BIOCHEMICAL
Vol. 168, No. 2, 1990 April 30, 1990
PHOSPHOLIPID
SYNTHESIS
PHOSPHOLIPASE
de Biochimie
*Laboratoire
Received
January
OLIVIER,
CHU St-Antoine,
de Technologie
BY EXTRACELLULAR
A2 IN ORGANIC
P. PERNAS, J.L. Laboratoire
AND BIOPHYSICAL RESEARCH COMMUNICATIONS Pages 644-650
M.D.
SOLVENTS
LEGOY* AND G. BEREZIAT
URA CNFt+S 217,27 rue Chaligny
Enzymatique, Universite de Technologie 60200, COMPIEGNE, FRANCE
75012, PARIS, FRANCE
de Compiegne,
BP 649
5, 1990
The catalytic activity of extracellular phospholipase A2 was studied in low polarity solvents where hydrolytic enzymes have been demonstrated to catalyze synthesis reactions. It was demonstrated that extracellular phospholipase A2 can catalyze the esterification of lysophosphatidylcholine with oleic acid. Up to 6.5% of lysophosphatidylcholine can be esterified into phosphatidylcholine. This activity requires a preincubation of the enzyme in a pH 9 aqueous solution containing calcium, before the incubation in the non-aqueous solvent. No transfer of fatty acid between a phospholipid and a lysophospholipid or between two phospholipids was observed. These results may be useful in understanding the function of the membrane phospholipase A2 which may catalyze acylation or deacylation depending on the local physico-chemical @ 1990 Academic Press, hc. environment.
Phospholipase
A2 (EC 3.1 J.41 is a hydrolytic
and bee venoms, pancreatic
enzyme present in various secretions (snake
juice), in cell membranes
enzymes catalyze the hydrolysis
of the 2-acyl ester
(l), and in fungi
and yeasts (2). These
bond of sn3-phosphoglycerides
forming
fatty
acids and lysophospholipids. The structures related (3). In particular,
of all the PLA2 known the structures
until now have been demonstrated
to be closely
of the active sites of both soluble and known membrane-
bound PLA2 are very similar (4,5). Many workers thermodynamically
have shown that hydrolytic
favourable
conditions:
enzymes can catalyze synthesis
reactions
that is, in the absence of water (6 - 9).
In this work, we show that soluble PLA2 can catalyze the synthesis of phospholipids
in non polar
solvents.
ABBREVIATIONS: PLA2: phospholipase ethanolamine; LPC: lysophosphatidylcholine; platelet-activating factor. COO6-291X&Q $1.50 Copyright 0 1990 by Academic Press, Inc. All rights of reproduction in any form reserved.
in
A2; PC: phosphatidylcholine; PE: LPE: lysophosphatidylethanolamine;
644
phosphatidylPAP-acether:
Vol.
BIOCHEMICAL
168, No. 2, 1990
AND BIOPHYSICAL
EXPERIMENTAL
RESEARCH COMMUNICATIONS
PROCEDURES
Enzvme preincubation Extracellular PLA2 from various origins (Nqju lraja venom, bee venom, porcine pancreas, bovine pancreas, Streptomyces uioluceoruber) were furnished by Sigma (St Louis, MO). For a typical experiment, 4.5 U of PLA2 was dissolved in 150 pl buffer, and the solution was lyophilized for 2 hours. Where not specified Nqju no&z venom PLA2 was used. Substrate quantities and buffers used are written in “results”. Reaction procedure LPC, LPE and egg yolk PC were purchased from Sigma and oleic acid from Aldrich (France). Tritium-labelled oleic acid (specific activity = 5 Wmmol) and 1-palmitoyl2[I4Cl-palmitoyl PC (specific activity = 55 mCi/mmol) were furnished by Amersham International (UK). The solvents used were of the highest purity available and were dessicated on 3 A molecular sieves. No water was detectable in these solvents by the Laitinen and Harris modification of Fischer’s method (10). LPC and oleic acid were dispersed in the organic solvent and 150~1 of this suspension were added to the lyophilized PLA2. The reaction was performed in a 2 ml screw-cap glass vial, with vigorous rotatory stirring, at 37’C in a dry incubator. The mixtures obtained were slightly turbid at room temperature but became clear at 37-C under agitation. Thus the enzyme was suspended in the organic solvent which contained dissolved substrates at 37-C. Periodically, 3 pl aliquots of the reaction mixture were withdrawn and assayed. The reaction products were separated on a silica gel thin layer plate (Schleicher and Schuell, FRG) with a developing system of chIoroformfmethanol/water (65:25:4, V/V/V). Fractions were visualized by iodine vapor and assayed for radioactivity with an Intertechnique SL 3000 liquid scintillation counter. m activitv determination in aqueous medium Fluorescent substrate (l-palmitoyl 2-(lo-pyrenyldecanoyl)~sn glycero 3-monomethyl phosphatidic acid) was furnished by RSV Chemicals and delipidated BSA by Sigma. The hydrolytic activity of the PLA2 was determinated with a Jobin-Yvon JY3 spectrofluorimeter according to Radvanyi et al. (11).
