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Platelet activating factor-acetylhydrolase in insectsIdentification and partial characterization of a 1-alkyl-2-acetyl-sn-glycero-3-phosphocholine acetylhydrolase in a cell-free system of Heliothis.

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Archives of Insect Biochemistry and Physiology 237-45 (1988)
Platelet Activating Factor-AcetyIhydrolase in
Insects: Identification and Partial
Characterization of a 1-Alkyl-2-Acetyl-snG l ycero-3-Phosphocholine Acety Ihydrolase in a
Cell-Free System of Heliothis
Edward N. Lambremont, Boyd Malone, and Fred Snyder
Nuclear Science Center, Louisiana State University, Baton Rouge (E. N . L.) and Biological
Chemist y Laboratoy, Medical and Health Sciences Division, Oak Ridge Associated Universities,
Oak Ridge, Tennessee (B.M., F.S.)
A specific acetylhydrolase that inactivates platelet activating factor (PAF; 1alkyl-2-acetyl-sn-glycero-3-phosphocholine),a potent cellular mediator in
mammalian cells, b y removal of the sn-2 acetyl moiety, has been found in the
cytosolic fraction of several postemhryonir developmental stages and specific
tissues of the corn eanvorm, Heliothis zea (Boddie). Effects of magnesium,
calcium, EGTA, deoxycholate, dithiothreitol, diisopropylfluorophosphate, egg
phosphatidylcholine, and an acylacetyl-glycerophosphocholine show that
hydrolysis of the acetate moiety is due t o a specific acetylhydrolase for PAF.
The activity does not appear to b e due t o a typical cellular phospholipase A2
that utilizes phospholipid substrates with a long-chain acyl group at position
sn-2 o f glycerol. Specific activities and properties of the acetylhydrolase from
this insect match closely with those described from tissues of vertebrate
animals.
Key words: lipid metabolism and enzymes, ether-linked lipids, inactivation of bio-active
lipids and cell mediators
INTRODUCTION
The occurrence of lipids containing an ether bond has been studied extensively since the early 1920s in a wide variety of lower and higher animals [l].
Acknowledgments: The insects used in this study were kindly provided by Dr. J.B. Graves of
the Department of Entomology, Louisiana State University, Baton Rouge, and Dr. Ed King,
USDA, Stoneville, MS. We thank Dr. M.C. Cabot, Dr. T-c Lee, and M.L. Blank for procedural
suggestions, support, and the gift of several lipid compounds, and Yvonne Thomas for
technical support. This research was jointly supported by Louisiana State University and Oak
Ridge Associated Universities’ Medical and Health Sciences Division and University Programs
Division. Work at the Medical and Health Sciences Division was supported by the Office of
Energy Research, US Department of Energy (Contract No. DE-AC05-7604000033).
Received July 29,1987; accepted December 1,1987.
Address reprint requests t o Dr. E.N. Lambremont, Nuclear Science Center, Louisiana State
University, Baton Rouge, LA 70803-5820.
0 1988 Alan R. Liss, Inc.
38
Lambremont, Malone, and Snyder
Until the late 1960s the biosynthesis of ether-linked lipids was unknown,
although their structure had been established. It was not until the late 1960s
and into the 1970s that ether-linked glycerolipids came under intense research when they were found to occur in elevated amounts in certain neoplasms and appeared to be associated with biomembranes [2].
Alkyldihydroxyacetone-P synthase, the enzyme that catalyzes the formation
of the ether bond in lipids, was discovered in 1969 [2]. Then in 1979, a
semisynthetic acetylated alkylglycerophosphocholine was shown to induce
potent pharmacologic actions that were identical to an antihypertensive principle isolated from kidney medulla called "APRL" by Muirhead in Blank et
al. [3] and "platelet activating factor," or PAF*, by Benveniste et al. [4] and
Demopoullos et al. [5].
Research interest in the metabolism and pharmacology of PAF has concentrated on higher animals. While this is quite understandable from a medical
point of view, it is also of interest in the pursuit of comparative biochemical
phylogeny to know whether other animal groups also contain and metabolize
such bioactive lipids. Because insects are known to contain ether-linked
lipids, including the alkyl-acyl type of glycerophosphocholines [6,q that are
precursors of PAF, and because insects readily synthesize ether-linked lipids
from other constitutents [8-lo], we decided to determine whether insects
contained enzymes involved in the metabolism of PAF as do vertebrate
animals. Of several possible choices to begin such a line of investigation, we
elected first to determine if a cell-free enzyme system from an insect could
inactivate PAF by removal of the acetate grouping. Hydrolysis of the acetate
group from the sn-2 position of PAF by an acetylhydrolase produces l-alkyl2-lyso-GPC, which is an inactive molecule when compared with similar
concentrations of PAF [ll]. The experiments described in this report demonstrate that insect tissues do indeed contain a PAF acetylhydrolase.
