Biochimica et Biophysica Aria. I ~26(1992) 206-214 © 1992ElsevierScience PublishersB.V. All rightsreserved0¢~)5-2760/92/$05.00
206
BBALIP 53925
Inhibition of phospholipase A 2 by c/s-unsaturated fatty acids: evidence for the binding of fatty acid to enzyme R a m e s h R a g h u p a t h i t a n d R i c h a r d C. F r a n s o n Department of Bi~'hemistry and Molecular Biophysics, I.'irginia Cmnmonweolth Unit'ersity, Richmond, VA (USA)
(Received I I October 1991) (Revised manuscriptreceived28 January 1992)
Key words: PhospholipaseA2 inhibition;c/s-Unsaturatedfatty acid; Enzyme-fattyacid interaction; Enzymc-inhibitor-substrate complex Calcium-dependent phospholipases A: are markedly inhibited in vitro by cis-unsaturatcd fatty acids (CUFAs) and to a much lesser extent by trans-unsaturated or saturated fatty acids. Thus, CUFAs may function as endogenous suppressors of lipolysis. To better understand the mechanism of inhibition, kinetic analysis, fluorescence spectroscopy and gel permeation chromatography were employed to demonstrate that CUFAs interact with a highly purified Ca2+-dependent phospholipase A 2 from Naja mossambica mossambica venom. Arachidonate inhibited hydrolysis of both [IJ4C]oleatc-labellcd, autoclaved Escherichia coli and [IJ4C]linolcatc-labelled phosphatidylcthanolamine in aib apparent competitive manner. When subjected to gel permeation chromatography, [3H]arachidonatc, but not [3H]palmitate, comigrated with the cnzymc. Arachidonic and other CUFAs increased the fluorescence intensity of the enzyme almost 2-fold in a dose-dependent fashion (50 ttM = 180% of control); methyl arachidonate was without effect. Saturated fatty acids had only a modest effect on enzyme fluorescence (50 gM = 122% of control). Concentrations of arachidonatc that inhibited in vitro enzymatic activity by almost 80% did not alter binding of phospholipase A 2 to the E. colt substrat¢. Collectively, these data demonstrate that, while CUFA~ selectively bind to the enzyme, they do not influence phospholipase Aa-substrate interaction. Inhibition of in vitro phospholipase A 2 activity by CUFAs may be mediated by the formation of an enzymatically inactive enzyme-subs~rate-inhibitorcomplex.
imtroduetion Cellular phospholipases A 2, which mediate fatty acid release from the so-2 position of membrane phospholipids, are carefully regulated by endogenous factors such as available calcium [1], glucocorticoidinduced proteins (iipocortins, Ref. 2), phospholipaseactivating protein [3] and undefined cdtosolic components from adrenal medulla [4], rat liver [5] and rabbit neutrn.nhil~ [61. Understanding the regulation of pho~;-
i Present address: CNS Injury Lab, Sergical Research Center, Department of Surgery, University of Connecticut Health Center. Farmington.CT 06032, USA. Abbreviations: PLA2, phospholipase A2; CUF?,~, cis-unsaturated fatty acids; Bistris, 2-[big2-hydro~ethylIamino]-24hydroxymethyl)propane-l,3-dio!: Hepes, 442-hydroxyt.thyl)-I-piperazineethanesulfonie acid: DMSO. dimethyl sulfoxide; PE. phosphatidylethanolamine.; PC, pho;phalid1,'lch~inc; SDS.PAGE. -:,or2~il:.3:. dod¢cyl ~ulfate-lmlyacr¥1amide ge; electrophoresis. Correspondence: R.C. Franson, Department of Biochemistry and Molecular niophy~i,:~.Virgini:~CommonwealthUni,:er~i:y. ~ x 614. MCV Station. Richmond,VA 23298-0614, USA.
pholipase A a activity by endogenous factors is iml~or tant because ot the role this enzyme plays in a variety of cellular metabolic processes including inflammation [7] and exocytosis [8]. We [9,10] and others [11] have frequently found increased phospholipase A a activity in extracts of cells or during enzyme purification. Thus, acidification of human neutrophil homogenate results in a lfl-fold increase in phosphclipase A e activity [10], Similarly, BaiIrtu and Cheung [ll] observed a 20.fold increase in human platelet phospholipase A 2 activity during purification, in both instances, phosphoiipase A 2 inhibitory activity was largely due to unesterified cis-unsaturated fatty acids (CUFAs) in the cell extracts. lnhibitio~l of phospholipase A2 activity in vitro was observed at levels well below the critical mieellar concentration of CIJFAs [10,12], while similar concentrations of trans.unsaturated and saturated fatty acids hdd littl~ ot no effect. Since high levels (0.1-12 raM) of unesterified CUFAs are found in human platelets, neutrophils and extrecellular fluids [10,13], questions re2arding the mechanism of endogenous suppression of phospholipases by fatty acids have been raised.
207 Lack of relief of inhibition by increasing substratc concentration led Ballou and Cheung to conclude that the CUFAs associate directly with the human platelet pbospholipase A 2 [14]. Lister et al. [15] reported that arachidonic a~id competitively inhibits a partially purified membrane-bound phospholipase A 2 from the P388DI macrophage-like cell line. These observations provide indirect evidence that inhibitory CUFAs associate directly with the enzyme and, therefore, may act as endogenous suppressors of phospholipase A 2. Since ceU-derived pbospholipase A : are, in general, available in limiting amounts, we have used a highly purJfled snake venom phospholipase A 2 to demonstrate that, while inhibitory CUFAs associate with the enzyme in the absence of substrate, they do not alter binding of enzyme to substrate. These data reinforce the complex nature of the inhibition of phospholipase A2 activity by unesterified CUFAs. Materials and Methods Materials Palmitic, stearic, oleic, arachidic and arachidonic acids and methyl oleate were purchased from Supelco, Bellefonte, PA. Methyl arachidonate, bovine serum albumin (fatty acid free, fraction V), cytochrome c (horse heart, type VI), Naja mossambica mossambiea venoni and Sephadex G-25 (50-150 p.) were purchased from Sigma Chemicals, St. Louis, Me. [5,6,8,9,11,12, 14,16-~H]arachidonic acid (100 Ci/mmol), [114C]arachidonic acid (51.3 mCi/mmoi) and [9,10•~H]palmitic acid (~,0 Ci/mmol) were purchased from New England Nuclear Research Products, Boston, MA. 1 Pa!-:~[:oyl-2-[1- '~C]linoleoyl-sn-glycero-3-phosphoryl ethanolamine (56 mCi/mmol) was purchased from Amersham and non-radioactive egg phosphatidylethanolamine w:~s from Avanti Polar Lipids, Birmingl.~am, AL. Precoated thin-layer chromatography plates (silica gel G)were purcl~sc~ fre~ _nr;.nkmann Instruments, Wcstbury, NY. All solvents were of reagent grade. Methods Phospholipase A2. Due to the fact that the N, moss. mossambica phe~pholipase A , q,p l = 9.6)obtained from Sigma Chemicals was contaminated with several proteins based on sodium dodecyl suifate-polyacrylamide gel electropholesis (unpublished observations), the isozyme was p~;rified re, homogeneity from the crude venom using the methods described by Joubert [116]. Purity and i.~electric point were assessed by SDSPAGE [17] and PAG-isoelectric focussing [18]. Assay of phospholip¢se A: activity. Phospholipase .A.2 activity was determir_ed by established procedures using [1-'~C]oleate-labelled, autoclaved E. coil as ~ebstrate [19], Reaction mixtul-es (0,5 ml) containeG 25
mM llcpes (pll 7.0), 5.0 mM CaCI 2, 1.4" 10~ [114C]oleate-labelled E. coil (8.9 nmols phospholipid, 7-9000 cpm). For assays utilizing liposomal substrate, PE ill CHCI~ was dried under a stream of N 2 and then resuspended in water by probe sonication for 20 s; reaction mixtures contained 10 nmols phospholipid and 5.o8000 cpm. The amount of enzyme and time of incubation at 3"PC w~re adjusted to produce 10-15% hydrolysis. Fatty acids were added as solutions of dimethyl sulfoxide (4% of reaction volume); concentrations of DMSO < 5% had no effect on enzymatic activity under these conditions. White, flocculent precipitates were observed when concentrations of saturated fatty acids exceeded 100 p.M; thus, inhibition profiles for the less inhibitory fatty acids could not be extended. Reactions were stopped with chloroform/methanol (1:2, v/v). Lipids were extracted by the method of Bligh and Dyer [20] and the lipid species were separated by thin-layer chromatography on silica gel G plates ,1sing a petroleum ether/diethyl ether/acetic acid (80: 20: 3, v/v) system, Two regions, visualized by exposure to iodine and corresponding to free fatty acid and origin (phospholipid + lysophospholipid), were scraped into vials and radioactivity was measured by liquid scintillation spectroscopy. Phospholipase