56
~~oc~~~~c~et Biophysicu Acta, 1125 (1992) X-61 0 1992 Eisevier Science Publishers B.V. All rights reserved UOOS-2760/92/$05.00
BBALIP 53872
The hydrolysis of natural phosphatidylethanolamines by phospholipase A, from rat serum: a degree of selectivity is shown for docosahexaenoate release R.R. Baker and H.-y. Chang Dicision of Neurology, Department of Medicine Unicersiiy of Toronto, Toronto (Canada)
(Received 16 July 1991) (Revised manuscript 2 October 1991)
Key words: Phospholipase A,; Docosahexaenoate;
Phosphatidylethanolamine;
Arachidonate; Serum; (Rat)
of phospholipase A, from serum was evaluated using radioassays and mass analyses of fatty acids liberated from phosphatidylcholine and phosphat~dylethanolamine. These natural phospholipid substrates were labelled at the sn-2 position with radioactive oleate, linoleate and arachidonate. The rates of reiease of fatty acids were compared with their abundance at the sn-2 position of these phospholipid substrates. While there was little or no selectivity in the liberation of these fatty acids from phosphatidylcholine, there was some evidence for a preferential release of arachidonate with respect to linoleate from phosphatidylethanolamine. Mass analyses of free fatty acid products revealed that docosahexaenoate was consistently liberated at levels that exceeded its abundance at the sn-2 position of phosphatidylethanolamine. Three different, natural phosphatidylethanolamines with varying levels of docosahexaenoate showed a 1.2-l&fold enrichment of this polyunsaturate in the free fatty acid products compared with its abundance at the 32-2 position. This preference could also be shown when phosphatidyiethanolamine was mixed with synthetic phosphatidylcholine as co-sonicated substrates. This preferential release of docosahexaenoate by serum phospholipase A, is of considerable significance in the nervous system which is enriched in this polyunsaturate. The potential competition between liberated docosahexaenoate and arachidonate may be of fundamental importance in the response of brain to hemorrhage. The selectivity
Introduction Phospholipase A, is of importance because it will release arachidonic acid from cellular phospholipids [I-S]. A preference for arachidonate has also been demonstrated using the enzyme from several sources with purified phosphatidylcholine and/or phosphatidylethanolamine in vitro [ti-131. In our studies we are interested in hemorrhage in brain because of the presence of phospholipase A, in serum and the potential for hydrolysis of phosphoIipids of neuronal membranes following bleeding in the tissue. We have shown that synaptosomes, isolated from brain, when incubated with serum will release a number of fatty acids of which docosahexaenoate is the most prominent [14]. Recently we have extensively purified phospholipase A, from
Abbreviations: amine.
PC, phosphatidylcholine;
PE, phosphatidylethanol-
Correspondence: R. Roy Baker, Clinical Science Division, 6368 Medical Sciences Building, University of Toronto, Toronto, Ontario, Canada, M5S lA8.
rat serum and characterized the activity with respect to the use of phospholipid substrates, Ca2+ requirement, pH optimum and product inhibition [15]. In the following studies we have investigated the selectivity of phospholipase A 2 from rat serum using natural populations of phosphatidylethanolamines or phosphatidylcholines by following individual fatty acid release in radioassay and mass analysis. These assays thus monitored competitions between molecular species. We have found that there is a degree of preference shown for the release of docosahexaenoate from phosphatidylethanolamine because this polyunsaturate was found in significantly higher abundance in the free fatty acid products than in the original substrate. This could be of considerable importance in brain hemorrhage because nervous tissue is remarkably enriched in this fatty acid, and competition between arachidonate and docosahexaenoate may determine the severity of the resulting pathological response. Materials and Methods Sigma (St. Louis, MO) provided the ATP and snake venom (Crotalu~ a~a~a~te~). Pig liver phosphatidyI-
57 ethanolamine, 1-acyl-sn-glycero-3-phosphoethanolamine and 1-acyl-sn-glycero-3-phosphocholine were supplied by Serdary Research Labs (London, Ontario). 1-Palmitoyl-2-oleoyl species of phosphatidylcholine was purchased from Avanti Polar Lipids (Birmingham, AL). Boehringer-Mannheim (Dorval, Quebec) provided coenzyme A and Nu Chek Prep (Elysian, MN) supplied oleic acid. DuPont (Mississauga, Ontario) provided the [l- “C]oleic acid (57 mCi/mmol), [l- “Cllinoleic acid (51.7 mCi/mmol) and [5,6,8,9,11,12,14,15“Hlarachidonic acid (76 Ci/mmol). Male Wistar rats (300-375 g) were purchased from Charles River Canada (LaSalle, Quebec). Mandel Scientific (Rockville, Ontario) supplied the thin-layer plates (Analtech G and H, 0.25 mm). Amicon Canada Ltd. (Oakville, Ontario) provided the Centricon 10 membrane units for filtration. Methods Preparation of radioactive substrates. Phosphatidyl-
choline or phosphatidylethanolamine was prepared in incubations containing 1-acyl lysophospholipid, coenzyme A, ATP, Mg2+, radioactive fatty acid and rat liver microsomes [15]. Radioactive phosphatidylcholine and phosphatidylethanolamine were isolated by thinlayer chromatography [15], and snake venom hydrolyses showed that more than 97% of the incorporated radioactive fatty acid was localized at the sn-2 position (for [‘4C]oleate labelled phosphatidylethanolamine this value was 88%). Preparation of unlabelled phosphatidylethanolamine.
