared and stored at - 20°C. A 40/.tl aliquot was dried under nitrogen and sub,scquently resuspended by son)cation in reaction buffer~ The reaction was initiated by addition of microsomes to a final concentration of 1 m g / m l protein, and allowed to incubate at 37°C for 60 min. Synthesis was tcrm;r~,ated by addition of 1 mt of chlo~ ~form/ isopropanol/6 M HCi (1011:50: !, v/v} (soh, ent B), followed by vigorous vortexing and centrifugation. The lipid-containing lower phase was separated, and evaporated trader nitrogen. The ~H-labelled PI was purified from other phospholipids as described belew for exchange-labeled PI. For collection of [~H]iysoPl the lipids were resuspended in 40 t~l methanol and spotted at the origin of a C-8 reversed-phase TLC plate which was developed twice !o 17 cm in 50~,.;~ ethanol. A tysoPl

For the phospholipase A , digestions, the dried lipids were rcsuspcnded in ii.)0 mM Hepes buffer (pH 7.4). containing 10 mM CaCI-, and 0.01% Triton X-100 in a total volume of 2(1(I t.tl, 12 units of pancreatic phospholipase A-, were added and the samples were incubated at 3TC for 2 h. Lipids were recovered by extraction with I ml solvent B. After eentrifugation to separate the phases, the organic (lower) phase was dried under nitrogen and resuspended by son)cation in 411 tsl of methanol. For lipasc digestions to determine fatty acid composition, the dried lipid was sonicated in 100 mM Hepes buffer (pH 7.4), containing 111 mM CaCI, an~! (I.(11% Triton X-I()0 in a total volume of 2011 ,ttl. Following addition of 450t) units of Rhizopus arrhizus lipase (specific fi~r the l-position of phospholipids), the samples were incubated at 37°C for 2 h, and iipids were extracted as described above for PLA2 digestions.

Preparation of 2-acyt iysoP! from ~:~'change-habe,led PI 5" 10" dpm of exchange-labelled or dipalmitoyl P! was suspended by son)cation in 50 mM Hepes (pH 7.4) containing I0 mM CaCI 2. 45011 units of Rhizopus arrhizus lipase were added and the Pi was digested at

294 37°C for 30 rain. The resu!ti,-j lysoPl v,er eeparatcd from PI by extraction as described below. Following chloroform/methanol/water separation the aqueous fraction was collected by centrifugation and partiaily dried under nitrogen to a final concentration cff ap prox. 20000 dpm of lysoP! per 10 /,1 of watel. The lysoPl was kept on ice and was added to reactions in this form, within an hour following digestion.

Separation of 1"1 and lysoPl by extraction Following in vitro synthesis as described above, 0.2 m! of reaction was extracted with 720 #t chloroform/ methanol (1:2, v/v), 250 #1 of chloroform and 250 #1 of water added sequentially with vortexing between additions (referred to as solvent D). The organic phase containing the labeIied PI was dried under nitrogen. When [3H]inositol or aqueous metabolites were present, the aqueous phase containing the tysoPl was reduced briefly under nitrogen and 500 #1 of watersaturated butanol was added to form two phases. Following centrifugation the resulting butanol phase was dried under nitrogen to yield lysoPl. Otherwise, the aqueous phase following chloroform/methanol/water separation was evaporated to dryness.

Recersed-phase thin-layer chromatography Reversed-phase C-8 TLC plates were activated at I25°C for 30 rain prior to sample application. Samples were applied as 1 cm streaks, and plates were developed twice in 50% ethanol/water until the solvent front was t7 cm from the origin. In this system, PI, PIP and PIP 2 all remain at or near the origin, water-soluble inositol metabolites migrate to the top of the plate, and lysoPI species migrate with an R F of approx. 0.4. In experiments to quantitate PI formation from lysoPI, plates were developed once in 60% ethanol/water to 8 cm from the origin. For fluorography, dried plates were treated with EN3HANCE spray and exposed to Kodak XAR-5 film. To quantitate radiolabelled lipids, 1 cm bands were scraped into scintillation vials, 5 ml of ReadiSafe was added and samples were counted in a Beckman LS 3801 scintillation counter.