RESULTS In order to verify the stability with no substrate.
At various
of the enzyme, PLA2 was incubated
times, the solvent was evaporated
at 37°C in solvents
and the residue
dissolved in
10 mM Tris / HCl pH 9, 10 mM CaC12 buffer. The PLA2 activity was then measured. activity could be observed after a 24 h incubation (80:20, VNl or toluene/methanol(80:20,
in benzene,
chloroform,
No loss of
chloroform/methanol
VN) or even after a one week incubation
in toluene.
Up to 6.5% of the LPC was acylated to form PC in the first 24 h (fig. 1) in the presence of PLA2 with toluene or benzene as solvent. No synthesis was observed in the absence of PLA2. The initial
velocity
of the
reaction,
measured
1 nmol h-I Q.tg pro++1. The ratio between
during
the synthetic
activity in water was 1.5X10T4, since the hydrolytic this study was measured as 0.11 pmol min-l Increasing chloroform
resulted
the polarity
5 hours,
activity in toluene
was
approximately
and the hydrolytic
activity of Naju naju venom PLA2 used in
(pg prot)-l.
of the solvent
in a suppression
the first
by adding
methanol
to toluene
or by using
of the PC synthesis as described in other studies (12) for
other synthesis reactions catalyzed in organic solvents. 645
Vol.
168,
No.
2, 1990
BIOCHEMICAL
AND
BIOPHYSICAL
36
40
RESEARCH
0 0
12
24
TIME
COMMUNICATIONS
0 60
72
(hours)
FIGURE 1: Formation of 3H-PC ( 0 venom PIA2. Formation to 10 mM Tris / HCl pH was composed of 70 mM 150 pl of toluene. Values
Use PI&
from
and porcine pancreatic
of bovine Nuju
1 from LPC and 13Hl-oleic acid in the presence of No& najn of [3Hl-PC ( 0 ),in the absence of PLA2. The enzyme was exposed 9, 10 mM CaC12 buffer before lyophilization. The reaction mixture LPC, 700 mM oleic acid, 40 mCi 13Hl-oleic acid, 4.5 U of PIA in are the means of duplicates from a typical experiment.
naju
venom
(table
1). The
PLA2 resulted porcine
enzyme
in a lower yield of PC than with was
less
efficient
in
catalyzing
synthesis than the bovine enzyme, and the activity of bee venom and Streptomyces
PC
violaceoruber
PLA2 in toluene and benzene was minimal. Increasing
the
enhanced the initial possible Higher
concentration
the LPC concentration
(fig. 2B). When the reaction was performed and at an oleic acid&PC synthetic
concentration
and
TABLE
substrates
up
to
140 mM
above 140 mM because of solubility
ratios enhanced both the final yield and the initial
acid&PC
The
of both
in
toluene
velocity of the acylation reaction and the final yield of PC (fig. 2A). It was not
to increase oleie
equimolar
pH
limitations.
velocity of the reaction
in dessicated oleic acid, i.e. in the absence of toluene
ratio of 40, no PC synthesis was observed (fig. 2B). activity of the
of PLA2 preincubation
1: Comparison
of various
displayed and
a strong
lyophilization
dependence buffer.
Initial
sources of PLA2 for the formation PLAz
on the velocity
calcium and
of 13H&PC
source
incubation time
Nqia
nqjb
venom
bovine
porcine
bee
pancreas
pancreas
venom
Streptomyces violaceoruber
3h
1.8 %
0.80 96
0.45 %
0.40 %
0.50 95
24 h
6.5 96
3.3 %
1.3 96
0.60 %
0.70 46
Data are reaction yields; they represent Reaction conditions are as in fig. 1.
the proportion
646
of LPC transformed
into PC.
final
Vol.