Phosphatidylethanolamine was isolated from extracts of rat liver microsomes or rat brain synaptosomes, prepared by the method of Whittaker and Barker [16]. Phosphatidylethanolamine was isolated by thin-layer chromatography [15]. Preparation of phospholipase A,. The enzyme was extensively purified from rat serum by Sephadex G-100 chromatography [153 as described by Aarsman et al. [17]. The enzyme activity was concentrated using Amicon Centricon 10 membranes. Phospholipase A, assays. The radioactive assays contained 100 mM Hepes (pH 7.41, 10 mM CaCl,, 0.02% (w/v) sodium deoxycholate, 12-40 PM labelled phospholipid (suspended in water by sonication) and enzyme (0.3-0.4 pg protein) in a final volume of 0.25 ml. Assays were carried out at 37°C for 3-4 min for phosphatidylethanolamine and for lo-15 min for phosphatidylcholine. Incubations were terminated using 5 ml of chloroform/ methanol (2 : 1, v/v> and carrier free fatty acid was added. Lipids were extracted by the method of Folch et al. [181, free fatty acids were isolated by thin-layer chromatography, eluted from the gel and counted [153. The non radioactive assays were similar to those described above but contained 1.25-1.3 mM sonicated
phosphatidylethanolamine and l-3 pg of enzyme. The incubations were carried out for 5-20 min. No carrier was added and the free fatty acids were isolated by thin-layer chromatography and methylated to give fatty acid methyl esters. The methyl esters were quantitated using gas chromatography with methyl heptadecanoate as internal standard [19]. Controls were carried out in the absence of enzyme or with solvent added to the complete assay mixture at time zero. Protein and phosphate determinations. Protein was determined by the method of Schaffner and Weissmann [20] using bovine serum albumin as standard. Phospholipid phosphate assays were carried out by the method of Bartlett [21]. Results Phospholipase A, release of radioactive fatty acids from phospholipids
When radioactive phosphatidylcholine or phosphatidylethanolamine was incubated with phospholipase A, from serum a linear release of radioactive fatty acid was seen with time. Complete snake venom hydrolyses of the phosphatidylcholine substrates showed that arachidonate, linoleate and oleate were present at 40.8, 38.6 and 7.8 mol% of the sn-2 fatty acids. Arachidonate and linoleate were almost exclusively found in the second position. The rates of release of radioactive oleate, linoleate and arachidonate, from 40 PM phosphatidylcholine in the presence of phospholipase A, from serum were 9.3 + 1.0, 55.6 + 8.2 and 50.1 + 4.9 nmol/min per mg protein, respectively (mean + S.D., n = 4-8). At lower concentrations of phosphatidylcholine the relative rates of release of the three fatty acids were similar to those given above. Complete snake venom hydrolyses of the phosphatidylethanolamine substrates showed arachidonate, linoleate and oleate present at the sn-2 position at 49.6, 24.4 and 5.5 mol%, respectively. Again, arachiTABLE I Hydrolysis of radioactive phosphatidylethanolamine
by phospholipase
A2
Phospholipase A, was isolated from serum and incubated in the presence of 100 mM Hepes (pH 7.4), 10 mM CaCI,, 0.02% (w/v) sodium deoxycholate and 12-40 pM phosphatidylethanolamine labelled in the sn-2 position with [‘4C]oleate, [‘4C]linoleate or [3H]arachidonate. Rates of release of fatty acid are given as nmol/min per mg protein and are expressed as meansfS.D. of 3-9 separate determinations. Concentration (PM)
Fatty acid release 18:l
18:2
20:4
12 20 40
16.1+ 2.6 20.5 f 3.1 25.2k3.4
117k 12.9 135 f 14.7 170+ 19.4
263 f 15.4 383 + 20.8 565 f 58.4
donate and linoleate were almost exclusively w-2 fatty acids. Table I shows the rates of release of the radioactive fatty acids from phosphatidylethanoIamine. Arachidonate was released most rapidly and activity ratios, arachidonate/litloleate, varied from 2.3 at 12 PM substrate to 3.3 at 40 PM substrate For each labelled phosphatidylethanolamine, rates of fatty acid release were higher than those for the corresponding phosphatidylcholine substrates.
TABLE
III
Pb~~spb~~lipase A,
from serum was incubated
using 1.25 mM pb~sphatjdyletbanol~mine In the incubations
as described in Table
312 nmol of phosphatidylethanolamjnc SII - 2 in phosphatidplethanolamine
by gas chromatography SD.
of the number
as described
in Table
times 15-20
TABLE
11
Phospholipase
in
A,
from serum was incubated
as described
the presence of 1.3 mM phosphatidytethanolamine
Each incubation
had 325 nmol of ph(~sphatidylethan~~~amine.
fatty acid products were isotated. methyiated ~bromato~r~phy. ethanolamine titated
Values
and quantitated
Free by gas
The fatty acids at the X~Z-2position of pbospbatidyl-
are means+S.D.
The venom hydrolyses went to comof 3 to 4 separate
determinations.
The free fatty acid columns are values taken with increasing incubation times 6-20
Fatty acid
mm). Distribution
(mol%) PE (~~1-2)
free fatty acids
16:O
0.5 + 0.7
1.2rt:0.h
0.9 + 0.7
lb: I
0.8+0.9
0.5 IO.4
0.6 + 0.3
0.5 f 0.1
1X:0
ft.3 i 0.2
2.3.k 1.4
0.7+
K3+0,t
I.1
0.9 * 0.2
IX: I IX:2
12.72
1.0
I2.6tO.6
I2.5 + 0.6
11.0+0.l
24.2+
I.6
23.9 ?r 0.8
24.0 f 0.8
20.3 t Cr.2
20:4t?t -6)
45.0 i-0.x
J-5.8&2.1
49.3 + 3.3
55.5 2 0.2
22 : 4(11 - 6)
3.22
1.2
2.0 -1_0.5
1.9kO.4
2.9+0.2
: xrf - 6) 22 : 5(t1- 3) 2Z:h(n -3)
3.x*
1.6
2.7 & 0.9
2.3 + 0.4
1.6+_0.1
xi+
I.1
3.7i: I.6
2.8 + 0.6
3.7 + 0.5
5.1 IO.7
3.4 f 0.2
22
Tota
(nmol)
I
were reieased using snake venom hydrolysis and quan-
by gas chromatography.
pletion.
in Table
from pig liver.
h.2& 1.3 25.1+
I.3
5.4 IO.8 56.4 sr 7. t
91.2+_7.6
fatty
were analyzed
II. Values are means*
of ~i~iern~;n~~tj~~nsshown in parentheses.
free fatty acid columns are values taken with increasing
When phosphatidylethanolamine from pig liver was completely digested with snake venom, the fatty acids at sn position 2 were isolated, and their composition is shown in Tabfe II. Arachidonate (56 moI%) was the most prominent of the sn-2 fatty acids, followed by linoieate (20 mol%) and oleate (11 mol%). Docosahexaenoate was a minor component at sn-position 2. The fatty acids with two or more doubfe bonds were almost exclusiveIy s/r-2 fatty acids. When this Phosphatidyletha~oIami~e was incubated with phospholipase A, from serum, docosahexaenoate was found in greater abundance in the free fatty acid products Ci-6 mol%l than in the original substrate (3.4 moI% at srz-21 tP < 0.01, Students t-test). This difference was most notable when the extent of hydroIysis was smallest. With increasing incubation time the extent of hydrolysis of substrate ranged from 7.7 to 28.1%. Arachidonate was present in the free fatty acid
was avail-
able for hydrolysis. Free fatty acid products and the constituent acids at position
1
from rat liver microsomes.