Assay for the acylation of 2-acyl lysoPl Unless otherwise noted the assay was performed in 50 mM Tris/50 mM Mes buffer (pH 7.7 at 37°C) or 20 mM Hepes (pH 7.4) in the presence of 50 ~M CoA and 100 ~g/ml microsomal protein ia a total volume of 200 t.tl. 30000-60000 dpm of 2-acyl lysoP! were added and the reaction incubated at 37°C for 1-5 rain. The mixture was quenched with 1 ml solvent B and PI quantitated by C-8 reversed-phase TLC as described above.

Assay for the incorporation of ['~H/arachidonate into PI The assay was performed in a total volume of 200 ~1 of buffer A containing 1 mg/mi microsomal protein,

19',2' /zM lya¢,PI (~,igma soybean), ard 0.05 ~Ci [3H]arachidonic acid. Micro.~or.'es were added to the background reaction after the addition of organic solvents. 1 mM ATP and/or 1 mM CoA were used to stimulate the acylation reaction. The reaction was incubated at 37 °C for 30 rain and quenched by the addition of 1 ml solvent B. Following centrifugation the organic phase was dried under nitrogen and resuspended in 50 tzl of methane! by sonication. The lipids were spotted at the origin of a prebaked silica gel G plate and the plate was developed twice to 17 cm in hexanes/anhydrous ether/glacial acetic acid (70: 30: I, v/v). In this system PI migrates with an R~ = 0, arachidonic acid with an R~ = 0.9. The plate origin was scraped into scintillation vials and counted in 5 ml of ReadiSafe to quantitate the incorporation of [SH]arachidonate into PI.

Phosphate analysis Exchange labelled or dipalmitoyl PI was dried under nitrogen. As a control, a stock solution of soybean PI (Sigma) standard was made to 6.6 mg/ml in chloroform:methanol. Aliquots of 25, 50 and 100 ::! were dried under nitrogen for digestion. All were resuspended in 100/.tl of 50 mM Hepes (pH 7.4), containing 10 mM CaCI 2. An equal volume of 4 N H2SO 4 was added and the samples heated at 160°C for 2 h in marble-capped tubes. As a control 100 p,I of the above buffer was digested as well. Free phosphate was determined by the method of Baykov et al. [19]. Free phosphate standards and P! standards were linear over the range of concentrations tested (data not shown).

Results

Multiple mechanisms exist to acylate lysoPl in rat liver microsomes Three distinct mechanisms have been proposed to explain the acylation of lysophospholipids and are potentially responsible for the fatty acid reacylation step of the remodelling of PI. In the first, termed ATP- and CoA.dependent acylation, fatty acyl CoAs are synthesized by the action of fatty acyl CoA synthetase. This reaction requires both ATP and CoA. The fatty acyi CoAs then serve as substrates for the acylation of lysolipids, such as lysoPI, by acyltransferascs. In the second form of aeylation, CoA-dependent, ATP-independent acylation, fatty acyl CoAs are released from phospholipids directly in the CoA-bound form by the reverse reaction of an acyltransferase [20-22]. This reaction requires only CoA. In the third form, cofactor-independent acylation, the fatty acid released from a phospholipid lay phospholipase cleavage is donated directly to lysoP! instead of to a water acceptor [22]. To explore the mechanisms in the microsomal sys-

295 i.cm invoi~ed in the a,.xm,,vn:" of IysoPl. wc added [3H]lysoP1 to rat liver microsomes in the presence or absence of cofactors likely to modulate this reaction, and assayed the formation of [3H]P1 by thin-layer chromatography (Fig. 1). In the absence of added cofactors (control reaction), no acylation of PI occurred over background. Formation of PI in the presence of A T P alone was slightly above background. Addition of CoA stimulated the acylation of approx. 15e/- of the added [3H]lysoPl, The addition of CoA and A T P together resulted in an insignificant stimulation of P! formation over CoA alone, These data suggest that 1-acyl lysoPl can be acylated in a C o A - d e p e n d e n t , ATP-independent manner. Further elucidation of this mechanism was dependent upon the demonstration that the microsomes do not have any A T P or CoA as stored. The microsomes were dialyzed against buffer, in dialysis tubing with a 6000-8000 molecular weight cutoff, overnight at 4 ° C to remove A T P and CoA, Dialyzed microsomes were still capable of lysoPi acylation, p e r f o r m e d as detailed above. Although the a m o u n t of PI formed was about 1 / 4 t h that observed with undialyzed microsomes, due to the loss of enzyme activity, the relative P! formation was similar to that shown in Fig. 1, suggesting that the PI formed in the presence of CoA alone is not due to A T P present in the microsomes. T h e C o A - d e p e n d e n t , A T P - i n d e p e n d e n t mechanism of lysoPl aeylation was further evaluated by following