A 2 activity is e~:pressed as percent of substrate hydrolysed. All values are the average of duplicate determinations corrected for non-enzymatic hydrolysis ( < 1.5%) and are representative of three separate experiments. Fluorescence measurements. The relative fluorescence intensity of N. moss. mossambica phospholipase A 2 with and without added fatty acids was monitored at room temperature using a Shimadzu RF-5000 spectrofluorophotometer. The enzyme (2.5 Fg ffi 0.18 nmols) was mixed wiih the indicated concentration of fatty acid in DMSO (1%, v/v) in the presence of 5 mM CaCl: and 10 mM Bistris (pH 7.0). Samples in quartz microcuvettes (3-ram pathlength) were excited at ?80 nm (slit width = 5 rim) and the emission at 342 n ~ was monitored. Enzyme-fatty acid samples were corrected for internal fluorescence of the fatty acid and net quenching was calculated as a percentage ef the fluorescence intew.ity of N. mo~s. mossambica phospholipase A 2 in the absence of fatty acid, Gel permeation chromatography. Gel permeation chromatography was performed using Sephadex G-25 (0.9 x 12 cm) equilibrated with 25 mM Hepes (pH 7.0). The column was washed extensively with 2% horse serum and then with buffer. A stock solution of [3H]arachidonic acid containing 22 ~Ci/ml (0.2 umol/ml) was prepared by removing organic solvent under nitrogen and dissoh, ing the fatty acid in DMSO. Aliquots (4% of reaction volume) of this solution containing approx. 200000 cpms ~ere incubated with either water, N. moss. mossambica phospholipase A 2 (81.5/tg ffi ~.8 nmoD, fatty acid-free bovine serum albu-
208
min (fraction V, 25 # g = 0.38 nmol) or cytochrome c (250 p.g = 18.12 nmol) in the presence of 25 mM Hepes (pH 7.0) and 1 mM CaCi~ in a final volume of 0.5 ml. Each sample was incubated for 3 h at 37°C and then applied to the Sephadex G-25 column; fractions were analyzed for radioactivity and either protein content a n d / o r phospholipase A 2 activity. The column was washed extensively between sample runs with 25 mM Hepes (pH 7.0), until the eluted radioactivity was negligible. Experiments with [3H]palmitic acid (stock solution = 462 /zCi/ml, 0.7 n m o l / m l ) were conducted as described for arachidonate. Typically, 15-30% of protein and 70-75% of radioactivity were recovered from the column. Enzyme - substrate binding assay. Assays were routinely performed in a total volume of 0.5 ml containing 25 mM Bistris (pH 7.0), 1 mM CaCI 2, autoclaved E. coil (7.109 cells, 74 nmols phospholipid) and 5 ng (0.36 pmol) phospholipase Ae. Arachidonic acid in DMSO was added (4% of total -volume), the reaction mixtures were incubated at 37°C for 20 minutes and centrifuged at 15 000 × g for 10 min. The supernatant fraction was assayed for phospholipase A 2 activity by diluting an aliquot to obtain a non-inhibitory concentration of arachidonic acid in the enzyme assay (_< 0.1/~M); since the enzyme was concomitantly diluted, the time of incubation in the assay was adjusted to obtain 8-10% hydrolysis. Control tubes contained either enzyme alone or enzyme and fatty acid. Binding o f fatty acid to E. coli. A stock solution of [1-'4C]arachidonie acid (0.05/zCi/ml) was prepared by dissolving the fatty acid in DMSO after the organic solvent was removed under nitrogen. Aliquots of the solution containing 3000-3500 cpms (20-30 pmoi) were mixed (4% of reaction volume) with unlabelled, autoclaved E. coil membranes (34 nmol phospholipid) in the presence of 5 mM CaCI2 and 25 raM Hepes (pH 7.0). Samples (0.5 ml) were incubated at 37°C for 15 mm and centrifuged at 15 000 × g for 10 rain to sediment the bacteria. Both pellet and supernatant fractions were counted by liquid scintillation. Control sampies did not contain E. co/i membranes. Results
Inhibition o f phospholipase A 2 by fatty acids The hydrolysis of [l-t4C]oleate-labelled, autoclaved E. coil by the purified N. moss. mossambica phosphoiipase A 2 ( p l = 9.6) was inhibited in a dose-dependent manner by c/s-unsaturated fatty acids (Fig. 1). Oleic and arachidonic acids had almost identical inhibitory profiles with 1C5o values of 1.5 /LM and 2.5 /zM, respectively; at concentrations > 1 0 / z M almost complete inhibition was observed. In contrast, the saturated fatty acid arachidate, inhibited phospholipase A z activity only 20% at 10/zM and had an ICso = 118
~ so] o
o o.~
1.o
1o.o FATTY ACID (p.M)
1oo.o
Fig. 1. Fatty acid inhibition of hydrolysis of E, c o l i by IV. moss, mossambica phospholipas¢ A 2. Fatty acids dissolved in DMSO were
added (4% of reaction volume) at the indicated concentrations. Control enzymaticactivities(in absence of fatty acid) were in the range of 10-15% substrate hydrolysis,e, oleate (cisl8: I); &, arachidonate (eis20:4); o. methyl oleate: A arachidate (20:0). Inset: Fatty acid inhibition of PE hydrolysis by N. moss. mossambica phospholipaseA 2. Fatty acids were added as described above; control activities(in the absence of fatty acids) were in the range of 10-15% substrate hydrol~,,~is.A, arachidonate; z~. arachidate. All values represent the average of three separate experiments(duplicate determinations).
/.tM, a 50-fold difference with respect to arachidonic acid. Similar results were obtained with palmitic and stearic acids (data not shown). A free carboxyl group was required for inhibition since the methyl ester of oleic acid had very low inhibitory activity (IC5o = 128 #,M). Almost identical results were obtained (both qualitative and quantitative) when liposomes of phosphatidylethanolamhlc were us¢~~. as substrate (Fig. 1, inset); the ICsn for arachidonate was 4.5 g M , while arachidate was without effect. Fig. 2 demonstrates the effect of substrate on the inhibition of phospholipase A 2 by arachidonic acid. When the substrate concentration was varied from 5 /zM to 500 p.M (for E. cold and 5 / z M to 300 p.M (for PE) in the absence of fatty acid, typical hyperbolic curves was obtained; in the presence of inhibitory concentrations of arachidonic acid (2.5-50 ~M), the velocity-substrate profiles obtained were consistent with the model for competitive inhibition (Ref. 21, data not shown). The apparent competitive nature of the inhibition by araehidonic acid of phospholipase A2-mediated hydrolysis of both membranous E. coil (Fig. 2A) and liposomal PE (Fig. 2B) is evident from the double reciprocal plots. Similar results were obtained with oleic acid (data not shown). These data are consistent with those obtained by Lister et al. [15] for the inhibition, by arachidonic acid, of the hydrolysis of phosphatidylcholine-Triton micelles by partially purified macrophage phospholipase A 2. These data suggest that inhibition of phospholipase A 2 activity by CUFAs is relatively independent of the physical state of the phos-
209 pholipid substrate, since three different substrates, E. coli, liposomal PE and micellar PC, yielded comparable results. Moreover, the fact that 2.5 /tM arachidonate inhibited enzymatic activity to similar extent (25-30%) over a 100-fold substrate concentration range for both membranous and liposomal substrates (data not shown) diminishes the likelihood that non-specific, hydrophobic association between substrate and fatty acid can account for the observed results. Interaction o f arachidonic acid with phospholipase A z Sephadex G-25 gel permeation chromatography was employed to demonstrate interaction between inhibitory fatty acids and snake venom phospholipase A 2 (Fig. 3). In the absence of protein, both [3H]arachidonic and [3H]palmitic acids ¢luted at the total volume of the column (Fig. 3A and C). When arachidonic acid (2 pmols) was preincubated with the enzyme (5.8 nmol) and applied to the column, 2% of the total radioactivity was recovered in the fractions containing enzymatic
.E E o E E
e,~ v~ B
3O
i
0
55
70
l/s (~,U) Fig. 2. Double-reciprocal plots of iuhibitlon of N, moss. mossambiea phospholipase A 2 by eb-arachidonie acid as a function of substrate
concentration. All ~alues represent the average of two separate experiments(duplicate determinations),(A) Hydrolysisof E. coil by phospholipase A 2 was measured in the presence of 0 p.M (t), 2,5 p.M (v), 5 p.M (v), t0/aM (Q) and 50 p.M (11) arachidonic acid added as DMSOsolutions(4% of reactionvolume).(B) Hydrolysisot PE liposomesby pbosphohoaseA2 was measured in the presence of 2.5 p.M, 5 p.M, l0 p.M and 50 ~M arachidonicacid. Symbolssame as in (A).