The
incubation
mm),
products at a lower abundance (45-49 moI%) than was seen for this fatty acid in the phosphatidylethanolamine substrate (56 mol%) (P < 0.005, Students t-test). Linoleate was found at a higher percentage level in the free fatty acid products (24 mol%,t than in the phosphatidylethanoIam~ne (20 mot%) (P < O.05, Students t-test). Table 111 shows that phosphatidylethanolamine isolated from rat liver microsomes had a higher content of docosahexaenoate than phosphatidylethanolaminc from pig liver as shown in the analysis of sn-2 fatty acids produced by complete snake venom hydrolyses. Arachidonate was still the predominant srr-2 fatty acid (47 mol%) but for this source of phosphatidylethanolamine docosahexaenoate made up 25 mol% of the ~1-2 fatty acids, foflowed by linoleate at 19 mol%. Each of these fatty acids was found virtuahy exctusivefy at the m-2 position” When this phosphatidylcthanolamine was incubated with serum phospholipase A 2 docosahexaenoate was more abundant in the free fatty acid products than in the sn-2 fatty acids of the original substrate. In the free fatty acids the proportion of docosahexaenoate ranged from 38 rnol% at the lowest level of substrate hydrolysis to 33 mol% at the highest extent of hydrolysis. This was 32 to 53% higher than its abundance at the sn-2 position in the phosphatidylethanolamine substrate CP < 0.001, Srudents t-test). The extent of phosphatidytethanolamine hydroIysis ranged from 8.2 to 18.5%.
59 TABLE
IV
Hydrolysis of phosphatidylethanoluminr by phospholipasr A, Phospholipase
A,
using 1.25 mM
from serum was incubated phosphatidylethanolamine
somes. Each incubation Free
fatty
position
products
as described
separate
from
and constituent
in Table I
rat brain
in Table
determinations.
Distribution
fatty
were II.
The
values taken with increasing incubation Fatty acid
as described
synapto-
had 312 nmol of phosphatidylethanolamine.
of phosphatidylethanolamine
matography three
acid
from rut brain synap!osomrs
Values free
acids at the
analyzed
are means+
fatty
acid
times (5-20
sn-2
by gas chroS.D.
columns
mini.
(mol%) PE (sn-2)
free fatty acids Ih:O
2.5 * 0.4
1.7*o.n
1.2+0.9
2.3 i 0.8
16: I
0.3 f 0.3
_
0.1 kO.1
0.5 * 0.5
l8:O
0.8 + 0.7
0.2kO.3
-
0.9 +0.x
l8:I
9.6 * 0.4
9.5 * 0.7
IX:2
0.3 i_ 0.4
8.0 + 0.3 _
8.9 If- 0.4 _
23.0 + 1.3
21.7t0.7
0.1 +0.2 24.4kO.4
20:4(n
-6)
22:4(n
-6)
22:5(n
-3)
tr
22:fXn
-3)
61.9?
I.9
64.0+
1.3
n1.9*0.9
22.x+
1.4
34.0&
I.9
44.1+
Total (nmol)
2.9 + 0.2
of are
3.1 kO.7 tr
3.OkO.7
28.3 + 1.0 6.6+
1.0
l.7+0.2
tr
50.9 f 0.2
I.1
Arachidonate was found at lower levels in the fatty acid released by the enzyme (37-40 mol%) compared with its abundance at the sn-2 position (47 mol%) (P < 0.001, Students t-test). The molar ratio of docosahexaenoate: arachidonate in the free fatty acid products was 0.8-1.0 in comparison with a corresponding value of 0.5 found in the sn-2 fatty acids of the phosphatidylethanolamine. Lower levels of docosapentaenoate(22 : 5(n - 3)) were found in the free fatty acids when compared with the original sn-2 fatty acids. Rat brain synaptosomes have phosphatidylethanolamine which is particularly enriched in docosahexaenoate, and this is shown in Table IV by analysis of the sn-2 fatty acids liberated by snake venom (complete hydrolysis). Docosahexaenoate made up 51 mol% of the sn-2 fatty acids, followed by arachidonate at 28 mol% and docosatetraenoate (22 : 4(n - 6)) at 7 mol%. When this phosphatidylethanolamine was incubated with phospholipase A, from serum, docosahexaenoate dominated the free fatty acid products, occurring at 62-64 mol% which was 1.2 to 1.3-times its level in the sn-2 fatty acids of the phosphatidylethanolamine substrate (P < 0.001, Students t-test). With increasing incubation time the extent of hydrolysis ranged from 7.3% to 14.1%. Arachidonate was found at lower levels in the free fatty acid products than at the 02-2 position in the substrate (P < 0.005, Students t-test). In the free fatty acid products the molar ratio docosahexaenoate : arachidonate was 2.5-2.9 in comparison with a value of 1.8 found at the sn-2 position in phosphatidylethanolamine.
Free fatty acid release from mixtures of phosphatidylethanolamine and phosphatidylcholine
The serum phospholipase A, was incubated with a mixed substrate composed of phosphatidyiethanolamine from rat liver microsomes and a synthetic phosphatidylcholine which contained only palmitate and oleate as its fatty acids. Although this phosphatidylcholine was nominally 1-palmitoyl-2-oleoyl approx. 25 mol% of each fatty acid was localized to the alternate position by snake venom hydrolysis. The two phospholipids were mixed in chloroform in a 1 : 1 molar ratio, dried under nitrogen and sonicates prepared. After a 10 min incubation with this mixed substrate, the free fatty acid products (36.6 f 2.2 nmol) contained oleate (19.0 f 2.4 mol%) and palmitate (8.7 + 2.1 mol%) as well as arachidonate (32.1 f 0.4 mol%) and docosahexqenoate (23.1 + 3.2 mol%), (means + S.D., n = 4). If the oleate and palmitate products are deleted from the total free fatty acid products (as palmitate and oleate come principally from the phosphatidylcholine substrate) the remaining fatty acids are derived from the hydrolysis of phosphatidylethanolamine. The content of docosahexaenoate among these was 32 mol%, a value which is significantly higher than the abundance of this polyunsaturate at the sn-2 position of phosphatidylethanolamine (P < 0.01, Student’s t-test). Discussion
Using the method of Aarsman et al. [17] we have recently isolated a phospholipase A, from rat serum [15]. The enzyme contained one component of molecular mass 16 kDa and a second which likely consisted of an aggregate of the first. The enzyme had an absolute requirement for Ca*+ and a pH optimum of 7.4. Phosphatidylethanolamine was a better substrate than phosphatidylcholine and the hydrolysis of the latter substrate was inhibited by the addition of free fatty acids and stimulated by the presence of bovine serum albumin (fatty acid-free). In the present work we have investigated the selectivity of the phospholipase A, from serum with respect to the release of individual fatty acids from phosphatidylcholine and phosphatidylethanolamine. In a number of previous studies [6-121 this selectivity was evaluated using different individual phospholipid species. This approach has been recently criticized by Schalkwijk et al. [22] who demonstrated that specificities seen for pure arachidonate bearing species may only reflect differences in the lipid packing of individual phospholipid species in vesicles. To avoid such problems we have used natural phospholipid substrates, containing a mixture of different species. The release of different fatty acids by phospholipase A, can then be monitored by mass analysis or radioassay using one substrate containing a variety of molecular species. This ap-