MATERIALS AND METHODS
Chemicals
CHAPS, EGTA, deoxycholic acid (sodium salt), dithiothreitol (Cleland's
reagent), DFP, and unlabeled l-hexadecyl-2-acetyl-sn-glycero-3-phosphocholine were from Sigma Chemical Co., St. Louis, MO. L-cd'hosphatidylcholine
(egg yolk lecithin) was from Calbiochem-Behring Corp., LaJolla, CA. The 1palmitoyl-2-acetyl-sn-glycero-3-phosphocholine
was provided by M. Blank.
Its purity was determined just before use to be greater than 99% by TLC on
silica gel H in a solvent system of ch1oroform:methanol:ammonium hydroxide (65:35:8). Lobster hemolymph was from Grand Island Biological Co.,
Grand Island, NY. All other chemicals were of reagent grade quality.
Labeled PAF, l-hexadecyl-2-[3H]-acetyl-sn-glycero-3-phosphocholine,
was
prepared by the method of Blank et al. [ll]; it had a radiochemical purity in
*Abbreviations: 3-[(3-cholarnidopropyI) dirnethyl-arnmoniol-I-propanesulfonate
= CHAPS; diisopropylfluorophosphate = DFP; ethyleneglycol-bis-(beta-arninoethylether) N, "-tetraace-
also shortened to alkylacetyl
tic acid = EGTA; l-alkyl-2-acetyl-sn-glycero-3-phosphocholine,
CPC = PAF, platelet activating factor.
PAF-Acetylhydrolase in Heliothis
39
excess of 99% as shown by TLC on Silica gel H plates in a system of
ch1oroform:methanol:glacial acetic acid:water (50:30:8:6).
Insects and Enzyme Preparations
Insect material was obtained from laboratory stocks reared on artificial
diets. Cytosols were prepared from freshly dissected rat kidney and from
third instar larvae, mature sixth instar larvae, newly molted (untanned)
pupae, tanned pupae, pupal fat bodies, and adult flight muscles of Heliothis
zea (Boddie) (Insecta, Lepidoptera: Noctuidae). To each gram of insect material, 9 ml of ice cold 0.25 M sucrose were added. After a brief mincing of the
tissues with sterile scissors, the mascerate was homogenized with four to
five strokes in a motor-driven Teflon-Pyrex tissue grinder in an ice bath. Cell
debris, nuclei, and mitochondria were pelleted at 15,OOOg for 10 min in a
refrigerated centrifuge; the pellet was discarded. Microsomes were then
pelleted from the supernatant fraction at lO0,OOOg for 60 min, and the supernatant (cytosolic fraction) was used for all enzyme analyses. The cytosols
were subdivided into 0.5-ml portions and transferred to ice-cold glass vials
(with Teflon-lined screw caps) and immediately frozen (-20°C). In some
initial experiments, freshly prepared cytosols were shown to have enzyme
activities comparable with those obtained with the frozen samples.
Pupal hemolymph was obtained by puncturing the cuticle at an intersegmental suture of a wing cover with a 50-pl glass capillary pipette. The clear
hemolymph was easily drawn by capillary action into the pipette, and with
very gentle pressure on the sides of the pupa, volumes in excess of 50 p1 per
insect were obtainable. Pooled hemolymph samples were kept on ice, diluted
with 10 volumes of 0.25 M sucrose, centrifuged very briefly in the cold to
sediment fat body cells, and then frozen in 0.5-ml aliquots as above.
Postmitochondrial supernatants of fat bodies were prepared after cutting
off the head end of the pupa and gently extruding the fat body into cold 0.25
M sucrose. The tissue was gently homogenized as above and centrifuged in
the cold at 12,SOOg for 10 min. The supernatant fluid was diluted with 20
volumes of cold 0.25 M sucrose and frozen as above. Protein concentrations
in all cytosols and tissue preparations were measured by the Lowry et al.
method [12].
Enzyme Assay
Enzyme activity was determined by adaptations of the methods of Blank
et al. [ll] and Cabot et al. [12].