BIOCHEMICAL
168, No. 2, 1990
:f
/
20
40
t=24h
60
80 100 concentrations
Substrate
A
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
1
120 140 (mM)
P Oleic
acid/LPC
molor
3
4
5
6
7
8
: 9
: 10
: 11
: 12
PH
ratio
(LPC) - 70 mM
FIGURE 2: A: Effect of substrate concentration on PC synthesis for an oleic acid/LFC molar ratio = 1. B: Effect of oleic acid&PC Other reaction conditions
molar ratio on PC synthesis are as in fig. 1.
for a LPC concentration
of 70 mM.
FIGURE 3: A: Effect of the calcium concentration to which the enzyme was exposed before lyophilization on the PC synthesis. The buffers were 10 mM Tris! HCl pH 9 with 50 mM EDTA or various concentrations of CaCl2 (1 to 100 mM). Other reaction conditions are as in fig. 1. B: Effect of the pH to which the enzyme was exposed before lyophilization on the PC synthesis. The buffers were 10 mM Tris / HCl pH 6 to 9 with 10 mM Ca or 10 mM acetic acid I sodium acetate pH 4 and 5, with 10 mM Ca. Other reaction conditions are as in fig. 1.
yield were maximal in 5mM Caz+ (fig. 3A). PC synthesis was inhibited presence
of EDTA
differently
in the lyophilization
by the pH of the preincubation
pH of the lyophilization dependent
velocity
buffer was between pH 9.0 and 11.0, but the maximal
set of experiments,
[2-I4C]-dipalmitoyl-PC) position
of PC was
PC was
degraded
8 hours
and final yield
PLA2 was added to mixtures
No interesterification
slowly, producing
or transesterification
free palmitic
of incubation.
647
conversion was not
pH 12.0 (fig. 3B). of PC / PE or PC / LPE (1 pCi
in toluene and the fate of the fatty acid initially studied.
were affected
buffer. The rate of PC synthesis was maximal when the
on the pH in the range tested except at the denaturing
In another
after
buffer. Initial
at 100 mM Ca2+ and in the
was
incorporated observed.
acid (data not shown). Equilibrium
in sn2 However
was reached
Vol.
BIOCHEMICAL
168, No. 2, 1990
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
DISCUSSION The present work provides evidence for PC formation
as a result of PL&
catalysis in
organic solvent. First, no PC synthesis was observed in the absence of PLA9 and the initial synthesis
increased
calcium
with
increasing
and pH dependent,
hydrolysis.
Furthermore,
no transacylase 14)(result
substrate
with
the same optimal
after inhibition
Second, the PC synthesis
calcium
concentration
of Naja nuju PLA9 with an anti-PL&
activity in aqueous medium
not shown). Therefore,
concentration.
was observed (as measured
it is unlikely
rate of was
and pH as for antiserum
by Colard’s
that the observed PC synthesis
(131,
method,
is due to a
contaminant.
The present work has implications Water
is absolutely
required
for the role of water in PLA9 activity.
for catalysis
and for maintainance
of the structure
of
enzymes. In the media used in the present study, water was not detectable, but was likely to be present in trace amounts. Since, in toluene, hydrolysis occurred
when
PLA9
was added to phospholipid
necessary to the reaction is enzyme-associated, polarity
medium,
this water
remains
might suppress the enzymatic Zaks and Klibanov
rather than trans- or inter-esterification
mixtures,
we can assume that
which is not eliminated
of enzyme-associated
activity in organic solvent was dependent
of preincubation
phenomenon
active conformation.
is thermodynamically
as “pH-memory”
by increasing
Renaturation
time is sufficient.
aqueous media (171, which renaturation
may explain
by Zaks and
This previously
of the enzyme from an inactive
activity is immediately
of the enzyme
to pH 4.0.
to acid may occur more slowly in organic
that Zaks and Klibanov
for the enzymes they studied. 648
to an
possible but the kinetics may differ in
the pH to 9.0 after a 15 min exposure
of the enzyme after exposure
to which the
show that the catalysis of synthesis is
pH, if the incubation
is possibly due to a transition
This transition
that initial
on the pH of the last aqueous solution
organic solvents and in water. In fact, in an aqueous solution, hydrolytic restored
solvents
as proposed by
water is consistent with the observation
(15,16) for lipase and proteases. Our experiments
undescribed
polar
the linked water molecules,
enzyme was exposed. Such an effect has also been described
possible regardless
In a low
(15).
The importance
Klibanov
by lyophilization.
associated with the enzyme. In contrast,
activity by dissolving
the water
than in
(15, 161, did not observe any
Vol.