preach has been used by Clark et al. [5] to demonstrate a marked arachidonate selectivity for a high molecular weight cytosolic phosp~loIipase A, using U93T cellular membranes as substrates. One disadvantage with a mixture of species is that ind~vjduals will not have precisely the same makeup of sn-1 position fatty acids. However, a selectivity for the sn-1 position by phospholipase A, from other sources has not been demonstrated [23]. Our own work has also shown little difference in the rates of release of oteate, linoleate or arachidunate from microsomal ~hos~hatidyi~holine that cannot be predicted from their abundance at the sn-2 positian. Our mass analyses of fatty acids released from phosphatidyl~thanolamine by the ~hospho~ipase A, from serum are expressed in terms of percentage composition of free fatty acids and are directly compared with the percentage of fatty acids available at the m-2 position of the phospholipid substrate. These latter data come from complete snake venom hydrotysis of the phos~ho~~~id. Selectivity wilf thus be shown by differences in fatty acid composition for the free fatty acid products in comparison with the ~1-2 position of the original substrate. It should be considered that farge differences seen in the use of individual phospholipid species by phospholi~ase A, can be lost when such species are present together in the same substrate vesicle [22]. Thus, a fatty acid found at higher abundance in the fret fatty acid products than at the 97-Z position in the original natural substrate is a significant finding and has particular relevance to the attack of cetlular membranes by phospholipase A 2. tt was apparent that the phospho~ipase A, from serum had little or no specificity for the release of fatty acids from sn-position 2 in phosphatidylcholine. However, comparing radioactive arachidonate and linoleate fiberation from 40 PM ~hosphatidylethanalamine~ arachidonate was released at rates which exceeded the 2: i molar ratio seen for arachidonate: Iinoleate at sn-position 2. This apparent selectivity for arachidonate was not seen in the mass analyses of fatty acids released from tmlabelled phosphatidylethanolamines at higher substrate concentrations. Using three different untabelled ~hos~hat~dyietha~o~am~nes we found that it was not arachidonate but docosahexaenaote that was consistently preferentially released from the sn-2 position. This enrichment of docosahexaenoate in the product free fatty acids ranged from 1.2 to M-times the abundance of this highly unsaturated fatty acid at the sn-2 position in the original phosphatid~~~ethano~amine substrate. The greatest enrichment was seen with pig liver phosphatidylethanolamine which had the smallest level of docosahexaenoate among its fatty acids. The greatest effect on a mass basis was seen with the synaptosomat phosphatidy~ethanolamine which had docosahexaenoate as its principal SE-2 fatty acid. In
contrast, arachidonate made up a smaller component of the free fatty acid products in comparision with its IeveIs at the sn-2 position in the substrate phosphoIipid. Pure Phos~hat~dy~ethanoI~mine sonicates tikeiy occur in hexagonal phase and this can account, to a large extent, for their superiority over phosphatidylcholine sonicates which assume a bilayer configuration [23]. As phosphatidylcholine and phosphat~dylethanolamine occur approximately in a L: 1 moiar ratio in synaptosomes, we mixed ~hosphatidylethano~amine with synthetic phosphatidylcholine (bearing only palmitate and oleate) in this proportion and incubated the mixture with the enzyme. We found that even with this mixed substrate the docosahexaenoate was released preferentially and occurred at a higher Ievel in the free fatty acid than in the sn-2 position of phosphatidylethanolamine. This suggested that a preferential release of docosahexaenoate could occur from phosphatidylethanolamine within a phos~hoiipid bilayer, Docosahexaenoate is of interest to us because of its enrichment in synaptosomes and other neural membranes, including retina [24]. We have demonstrated that when synaptosomes are incubated with serum there is a release of free fatty acids among which docosahexaenoate is a major component [14]. We have atso found in incubations with synaptosomes that phosphohpase AZ from serum will release docosahexaenoate as the principal free fatty acid (Z 40 mol%/. Docosahexaenoate is of importance because of its potential conversion to ‘docosanoids’, and its competition with arachidonic acid in various aspects of lipid metabolism [25,25]. Human grey matter phosphat~dy~ethanolamine is also enriched in docosahexaenoate t27] and the potential for this fatty acid to moderate the effects of the metabolites of arachidonate is of particular significance in pathologyi. For example docosahexaenoate has been found to reverse contractions in rat aorta induced by arachido~ate E28]. Thus, the interaction of serum phospholipase A, with brain membranes and the relative release of these polyunsaturates is of particular significance in brain hemorrhage.
This work was supported by a grant from the Heart and Stroke Foundation of Ontario.
I Mahadevappa, V.G. and Holub, B.J. (1986) Biochem. Biophys. Res. Commun. 134, 1327-1333. 2 Purdon, A.D., Patelunas, D. and Smith, J.B. (1’387) Biochim. Biophys. Acta 920, 205-214. 3 Chitton, F.H.. Elks, J.M., Olson, S.C, and Wykle, R.L. (19845 5. Biot. Chem. 259, i?OI4-i2019.
61 4 Tessner, T.G., Greene, D.G. and Wykle, R.L. (1990) J. Biol. Chem. 265, 21032-21038. 5 Clark, J.D., Lin L.L., Kriz, R.N., Ramesha, C.S., Sultzman, L.A., Lin, A.Y., Milona, N. and Knopf, J.L. (1991) Cell 65, 1043-1051. 6 Alonso, F., Henson, P.M. and Leslie, C.C. (1986) Biochim. Biophys. Acta 878, 273-280. 7 Ballou, L.R., Dewitt, L.M. and Cheung, W.Y. (1986) J. Biol. Chem. 261.3107-3111. 8 Kim, D.K., Kudo, I. and Inoue, K. (1988) J. Biochem. 104, 492-494. 9 Diez, E. and Meng, S. (1990) J. Biol. Chem. 265, 14654-14661. 10 Clark, J.D., Milona, N. and Knopf, J.L. (1990) Proc. Nat. Acad. Sci. USA 87, 7708-7712. 11 Kim, D.K., Kudo. I., Fujimori, Y., Mizushima, I-I., Masuda, M., Kikuchi, R., Ikizawa, K. and Inoue, K. (19901 J. Biochem. 108, 903-906. 12 Kim, D.Y., Suh, P.G. and Ryu, S.H. (1991) Biochem. Biophys. Res. Commun. 174, 1899196. 13 Wijkander, J. and Sundler, R. (1989) FEBS Lett. 244, 51-56. 14 Baker, R.R. and Lob, Z.D. (1990) Biochem. Cell Biol. 68, 148153. 15 Baker, R.R. and Chang, H.-y. (1991) Biochem. Cell Biol. 69, 358-365. 16 Whittaker, V.P. and Barker, L.A. (1972) Methods Neurochem. 12, l-52.