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o Fig. I. The acylation of lysoPI is dependent on the addition of ('oA or ATp and CoA. Washed rat liver microsomcs (2(tt)/,tg/ml protein) we", ,cubated with a 51) mM Tris/50 mM Mes buffer (pl-I 7.7 at 3"1 ..), and 351}(11)dpm of lysoPI synthesized from dipalmitoyl ('DPDAG as described in M~,teriats and Methods. in a total volume of 250 #1. To the designated reactions either 1 mM ATP, 1 mM (.k~Aor both were added. The reactions were incubated t5 rain at 37 ° C and quenched with ! ml solvent B. The organic phase was dried and chromatographed on C-8 TLC to 8 cm as described in Materials and Methods. The origins were scraped into scintillation vials and counted in Readi-Safe. The reactions were performed in triplicate and the data is presented as the mean dpm. Standard deviations 195"; confidence limit) are shown. This experiment has been repeated three times with similar results.

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u ..o Fig. 2. The incorp~ration of I~H]arachidonate into lysoPI is dependent on the addition of broth ATP and CoA. Rat liver microsomes(I rag/rot) ~,'crc incubated ~:ith buffer A, 0.115 t.tCi [3tt]arachidonate, and 1111)p M Sigma soybean lysoP! plus the indicated cufactors at a concentration af I mM for 31) rain at 37 °C. Reactions were terminated by the addition of Imt ~dvent B and the organic phases were dried under nitrogen. Lipids ~'ere rcsuspended in 51)/,tl of methanol and spotted on a prebaked silica gel G plate which was de,.'eloped as described in Materials and Methods. The plate origins were ~raped and cuunted in 5 ml of Readi-Sale scintillant. Shaded bars represent dpm of PI labelled with undialyzed microsomes, hatched bars with microsomcs dialyzed o'¢ernight against 20 mM Hepes (pH 7.-I). as described in Materials and Methods. This experiment was repealed twice with the same results.

the incorporation of [3H]arachidonate into PI in the presence or absence of :ofactors. LysoPl and [~H]arachidonie acid were a d d e d to microsomes in the presence of Mg-" ". T h e incorporation of counts into PI was assayed by thin-layer chromatography (Fig. 2). The addition of both CoA and A T P resulted in the incorporation of approx. 41)000 dpm of arachidonate into PI. The absence of either A T P or CoA or both, however, completely prevented the incorporation of fatty acid into the phospholipid. Dialyzed and undialyzed microsomcs exhibited identical results. The data argue that little, if any, A T P or CoA is present in the microsomes as stored. The lysoPI added in the above experiments was approx. (~)G l-aeyl lysoPl, which is inherently more stable than the 2-acyl isomer (23]. It was also of interest to study mechanisms of acylation of 2-acyi lysoPl, that most likely reflect the incorporation of stearic acid into PI. 2-acyl lysoPI was prepared as described in the Methods section and its aeylation assayed in rat liver microsomes previously shown to be free of ATP and CoA (Fig. 3). T h e r e was a small but signific~,nt production of PI in the absence of a d d e d cofactors. Addition of ATP alone caused somewhat more lysoPl to be acylated while the addition of CoA alone caused a nearly 12-fold stimulation of P! formation. The addition of CoA and A T P caused a 16-fold stimulation of