activity (Fig. 3B). In similar experiments with up to 40 pmols [3H]palmitic acid, no m~asurabl¢ radioactivity was observed in the fractions containing phospholipase A2 activity (Fig. 3D). The same distribution profiles were obtained in the presence or absence of calcium (unpublished observations). When [3H]arachidonate or [3H]palmitate were fractionated in the presence of 25 p.g defatted bovine serum albumin (molar ratio of protein to fatty acid = 190 to 1), 56% and 65%, respectively, of the radioactivity was recovered in the albumin fractions (data not shown). Radioactive fatty acid did not comigrate with protein when fractionated with 250 ~ g cytochrome c (data not shown). These results demonstrate that inhibitory CUFAs but not the relatively non-inhibitory saturated fatty acids interact with the enzyme in a calcium-independent manner. Fluorescence was used to demonstrate the direct interaction between C U F A s and snake venom phospholipase A a. O n excitation at 280 nm, the N. moss. mossambica phospholipase A 2 exhibited a broad fluorescence spectrum, with maximal emission at 342 nm (Fig, 4A). As seen in Fig. 4B, the relative fluorescence was enhanced linearly by increasing concentrations of c/s-arachidonic acid indicating that the fatty acid interacts with the enzyme; no further increase in the fluorescence intensity of the enzyme was observed at concentrations of arachidonic acid > 50/~M (data not shown). The addition of 50 p.M arachidonic acid (molar ratios of fatty acid to phospholipase A 2 = 40 to 1) enhanced the relative fluorescence by 80% (Fig. 4A and B, dashed lines). A similar dose-dependent increase in fluorescence intensity was observed with other inhibitory CUFAs irrespective of their length or degree of unsaturation (data not shown). In contrast, similar concentrations of the non-inhibitory fatty acid, arachidate (Fig. 4A and B, dotted lines) had only a modest effect on the relative fluorescence intensity of the enzyme (50 ~ M = 122% of control), while methyl arachidonate (Fig. 4A and B, dashed-dotted lines) was without effect. The effects of the various fatty acids on the fluorescence of the enzyme were independent of the presence of 5 mM calcium (unpublished observations). The increase in the fluorescence intensity and a modest blue shift (3-5 nm) selectively induced by inhibitory C U F ~ indicates an increased hydrophobic environment of the reporter tryptophan(s) of the enzyme. Interaction o f phospholipase A 2 with substrate Because CUFAs associate with the isolated enzyme, the ability of CUFA-bound phospholipase A 2 to subsequently bind substrate was examined. When snake venom phospholipase A 2 (0.36 pmol) was incubated with E. coil (74 nmols phospholipid) in the presence of 1.0 mM CaCI 2 and then centrifuged to sediment the bacteria, 35% of the enzyme activity remained in the
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Fig. 3. Scphadex G-Z5 chromatography of [ 3H]arachidonic a:M (panel A), rachidonatc-N, moss. mossambica phospholipasc A 2 (panel B), [SH]palmitic acid (panel (2) and palmitatc*phospholipase A 2 (panel D). [SH]arachidonic acid (2 pmols, 200000 cpms) was incubated for 3 h at 3"PC in the absence (panel A) and presence of phospholipa,*¢ A2 (panel B). $ ,ailar analyses were conducted with [~Hlpalmitate (40 pmols, 1.5.106 cpm) in the absence (panel C) and presence (panel D) of phospholipase A 2. Fractions were analyzed for radioactivity (0) and enzymatic activity ( • ) . Values are representative of two independent determinations per experiment. s~:pernatant f r a c t i o n relative to c o n t r o l (Table I, phosp h o l i p a s e A 2 i n c u b a t e d in the a b s e n c e o f E. colO. N o a p p r e c i a b l e i n c r e a s e in e n z y m a t i c activity in the s u p e r TABLE l Effect of arachidonic acid on N. moss. mossambica phospholipase A 2 - E. coil interaction
Phospholipas¢ A z (PLA.2, 5 n£, 0.36 Drools) was incubated for 20 rain at 37°C with (A) 25 mM Bistris (pH 7.0), 1 mM CaCI 2 (burrer 1); (B) buffer I containing 50/,~M arachidonic acid (AA) (in DMSO); (C) buffer I containing E. coil (74 nmols phospholipid); (D) buffer 1 containing 50 v.M arachidonate and E. coil (74 nmols phospholipid). The sample (0.5 ml) was centrifuged as described in Materials and Mctbods to remove the bacteria and the supernatant fraction was assayed for enzymatic activity under conditions such that the concentration of arachidunic acid was ~0.1 /~M. Supernatant fraction
Activity (nmol h - z)
(A) PLA 2 (B) PEA 2 +AA
5.04 +0.00 4.56+0.60 1.74±0.60
(C) PLA 2 + E. coil ( D ) PLA 2 + E. coil + A A
2.16±0.06
n a t a n t fraction w a s o b s e r v e d w h e n e n z y m e w a s incub a t e d with E. coil in the p r e s e n c e o f sufficient a r a c h i d o n i c acid (50 g M ) to inhibit 8 0 % o f the e n z y m a t i c activity ( u n p u b l i s h e d observations), s u g g e s t i n g t h a t C U F A s did n o t p r e v e n t e n z y m e f r o m b i n d i n g t h e substrate. P r e i n c u b a t i n g s u b s t r a t e (148 v,M p h o s p h o l i p i d )
TAn-LE !l Binding of arachidonic acid to E. coil
[1J4C]Arachidonic Acid (AA, 20 pmols, 3355 cpms), added as DMSO solution, was incubated for 10 rain at 3"PC with (A) 20 mM Hepes (pH 7.0), 5 mM CaCl 2 (buffer l); (B) buffer I containing E. coil (34 nmols pbospholipid). The sample (0.5 ml) was centrifuged as described in Materials and Methods and the radioactivity in both pellet and supernatant fractions were measured. Sample
[i - 14C]AA [I-t4C]AA+ E. c o i l
Radioactivity (cpm) supematant fraction
pellet
total
1426 ± 255 598+288
962+2
1426 1560
211
/ \ .=,
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~so ), (nrn~:
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i5 . FATTYACID (/.LM)
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Fig. 4. Effect of 20-carbonfany acidson the fluorescenceemissionof ,IV. moss. mossambica phospholipaseA z. (A) Emissionspectraof N. moss. mossambica phosphollpaseA2 (0.16 nrnols, ) mixed
with 25 nmols (SO /LM) cb-arachidonie acid (------), melhyl arachidonate ( . . . . . ) and arachidic acid ( ...... ). Spectra were recordedfrom 300 nm to 420 nm. The emissionspectrumfor methyl arachidonate is almost completely superimposedby that of the eytzyme control. (B) Fluorescence of N. moyy. mossombica (0.16 nmols) was measuredin the presenceof indicatedconcentrationsof cb-arachidonate(------), methyl arachidonate(. . . . ) and arachidate ( ...... ) as describedin Material~and Methods.Sampleswere excitedat 280 nm and emissionintensityrecordedat 342 nm. Values are an averageof duplicate determinations of four independent experiment:~. with arachidonic acid (50/~M) did not alter the distribution of the enzyme upon centrifugation (d~ta not shown). These data suggest that binding of the CUFAs to either enzyme or substrate do not affect their subsequent interaction.
Interaction of fatty acids with substrate It is well established that fatty acids associate with membrane bilayers [22,23]. To determine whether inhibitory fatty acids associate with substrate, [114C]arachidonate (20 pmols) was incubated with E. coil (34 nmols) in the presence of 5 mM CaCIz; 62% of the radioactivity associated with the bacterial pellet, even though E. coli phospbolipid was present in a 1500-fold molar excess of the (Table ll). Interestingly, this distribution of radioactivity was indepetident of the presence of calcium (data not shown). In similar experiments, 60-65% of [1-t4C]palmitate (25 pmols) sedimented with the E, coli membranes (unpublished observations), possibly because saturated fatty acids are naturally more hydrophobic than their c/s-unsaturated analogues. Discussion These results demonstrate that c/s-unsaturated fatty acids (CUFAs) behave as apparent competitive in-
hibitors of Ca2+-dependent phospholipases A 2 and although CUFAs bind directly to the isolated enzyme, they do not affect enzyme-substrate interaction, Thus, CUFAs may compete with the substrate for the active site on the enzyme to suppress ip. vitro and in situ phospholipase A 2 activity, In addition to their potential role as endogenous suppressors, elevated levels of CUFAs released via catalysis may function as endproduct inhibitors and may contribute to the non-linear kinetics characteristic of interfacia! catalysis and lipolysis [6,15,24]. Our results are consistent with previous studies in which calcium.dependent phospholipases A2 from human neutrophils [10], human platclets [9,11], P388D1 macrophage-like cells [15] and rat heart [25] were inhibited by CUFAs, and only to a much lesser extent by methylated CUFAs, trans.un,~aturated or saturated fatty acids. Interestingly, we have found that CUFAs are not potent inhibitors of the non-calcium-dependent phospholipase A 2 isolated from the adrenal medulla (ReL 26, unpublishcJ observations). Collectively, these observations s-ggest that inhibition by CUFAs may be a property common to Ca2+-dependent phospholipases A2, as previously noted by Lister et al. [15]. Inhibition of the phospholipases A 2 occurred at concentrations of CUFAs well below their critical micellar concentrations (40-60/~M in pure solution) [10,12], indicating that it is not the detergent properties of micelles that affect pbospholipase A 2 activity. Baliou and Cheung postulated that CUFAs inhibited platelct phosphoiipase A z activity by associating with the enzyme since inhibition was not diminished by increasing concentrations of the phosphatidylcboline substrat¢ [14]. Lister et al. [15] developed a kinetic model to demonstrate that arachidonic acid competitively inhibited hydrolysis of micellar and liposomal dipaimitoyI-PC by the calcium-dependent phospholipase A 2 from P388D1 cells. Our data (Fig. 2) illustrating the apparent competitive nature of inhibition (by CUFAs) of the hydrolysis of both biomembranous and liposomal substrates is in agreement with the observations of Lister et al. [15]. Furthermore, our results (Figs. 3 and 4) demonstrating a direct association between CUFAs and the N, moss. mossambica phospholipase A 2 ( p l = 9.6) provide experimental evidence that CUFAs may inhibit enzymatic activity by direct association with the enzyme. On the other hand, Yasuda et al. [2?] have recently reported that the hydrolysis of pbosphatidylcboline by the pbospholipase A 2 purified from rat stomach was inhibited in a non-competitive manner by c/s-arachidonic acid; this apparent discrepancy may be due to the inability of the gastric/ pancreatic enzymes to efficiently penetrate phospbolipid bilayers [28]. The sharp contrast between the biological effects of c/s- vs. trans.unsaturated and saturated fatty acids have