The complete enzyme reaction system contained l-he~adecyl-2-[~H]-acetyl
GPC (10 nmol and 0.25 pCi 3H) and enzyme protein in a final volume of 0.5
ml of 0.1 M phosphate buffer, pH 8.0. Duplicate incubations were done in
screw-capped polypropylene tubes for 5 min at 37°C. Protein concentrations
of the enzyme preparations in each incubation ranged from 50-350 pg.
Labeled acetate released by the active enzyme was compared with 3Hradioactivities in water blanks or heat-denatured cytosols or tissue preparations
prepared in sealed screw-capped vials placed for 10 min in a boiling water
bath. The precipitated proteins in the boiled preparations were resuspended
40
Larnbremont, Malone, and Snyder
by gentle homogenization in a Teflon-Pyrex tissue grinder before use in the
assays as blanks.
RESULTS AND DISCUSSION
Presence of an Alklyacetylhydrolase in Insects
Alkylacetyl-GPC:acetylhydrolase activity was found in all insect stages
examined and, except for hemolymph, was easily detectable in two specific
tissues, namely the fat body and flight muscle (Table 1).While typical values
for the insect preparations were approximately 3 to 4 times less than that
observed in rat kidneys, it is clear that an enzyme capable of inactivating PAF
is present. Typical values for the acetylhydrolase in insects ranged between
0.3 to 1.3 nmol of acetate released per min per mg protein, with mean values
of 0.7 for most larval and pupal cytosols. Of three hemolymph preparations
tested, two showed no enzyme activity at all, and the other had a very low
activity, possibly due to the presence of contaminating fat body cells. Assays
of two samples of frozen lobster hemolymph also showed no acetylhydrolase
activity. To our knowledge, these findings constitute the first available information on the presence of any enzyme capable of metabolizing PAF for any
insect or invertebrate animal.
Optimum activity of acetylhydrolase from Heliothis occurred over a broad
pH range (6.0 to S.9), with no sharp peak noted. Increases in activity above
pH 8.0 were less steep than those at the lower pH levels, which indicates
there is a leveling off of activity at higher pH. Activities were linear with
TABLE 1. Alkylacetyl-GPC Acetylhydrolase
Activities in Cystosolic Fractions at Various
Developmental Stages in Tissues of Heliothis
as Compared With the Enzyme Activity in
Rat Kidnev*
Enzyme source
Rat
Kidney (5)
Heliot his
Larva
Third instar (2)
Sixth instar (13)
Pupa
Untanned (2)
Tanned
Fat body
Hemolymph
Adult flight muscle
Acetate liberated
(nmol acetate
min-l protein-*)
2.52
0.93
1.03 f 0.98
0.72 f 0.16
0.57 rt 0.25
0.61 f 0.39
0.93
0.0 f 0.18
0.97
*All preparations were of cytosolic origin except
postmitchrondrial supernatants from the fat body
and hemolymph; see Materials and Methods. The
number of experiments are in parentheses; each
experiment represents duplicate incubations.
Values are means of the incubations f SD.
PAF-Acetylhydrolase in Heliothis
41
protein concentration up to about 370 pg and with incubation time up to 5
min. All subsequent incubations, therefore, were done at pH 8.0 for 5 min
and at protein concentrations equal to or below 350 pg per incubation.
Acetylhydrolase appears to be quite stable during long-term storage. Activity in cytosolic fractions stored at -20°C for more than 2 years was the
same as fresh or freshly frozen preparations.
Characterization of Properties of Acetylhydrolase in Insects
While finding alky1acetyLGPC:acetylhydrolase in Heliothis is of itself both
interesting and significant, it was next necessary to characterize the enzyme's
properties in greater detail. Although activity was detected in a variety of
developmental stages, most of our work was done with the easily obtainable
cytosol from the mature sixth larval instar. A key question was first to
determine whether the enzyme was specific for alkylacetyl-GPC or whether
it was essentially a phospholipase A2 that could utilize both diacyl phospholipids as well as PAF. To determine such specificity, we evaluated divalent
metal ion requirements, chelators, and other factors known to influence the
activity of phospholipase A2 from other animal sources [lo, 121. The results
are summarized in Table 2.
The addition of either Ca2+ or Mg2+ had a slight stimulatory effect.