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Fig. 3. The acylation of 2-acyl lysoP! depends on the addition of acylation cofactors. Washed rat liver microsomes (201")#g/ml prorein) were incubated with a 50 mM Tris/50 mM Mes buffer (pH 7.7 al 37 o C), and 10[)000 dpm of lysoPl synthesized by deacylation of exchange-lab(led PI as described in Malerials and Methods, in a total volume of 250 ul. To the designated reactions either 1 mM ATP. 1 mM CoA or both were added. The reactions were incubated 15 rain ~,t 37°C and quenched with 1 ml solvent B. The organic phase was dried and chromatographed on C-8 TLC to 8 cm as described in Materials and Methods. The origins were scraped into scintillation vials and counted in Readi-Safe. The reactions were perlk)rmed in triplicate and the data is presented as the mean dpm. Standard devialions (95% confidence limit) are shown. This experiment has been repeated twice with similar results. PI production over control levels. This r e p r e s e n t e d a barely significant increase over C o A alone yet has been reproduced in many repetitions of the experiment. This may suggest the presence of A T P - d e p e n d e n t and independent mechanisms to acylate 2-acyl lysoPl. The acylation of lysoPl in the presence of A T P alone is possibly duc t o the presence of trace amounts of C o A in the microsome which were not detected by the arachidonate incorporation assay. If this were true, however, one would expect higher control levels versus the blank values due to A T P - i n d e p e n d e n t acylation. Therefore, the significance of thc PI formation in the absence of addcd C o A is not yet clear.

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Fig. 4. The pH dependence of the acylation of 2-acyl lysoPI. [~HJ2acyl lysoPI (60000 dpm) was added to reactions containing prewarmed 50 mM Tris/50 mM Mes buffers of lhe indicated pH. 50 ~M CoA and 100 u.g/ml microsomal protein in a total volume of 200 #1. Reactions were incubated at 37 °C for 2 rain and quenched with ! ml solvent B. PI synthesis was quantitated by C-8 TLC described in Materials and Methods. The data is presented as dpm of PI synthesized minus blank values (determined by incubation with quenched microsomes). was d e t e r m i n e d using a Tris-Mes buffer at all p H s (Fig. 4). T h e p H o p t i m u m a p p e a r e d to be b e t w e e n p H 7.5 and 8.0, with very little activity at the more acidic pHs. T h e r e was a small d e c r e a s e in acylation activity at the higher pHs. T h e divalent cation d e p e n d e n c e of the A T P - i n d e p e n d e n t acylation o f 2-,,eyl lysoP! was examined by assaying acylation of [3H]2-acyl lysoPl in rat liver miI'I 9 + 2

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In order to examine the characteristics of the enzymc that acylates 2-acyl lysoPl in an A T P - i n d e p e n dent manner we optimized the incubation with respect to protcin, time, pH, d!valent cations and substrates. Tnc reaction was found to be linear with protein up to 10l) # g / m l protein (data not shown). 100 # g / m i was used in all subsequent experiments. Using 10() ~ g / m l protein and 5(i /zM added CoA, the acylation of 2-acyl lysoPI was found to bc linear for 2 min (data not shown). In subsequent experiments the reaction was stopped at 1 rain. to ensure lincarity with respect to both time and protein concentration. Thc pH d c p c n d c n e c of the acylation of 2-acyl lysoPl

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Fi~. 5. The divalent cation dependence for the acylation o[ ~-acyl lysoPI. Rat liver microsomes (100 #g/roll were incubated with 50 ~M CoA in 20 mM Hepes (pH 7.4) for 1 rain with the indicated additions. Either 0-5 mM MgCI2. (I-i0 mM CaCI., (in the presence of 2 mM MgCI_,), I mM ZnCI, (in the presence of 2 mM MgCI2). or and Ill mM EDTA were added Io the incubation mixtures. Control reactions refer to the addition of no metals or EDTA except in the case of Ca-'+ and Zn ~" where the control contained 2 mM Mg2÷. Blanks were performed with quenched micmsomes. Synthesis of P! was quantituled as in the legend for Fig. 4.