BIOCHEMICAL
168, No. 2, 1990 Calcium,
which
microenvironment conformational
is
insoluble
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
in
apolar
solvents,
of the enzyme when in toluene. Moderate
might
concentrations
change in PLA2 in water (18) which may be maintained
hand, high concentrations of the substrates
accumulate
in
the
of calcium induce a
in toluene. On the other
of calcium may create an ionic shield around PLA2 and impede access
to the enzyme.
PLA2 from all the sources we have tested synthesize PC, but we observed differences their catalytic behavior. The differences between porcine and bovine pancreatic due to preparation
procedures,
but they may be due to more fundamental
enzymes. For example, Dijkstra hydrolytic
properties,
et al. (19) demonstrated
in
PLA2 are possibly
differences between the
that these two enzymes exhibit different
due to the presence of two phospholipid
substrate-binding
sites on porcine
PLA2.
In light of the present study, as membranes PLA2 in cell membranes membranes
implies
cannot be ruled out. Tbe very low concentration
a tight
involved in the membrane least three pathways neither
coupling phospholipid
(20,21,22),
between
transacylase
remodeling.
and PLA2
Tbe transacylation
role for
of lysophospholipids activities,
which
in are
can be achieved by at
one of which being ATP and CoA independent
(22). Importantly,
coenzyme A nor ATP are necessary to the synthesis of PC in organic solvents.
Finally, in the present study, esteritication of PLA2
are non aqueous media, a synthetase
to synthesize
Such applications
various phospholipids
of up to 6.5% LPC has been observed. The use
of biological
require a study of the stereospecificity
interest
can therefore
be suggested.
of tbe synthesis.
REFERENCES 1 2 3 4
- Van den Bosch, H. (1980) Biochim. Biophys. Acta 604,191-246 - Okumura, T., Sugatani, J., Saito, K (1981) Arch. Biochem. Biophys. 211,419-429 - Dufton, M. J., and Hider R. C. (1983) Eur. J. Biochem. 137,545-551 - Seilhamer, J., Pruzanski, W., Vada, P., Plant, S., Miller, J. A., Kloss, J., and Johnson L. K (1989) J. Biol. Chem. 264,5335-5338 5 - Kramer, R., Hession, C., Johansen, B., Hayes, G., MC Gray, P., Chow, E. P., Tizard, R., and Pepinsky R. B. (1989) J. Biol. Chem. 264.5768-5775 6 - Dastoli, F. R., and Musto, N.A, and Price, S. (1967) Arch. Biochem. Biophys. 118,163-165 7 - Cremonesi, P., Carrea, G., Sportoletti, G., and Antonini, E. (1973) Arch. Biochem. Biophys. 159,7 8 - Martinek, K, Levashov, A V., Khmelnitsky, Y. L., Klyachko, N. L., and Berezin, I. V. (1982) Science 218,889-891 9 - Bello, M., Thomas, D., Legoy, M. D. (1987) Biochem. Biophys. Res. Commun. 146,361-367 649
Vol.
168,
No.
2, 1990
BIOCHEMICAL
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
10 - Laitinen, H.A., and Harris, W.E., Chemical analysis, 2nd Ed., pp 361-363, McGraw-Hill, New-York 11 - Radvanyi, F., Saliou, B., Lemhezat, M. P., and Bon, C. (1989) Anal. Biochem. 177,103-109 12 - Laane, C., Boeren, S., Vos, K, Veeger, C. (1987) Biotechnol. Bioeng. 30,80-87 13 - Masliah, J., Kadiri, C., Pepin, D., Rybkine, T., Etienne, J., Chambaz, J. and Bereziat, G. (1987) FEBS Lett. 222,11-16 14 - Colard, O., Breton, M., Bereziat, G. (1984) Biochim. Biophys. Acta 793,42-48 15 - Zaks, A, and Klibanov, A. M. (1988) J. Biol. Chem. 263,3194-3201 16 - Zaks, A., and Klibanov, A. M. (1965) Proc. Natl. Acad. Sci. USA82,3192-3196 17 - Poole, P. L. and Finney, J. L. (1983) Int. J. Biol. Macromol. 5,308-310 18 - Pieterson, W. A., Volwerk, J. J., and De Haas, G. H. (1974) Biochemistry 13,1439-1445 19 - Dijkstra, B.W., Renetseder, R., Kalk, KH., HOI, W.G.J., and Drenth, J. (1983) J. Mol. Biol. 168,163-179 20 - Lands, W.E.M., Merkl, I. (1963) J. Biol. Chem. 238,898-904 21 - Irvine, R. F.,and Dawson, R. M. C. (1979) Biochem. Biophys. Res. Commun. 91,1399-1405 22 - Kramer, R. M., and Deykin, D. J. (1983) Biol. Chem. 258,13806-13811
650
Vol. 168, No. 2, 1990 April 30, 1990
PHOSPHOLIPID
SYNTHESIS
PHOSPHOLIPASE
de Biochimie
*Laboratoire
Received
January
OLIVIER,
CHU St-Antoine,
de Technologie
BY EXTRACELLULAR
A2 IN ORGANIC
P. PERNAS, J.L. Laboratoire
AND BIOPHYSICAL RESEARCH COMMUNICATIONS Pages 644-650
M.D.