212 been attributed to the fact that CUFAs preferentially partition into the 'fluid' domain of membranes creating disorder, while trans-unsaturated and saturated fatty acids partition into the 'gel' domain and do not induce disorder [22,23]. We believe that CUFA inhibition of in vitro phospholipase A 2 activity is not due to differences in partitioning of fatty acids into the phospholipid bilayer. Two lines of evidence support this view. First, we [10,25] and others [11,25,27,29] have consistently reported selective inhibition of l~hospholipase A 2 activity by CUFAs irrespective of the type of substrate. Thus, CUFAs (but not saturated fatty acids) inhibited hydrolysis of liposomal PE (Fig. 1), liposomal PC [11,15,27], PC-detergent mixed micellcs [29] and membranous E. coli (Ref. 10, 25 and Fig. 1) substrates. In fact, assays with liposoCnal dipalmitoyI-PC as substrate [15] were performed at 370C, a temperature at which the phospholipid is primarily in the 'gel' phase. According to Karnovsky's model, only saturated and trans-unsaturated fatty acids should associate with this phase of the lipid bilayer [23]. The inhibitory profile of CUFAs with PE as substrate were identical to that obtained with other bilayer substrates; PE packs in a non-bilayer, hexagonal array. Interestingly, the appareut K i for inhibition by arachidonie acid for both E. coli and PE was 5/.tM, which is identical to the value reported by Lister et al. [15] using PC as substrate. In addition, reports of formation of 'defect' sites [30] and 'domains' [31] in membranes by products of phospholipase A ~ action are observed only in ternary codispersions of lysophospholipid, fatty acid and phospholipid and not in binary mixtures of either product and phospholipid [30]. Collectively, these data provide strong support for the independence of CUFA inhibition on eitrler the physical state of the substrate (i.e., packing) or the chemical nature of the phospholipids used. Hence, the selective inhibition by CUFAs could involve a preferential association of inhibitory fatty acid and enzyme. The natural hydrophobic affinity of fatty acid for membranes, however, does not preclude the possibility of a 'partitioning' of fatty acid between enzyme and substrate. Jain et al. [24] postulated that certain lipophilic inhibitors could exist predominantly in the lipid bilayer and interact with enzyme, thereby competing with substrate for binding to the catalytic site. Such a model was demonstrated by de Haas and co-workers using non-hydrolyzable acylamino phospholipid analogues [32]. In their studies, the K~ for enzyme-inhibitor association was 2-3-fold higher than that for binding of enzyme to phospholipid [32,33]. Our data demonstrating that inhibitory concentrations of arachidonic acid had little to no effect on the interaction between phospholipase A 2 and E. coli substrate (Table I), is consistent with this hypothesis. Based on the crystal structure of a complex between Naja naja atra
phospholipas¢ A 2 and a non-hydrolyzabl¢ phosphonate phospholipid, Scott et al. [34] suggested that the active site was embedded in a hydrophobic ~ehannel' which facilitates the diffusion of a phospholipid molecule from the interface to the active site and binds the sn-2 alkyl substituent of the phospholipid during catalysis. It is, therefore, possible that CUFAs may similarly diffuse from the membrane bilayer interface into the active site to preve.nt productive association, i.e., catalysi.~. However, the precise nature of the interaction between a long chain CUFA and enzyme is speculative at best in view of the complex interactions involving multiple protein domains with substrate in interfacial catalysis [35i. it is well known that the 'quality of the interface' is critical to interfacial catalysis [35] and that subtle conformational and chemical differences in substrate phospholipid a n d / o r enz~'me play a crucial role in expression of phospholipase A 2 activity [36-38]. Thus, inhibitors such as calpactin-lipocortin [39], quercetin [40] and local anesthetics [41] interact non-specifically with the substrate and serve as examples of inhibition by 'substrate depletion' [24,39,42]. The active site of pancreatic and snake venom phospholipases A 2 is in a hydrophobic environment that presumably participates in orienting the 2-acyl ester bond for the subsequent hydrolysis [43]. Structure-activity studies witil the inhibitory sn-2-acylamino phospholipid analogues suggested that the binding site for the sn-2 substituent could optimally accommodate alkyl chains 14-carboas long [33]. After the catalytic event, the active site of the phospholipas¢ A 2 is in some transient manner intimately associated with the catalytic product, CUFA. Inhibitory CUFAs may occupy the active site of the enzyme thus preventing interaction with the substrate. Fluorescence has been previously used in our laboratory to demonstrate direct interaction between snake venom phospholipase A 2 and in vitro inhibitors such as retinal [40], aristolochie acid [44] and the prostaglandin polymer, PGB x [45]. Interestingly, while these inhibitors quenched the relative fluorescence of the IV. moss. mossambica phospholipase A 2, CUFAs enhanced the intrinsic fluorescence of the enzyme. These results indicate that CUFAs have modified the environment of at least one of the three tryptophan residues (at positions 19, 20 and 60) in this enzyme. Trp-60 is presumably homologous to Tyr-69 on the porcine pancreatic phospholipase A a [46] which, based on X-ray diffraction analysis, is involved in interaction with the substrate [47]. The crystal structure of the monomeric Naja naja atra phospholipase A 2 contains a tryptophan (Trp-19) at the roof of the hydrophohic channel, which is closely associated with the sn-2 alkyl substituent [48]. The increase in fluorescence intensity (Fig. 4) is indicative of increased hydrophobicity in the environment of tryptophan [49]. In contrast,
213 the naturally more hydrophobic saturated and methylated fatty acids hap minimal effect on the fluorescence intensity of the enzyme, indicating that hydrophobicity of fatty acid per se was not responsible for the observed changes in enzyme fluorescence. Furthermore, circular diehroism studies demonstrated that, unlike the effect of the inhibitor, aristolochic acid, which increased the a-helical content of the phospholipase A 2 [44], inhibitory C U F A s did not affect the secondary structure of the snake venom phospholipase A 2 (unpublished observations). Fatty acids could interact with lipid binding domains of the enzyme either in a non-covalent or covalent m a n n e r [29,50,51]. Pliiekthun and Dennis demonstrated that the rate of hydrolysis of mixed micelles by the pancreatic phospholipase A 2 was enhanced, in a presumably non-covalent manner, by the addition of oleic acid [29]. However, recent studies have shown that autoeatalytic acylation of lysine residues (Lys-7 and Lys-10) adjacent to the active site dramatically activates snake venom phospholipase A 2 [50]. Similarly, De H a a s and eoworkers have d e m o n s t r a t e d that monoacylation of the pancreatic phospholipase A 2 (Lys-10 or L y s - l l 9 ) resulted in an increased affinity of enzyme for phospholipid substrate, but did not affect enzymatic activity per se [51]. In our hands, C U F A s consistently inhibited in a d o s e - d e p e n d e n t m a n n e r the hydrolysis of both m e m b r a n o u s E. coil and liposomal P E by all calcium-dependent phospholipases A2 tested (results similar to Fig. 1, data not shown). These observations are indicative of the complex nature of the association of C U F A s with phospholipases A zO u r results illustrate that C U F A s inhibit calciumd e p e n d e n t phospholipases A 2 by selectively interacting with the enzyme. In this regard, calcium-dependent phospholipases A 2 may resemble the intracellular, but non-catalytic, fatty acid binding proteins which exhibit specificity for C U F A s (for review see Ref. 52), The natural association of C U F A s with e n d o g e n o u s phospholipases A 2 to suppress enzymatic activity may imply a unique mode for activation of enzymatic activity as well. Thus, we have shown that autoxidation of cisarachidonic acid substantially diminished its ability to inhibit myocar:lial phospholipase A 2 activity [25]. Increased phospholipase A 2 activity, concomitant with an increase in lipid peroxidation has been observed during pro-oxidant injury such as isehemia [53]. While it is generally believed that pro-oxidants promote phospholipase A2 m e d i a t e d hydrolysis because oxidized phospholipids are more susceptible to hydrolysis [5456], our results [25] suggest that oxidation of unesterifled C U F A s may also activate latent enzyme. A n understanding of how physical p a r a m e t e r s and chemical events such as ischemia and peroxidation influence e n z y m e - C U F A - s u b s t r a t e interaction may provide badly n e e d e d clues as to the role and regulation of phospholipases A 2 in normal and pathologic processes.
Acknowledgement These studies have been supported by a NIH Grant DX 34558. References 1 van den Bosch, H. (1980) Biochim. Biophys. Acta 604, 191-246. 2 Flower, RJ. (1984) in Advances in Inflammation Research (Weismann, G., ed.), pp. 1-34, Raven Press, New York. 3 Clark, M., Conway, T, Schorr. R. and Cooke, S. (1987) J. Biol. Chem. 262, 4402-4406. 4 Bartolf, M. and Franson, R.C. (1987) Biochim. Biophys. Acta 917, 308-317. 5 Weglicki, W.B., Ruth, R.C., Owens, K,, Griffin, H.D. and Waite, B.M. (1974) Pip,:him. Biophys. Acta 337, 145-152. 6 Franson, R., Patriarca, P. and Elsbach, P. (1974) J. Lipid Res. 15, 380-388. 7 Vishwanath, B.S., Fawzy, A.A, and Franson, R.C. (1988) Inflammation 12, 549-561. 8 Creutz, C.E. (1981) I. Cell Biol. 91. 247-256. 9 Raghupathi, R. and Franson, R.C. (1987) Fed. Proc. 47, AI366. 10 M~rki, F. and Franson, R, (1986) Bioehim. Biophys. Acta 879, 149-156. II Ballou, L.R. and Cheung, W.Y. (1983) Proc. Natl. Acad. Sci. USA 80, 5203-5207. 12 Klevens, H.B. (1953) J. Am. Oil Chem. Soc. 30. "/4-80. 13 Bills. T.K., Smith, J.B. and Silver, MJ. (1977) J. Clin. Invest. b0, 1-6. 14 Ballou, L.R. and Cheung, W.Y. (1985) Proc. Natl. Acad. Sci. USA ~2, 371-375. 15 Lister, M.D., Deems, R.A., Watanabe, Y., Ulevitch, RJ, and Dennis E,A. (1988) J. Biol. Chem. 263, 7506-7513. 16 Joubert, FJ. (1977) Biochim. Biophys, Acta 493, 216-227. 17 Lacmm:i, U.K. (1970) Nature 227, 680-685. 18 O'FarrelL P.H. (1975)J. Biol. Chem. 250, 4007-4021. 19 Patriarca, P., Beekerdite, S. and Elsbach, P. (1972) Biochim. Biophys. Acta 260, 593-600. 20 Bligh, E.G. and Dyer, WJ. (1959) Can. J. Biochem. Physiol. 37, 911-917. 21 Segel, 1.H. (1976) in Biochemical Calculations, pp. 246-273, L Wiley and Sons, New York. 22 Klausner, R.D., Kleinfeld, A.M., Hoover, K.L and Karnovs~, MJ. (1980) J. Biol. Chem. 255, 1286-1295. 23 Karnovsk2~, M.J., Kleinfeld, A.M., Hoover, R,L. and Klausner, R.D. (1982) J. Cell Biol. 94, I-6. 24 .lain, M.K., Yuan, W. and Gelb, M.H. (1989) Biochem. 28, 4135-4139. 25 Franson, R.C., Harris, L.H. and Raghupathi, R. (19.5t})MoL Cell. Biochem. 88, ' 55-159. 26 Bartolf, M.B. and Franson, R.C. (1990) Biochim. Biophys. Acta 1042, 247-254, 27 Yasuda, T., Hirohara, J., Okumura, T. and Saito, K. (1990) Biochim. Biophys. Acta 1046, 189-194. 28 Mansbach, C.M. (1990) Gastroenterology 98,1369-82. 29 Pliickthun, A. and Dennis, E.A. (1985) J. Biol. Chem, 260, 11099-11106. 30 Jain, M.K., Esmond, M.R., Verheij, H.M., Apitz-Castro, R., Dijkman, R. and de Haas, G,H, (1982) Biochim. 8ophys, Acta 688, 341-348. 31 Grainger, D.W, Reichert, A., Ringsdorf, H. and Salesse, C. (1989) FEBS Len. 252, 73-82. 32 de Haas, G.H,, Dijkman, R., van Oort, M.G. and Verger, R, (1990) Biochlm, Biophys. Acta 1043, 75-82. 33 de Haas, G.H., Diikman, R., Ransac, S. and Verger, R, (1990) Biochim. Biophys. Acta 1046, 249-257.