297 crosomes in the presence of CoA (Fig. 5). The addition of 1 and 10 mM EDTA to the acyiation mixture slightly stimulated the formation of PI demonstrating the divalent cation-independence of this process. The slight stimulatory effect is most likely due to the chelation of heavy metals like Zn 2. and Hg -~ that inhibit the activity. Indeed, the addition of 1 mM Zn -'÷ to the reaction almost completely abolished the acylation activity, suggesting that a sulfhydryl group may be essential for the enzyme activity. Moreover, preincubation of rat liver microsomes with 50 mM NEM reduced the synthesis of PI from [3H]lysoPl in the presence of CoA alone to 15% of levels without the addition of NEM (data not shown), further suggesting that the enzyme catalyzing this reaction has an active sutfhydryl group. The addition of 0.1 to 5 mM Mg z+ did not significantly affect the activity. The addition of 0.1 and 1 mM Ca z÷ did not affect the activity, although 10 mM Ca ~-* produced some inhibition. The substrate toncentration dependence "for I-position acylation from microsomal lipid sources was assayed for both CoA and lysoPl. Analysis of the CoA concentration dependence (Fig. 6) yielded an apparent K m of 14/.~M CoA. Analysis of 2-acyl lysoPl concentration dependence yielded an apparent K m of 30/.tM (Fig. 7), Because the substrate 2-acyl lysoPl is unstable, efforts were made to reduce the rate of acyl migration during its preparation. The substrate was typically used within 1 h of preparation, In order to demonstrate aeylation at the l-position, the ['~H]PI resulting from acylation of 2-acyl lysoPl was separated from the [3H]lysoP! substrate by extraction. One third of this

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Fig. 6. The CoA dependence for the atS'lation of 2-acyl lysoPi. Rat liver microsomcs (IIX) p g / m D were incubated in 21) mM Hepes (pH 7.4) containing 2 mM MgCI z with the indicated concentration of CoA and 300110 dpm of ['~H]2-acyl lysoPI for 5 rain. The reactions

were quenchedwith I ml solvent B and PI synthesiswas quantitated by C-8 TLC described in Materials and Methods. Data are presented as dpm of P! formed minus a blank (quenched microsomes) value in double-reciprocal plot form. This experiment was repeated twice

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Fig. 7. The 2-acyl iysoPl concentration dependence for its acylation by rat liver microsomes to form PI. Rat liver microsomes (100 ~ g / m O were incubated with 50 p.M CoA. and the indicated concentration of 2-acyl lysoP1 in 50 mM Hepes (pH 7.4), for ! rain at 37 °C. 1 ml of solvent B was added and the organic phase dried under nitrogen foltov~'ing centrifugation. The s~.,'nthesis of Pi v,'as quantitared by C-8 TLC as in Materials and Methods. For each [~H]lysoPI concentralion a blank (quenched microsomes) was used and the blank dpm were ~ubtracted from the results before plotting. The data art: presented in double-reciprocal plol form. This experiment was performed twice with the same results.

product was chromatographed on a C-8 TLC plate (Fig. 8). The absence of lyso-Pl in this fraction (lane 6) demonstrates that all the substrate was removed by extraction. The remaining PI was digested with either a phospholipase A 2 (PLA_O, which removes the 2-position fatty acids from P! to generate lysoPls differing in their l-position fatty acid, or with a lipase specific for the l-position. Treatment of PI with this lipase removes the 1-position fatty acid, generating a set of iysoPI species that differ in their 2-position fatty acid. In this way it is possible to examine the fatty acid composition of the PI in either position. The digestion of PI with PLA, (lane 4) gave rise to 2 major lysoP1 species previously identified as l-oleoyl (uoper band) and l-stearoyl (lower band)lyso?i~ [18]. Digestion with tipase (lane 5) gave rise to 1- and 2-paimitoyl lysoPl and some 2-oleoyl lysoP1 as well. The 2-acyl lysoPl substrate used for the reacylation experiment was produccd by l-lipase treatment of PI synthc'sized from dipalmitoyl CDP-DAG. An aliquot of the 'dipalmitovi Pi was treated in the same manner as me reacyiateti ~i (Ianes 1, 2, and 3 correspond to lanes 6, 5 and 4). These results illustrate that the 2-position fatty acids were not altered by the removal and subsequent reacylation of the I-position fatty acids under ATP-independent conditions. In contrast, the l-position fatty acids (lanes 3 and 4) were altered under these conditions. The palmitate in the l-position of P! was removed by lipase treatment and no palmitate was introduced into this position during acylation. The fatty acids which have been incorporated appear to be predominantly stearic acid, the lower band, and oleic acid, the upper band. The incorporation of these fatty acids would be expected during fatty acid remodelling of PI, based on