17 Aarsman, A.J., Roosenboom, C.F.P., Van Geffen, G.E.W. and Van den Bosch, H. (1985) Biochim. Biophys. Acta 837, 288-298. 18 Folch, J., Lees. M. and Sloane-Stanley, G.H. (1957) J. Biol. Chem. 226, 497-509. 19 Baker, R.R. and Loh, Z.D. (1985) Can. J. Biochem. Cell Biol. 63, 118331188. 20 Schaffner, W. and Weissmann, C. (1973) Anal. Biochem. 56, 502-S 14. 21 Bartlett, G. (1959) J. Biol. Chem. 234, 466-468. 22 Schalkwijk, C.G., Marki, F. and Van den Bosch, H. (lY90) Biochim. Biophys. Acta 1044, 139-146. 23 Van den Bosch, H., Aarsman, A.J., Van Schaik, R.H.N.. Schalkwijk, C.G., Neijs, F.W. and Sturk, A. (1990) Biochem. Sot. Trans. 18, 781-785. 24 Rodriguez de Turco, E.B., Gordon, W.C., Peyman, G.A and Bazan, N.G. (19YO) J. Neurosci. Res. 27,522-532. 2.5 Bazan, N.G. (1990) in Nutrition and the Brain (Wurtman, R.J. and Wurtman, J.J., eds.), Vol. 8, pp. l-24, Raven Press, New York. 26 Bouroudian, M., Nalbone, G., Grynberg, A., Leonardi, J. and Lafont, H. (1990) Mol. Cell Biochem. 93, 119-128. 27 O‘Brien, J.S., Fillerup, D.L. and Mead, J.F. (19641 J. Lipid Res. 5.329-338. 28 Engler, M.B., Karanian, J.W. and Salem, N., Jr. (1990) Eur. J. Pharmacol. 185, 223-226.
~~oc~~~~c~et Biophysicu Acta, 1125 (1992) X-61 0 1992 Eisevier Science Publishers B.V. All rights reserved UOOS-2760/92/$05.00
BBALIP 53872
The hydrolysis of natural phosphatidylethanolamines by phospholipase A, from rat serum: a degree of selectivity is shown for docosahexaenoate release R.R. Baker and H.-y. Chang Dicision of Neurology, Department of Medicine Unicersiiy of Toronto, Toronto (Canada)
(Received 16 July 1991) (Revised manuscript 2 October 1991)
Key words: Phospholipase A,; Docosahexaenoate;
Phosphatidylethanolamine;
Arachidonate; Serum; (Rat)
of phospholipase A, from serum was evaluated using radioassays and mass analyses of fatty acids liberated from phosphatidylcholine and phosphat~dylethanolamine. These natural phospholipid substrates were labelled at the sn-2 position with radioactive oleate, linoleate and arachidonate. The rates of reiease of fatty acids were compared with their abundance at the sn-2 position of these phospholipid substrates. While there was little or no selectivity in the liberation of these fatty acids from phosphatidylcholine, there was some evidence for a preferential release of arachidonate with respect to linoleate from phosphatidylethanolamine. Mass analyses of free fatty acid products revealed that docosahexaenoate was consistently liberated at levels that exceeded its abundance at the sn-2 position of phosphatidylethanolamine. Three different, natural phosphatidylethanolamines with varying levels of docosahexaenoate showed a 1.2-l&fold enrichment of this polyunsaturate in the free fatty acid products compared with its abundance at the 32-2 position. This preference could also be shown when phosphatidyiethanolamine was mixed with synthetic phosphatidylcholine as co-sonicated substrates. This preferential release of docosahexaenoate by serum phospholipase A, is of considerable significance in the nervous system which is enriched in this polyunsaturate. The potential competition between liberated docosahexaenoate and arachidonate may be of fundamental importance in the response of brain to hemorrhage. The selectivity
Introduction Phospholipase A, is of importance because it will release arachidonic acid from cellular phospholipids [I-S]. A preference for arachidonate has also been demonstrated using the enzyme from several sources with purified phosphatidylcholine and/or phosphatidylethanolamine in vitro [ti-131. In our studies we are interested in hemorrhage in brain because of the presence of phospholipase A, in serum and the potential for hydrolysis of phosphoIipids of neuronal membranes following bleeding in the tissue. We have shown that synaptosomes, isolated from brain, when incubated with serum will release a number of fatty acids of which docosahexaenoate is the most prominent [14]. Recently we have extensively purified phospholipase A, from
Abbreviations: amine.
PC, phosphatidylcholine;
PE, phosphatidylethanol-
Correspondence: R. Roy Baker, Clinical Science Division, 6368 Medical Sciences Building, University of Toronto, Toronto, Ontario, Canada, M5S lA8.
rat serum and characterized the activity with respect to the use of phospholipid substrates, Ca2+ requirement, pH optimum and product inhibition [15]. In the following studies we have investigated the selectivity of phospholipase A 2 from rat serum using natural populations of phosphatidylethanolamines or phosphatidylcholines by following individual fatty acid release in radioassay and mass analysis. These assays thus monitored competitions between molecular species. We have found that there is a degree of preference shown for the release of docosahexaenoate from phosphatidylethanolamine because this polyunsaturate was found in significantly higher abundance in the free fatty acid products than in the original substrate. This could be of considerable importance in brain hemorrhage because nervous tissue is remarkably enriched in this fatty acid, and competition between arachidonate and docosahexaenoate may determine the severity of the resulting pathological response. Materials and Methods Sigma (St. Louis, MO) provided the ATP and snake venom (Crotalu~ a~a~a~te~). Pig liver phosphatidyI-
57 ethanolamine, 1-acyl-sn-glycero-3-phosphoethanolamine and 1-acyl-sn-glycero-3-phosphocholine were supplied by Serdary Research Labs (London, Ontario). 1-Palmitoyl-2-oleoyl species of phosphatidylcholine was purchased from Avanti Polar Lipids (Birmingham, AL). Boehringer-Mannheim (Dorval, Quebec) provided coenzyme A and Nu Chek Prep (Elysian, MN) supplied oleic acid. DuPont (Mississauga, Ontario) provided the [l- “C]oleic acid (57 mCi/mmol), [l- “Cllinoleic acid (51.7 mCi/mmol) and [5,6,8,9,11,12,14,15“Hlarachidonic acid (76 Ci/mmol). Male Wistar rats (300-375 g) were purchased from Charles River Canada (LaSalle, Quebec). Mandel Scientific (Rockville, Ontario) supplied the thin-layer plates (Analtech G and H, 0.25 mm). Amicon Canada Ltd. (Oakville, Ontario) provided the Centricon 10 membrane units for filtration. Methods Preparation of radioactive substrates. Phosphatidyl-
choline or phosphatidylethanolamine was prepared in incubations containing 1-acyl lysophospholipid, coenzyme A, ATP, Mg2+, radioactive fatty acid and rat liver microsomes [15]. Radioactive phosphatidylcholine and phosphatidylethanolamine were isolated by thinlayer chromatography [15], and snake venom hydrolyses showed that more than 97% of the incorporated radioactive fatty acid was localized at the sn-2 position (for [‘4C]oleate labelled phosphatidylethanolamine this value was 88%). Preparation of unlabelled phosphatidylethanolamine.