214 34 Sc
206
BBALIP 53925
Inhibition of phospholipase A 2 by c/s-unsaturated fatty acids: evidence for the binding of fatty acid to enzyme R a m e s h R a g h u p a t h i t a n d R i c h a r d C. F r a n s o n Department of Bi~'hemistry and Molecular Biophysics, I.'irginia Cmnmonweolth Unit'ersity, Richmond, VA (USA)
(Received I I October 1991) (Revised manuscriptreceived28 January 1992)
Key words: PhospholipaseA2 inhibition;c/s-Unsaturatedfatty acid; Enzyme-fattyacid interaction; Enzymc-inhibitor-substrate complex Calcium-dependent phospholipases A: are markedly inhibited in vitro by cis-unsaturatcd fatty acids (CUFAs) and to a much lesser extent by trans-unsaturated or saturated fatty acids. Thus, CUFAs may function as endogenous suppressors of lipolysis. To better understand the mechanism of inhibition, kinetic analysis, fluorescence spectroscopy and gel permeation chromatography were employed to demonstrate that CUFAs interact with a highly purified Ca2+-dependent phospholipase A 2 from Naja mossambica mossambica venom. Arachidonate inhibited hydrolysis of both [IJ4C]oleatc-labellcd, autoclaved Escherichia coli and [IJ4C]linolcatc-labelled phosphatidylcthanolamine in aib apparent competitive manner. When subjected to gel permeation chromatography, [3H]arachidonatc, but not [3H]palmitate, comigrated with the cnzymc. Arachidonic and other CUFAs increased the fluorescence intensity of the enzyme almost 2-fold in a dose-dependent fashion (50 ttM = 180% of control); methyl arachidonate was without effect. Saturated fatty acids had only a modest effect on enzyme fluorescence (50 gM = 122% of control). Concentrations of arachidonatc that inhibited in vitro enzymatic activity by almost 80% did not alter binding of phospholipase A 2 to the E. colt substrat¢. Collectively, these data demonstrate that, while CUFA~ selectively bind to the enzyme, they do not influence phospholipase Aa-substrate interaction. Inhibition of in vitro phospholipase A 2 activity by CUFAs may be mediated by the formation of an enzymatically inactive enzyme-subs~rate-inhibitorcomplex.
imtroduetion Cellular phospholipases A 2, which mediate fatty acid release from the so-2 position of membrane phospholipids, are carefully regulated by endogenous factors such as available calcium [1], glucocorticoidinduced proteins (iipocortins, Ref. 2), phospholipaseactivating protein [3] and undefined cdtosolic components from adrenal medulla [4], rat liver [5] and rabbit neutrn.nhil~ [61. Understanding the regulation of pho~;-
i Present address: CNS Injury Lab, Sergical Research Center, Department of Surgery, University of Connecticut Health Center. Farmington.CT 06032, USA. Abbreviations: PLA2, phospholipase A2; CUF?,~, cis-unsaturated fatty acids; Bistris, 2-[big2-hydro~ethylIamino]-24hydroxymethyl)propane-l,3-dio!: Hepes, 442-hydroxyt.thyl)-I-piperazineethanesulfonie acid: DMSO. dimethyl sulfoxide; PE. phosphatidylethanolamine.; PC, pho;phalid1,'lch~inc; SDS.PAGE. -:,or2~il:.3:. dod¢cyl ~ulfate-lmlyacr¥1amide ge; electrophoresis. Correspondence: R.C. Franson, Department of Biochemistry and Molecular niophy~i,:~.Virgini:~CommonwealthUni,:er~i:y. ~ x 614. MCV Station. Richmond,VA 23298-0614, USA.
pholipase A a activity by endogenous factors is iml~or tant because ot the role this enzyme plays in a variety of cellular metabolic processes including inflammation [7] and exocytosis [8]. We [9,10] and others [11] have frequently found increased phospholipase A a activity in extracts of cells or during enzyme purification. Thus, acidification of human neutrophil homogenate results in a lfl-fold increase in phosphclipase A e activity [10], Similarly, BaiIrtu and Cheung [ll] observed a 20.fold increase in human platelet phospholipase A 2 activity during purification, in both instances, phosphoiipase A 2 inhibitory activity was largely due to unesterified cis-unsaturated fatty acids (CUFAs) in the cell extracts. lnhibitio~l of phospholipase A2 activity in vitro was observed at levels well below the critical mieellar concentration of CIJFAs [10,12], while similar concentrations of trans.unsaturated and saturated fatty acids hdd littl~ ot no effect. Since high levels (0.1-12 raM) of unesterified CUFAs are found in human platelets, neutrophils and extrecellular fluids [10,13], questions re2arding the mechanism of endogenous suppression of phospholipases by fatty acids have been raised.
207 Lack of relief of inhibition by increasing substratc concentration led Ballou and Cheung to conclude that the CUFAs associate directly with the human platelet pbospholipase A 2 [14]. Lister et al. [15] reported that arachidonic a~id competitively inhibits a partially purified membrane-bound phospholipase A 2 from the P388DI macrophage-like cell line. These observations provide indirect evidence that inhibitory CUFAs associate directly with the enzyme and, therefore, may act as endogenous suppressors of phospholipase A 2. Since ceU-derived pbospholipase A : are, in general, available in limiting amounts, we have used a highly purJfled snake venom phospholipase A 2 to demonstrate that, while inhibitory CUFAs associate with the enzyme in the absence of substrate, they do not alter binding of enzyme to substrate. These data reinforce the complex nature of the inhibition of phospholipase A2 activity by unesterified CUFAs. Materials and Methods Materials Palmitic, stearic, oleic, arachidic and arachidonic acids and methyl oleate were purchased from Supelco, Bellefonte, PA. Methyl arachidonate, bovine serum albumin (fatty acid free, fraction V), cytochrome c (horse heart, type VI), Naja mossambica mossambiea venoni and Sephadex G-25 (50-150 p.) were purchased from Sigma Chemicals, St. Louis, Me. [5,6,8,9,11,12, 14,16-~H]arachidonic acid (100 Ci/mmol), [114C]arachidonic acid (51.3 mCi/mmoi) and [9,10•~H]palmitic acid (~,0 Ci/mmol) were purchased from New England Nuclear Research Products, Boston, MA. 1 Pa!-:~[:oyl-2-[1- '~C]linoleoyl-sn-glycero-3-phosphoryl ethanolamine (56 mCi/mmol) was purchased from Amersham and non-radioactive egg phosphatidylethanolamine w:~s from Avanti Polar Lipids, Birmingl.~am, AL. Precoated thin-layer chromatography plates (silica gel G)were purcl~sc~ fre~ _nr;.nkmann Instruments, Wcstbury, NY. All solvents were of reagent grade. Methods Phospholipase A2. Due to the fact that the N, moss. mossambica phe~pholipase A , q,p l = 9.6)obtained from Sigma Chemicals was contaminated with several proteins based on sodium dodecyl suifate-polyacrylamide gel electropholesis (unpublished observations), the isozyme was p~;rified re, homogeneity from the crude venom using the methods described by Joubert [116]. Purity and i.~electric point were assessed by SDSPAGE [17] and PAG-isoelectric focussing [18]. Assay of phospholip¢se A: activity. Phospholipase .A.2 activity was determir_ed by established procedures using [1-'~C]oleate-labelled, autoclaved E. coil as ~ebstrate [19], Reaction mixtul-es (0,5 ml) containeG 25
mM llcpes (pll 7.0), 5.0 mM CaCI 2, 1.4" 10~ [114C]oleate-labelled E. coil (8.9 nmols phospholipid, 7-9000 cpm). For assays utilizing liposomal substrate, PE ill CHCI~ was dried under a stream of N 2 and then resuspended in water by probe sonication for 20 s; reaction mixtures contained 10 nmols phospholipid and 5.o8000 cpm. The amount of enzyme and time of incubation at 3"PC w~re adjusted to produce 10-15% hydrolysis. Fatty acids were added as solutions of dimethyl sulfoxide (4% of reaction volume); concentrations of DMSO < 5% had no effect on enzymatic activity under these conditions. White, flocculent precipitates were observed when concentrations of saturated fatty acids exceeded 100 p.M; thus, inhibition profiles for the less inhibitory fatty acids could not be extended. Reactions were stopped with chloroform/methanol (1:2, v/v). Lipids were extracted by the method of Bligh and Dyer [20] and the lipid species were separated by thin-layer chromatography on silica gel G plates ,1sing a petroleum ether/diethyl ether/acetic acid (80: 20: 3, v/v) system, Two regions, visualized by exposure to iodine and corresponding to free fatty acid and origin (phospholipid + lysophospholipid), were scraped into vials and radioactivity was measured by liquid scintillation spectroscopy. Phospholipase A 2 activity is e~:pressed as percent of substrate