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reaeylated

Pl

Fig. 8. The incc)rporation of stcarate and pit.ate into PI during 2-acyl ]ys,PI rcacylali~)n in rat liver microsomcs. Rat liver mierosomes (100 # g / m l ) were incubated with 50/~M CoA. and I. 10¢' dpm [~l-I]2-acyl I.v.soPl prepared by digesting PI labelled by de novo .synthesis from dipalrniloyl CDP-DAG with Rhizolms arrhizus lipase as described in Matcri:ds and Methods. in I(lll mM ph~sphale buffer (pll 7A) for 15 rain. in a Iotal volume of 400 /.tlr The reaction was quenched by the additi~m of 1 mt chloroforn~/methanol/b M tlCI ( 1 1 1 ( } : 5 0 : 1 , v/v) and the organic phase was dried, re.suspended in 40 ~1 methanol and spotted on a (.'-8 TLC plalc, dcvel()pcd in 6~)~ elhanol. The origin was scraped and elated exhaustivcJy wilh chloroform/methanol/6 M tlC'l (1(1[):5[): 1. v/v). The dried lipids were then resuspendcd in 600 /zl of 5~] mM ]lopes (plt 7.4)conlaining III mM CaCI:. I. Ill ~' dpm of Pl synthesized from dipalmimyl CDP-DAG was dried and resuspcndcd in the same buffer. Both .sels o|" lipids were cilher digested ~villl Rhi:opu.~ arrhiz,s lipasc, pancrealic PLA a. ~r no enzyme a.s described in Malerials and Metht~ds. The rec¢wered lipid products ~.re chrt)malographed on (--'-8reversed-phase TL(' as described and ihc plate was sprayed with Enattance arid expensed to Kodak XAR film lot 7 days al - 71) ° C.

the fatty acid composition of steady-state rat liver PI [1.4-8]. In addition, previous experiments using this system demonstrated that dipaLmitoyi PI was remodelled to incorporate stcarate and olcate into the i-position [18]. Discussion

The predominant fatty acid composition of the phosphoinositidcs consists of arachidonic acid in the

2-position and stearic acid in the l-position [4.24.25]. This structure predominates in rat liver [8]. bovine brain [4], human platclets [26] and pig lymphocytes [27]. Fatty acid composition analysis of the precursors of P! revealed that phosphatidic acid is rclati~,ely enriched in palmitate in the l-position and oleate and linoleate in the 2-position. and relatively depleted in 1-stearate and 2-arachidonate [28]. Analysis of phosphoinositide fatty acid composition following injection of rats with [taC]glycerol, [32p], or ['~H]inositol revealed that the mono- and dicnoic forms of PI were preferentially labeled during initial Pi synthesis [1,2]. The labelling of these species decreased concommitant with the time-dependent increase in the labelling of the tetraenoic class. This led to the hypothesis that PI was remodelled following its synthesis by cycles of deacylation and rcacylation to incorporate new fatty acids into the lipid, Although the incorporation of arachidonate has been the focus of intense investigation, the mechanisms and significance of stearate incorporation have received less attention [16]. The preferential pairing of 2-arachidonate with stearate in the t-position suggests that l-position remodelling plays some role in the generation of the predominant form of PI in almost all mammalian tissues [29]. Although palmitic acid and stearic acid are fairly common constituents of the 1-position of the monoenoic and dienoic classes of PI (that arise by de novo synthesis) greater than 95% of the 2-arachidonate of the tetraenoic class is associated with stearic acid in the l-position [30]. Moreover, this exclusive pairing of 2-arachidonate and l-stearate appears to be unique to PI. Arachidonic acid in the 2-position of phosphatidylcholine was found to be paired with almost equal amounts of 1-palmitate and l-stearate [8]. Additionally, the pairing of 2-arachidonate with 1-stearate was found to be vaore restrictive when araehidonate was in the 2-position of P! than when oleate was found in this position [4,8], further suggesting that the remodelling of PI in the 1-position is critical and may even precede the incorporation of arachidonate. in this manuscript we present evidence that lysoPl can be acylated by rat liver microsomes in an ATP-independent, CoA-dependent manner. This reaction, the reverse of that eatalyzed by acyl-CoA:lysophosphoglyceride acyltransferase [20], releases fatty acids from phospholipids in their activated CoA form and requires CoA but not ATP. This reaction has been shown to be important in the transfer of sn-2 fatty acids (primarily arachidonic acid) from phosphatidylcholine to l-acyl, 2-1ysophosphatidylethanolamine, 1-alkyl, 2-1ysophosphatidylethanolamine and l-acyl, 2-1ysophosphatidylserine [20,21,31-36]. The addition of l-or 2-acyl lysoPI to rat liver microsomes resulted in similar patterns of cofactor dependence, indicating that acylation at the l-position of lysoPl occurs by an ATP-independent