However EGTA, which chelates Ca2+and which would have depressed the
activity of a typical cellular phospholipase A2, actually showed a slight
acetylhydrostimulation of the l-alkyl-2-acetyl-sn-glycero-3-phosphocholine
lase in the cytosol of Heliothis larvae. Dithiothreitol had no effect on the
enzyme activity, whereas there appeared to be a slight inhibition (82% of the
control) by deoxycholate, which agrees very closely with 80% of control
reported by Blank et al. [ll]for the acetylhydrolase from rat liver. Diisopropylfluorophosphate (1mM), a known inhibitor of the acetylhydrolase from
rat liver [ll], reduced activity in the Heliothis cytosol to about one-fourth of
control values. These combined observations strongly suggest that the properties of the enzyme that hydrolyzes the acetate moiety of PAF are not the
same as a typical cellular phospholipase A2.
TABLE 2. Characteristics of Alkylacetyl-GPC Acetylhydrolase Activity in
the Cvtosolic Fraction of Heliothis (Last Instar Larvae)*
Addition
None
Mg2+ (10 mM)
Ca2+ (10m ~ )
Deoxycholate (0.1 mM)
Dithiothrietol (1mM)
EGTA (2 mM)
DFP (1 mM)
DFP (10 mh4)
Acetate liberated
(nmol acetate
min-l protein-')
0.78
0.91
0.77
0.64
0.76
0.94
0.21
0.20
% of
Control
f 0.18
-
k 0.08
117
99
82
97
120
27
26
f 0.11
f 0.13
0.09
f 0.05
k 0.16
+ 0.11
+
*Values are based on two separate experiments of duplicate incubations
and represent the mean of the four incubations f SD. Each incubation
contained 350 pg protein.
42
Lambremont, Malone, and Snyder
Effects of Other Lipids as Potential Substrates for the Acetylhydrolase
To confirm the specificity of the enzyme activity further, the effect of
egg lecithin (L-alpha-phosphatidylcholine)in the presence of several solvents
and solubilizing agents was used as a possible competitive inhibitor of the
alkylacetyl-GPC:acetylhydrolase in the cytosol of Heliothis larvae (Table 3).
Three of the solubilizing agents (ethanol, acetone, and CHAPS) depressed
the enzyme activity, but no further reduction of activity was noted when
either 2 or 14 mM egg phosphatidylcholine was included. In fact, slight but
probably insignificant stimulation appeared to occur in some instances when
egg lecithin was added to the standard incubations. Propyleneglycol, used
by Snyder and Pfleger [14] to solubilize fatty acids, showed a slight stimulation of acetylhydrolase activity. However, when propyleneglycol was used
in conjunction with 2 or 14 mh4 egg phosphatidylcholine, the enzyme was
unaffected. On the other hand, when a phosphatidylcholine (14 mM) with
an acetyl group in the sn-2 position (l-acyl-2-acetyl-sn-glycero-3-phosphocholine) was incubated under the same conditions, the acetylhydrolase activity
decreased to approximately one-third of control values. This observation,
and the fact that egg phosphatidylcholine did not compete for the enzyme
TABLE 3. Effects of Phosphatidylcholine (PC) and Various Solubilizing
Agents on the Activity of Alkylacetyl-GPC Acetylhydrolase From
Heliothis (Last Larval Instar)*
Addition
Egg phosphatidylcholine
None
Ethanol (10 pl)
+2 mM PC
+14 mM PC
Acetone (10 pl)
+2 mM PC
+14 mM PC
None
CHAPS (0.05%)
+2 mh4 PC
+14 mM PC
Propylenegly col (0.05%)
+2 mM PC
+14 mM PC
1-Palmitoyl-2-acet ylglycerophosphocholine
None
Ethanol (10 pl)
+2 mM acylacetyl GPC
+14 mM acvlacetvl GPC
Acetate liberated
(nmol acetate
m i d protein-*)
0.81
0.43
0.42
0.46
0.46
0.53
0.50
0.48
0.33
0.40
0.34
0.64
0.44
0.46
f 0.20
O h of
Control
f 0.03
f 0.07
f 0.08
k 0.07
i 0.01
f 0.01
f 0.13
100
53
52
57
57
65
62
100
69
83
71
133
92
96
1.18 k 0.20
1.06 + 0.44
1.02 f 0.22
0.73 k 0.20
100
90
86
62
0.01
f 0.11
k 0.20
*Values are based on three separate enzyme preparations; results in each
subsection of the table relate to control readings for each particular
preparation appropriate with additive indicated; each value represents the
mean of four incubations 5 SD when shown. Protein contents of the
cytosolic fractions were 370, 300, and 230 pg, respectively.