Phosphatidylethanolamine was isolated from extracts of rat liver microsomes or rat brain synaptosomes, prepared by the method of Whittaker and Barker [16]. Phosphatidylethanolamine was isolated by thin-layer chromatography [15]. Preparation of phospholipase A,. The enzyme was extensively purified from rat serum by Sephadex G-100 chromatography [153 as described by Aarsman et al. [17]. The enzyme activity was concentrated using Amicon Centricon 10 membranes. Phospholipase A, assays. The radioactive assays contained 100 mM Hepes (pH 7.41, 10 mM CaCl,, 0.02% (w/v) sodium deoxycholate, 12-40 PM labelled phospholipid (suspended in water by sonication) and enzyme (0.3-0.4 pg protein) in a final volume of 0.25 ml. Assays were carried out at 37°C for 3-4 min for phosphatidylethanolamine and for lo-15 min for phosphatidylcholine. Incubations were terminated using 5 ml of chloroform/ methanol (2 : 1, v/v> and carrier free fatty acid was added. Lipids were extracted by the method of Folch et al. [181, free fatty acids were isolated by thin-layer chromatography, eluted from the gel and counted [153. The non radioactive assays were similar to those described above but contained 1.25-1.3 mM sonicated
phosphatidylethanolamine and l-3 pg of enzyme. The incubations were carried out for 5-20 min. No carrier was added and the free fatty acids were isolated by thin-layer chromatography and methylated to give fatty acid methyl esters. The methyl esters were quantitated using gas chromatography with methyl heptadecanoate as internal standard [19]. Controls were carried out in the absence of enzyme or with solvent added to the complete assay mixture at time zero. Protein and phosphate determinations. Protein was determined by the method of Schaffner and Weissmann [20] using bovine serum albumin as standard. Phospholipid phosphate assays were carried out by the method of Bartlett [21]. Results Phospholipase A, release of radioactive fatty acids from phospholipids
When radioactive phosphatidylcholine or phosphatidylethanolamine was incubated with phospholipase A, from serum a linear release of radioactive fatty acid was seen with time. Complete snake venom hydrolyses of the phosphatidylcholine substrates showed that arachidonate, linoleate and oleate were present at 40.8, 38.6 and 7.8 mol% of the sn-2 fatty acids. Arachidonate and linoleate were almost exclusively found in the second position. The rates of release of radioactive oleate, linoleate and arachidonate, from 40 PM phosphatidylcholine in the presence of phospholipase A, from serum were 9.3 + 1.0, 55.6 + 8.2 and 50.1 + 4.9 nmol/min per mg protein, respectively (mean + S.D., n = 4-8). At lower concentrations of phosphatidylcholine the relative rates of release of the three fatty acids were similar to those given above. Complete snake venom hydrolyses of the phosphatidylethanolamine substrates showed arachidonate, linoleate and oleate present at the sn-2 position at 49.6, 24.4 and 5.5 mol%, respectively. Again, arachiTABLE I Hydrolysis of radioactive phosphatidylethanolamine
by phospholipase
A2
Phospholipase A, was isolated from serum and incubated in the presence of 100 mM Hepes (pH 7.4), 10 mM CaCI,, 0.02% (w/v) sodium deoxycholate and 12-40 pM phosphatidylethanolamine labelled in the sn-2 position with [‘4C]oleate, [‘4C]linoleate or [3H]arachidonate. Rates of release of fatty acid are given as nmol/min per mg protein and are expressed as meansfS.D. of 3-9 separate determinations. Concentration (PM)
Fatty acid release 18:l
18:2
20:4
12 20 40
16.1+ 2.6 20.5 f 3.1 25.2k3.4
117k 12.9 135 f 14.7 170+ 19.4
263 f 15.4 383 + 20.8 565 f 58.4
donate and linoleate were almost exclusively w-2 fatty acids. Table I shows the rates of release of the radioactive fatty acids from phosphatidylethanoIamine. Arachidonate was released most rapidly and activity ratios, arachidonate/litloleate, varied from 2.3 at 12 PM substrate to 3.3 at 40 PM substrate For each labelled phosphatidylethanolamine, rates of fatty acid release were higher than those for the corresponding phosphatidylcholine substrates.
TABLE
III
Pb~~spb~~lipase A,
from serum was incubated
using 1.25 mM pb~sphatjdyletbanol~mine In the incubations
as described in Table
312 nmol of phosphatidylethanolamjnc SII - 2 in phosphatidplethanolamine
by gas chromatography SD.
of the number
as described
in Table
times 15-20
TABLE
11
Phospholipase
in
A,
from serum was incubated
as described
the presence of 1.3 mM phosphatidytethanolamine
Each incubation
had 325 nmol of ph(~sphatidylethan~~~amine.
fatty acid products were isotated. methyiated ~bromato~r~phy. ethanolamine titated
Values
and quantitated
Free by gas
The fatty acids at the X~Z-2position of pbospbatidyl-
are means+S.D.
The venom hydrolyses went to comof 3 to 4 separate
determinations.
The free fatty acid columns are values taken with increasing incubation times 6-20
Fatty acid
mm). Distribution
(mol%) PE (~~1-2)
free fatty acids
16:O
0.5 + 0.7
1.2rt:0.h
0.9 + 0.7
lb: I
0.8+0.9
0.5 IO.4
0.6 + 0.3
0.5 f 0.1
1X:0
ft.3 i 0.2
2.3.k 1.4
0.7+
K3+0,t
I.1
0.9 * 0.2
IX: I IX:2
12.72
1.0
I2.6tO.6
I2.5 + 0.6
11.0+0.l
24.2+
I.6
23.9 ?r 0.8
24.0 f 0.8
20.3 t Cr.2
20:4t?t -6)
45.0 i-0.x
J-5.8&2.1
49.3 + 3.3
55.5 2 0.2
22 : 4(11 - 6)
3.22
1.2
2.0 -1_0.5
1.9kO.4
2.9+0.2
: xrf - 6) 22 : 5(t1- 3) 2Z:h(n -3)
3.x*
1.6
2.7 & 0.9
2.3 + 0.4
1.6+_0.1
xi+
I.1
3.7i: I.6
2.8 + 0.6
3.7 + 0.5
5.1 IO.7
3.4 f 0.2
22
Tota
(nmol)
I
were reieased using snake venom hydrolysis and quan-
by gas chromatography.