hydrolysed. All values are the average of duplicate determinations corrected for non-enzymatic hydrolysis ( < 1.5%) and are representative of three separate experiments. Fluorescence measurements. The relative fluorescence intensity of N. moss. mossambica phospholipase A 2 with and without added fatty acids was monitored at room temperature using a Shimadzu RF-5000 spectrofluorophotometer. The enzyme (2.5 Fg ffi 0.18 nmols) was mixed wiih the indicated concentration of fatty acid in DMSO (1%, v/v) in the presence of 5 mM CaCl: and 10 mM Bistris (pH 7.0). Samples in quartz microcuvettes (3-ram pathlength) were excited at ?80 nm (slit width = 5 rim) and the emission at 342 n ~ was monitored. Enzyme-fatty acid samples were corrected for internal fluorescence of the fatty acid and net quenching was calculated as a percentage ef the fluorescence intew.ity of N. mo~s. mossambica phospholipase A 2 in the absence of fatty acid, Gel permeation chromatography. Gel permeation chromatography was performed using Sephadex G-25 (0.9 x 12 cm) equilibrated with 25 mM Hepes (pH 7.0). The column was washed extensively with 2% horse serum and then with buffer. A stock solution of [3H]arachidonic acid containing 22 ~Ci/ml (0.2 umol/ml) was prepared by removing organic solvent under nitrogen and dissoh, ing the fatty acid in DMSO. Aliquots (4% of reaction volume) of this solution containing approx. 200000 cpms ~ere incubated with either water, N. moss. mossambica phospholipase A 2 (81.5/tg ffi ~.8 nmoD, fatty acid-free bovine serum albu-
208
min (fraction V, 25 # g = 0.38 nmol) or cytochrome c (250 p.g = 18.12 nmol) in the presence of 25 mM Hepes (pH 7.0) and 1 mM CaCi~ in a final volume of 0.5 ml. Each sample was incubated for 3 h at 37°C and then applied to the Sephadex G-25 column; fractions were analyzed for radioactivity and either protein content a n d / o r phospholipase A 2 activity. The column was washed extensively between sample runs with 25 mM Hepes (pH 7.0), until the eluted radioactivity was negligible. Experiments with [3H]palmitic acid (stock solution = 462 /zCi/ml, 0.7 n m o l / m l ) were conducted as described for arachidonate. Typically, 15-30% of protein and 70-75% of radioactivity were recovered from the column. Enzyme - substrate binding assay. Assays were routinely performed in a total volume of 0.5 ml containing 25 mM Bistris (pH 7.0), 1 mM CaCI 2, autoclaved E. coil (7.109 cells, 74 nmols phospholipid) and 5 ng (0.36 pmol) phospholipase Ae. Arachidonic acid in DMSO was added (4% of total -volume), the reaction mixtures were incubated at 37°C for 20 minutes and centrifuged at 15 000 × g for 10 min. The supernatant fraction was assayed for phospholipase A 2 activity by diluting an aliquot to obtain a non-inhibitory concentration of arachidonic acid in the enzyme assay (_< 0.1/~M); since the enzyme was concomitantly diluted, the time of incubation in the assay was adjusted to obtain 8-10% hydrolysis. Control tubes contained either enzyme alone or enzyme and fatty acid. Binding o f fatty acid to E. coli. A stock solution of [1-'4C]arachidonie acid (0.05/zCi/ml) was prepared by dissolving the fatty acid in DMSO after the organic solvent was removed under nitrogen. Aliquots of the solution containing 3000-3500 cpms (20-30 pmoi) were mixed (4% of reaction volume) with unlabelled, autoclaved E. coil membranes (34 nmol phospholipid) in the presence of 5 mM CaCI2 and 25 raM Hepes (pH 7.0). Samples (0.5 ml) were incubated at 37°C for 15 mm and centrifuged at 15 000 × g for 10 rain to sediment the bacteria. Both pellet and supernatant fractions were counted by liquid scintillation. Control sampies did not contain E. co/i membranes. Results
Inhibition o f phospholipase A 2 by fatty acids The hydrolysis of [l-t4C]oleate-labelled, autoclaved E. coil by the purified N. moss. mossambica phosphoiipase A 2 ( p l = 9.6) was inhibited in a dose-dependent manner by c/s-unsaturated fatty acids (Fig. 1). Oleic and arachidonic acids had almost identical inhibitory profiles with 1C5o values of 1.5 /LM and 2.5 /zM, respectively; at concentrations > 1 0 / z M almost complete inhibition was observed. In contrast, the saturated fatty acid arachidate, inhibited phospholipase A z activity only 20% at 10/zM and had an ICso = 118
~ so] o
o o.~
1.o
1o.o FATTY ACID (p.M)
1oo.o
Fig. 1. Fatty acid inhibition of hydrolysis of E, c o l i by IV. moss, mossambica phospholipas¢ A 2. Fatty acids dissolved in DMSO were
added (4% of reaction volume) at the indicated concentrations. Control enzymaticactivities(in absence of fatty acid) were in the range of 10-15% substrate hydrolysis,e, oleate (cisl8: I); &, arachidonate (eis20:4); o. methyl oleate: A arachidate (20:0). Inset: Fatty acid inhibition of PE hydrolysis by N. moss. mossambica phospholipaseA 2. Fatty acids were added as described above; control activities(in the absence of fatty acids) were in the range of 10-15% substrate hydrol~,,~is.A, arachidonate; z~. arachidate. All values represent the average of three separate experiments(duplicate determinations).
/.tM, a 50-fold difference with respect to arachidonic acid. Similar results were obtained with palmitic and stearic acids (data not shown). A free carboxyl group was required for inhibition since the methyl ester of oleic acid had very low inhibitory activity (IC5o = 128 #,M). Almost identical results were obtained (both qualitative and quantitative) when liposomes of phosphatidylethanolamhlc were us¢~~. as substrate (Fig. 1, inset); the ICsn for arachidonate was 4.5 g M , while arachidate was without effect. Fig. 2 demonstrates the effect of substrate on the inhibition of phospholipase A 2 by arachidonic acid. When the substrate concentration was varied from 5 /zM to 500 p.M (for E. cold and 5 / z M to 300 p.M (for PE) in the absence of fatty acid, typical hyperbolic curves was obtained; in the presence of inhibitory concentrations of arachidonic acid (2.5-50 ~M), the velocity-substrate profiles obtained were consistent with the model for competitive inhibition (Ref. 21, data not shown). The apparent competitive nature of the inhibition by araehidonic acid of phospholipase A2-mediated hydrolysis of both membranous E. coil (Fig. 2A) and liposomal PE (Fig. 2B) is evident from the double reciprocal plots. Similar results were obtained with oleic acid (data not shown). These data are consistent with those obtained by Lister et al. [15] for the inhibition, by arachidonic acid, of the hydrolysis of phosphatidylcholine-Triton micelles by partially purified macrophage phospholipase A 2. These data suggest that inhibition of phospholipase A 2 activity by CUFAs is relatively independent of the physical state of the phos-
209 pholipid substrate, since three different substrates, E. coli, liposomal PE and micellar PC, yielded comparable results. Moreover, the fact that 2.5 /tM arachidonate inhibited enzymatic activity to similar extent (25-30%) over a 100-fold substrate concentration range for both membranous and liposomal substrates (data not shown) diminishes the likelihood that non-specific, hydrophobic association between substrate and fatty acid can account for the observed results. Interaction o f arachidonic acid with phospholipase A z Sephadex G-25 gel permeation chromatography was employed to demonstrate interaction between inhibitory fatty acids and snake venom phospholipase A 2 (Fig. 3). In the absence of protein, both [3H]arachidonic and [3H]palmitic acids ¢luted at the total volume of the column (Fig. 3A and C). When arachidonic acid (2 pmols) was preincubated with the enzyme (5.8 nmol) and applied to the column, 2% of the total radioactivity was recovered in the fractions containing enzymatic
.E E o E E
e,~ v~ B
3O
i
0
55
70
l/s (~,U) Fig. 2. Double-reciprocal plots of iuhibitlon of N, moss. mossambiea phospholipase A 2 by eb-arachidonie acid as a function of substrate
concentration. All ~alues represent the average of two separate experiments(duplicate determinations),(A) Hydrolysisof E. coil by phospholipase A 2 was measured in the presence of 0 p.M (t), 2,5 p.M (v), 5 p.M (v), t0/aM (Q) and 50 p.M (11) arachidonic acid added as DMSOsolutions(4% of reactionvolume).(B) Hydrolysisot PE liposomesby pbosphohoaseA2 was measured in the presence of 2.5 p.M, 5 p.M, l0 p.M and 50 ~M arachidonicacid. Symbolssame as in (A).