PAF-Acetylhydrolase in Heliothis
43
activity, coupled with the failure of EGTA to inhibit and calcium ions to
stimulate activity, strongly imply that a phospholipase A2 is not responsible
for the release of [3H]-acetatefrom the substrate. Thus the acetylhydrolase in
the insect preparations appears to be specific for alkylacetyl GPC (or acylacetyl GPC).
Comparison With the Acetylhydrolase of Higher Animals
Since these observations constitute the first report of an alkylacetylGPC:acetylhydrolase from insects, comparisons can only be drawn from
what is known about the properties of this enzyme obtained from higher
animals. Such a comparison reveals a striking similarity between the acetylhydrolase of Heliothis and that found in vertebrates [11,13,15]. For example, a
broad pH range was reported for the acetylhydrolase from rat plasma and
rat kidney cortex with indistinct optima near 8.0, as is shown here for
Heliothis. EGTA was shown to be slightly stimulatory with the acetylhydrolase from rat tissues [ll], and EGTA similarlly stimulated the acetylhydrolase
in insects to the same extent, as reported here. Ca2+ and Mg2+ ions had
essentially no effect on the enzyme activities in rats, although with insect
preparations we observed a very slight stimulation of the acetylhydrolase
with Mg2+ only. We also noted that inhibition of acetylhydrolase activity by
deoxycholate in the insect system was essentially equal to that reported for
the rat enzyme preparations. Similarly, DFP, which inhibits the acetylhydrolase in rat liver to about 9% of control (1.1mM) [ll],inhibited the acetylhydrolase of Heliothis to 27% of control (1.0 mM).Phosphatidylcholine does not
appear to compete as a substrate with the alkylacetyl-GPC, because the
acetylhydrolase activities for the variety of insect tissues and at different
developmental stages reported here overlap broadly with those reported in
the literature for sera and plasma from rat [ll], reptile [16], or fish [13] and
for cytosolic fractions from liver and kidney of rats [ll], rabbit platelets and
rat alviolar macrophages [15], and human neutrophils and eosinophils [17,18].
Our results with insects differed from those reported for acetylhydrolase
in mammals only in three ways, namely, the slight Mg2+ ion stimulation,
the lack of any stimulatory effect by dithiothreitol, and the slight inhibitory
effect of ethanol. These differences are very minor and probably not of any
significance. We must conclude that Heliothis has a specific alkylacetyl-GPCacetylhydrolase (EC 3.1.1.48) in a variety of its cellular systems similar to the
enzyme found in higher animal sources [19].
Finding a specific alkylacetyl-GPC:acetylhydrolase in Heliothis therefore
suggests that insects might contain a compound similar or identical to the
potent alkylacetyl glycerophosphocholine (or PAF) mediator found in higher
animals [19]. What role such a biologically active ether-linked lipid might
have in insects if found is, of course, unclear. Because "platelet activating
factor" is known to possess a diverse number of potent biologic activities in
higher animals [3] (e.g., the induction of platelet and neutrophil aggregation,
degranulation reactions, hypotension, hypersensitivity, and the cellular release of other bioactive agents), it is conceivable that PAF has an even more
widespread role as a cellular mediator throughout the animal kingdom.
44
Lambremont, Malone, and Snyder
While the presence of the acetylhydrolase in Heliothis merely infers the
presence of PAF in this species, it is known that this and other insect species
contain ether-linked phosphoglycerides and that they possess metabolic
pathways necessary to provide at least the ether-lipid intermediates required
for PAF synthesis. For example, Heliothis has been shown to contain an
endogenous pool of alkylacetyl-GPC in all of its postembryonic developmental stages [6,7l. Heliothis is also capable of incorporating various radiolabeled precursors, such as acetate, glycerol, long-chain fatty acids, and longchain fatty alcohols into the alkylacetyl-GPC pool 18/91;in addition, it is able
to synthesize from fatty acids the fatty alcohol precursor needed to form the
ether bond at the sn-1 position of glycerolipids [lo].
Heliothis and other insect species could be interesting experimental models
to investigate the presence and synthesis of PAF compounds in nonvertebrate animals and to determine if they have physiologic or pharmacologic
roles in these species. Even if such acetylated phospholipids do not occur,
the significance of a specific alkylacetyl-GPC acetylhydrolase as described by
us raises intriguing questions about the possible role of this enzyme in
Heliothis. Obviously the phylogenic significance of ether-linked lipids and
their metabolism in insects warrant further study.