299

mcchanism. In addition, it is possible that a low level of cofactor-independent acylation occurs. By examining the fatty acid composition of the PI that results from the ATP-independent reacylation of 2-acyl lysoPi and comparing this with the fatty acid composition of the P! from which the 2-acyi lysoPl substrate was made, we have shown that CoA-dependent, ATP-independent acylation in this system occurs at the l-position. The fatty acids incorporated into this position are most likely stearate and oleate. The acyla(ion of PI in the l-position with primarily stcarate, and to a lesser extent oleate, is consistent with the observed fatty acid composition of PI in liver cells [8]. The characteristics of the ATP-independent acylation of 2-aeyl lysoPl are similar to those observed for the ATP-independent acylation of other lysolipids in the 2-position. We find that this reaction is calcium-independent [33,34], and exhibits a pH optimum of approx. 7.7 [33,21,22]. Interestingly, Pada!a and Reddy [21,22] find a second pH optimum at pH 4.5, however, we detect little or no activity at acidic pHs. We have determined a g , , for CoA of t4 ttM for l-position transacylation. This is an order of magnitude higher then the K m for CoA determined by others [3,*36] for 2-position acylation. The apparent K m for 2-acyl lysoP! in our system is 30/~M. This is in the same range as that determined for lysoPS (76 v,M) [34] and lysoPE (21-30 v,M) [36]. Interestingly, the l-position acylation of other iysolipids has been characterized in rat liver microsomes by examining the incorporation of acyl CoAs [37,38]. The acylation of l-lyso, 2-acyl phosphatidylserine exhibited at pH optimum of 7.i;, a maximum effect of acyl CoA addition at 20 p.M and the maximum acylation of l-iyso, 2-acyl phosphatidyiserine at 50 v.M [38], conditions similar to those we have determined. Interestingly, the CoA-depcndent, ATP-independent acylation of 2-acyl lysoPl occurs in the absence of added fatty acids, suggesting that the fatty acids must come from an endogenous microsomal donor. Possible sources include free fatty acids, fatty aeyl CoAs, phospholipids, neutral lipids or another lipid source. The microsomal source of the stearic acid is currently under investigation. Acknowledgments We thank Peter Elsbach for helpful discussions. This work was supported by grants from the NIH (DK33807 and BRSG S07 RR07065) A.R.S. is an Irma T. Flirsehl Scholar. References I Holub, B.J. and Kuksis. A. (19711J. Lipid Res. 12. 699-705. 2 Holub, B.J. and Kuksis, A. (19721 Lipids 7, 78-80.