activity (Fig. 3B). In similar experiments with up to 40 pmols [3H]palmitic acid, no m~asurabl¢ radioactivity was observed in the fractions containing phospholipase A2 activity (Fig. 3D). The same distribution profiles were obtained in the presence or absence of calcium (unpublished observations). When [3H]arachidonate or [3H]palmitate were fractionated in the presence of 25 p.g defatted bovine serum albumin (molar ratio of protein to fatty acid = 190 to 1), 56% and 65%, respectively, of the radioactivity was recovered in the albumin fractions (data not shown). Radioactive fatty acid did not comigrate with protein when fractionated with 250 ~ g cytochrome c (data not shown). These results demonstrate that inhibitory CUFAs but not the relatively non-inhibitory saturated fatty acids interact with the enzyme in a calcium-independent manner. Fluorescence was used to demonstrate the direct interaction between C U F A s and snake venom phospholipase A a. O n excitation at 280 nm, the N. moss. mossambica phospholipase A 2 exhibited a broad fluorescence spectrum, with maximal emission at 342 nm (Fig, 4A). As seen in Fig. 4B, the relative fluorescence was enhanced linearly by increasing concentrations of c/s-arachidonic acid indicating that the fatty acid interacts with the enzyme; no further increase in the fluorescence intensity of the enzyme was observed at concentrations of arachidonic acid > 50/~M (data not shown). The addition of 50 p.M arachidonic acid (molar ratios of fatty acid to phospholipase A 2 = 40 to 1) enhanced the relative fluorescence by 80% (Fig. 4A and B, dashed lines). A similar dose-dependent increase in fluorescence intensity was observed with other inhibitory CUFAs irrespective of their length or degree of unsaturation (data not shown). In contrast, similar concentrations of the non-inhibitory fatty acid, arachidate (Fig. 4A and B, dotted lines) had only a modest effect on the relative fluorescence intensity of the enzyme (50 ~ M = 122% of control), while methyl arachidonate (Fig. 4A and B, dashed-dotted lines) was without effect. The effects of the various fatty acids on the fluorescence of the enzyme were independent of the presence of 5 mM calcium (unpublished observations). The increase in the fluorescence intensity and a modest blue shift (3-5 nm) selectively induced by inhibitory C U F ~ indicates an increased hydrophobic environment of the reporter tryptophan(s) of the enzyme. Interaction o f phospholipase A 2 with substrate Because CUFAs associate with the isolated enzyme, the ability of CUFA-bound phospholipase A 2 to subsequently bind substrate was examined. When snake venom phospholipase A 2 (0.36 pmol) was incubated with E. coil (74 nmols phospholipid) in the presence of 1.0 mM CaCI 2 and then centrifuged to sediment the bacteria, 35% of the enzyme activity remained in the
210 30.0 30.0
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Fig. 3. Scphadex G-Z5 chromatography of [ 3H]arachidonic a:M (panel A), rachidonatc-N, moss. mossambica phospholipasc A 2 (panel B), [SH]palmitic acid (panel (2) and palmitatc*phospholipase A 2 (panel D). [SH]arachidonic acid (2 pmols, 200000 cpms) was incubated for 3 h at 3"PC in the absence (panel A) and presence of phospholipa,*¢ A2 (panel B). $ ,ailar analyses were conducted with [~Hlpalmitate (40 pmols, 1.5.106 cpm) in the absence (panel C) and presence (panel D) of phospholipase A 2. Fractions were analyzed for radioactivity (0) and enzymatic activity ( • ) . Values are representative of two independent determinations per experiment. s~:pernatant f r a c t i o n relative to c o n t r o l (Table I, phosp h o l i p a s e A 2 i n c u b a t e d in the a b s e n c e o f E. colO. N o a p p r e c i a b l e i n c r e a s e in e n z y m a t i c activity in the s u p e r TABLE l Effect of arachidonic acid on N. moss. mossambica phospholipase A 2 - E. coil interaction
Phospholipas¢ A z (PLA.2, 5 n£, 0.36 Drools) was incubated for 20 rain at 37°C with (A) 25 mM Bistris (pH 7.0), 1 mM CaCI 2 (burrer 1); (B) buffer I containing 50/,~M arachidonic acid (AA) (in DMSO); (C) buffer I containing E. coil (74 nmols phospholipid); (D) buffer 1 containing 50 v.M arachidonate and E. coil (74 nmols phospholipid). The sample (0.5 ml) was centrifuged as described in Materials and Mctbods to remove the bacteria and the supernatant fraction was assayed for enzymatic activity under conditions such that the concentration of arachidunic acid was ~0.1 /~M. Supernatant fraction
Activity (nmol h - z)
(A) PLA 2 (B) PEA 2 +AA
5.04 +0.00 4.56+0.60 1.74±0.60
(C) PLA 2 + E. coil ( D ) PLA 2 + E. coil + A A
2.16±0.06
n a t a n t fraction w a s o b s e r v e d w h e n e n z y m e w a s incub a t e d with E. coil in the p r e s e n c e o f sufficient a r a c h i d o n i c acid (50 g M ) to inhibit 8 0 % o f the e n z y m a t i c activity ( u n p u b l i s h e d observations), s u g g e s t i n g t h a t C U F A s did n o t p r e v e n t e n z y m e f r o m b i n d i n g t h e substrate. P r e i n c u b a t i n g s u b s t r a t e (148 v,M p h o s p h o l i p i d )
TAn-LE !l Binding of arachidonic acid to E. coil
[1J4C]Arachidonic Acid (AA, 20 pmols, 3355 cpms), added as DMSO solution, was incubated for 10 rain at 3"PC with (A) 20 mM Hepes (pH 7.0), 5 mM CaCl 2 (buffer l); (B) buffer I containing E. coil (34 nmols pbospholipid). The sample (0.5 ml) was centrifuged as described in Materials and Methods and the radioactivity in both pellet and supernatant fractions were measured. Sample
[i - 14C]AA [I-t4C]AA+ E. c o i l
Radioactivity (cpm) supematant fraction
pellet
total
1426 ± 255 598+288
962+2
1426 1560
211
/ \ .=,
/
i
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~
,,
~so ), (nrn~:
J, ,oo o
i5 . FATTYACID (/.LM)
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Fig. 4. Effect of 20-carbonfany acidson the fluorescenceemissionof ,IV. moss. mossambica phospholipaseA z. (A) Emissionspectraof N. moss. mossambica phosphollpaseA2 (0.16 nrnols, ) mixed
with 25 nmols (SO /LM) cb-arachidonie acid (------), melhyl arachidonate ( . . . . . ) and arachidic acid ( ...... ). Spectra were recordedfrom 300 nm to 420 nm. The emissionspectrumfor methyl arachidonate is almost completely superimposedby that of the eytzyme control. (B) Fluorescence of N. moyy. mossombica (0.16 nmols) was measuredin the presenceof indicatedconcentrationsof cb-arachidonate(------), methyl arachidonate(. . . . ) and arachidate ( ...... ) as describedin Material~and Methods.Sampleswere excitedat 280 nm and emissionintensityrecordedat 342 nm. Values are an averageof duplicate determinations of four independent experiment:~. with arachidonic acid (50/~M) did not alter the distribution of the enzyme upon centrifugation (d~ta not shown). These data suggest that binding of the CUFAs to either enzyme or substrate do not affect their subsequent interaction.
Interaction of fatty acids with substrate It is well established that fatty acids associate with membrane bilayers [22,23]. To determine whether inhibitory fatty acids associate with substrate, [114C]arachidonate (20 pmols) was incubated with E. coil (34 nmols) in the presence of 5 mM CaCIz; 62% of the radioactivity associated with the bacterial pellet, even though E. coli phospbolipid was present in a 1500-fold molar excess of the (Table ll). Interestingly, this distribution of radioactivity was indepetident of the presence of calcium (data not shown). In similar experiments, 60-65% of [1-t4C]palmitate (25 pmols) sedimented with the E, coli membranes (unpublished observations), possibly because saturated fatty acids are naturally more hydrophobic than their c/s-unsaturated analogues. Discussion These results demonstrate that c/s-unsaturated fatty acids (CUFAs) behave as apparent competitive in-
hibitors of Ca2+-dependent phospholipases A 2 and although CUFAs bind directly to the isolated enzyme, they do not affect enzyme-substrate interaction, Thus, CUFAs may compete with the substrate for the active site on the enzyme to suppress ip. vitro and in situ phospholipase A 2 activity, In addition to their potential role as endogenous suppressors, elevated levels of CUFAs released via catalysis may function as endproduct inhibitors and may contribute to the non-linear kinetics characteristic of interfacia! catalysis and lipolysis [6,15,24]. Our results are consistent with previous studies in which calcium.dependent phospholipases A2 from human neutrophils [10], human platclets [9,11], P388D1 macrophage-like cells [15] and rat heart [25] were inhibited by CUFAs, and only to a much lesser extent by methylated CUFAs, trans.un,~aturated or saturated fatty acids. Interestingly, we have found that CUFAs are not potent inhibitors of the non-calcium-dependent phospholipase A 2 isolated from the adrenal medulla (ReL 26, unpublishcJ observations). Collectively, these observations s-ggest that inhibition by CUFAs may be a property common to Ca2+-dependent phospholipases A2, as previously noted by Lister et al. [15]. Inhibition of the phospholipases A 2 occurred at concentrations of CUFAs well below their critical micellar concentrations (40-60/~M in pure solution) [10,12], indicating that it is not the detergent properties of micelles that affect pbospholipase A 2 activity. Baliou and Cheung postulated that CUFAs inhibited platelct phosphoiipase A z activity by associating with the enzyme since inhibition was not diminished by increasing concentrations of the phosphatidylcboline substrat¢ [14]. Lister et al. [15] developed a kinetic model to demonstrate that arachidonic acid competitively inhibited hydrolysis of micellar and liposomal dipaimitoyI-PC by the calcium-dependent phospholipase A 2 from P388D1 cells. Our data (Fig. 2) illustrating the apparent competitive nature of inhibition (by CUFAs) of the hydrolysis of both biomembranous and liposomal substrates is in agreement with the observations of Lister et al. [15]. Furthermore, our results (Figs. 3 and 4) demonstrating a direct association between CUFAs and the N, moss. mossambica phospholipase A 2 ( p l = 9.6) provide experimental evidence that CUFAs may inhibit enzymatic activity by direct association with the enzyme. On the other hand, Yasuda et al. [2?] have recently reported that the hydrolysis of pbosphatidylcboline by the pbospholipase A 2 purified from rat stomach was inhibited in a non-competitive manner by c/s-arachidonic acid; this apparent discrepancy may be due to the inability of the gastric/ pancreatic enzymes to efficiently penetrate phospbolipid bilayers [28]. The sharp contrast between the biological effects of c/s- vs. trans.unsaturated and saturated fatty acids have