LITERATURE CITED
1. Snyder F (editor): Ether Lipids: Chemistry and Biology. Academic Press, New York, 433
PP (1972).
2. Snyder F, Lee T-c, Wykle RL: Ether-linked glycerolipids and their bioactive species:
Enzymes and metabolic regulation. In: The Enzymes of Biological Membranes. Martonosi,
AN, ed. Plenum Publishing Co., New York, Vol. 2, pp 1-58 (1985).
3. Blank ML, Snyder F, Byers LW, Brooks B, Muirhead EE: Antihypertensive activity of an
alkyl ether analog of phosphatidylcholine. Biochem Biophys Res Commun 90, 1194 (1974).
4. Benveniste J, Henson PM, Cochrane CG: Leucocyte-dependent histamine release from
rabbit platelets. The role of IgE, basophils, and platelet-activating factor. J Exp Med, 136,
1356 (1972).
5. Demopoullos CA, Pinckard RN, Hanahan DJ: Platelet-activating factor: Evidence for 1-0alkyl-2-acetyl-sn-glycero-3-phosphocholine
as the active component (a new class of lipid
chemical mediators). J Biol Chem, 254, 9355 (1979).
6. Lambremont EN: Ether-bonded lipids of insects: A quantitative comparison of the glyceryl
ethers associated with ethanolamine and choline phosphoglycerides. Comp Biochem
Physiol, 41B, 337 (1972).
7. Lambremont EN, Wood R: Glyceryl ethers in insects: Identification of alkyl and alk-1-enyl
glyceryl ether phospholipids. Lipids 3, 503 (1968).
8. Lambremont EN: The in vivo synthesis of acyl- and ether-bonded phospholipids in Heliothis virescens. J Insect Physiol, 18, 581 (1972).
9. Lambremont EN: Glyceryl ethers in insects: Biosynthesis of ethanolamine phosphoglycerides containing alkyl and alk-1-enyl glyceryl ether linkages. Lipids 7, 528 (1972).
10. Lambremont EN: Lipid metabolism of insects: Interconversion of fatty acids and fatty
alcohols. Insect Biochem, 2, 197 (1972).
11. Blank ML, Lee T-c, Fitzgerald V, Snyder F: A specific acetylhydrolase for 1-alkyl-2-acetylsn-glycero-3-phosphocholine(a hypotensive platelet activating lipid). J Biol Chem, 256,
175 (1981).
12. Lowry OH, Rosebrough NJ, Farr AL, Randall RJ: Protein measurement with the Folin
phenol reagent. J Biol Chem, 293, 265 (1951).
13. Cabot MC, Faulkner LA, Lackey RJ, Snyder F: Vertebrate class distribution of l-alkyl-2acetyl-sn-glycero-3-phosphocholineacetylhydrolase in serum. Comp Biochem Physiol,
78B, 37 (1984).
PAF-Acetylhydrolasein Heliothis
45
14. Snyder F, Pfleger RC: Metabolism of alpha-alkoxy glyceryl monoethers in rat liver, in vivo
and in vitro. Lipids I, 328 (1966).
15. Snyder F, Lee T-c, Blank ML, Cabot MC, Malone B, Albert DH: Enzymatic pathways for
platelet activating factor. In: Platelet-Activating Factor and Structurally Related Ether
Lipids. Benveniste J, Arnoux B, eds. Amsterdam, The Netherlands, Elsevier Scientific
Publs. 253 pp (1983).
16. Lenihan DL, Greenberg D, Lee T-c: Involvement of platelet activating factor in physiological stress in the lizard, AnoZis curolinesis. Comp Biochem Physiol, 81C, 81 (1986).
17. Lee T-c, Malone B, Wasserman SI, Fitzgerald V, Snyder F: Activities of enzymes that
metabolize platelet-activating factor (l-alkyl-2-acetyl-sn-glycero-3-phosphocholine)
in neutrophils and eosinophils from human and the effect of a calcium ionophore. Biochem
Biophys Res Commun, 105, 1303 (1982).
18. Lee T-c, Snyder F: Function, metabolism and regulation of platelet activating factor and
related ether lipids. In: Phospholipids and Cellular Regulation. Kuo FJ, ed. CRC Press,
Inc., Boca Raton, FL, Vol. 2, pp 1-39 (1985).
Platelet activating factor: A biologically active phosphoglyceride. Annu Rev
19. Hanahan DJ:
Biochem 55, 583 (1986).
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