pletion.
in Table
from pig liver.
h.2& 1.3 25.1+
I.3
5.4 IO.8 56.4 sr 7. t
91.2+_7.6
fatty
were analyzed
II. Values are means*
of ~i~iern~;n~~tj~~nsshown in parentheses.
free fatty acid columns are values taken with increasing
When phosphatidylethanolamine from pig liver was completely digested with snake venom, the fatty acids at sn position 2 were isolated, and their composition is shown in Tabfe II. Arachidonate (56 moI%) was the most prominent of the sn-2 fatty acids, followed by linoieate (20 mol%) and oleate (11 mol%). Docosahexaenoate was a minor component at sn-position 2. The fatty acids with two or more doubfe bonds were almost exclusiveIy s/r-2 fatty acids. When this Phosphatidyletha~oIami~e was incubated with phospholipase A, from serum, docosahexaenoate was found in greater abundance in the free fatty acid products Ci-6 mol%l than in the original substrate (3.4 moI% at srz-21 tP < 0.01, Students t-test). This difference was most notable when the extent of hydroIysis was smallest. With increasing incubation time the extent of hydrolysis of substrate ranged from 7.7 to 28.1%. Arachidonate was present in the free fatty acid
was avail-
able for hydrolysis. Free fatty acid products and the constituent acids at position
1
from rat liver microsomes.
The
incubation
mm),
products at a lower abundance (45-49 moI%) than was seen for this fatty acid in the phosphatidylethanolamine substrate (56 mol%) (P < 0.005, Students t-test). Linoleate was found at a higher percentage level in the free fatty acid products (24 mol%,t than in the phosphatidylethanoIam~ne (20 mot%) (P < O.05, Students t-test). Table 111 shows that phosphatidylethanolamine isolated from rat liver microsomes had a higher content of docosahexaenoate than phosphatidylethanolaminc from pig liver as shown in the analysis of sn-2 fatty acids produced by complete snake venom hydrolyses. Arachidonate was still the predominant srr-2 fatty acid (47 mol%) but for this source of phosphatidylethanolamine docosahexaenoate made up 25 mol% of the ~1-2 fatty acids, foflowed by linoleate at 19 mol%. Each of these fatty acids was found virtuahy exctusivefy at the m-2 position” When this phosphatidylcthanolamine was incubated with serum phospholipase A 2 docosahexaenoate was more abundant in the free fatty acid products than in the sn-2 fatty acids of the original substrate. In the free fatty acids the proportion of docosahexaenoate ranged from 38 rnol% at the lowest level of substrate hydrolysis to 33 mol% at the highest extent of hydrolysis. This was 32 to 53% higher than its abundance at the sn-2 position in the phosphatidylethanolamine substrate CP < 0.001, Srudents t-test). The extent of phosphatidytethanolamine hydroIysis ranged from 8.2 to 18.5%.
59 TABLE
IV
Hydrolysis of phosphatidylethanoluminr by phospholipasr A, Phospholipase
A,
using 1.25 mM
from serum was incubated phosphatidylethanolamine
somes. Each incubation Free
fatty
position
products
as described
separate
from
and constituent
in Table I
rat brain
in Table
determinations.
Distribution
fatty
were II.
The
values taken with increasing incubation Fatty acid
as described
synapto-
had 312 nmol of phosphatidylethanolamine.
of phosphatidylethanolamine
matography three
acid
from rut brain synap!osomrs
Values free
acids at the
analyzed
are means+
fatty
acid
times (5-20
sn-2
by gas chroS.D.
columns
mini.
(mol%) PE (sn-2)
free fatty acids Ih:O
2.5 * 0.4
1.7*o.n
1.2+0.9
2.3 i 0.8
16: I
0.3 f 0.3
_
0.1 kO.1
0.5 * 0.5
l8:O
0.8 + 0.7
0.2kO.3
-
0.9 +0.x
l8:I
9.6 * 0.4
9.5 * 0.7
IX:2
0.3 i_ 0.4
8.0 + 0.3 _
8.9 If- 0.4 _
23.0 + 1.3
21.7t0.7
0.1 +0.2 24.4kO.4
20:4(n
-6)
22:4(n
-6)
22:5(n
-3)
tr
22:fXn
-3)
61.9?
I.9
64.0+
1.3
n1.9*0.9
22.x+
1.4
34.0&
I.9
44.1+
Total (nmol)
2.9 + 0.2
of are
3.1 kO.7 tr
3.OkO.7
28.3 + 1.0 6.6+
1.0
l.7+0.2
tr
50.9 f 0.2
I.1
Arachidonate was found at lower levels in the fatty acid released by the enzyme (37-40 mol%) compared with its abundance at the sn-2 position (47 mol%) (P < 0.001, Students t-test). The molar ratio of docosahexaenoate: arachidonate in the free fatty acid products was 0.8-1.0 in comparison with a corresponding value of 0.5 found in the sn-2 fatty acids of the phosphatidylethanolamine. Lower levels of docosapentaenoate(22 : 5(n - 3)) were found in the free fatty acids when compared with the original sn-2 fatty acids. Rat brain synaptosomes have phosphatidylethanolamine which is particularly enriched in docosahexaenoate, and this is shown in Table IV by analysis of the sn-2 fatty acids liberated by snake venom (complete hydrolysis). Docosahexaenoate made up 51 mol% of the sn-2 fatty acids, followed by arachidonate at 28 mol% and docosatetraenoate (22 : 4(n - 6)) at 7 mol%. When this phosphatidylethanolamine was incubated with phospholipase A, from serum, docosahexaenoate dominated the free fatty acid products, occurring at 62-64 mol% which was 1.2 to 1.3-times its level in the sn-2 fatty acids of the phosphatidylethanolamine substrate (P < 0.001, Students t-test). With increasing incubation time the extent of hydrolysis ranged from 7.3% to 14.1%. Arachidonate was found at lower levels in the free fatty acid products than at the 02-2 position in the substrate (P < 0.005, Students t-test). In the free fatty acid products the molar ratio docosahexaenoate : arachidonate was 2.5-2.9 in comparison with a value of 1.8 found at the sn-2 position in phosphatidylethanolamine.