212 been attributed to the fact that CUFAs preferentially partition into the 'fluid' domain of membranes creating disorder, while trans-unsaturated and saturated fatty acids partition into the 'gel' domain and do not induce disorder [22,23]. We believe that CUFA inhibition of in vitro phospholipase A 2 activity is not due to differences in partitioning of fatty acids into the phospholipid bilayer. Two lines of evidence support this view. First, we [10,25] and others [11,25,27,29] have consistently reported selective inhibition of l~hospholipase A 2 activity by CUFAs irrespective of the type of substrate. Thus, CUFAs (but not saturated fatty acids) inhibited hydrolysis of liposomal PE (Fig. 1), liposomal PC [11,15,27], PC-detergent mixed micellcs [29] and membranous E. coli (Ref. 10, 25 and Fig. 1) substrates. In fact, assays with liposoCnal dipalmitoyI-PC as substrate [15] were performed at 370C, a temperature at which the phospholipid is primarily in the 'gel' phase. According to Karnovsky's model, only saturated and trans-unsaturated fatty acids should associate with this phase of the lipid bilayer [23]. The inhibitory profile of CUFAs with PE as substrate were identical to that obtained with other bilayer substrates; PE packs in a non-bilayer, hexagonal array. Interestingly, the appareut K i for inhibition by arachidonie acid for both E. coli and PE was 5/.tM, which is identical to the value reported by Lister et al. [15] using PC as substrate. In addition, reports of formation of 'defect' sites [30] and 'domains' [31] in membranes by products of phospholipase A ~ action are observed only in ternary codispersions of lysophospholipid, fatty acid and phospholipid and not in binary mixtures of either product and phospholipid [30]. Collectively, these data provide strong support for the independence of CUFA inhibition on eitrler the physical state of the substrate (i.e., packing) or the chemical nature of the phospholipids used. Hence, the selective inhibition by CUFAs could involve a preferential association of inhibitory fatty acid and enzyme. The natural hydrophobic affinity of fatty acid for membranes, however, does not preclude the possibility of a 'partitioning' of fatty acid between enzyme and substrate. Jain et al. [24] postulated that certain lipophilic inhibitors could exist predominantly in the lipid bilayer and interact with enzyme, thereby competing with substrate for binding to the catalytic site. Such a model was demonstrated by de Haas and co-workers using non-hydrolyzable acylamino phospholipid analogues [32]. In their studies, the K~ for enzyme-inhibitor association was 2-3-fold higher than that for binding of enzyme to phospholipid [32,33]. Our data demonstrating that inhibitory concentrations of arachidonic acid had little to no effect on the interaction between phospholipase A 2 and E. coli substrate (Table I), is consistent with this hypothesis. Based on the crystal structure of a complex between Naja naja atra
phospholipas¢ A 2 and a non-hydrolyzabl¢ phosphonate phospholipid, Scott et al. [34] suggested that the active site was embedded in a hydrophobic ~ehannel' which facilitates the diffusion of a phospholipid molecule from the interface to the active site and binds the sn-2 alkyl substituent of the phospholipid during catalysis. It is, therefore, possible that CUFAs may similarly diffuse from the membrane bilayer interface into the active site to preve.nt productive association, i.e., catalysi.~. However, the precise nature of the interaction between a long chain CUFA and enzyme is speculative at best in view of the complex interactions involving multiple protein domains with substrate in interfacial catalysis [35i. it is well known that the 'quality of the interface' is critical to interfacial catalysis [35] and that subtle conformational and chemical differences in substrate phospholipid a n d / o r enz~'me play a crucial role in expression of phospholipase A 2 activity [36-38]. Thus, inhibitors such as calpactin-lipocortin [39], quercetin [40] and local anesthetics [41] interact non-specifically with the substrate and serve as examples of inhibition by 'substrate depletion' [24,39,42]. The active site of pancreatic and snake venom phospholipases A 2 is in a hydrophobic environment that presumably participates in orienting the 2-acyl ester bond for the subsequent hydrolysis [43]. Structure-activity studies witil the inhibitory sn-2-acylamino phospholipid analogues suggested that the binding site for the sn-2 substituent could optimally accommodate alkyl chains 14-carboas long [33]. After the catalytic event, the active site of the phospholipas¢ A 2 is in some transient manner intimately associated with the catalytic product, CUFA. Inhibitory CUFAs may occupy the active site of the enzyme thus preventing interaction with the substrate. Fluorescence has been previously used in our laboratory to demonstrate direct interaction between snake venom phospholipase A 2 and in vitro inhibitors such as retinal [40], aristolochie acid [44] and the prostaglandin polymer, PGB x [45]. Interestingly, while these inhibitors quenched the relative fluorescence of the IV. moss. mossambica phospholipase A 2, CUFAs enhanced the intrinsic fluorescence of the enzyme. These results indicate that CUFAs have modified the environment of at least one of the three tryptophan residues (at positions 19, 20 and 60) in this enzyme. Trp-60 is presumably homologous to Tyr-69 on the porcine pancreatic phospholipase A a [46] which, based on X-ray diffraction analysis, is involved in interaction with the substrate [47]. The crystal structure of the monomeric Naja naja atra phospholipase A 2 contains a tryptophan (Trp-19) at the roof of the hydrophohic channel, which is closely associated with the sn-2 alkyl substituent [48]. The increase in fluorescence intensity (Fig. 4) is indicative of increased hydrophobicity in the environment of tryptophan [49]. In contrast,
213 the naturally more hydrophobic saturated and methylated fatty acids hap minimal effect on the fluorescence intensity of the enzyme, indicating that hydrophobicity of fatty acid per se was not responsible for the observed changes in enzyme fluorescence. Furthermore, circular diehroism studies demonstrated that, unlike the effect of the inhibitor, aristolochic acid, which increased the a-helical content of the phospholipase A 2 [44], inhibitory C U F A s did not affect the secondary structure of the snake venom phospholipase A 2 (unpublished observations). Fatty acids could interact with lipid binding domains of the enzyme either in a non-covalent or covalent m a n n e r [29,50,51]. Pliiekthun and Dennis demonstrated that the rate of hydrolysis of mixed micelles by the pancreatic phospholipase A 2 was enhanced, in a presumably non-covalent manner, by the addition of oleic acid [29]. However, recent studies have shown that autoeatalytic acylation of lysine residues (Lys-7 and Lys-10) adjacent to the active site dramatically activates snake venom phospholipase A 2 [50]. Similarly, De H a a s and eoworkers have d e m o n s t r a t e d that monoacylation of the pancreatic phospholipase A 2 (Lys-10 or L y s - l l 9 ) resulted in an increased affinity of enzyme for phospholipid substrate, but did not affect enzymatic activity per se [51]. In our hands, C U F A s consistently inhibited in a d o s e - d e p e n d e n t m a n n e r the hydrolysis of both m e m b r a n o u s E. coil and liposomal P E by all calcium-dependent phospholipases A2 tested (results similar to Fig. 1, data not shown). These observations are indicative of the complex nature of the association of C U F A s with phospholipases A zO u r results illustrate that C U F A s inhibit calciumd e p e n d e n t phospholipases A 2 by selectively interacting with the enzyme. In this regard, calcium-dependent phospholipases A 2 may resemble the intracellular, but non-catalytic, fatty acid binding proteins which exhibit specificity for C U F A s (for review see Ref. 52), The natural association of C U F A s with e n d o g e n o u s phospholipases A 2 to suppress enzymatic activity may imply a unique mode for activation of enzymatic activity as well. Thus, we have shown that autoxidation of cisarachidonic acid substantially diminished its ability to inhibit myocar:lial phospholipase A 2 activity [25]. Increased phospholipase A 2 activity, concomitant with an increase in lipid peroxidation has been observed during pro-oxidant injury such as isehemia [53]. While it is generally believed that pro-oxidants promote phospholipase A2 m e d i a t e d hydrolysis because oxidized phospholipids are more susceptible to hydrolysis [5456], our results [25] suggest that oxidation of unesterifled C U F A s may also activate latent enzyme. A n understanding of how physical p a r a m e t e r s and chemical events such as ischemia and peroxidation influence e n z y m e - C U F A - s u b s t r a t e interaction may provide badly n e e d e d clues as to the role and regulation of phospholipases A 2 in normal and pathologic processes.
Acknowledgement These studies have been supported by a NIH Grant DX 34558. References 1 van den Bosch, H. (1980) Biochim. Biophys. Acta 604, 191-246. 2 Flower, RJ. (1984) in Advances in Inflammation Research (Weismann, G., ed.), pp. 1-34, Raven Press, New York. 3 Clark, M., Conway, T, Schorr. R. and Cooke, S. (1987) J. Biol. Chem. 262, 4402-4406. 4 Bartolf, M. and Franson, R.C. (1987) Biochim. Biophys. Acta 917, 308-317. 5 Weglicki, W.B., Ruth, R.C., Owens, K,, Griffin, H.D. and Waite, B.M. (1974) Pip,:him. Biophys. Acta 337, 145-152. 6 Franson, R., Patriarca, P. and Elsbach, P. (1974) J. Lipid Res. 15, 380-388. 7 Vishwanath, B.S., Fawzy, A.A, and Franson, R.C. (1988) Inflammation 12, 549-561. 8 Creutz, C.E. (1981) I. Cell Biol. 91. 247-256. 9 Raghupathi, R. and Franson, R.C. (1987) Fed. Proc. 47, AI366. 10 M~rki, F. and Franson, R, (1986) Bioehim. Biophys. Acta 879, 149-156. II Ballou, L.R. and Cheung, W.Y. (1983) Proc. Natl. Acad. Sci. USA 80, 5203-5207. 12 Klevens, H.B. (1953) J. Am. Oil Chem. Soc. 30. "/4-80. 13 Bills. T.K., Smith, J.B. and Silver, MJ. (1977) J. Clin. Invest. b0, 1-6. 14 Ballou, L.R. and Cheung, W.Y. (1985) Proc. Natl. Acad. Sci. USA ~2, 371-375. 15 Lister, M.D., Deems, R.A., Watanabe, Y., Ulevitch, RJ, and Dennis E,A. (1988) J. Biol. Chem. 263, 7506-7513. 16 Joubert, FJ. (1977) Biochim. Biophys, Acta 493, 216-227. 17 Lacmm:i, U.K. (1970) Nature 227, 680-685. 18 O'FarrelL P.H. (1975)J. Biol. Chem. 250, 4007-4021. 19 Patriarca, P., Beekerdite, S. and Elsbach, P. (1972) Biochim. Biophys. Acta 260, 593-600. 20 Bligh, E.G. and Dyer, WJ. (1959) Can. J. Biochem. Physiol. 37, 911-917. 21 Segel, 1.H. (1976) in Biochemical Calculations, pp. 246-273, L Wiley and Sons, New York. 22 Klausner, R.D., Kleinfeld, A.M., Hoover, K.L and Karnovs~, MJ. (1980) J. Biol. Chem. 255, 1286-1295. 23 Karnovsk2~, M.J., Kleinfeld, A.M., Hoover, R,L. and Klausner, R.D. (1982) J. Cell Biol. 94, I-6. 24 .lain, M.K., Yuan, W. and Gelb, M.H. (1989) Biochem. 28, 4135-4139. 25 Franson, R.C., Harris, L.H. and Raghupathi, R. (19.5t})MoL Cell. Biochem. 88, ' 55-159. 26 Bartolf, M.B. and Franson, R.C. (1990) Biochim. Biophys. Acta 1042, 247-254, 27 Yasuda, T., Hirohara, J., Okumura, T. and Saito, K. (1990) Biochim. Biophys. Acta 1046, 189-194. 28 Mansbach, C.M. (1990) Gastroenterology 98,1369-82. 29 Pliickthun, A. and Dennis, E.A. (1985) J. Biol. Chem, 260, 11099-11106. 30 Jain, M.K., Esmond, M.R., Verheij, H.M., Apitz-Castro, R., Dijkman, R. and de Haas, G,H, (1982) Biochim. 8ophys, Acta 688, 341-348. 31 Grainger, D.W, Reichert, A., Ringsdorf, H. and Salesse, C. (1989) FEBS Len. 252, 73-82. 32 de Haas, G.H,, Dijkman, R., van Oort, M.G. and Verger, R, (1990) Biochlm, Biophys. Acta 1043, 75-82. 33 de Haas, G.H., Diikman, R., Ransac, S. and Verger, R, (1990) Biochim. Biophys. Acta 1046, 249-257.
214 34 Sc