3 Akino. T. and Shimojo. T. (t97(I) Biochirn. Biophys. A','ta 21t=.

343-346. 4 [|tqub, B.J., Kuksis, A, and Thompson, ~,V. (1971)} J. I.ipid Res. I I. 558-564. 5 Kcough, KM.W., MacDonald, O. and Thompson, W. (1972) Biochim. Biophys. Acta 2711. 337-347. 6 Luthra, M.G, alid Shettawy. A. (1972) Biochem. J. 128. 587-595, 7 Wood, R, and Harlow, R,D, (19691 Arch. Biochem. Biophys. 135, 272-281. g i-tolub, B,J. and Kuksis, A.(1971)Can. J. Biochem. 49. l,M-7-1356. 9 MacDonald, G,, Baker, R.R. and Thompson. W. (19751 I. Neurochem. 24, 655-66I. 10 Luthra, M,G. and Shcltaw'/, A, 119761 J. Ncurt',chcm. 27, 151B15tl. t I Irvine. R.F. tlcmington, N, and Daw~m, R.M,C, (19771 Biochem J. 164, 277-2811. 12 Strickland, K,P., Sham, P. and Rao, R.|t. (19781 in Cyclitols and Phosphoinositides (F. Eisenberg, Jr. and W. W. Wells, eds,), lap. 2t11 - 214. Academie Press, New York. 13 Kcenan, R.W. and Hokin, L E . 11%4) J, Biol, Chem, 239, 21232129. 14 B;Iker, R.R, and Thompson, W. (It173) J. Biol. Chem. 248, 7t1611-7[t65. 15 Ihflub, B J, 119761 Lipids I I, I-5. 16 Holub, BJ. and Piekarski, J. (19791 Lipids 14, 529-532. 17 Darnell. J.('., Osterman, D.G. and Salt(el A.R. (IqOO) Bit~chim, Biophys. Acta like5. 260-278. t8 Dilrnell, J,('., Oslerman, D.G, and Salt(el A.R. (19~D Biochim. Biophys. Acla I1185, 27t~-2~41. 19 B;,ykov, A.A,, Evtushenko, O.A. and Avaeva, S,M. (19,~81 Anat. Biochem, 171.2~-270. 211 Irvine, R.F. :rod Dawson, R.C.M. (|9711) Bit~hem. Biophys. Res. Commun. gl, 13t}q-1405. 21 Reddy, P.V. and Schmid. H.ti.O. (1985) Biochem. Biophys. Res. Commun. 129, 38t-388. 22 Redtty, P.V, and Sehmid, tt,tt.O. (lq,q6) Biochim, Biophys. AeIa 879, 369-377. 23 Hill, E.E. and Lands, W.E,M. (19711) in Lipid Metabolism (S.J, WakiL ed.l. pp, 185-279, Academic Press, New York. 24 Thompson, 'iV. ( 19691 Biochim. Biophys. Acta 187, 150-153. 25 Holub. B.J, ;,nd Kuksis. A. 11978) Adv. Lipid Rcs. 16, I - I ! g , 26 Mahadevappa, V,G. and Holub, B.J. (19821 Biochim. Biophys. Aela 713, 73-79. 27 Sugiura, T, and Waku, K. (19841 Biochim. Biophys. Acta 796,

1911- 198. 28 PoK'~mayer, F,, Scherphof, G . L , Dubbelman, T.M.A.R., Van Golde, L M . G . and Van Deenea, E L M . 11969) Bioehim. Bi{~phys. Acla 176, 95-I t0, 29 |hflub, B.J. 11978) in Cyclitols and Phosphoinositidcs (W.W. Wells and F. Eisenberg, Jr., eds.t, pp. 523-534, Academic Press, New York. 311 |lolub. B,J. (1984)C~m, J, PhysM, Pharmacol, 62, I-8. 31 Trotter, J. and Ferber, E, 119811 FEBS Let(. 128, 237-241. 32 Trotter, J., Flesch, I., Schmidl, B. ~md Ferber, E, (1982) J. Biol. (.'hem. 257, 1816-1823. 33 Kramer, R.M. and Deykin, D. (1983~ J. Biol. Chem. LSB, 138116138t 1. 34 Kramer. R.M., Pritzker, C,R, and Deykin, D. (19841 J. Biol. ('hem. 25*4. 2403-2406. 35 Robinson, M., Blank, M.L. and Snyder. F, (19851 J. Biol. Chem. 2611, 7889= 78~15. 36 Nijssen, J.G. and Van den Bosch, tl. (19b161 Biochim. Binphys. Acla 875, 458-464. 37 Holub, B.J. (1981) Bkx:him. Biophys. Acta 664, 221-228. 38 Thompson, W. and Belina, H. (19861 Biochim. Biophys. Acta 876, 379-386.