Free fatty acid release from mixtures of phosphatidylethanolamine and phosphatidylcholine
The serum phospholipase A, was incubated with a mixed substrate composed of phosphatidyiethanolamine from rat liver microsomes and a synthetic phosphatidylcholine which contained only palmitate and oleate as its fatty acids. Although this phosphatidylcholine was nominally 1-palmitoyl-2-oleoyl approx. 25 mol% of each fatty acid was localized to the alternate position by snake venom hydrolysis. The two phospholipids were mixed in chloroform in a 1 : 1 molar ratio, dried under nitrogen and sonicates prepared. After a 10 min incubation with this mixed substrate, the free fatty acid products (36.6 f 2.2 nmol) contained oleate (19.0 f 2.4 mol%) and palmitate (8.7 + 2.1 mol%) as well as arachidonate (32.1 f 0.4 mol%) and docosahexqenoate (23.1 + 3.2 mol%), (means + S.D., n = 4). If the oleate and palmitate products are deleted from the total free fatty acid products (as palmitate and oleate come principally from the phosphatidylcholine substrate) the remaining fatty acids are derived from the hydrolysis of phosphatidylethanolamine. The content of docosahexaenoate among these was 32 mol%, a value which is significantly higher than the abundance of this polyunsaturate at the sn-2 position of phosphatidylethanolamine (P < 0.01, Student’s t-test). Discussion
Using the method of Aarsman et al. [17] we have recently isolated a phospholipase A, from rat serum [15]. The enzyme contained one component of molecular mass 16 kDa and a second which likely consisted of an aggregate of the first. The enzyme had an absolute requirement for Ca*+ and a pH optimum of 7.4. Phosphatidylethanolamine was a better substrate than phosphatidylcholine and the hydrolysis of the latter substrate was inhibited by the addition of free fatty acids and stimulated by the presence of bovine serum albumin (fatty acid-free). In the present work we have investigated the selectivity of the phospholipase A, from serum with respect to the release of individual fatty acids from phosphatidylcholine and phosphatidylethanolamine. In a number of previous studies [6-121 this selectivity was evaluated using different individual phospholipid species. This approach has been recently criticized by Schalkwijk et al. [22] who demonstrated that specificities seen for pure arachidonate bearing species may only reflect differences in the lipid packing of individual phospholipid species in vesicles. To avoid such problems we have used natural phospholipid substrates, containing a mixture of different species. The release of different fatty acids by phospholipase A, can then be monitored by mass analysis or radioassay using one substrate containing a variety of molecular species. This ap-
preach has been used by Clark et al. [5] to demonstrate a marked arachidonate selectivity for a high molecular weight cytosolic phosp~loIipase A, using U93T cellular membranes as substrates. One disadvantage with a mixture of species is that ind~vjduals will not have precisely the same makeup of sn-1 position fatty acids. However, a selectivity for the sn-1 position by phospholipase A, from other sources has not been demonstrated [23]. Our own work has also shown little difference in the rates of release of oteate, linoleate or arachidunate from microsomal ~hos~hatidyi~holine that cannot be predicted from their abundance at the sn-2 positian. Our mass analyses of fatty acids released from phosphatidyl~thanolamine by the ~hospho~ipase A, from serum are expressed in terms of percentage composition of free fatty acids and are directly compared with the percentage of fatty acids available at the m-2 position of the phospholipid substrate. These latter data come from complete snake venom hydrotysis of the phos~ho~~~id. Selectivity wilf thus be shown by differences in fatty acid composition for the free fatty acid products in comparison with the ~1-2 position of the original substrate. It should be considered that farge differences seen in the use of individual phospholipid species by phospholi~ase A, can be lost when such species are present together in the same substrate vesicle [22]. Thus, a fatty acid found at higher abundance in the fret fatty acid products than at the 97-Z position in the original natural substrate is a significant finding and has particular relevance to the attack of cetlular membranes by phospholipase A 2. tt was apparent that the phospho~ipase A, from serum had little or no specificity for the release of fatty acids from sn-position 2 in phosphatidylcholine. However, comparing radioactive arachidonate and linoleate fiberation from 40 PM ~hosphatidylethanalamine~ arachidonate was released at rates which exceeded the 2: i molar ratio seen for arachidonate: Iinoleate at sn-position 2. This apparent selectivity for arachidonate was not seen in the mass analyses of fatty acids released from tmlabelled phosphatidylethanolamines at higher substrate concentrations. Using three different untabelled ~hos~hat~dyietha~o~am~nes we found that it was not arachidonate but docosahexaenaote that was consistently preferentially released from the sn-2 position. This enrichment of docosahexaenoate in the product free fatty acids ranged from 1.2 to M-times the abundance of this highly unsaturated fatty acid at the sn-2 position in the original phosphatid~~~ethano~amine substrate. The greatest enrichment was seen with pig liver phosphatidylethanolamine which had the smallest level of docosahexaenoate among its fatty acids. The greatest effect on a mass basis was seen with the synaptosomat phosphatidy~ethanolamine which had docosahexaenoate as its principal SE-2 fatty acid. In
contrast, arachidonate made up a smaller component of the free fatty acid products in comparision with its IeveIs at the sn-2 position in the substrate phosphoIipid. Pure Phos~hat~dy~ethanoI~mine sonicates tikeiy occur in hexagonal phase and this can account, to a large extent, for their superiority over phosphatidylcholine sonicates which assume a bilayer configuration [23]. As phosphatidylcholine and phosphat~dylethanolamine occur approximately in a L: 1 moiar ratio in synaptosomes, we mixed ~hosphatidylethano~amine with synthetic phosphatidylcholine (bearing only palmitate and oleate) in this proportion and incubated the mixture with the enzyme. We found that even with this mixed substrate the docosahexaenoate was released preferentially and occurred at a higher Ievel in the free fatty acid than in the sn-2 position of phosphatidylethanolamine. This suggested that a preferential release of docosahexaenoate could occur from phosphatidylethanolamine within a phos~hoiipid bilayer, Docosahexaenoate is of interest to us because of its enrichment in synaptosomes and other neural membranes, including retina [24]. We have demonstrated that when synaptosomes are incubated with serum there is a release of free fatty acids among which docosahexaenoate is a major component [14]. We have atso found in incubations with synaptosomes that phosphohpase AZ from serum will release docosahexaenoate as the principal free fatty acid (Z 40 mol%/. Docosahexaenoate is of importance because of its potential conversion to ‘docosanoids’, and its competition with arachidonic acid in various aspects of lipid metabolism [25,25]. Human grey matter phosphat~dy~ethanolamine is also enriched in docosahexaenoate t27] and the potential for this fatty acid to moderate the effects of the metabolites of arachidonate is of particular significance in pathologyi. For example docosahexaenoate has been found to reverse contractions in rat aorta induced by arachido~ate E28]. Thus, the interaction of serum phospholipase A, with brain membranes and the relative release of these polyunsaturates is of particular significance in brain hemorrhage.
This work was supported by a grant from the Heart and Stroke Foundation of Ontario.
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