SOLVENTS
LEGOY* AND G. BEREZIAT
URA CNFt+S 217,27 rue Chaligny
Enzymatique, Universite de Technologie 60200, COMPIEGNE, FRANCE
75012, PARIS, FRANCE
de Compiegne,
BP 649
5, 1990
The catalytic activity of extracellular phospholipase A2 was studied in low polarity solvents where hydrolytic enzymes have been demonstrated to catalyze synthesis reactions. It was demonstrated that extracellular phospholipase A2 can catalyze the esterification of lysophosphatidylcholine with oleic acid. Up to 6.5% of lysophosphatidylcholine can be esterified into phosphatidylcholine. This activity requires a preincubation of the enzyme in a pH 9 aqueous solution containing calcium, before the incubation in the non-aqueous solvent. No transfer of fatty acid between a phospholipid and a lysophospholipid or between two phospholipids was observed. These results may be useful in understanding the function of the membrane phospholipase A2 which may catalyze acylation or deacylation depending on the local physico-chemical @ 1990 Academic Press, hc. environment.
Phospholipase
A2 (EC 3.1 J.41 is a hydrolytic
and bee venoms, pancreatic
enzyme present in various secretions (snake
juice), in cell membranes
enzymes catalyze the hydrolysis
of the 2-acyl ester
(l), and in fungi
and yeasts (2). These
bond of sn3-phosphoglycerides
forming
fatty
acids and lysophospholipids. The structures related (3). In particular,
of all the PLA2 known the structures
until now have been demonstrated
to be closely
of the active sites of both soluble and known membrane-
bound PLA2 are very similar (4,5). Many workers thermodynamically
have shown that hydrolytic
favourable
conditions:
enzymes can catalyze synthesis
reactions
that is, in the absence of water (6 - 9).
In this work, we show that soluble PLA2 can catalyze the synthesis of phospholipids
in non polar
solvents.
ABBREVIATIONS: PLA2: phospholipase ethanolamine; LPC: lysophosphatidylcholine; platelet-activating factor. COO6-291X&Q $1.50 Copyright 0 1990 by Academic Press, Inc. All rights of reproduction in any form reserved.
in
A2; PC: phosphatidylcholine; PE: LPE: lysophosphatidylethanolamine;
644
phosphatidylPAP-acether:
Vol.
BIOCHEMICAL
168, No. 2, 1990
AND BIOPHYSICAL
EXPERIMENTAL
RESEARCH COMMUNICATIONS
PROCEDURES
Enzvme preincubation Extracellular PLA2 from various origins (Nqju lraja venom, bee venom, porcine pancreas, bovine pancreas, Streptomyces uioluceoruber) were furnished by Sigma (St Louis, MO). For a typical experiment, 4.5 U of PLA2 was dissolved in 150 pl buffer, and the solution was lyophilized for 2 hours. Where not specified Nqju no&z venom PLA2 was used. Substrate quantities and buffers used are written in “results”. Reaction procedure LPC, LPE and egg yolk PC were purchased from Sigma and oleic acid from Aldrich (France). Tritium-labelled oleic acid (specific activity = 5 Wmmol) and 1-palmitoyl2[I4Cl-palmitoyl PC (specific activity = 55 mCi/mmol) were furnished by Amersham International (UK). The solvents used were of the highest purity available and were dessicated on 3 A molecular sieves. No water was detectable in these solvents by the Laitinen and Harris modification of Fischer’s method (10). LPC and oleic acid were dispersed in the organic solvent and 150~1 of this suspension were added to the lyophilized PLA2. The reaction was performed in a 2 ml screw-cap glass vial, with vigorous rotatory stirring, at 37’C in a dry incubator. The mixtures obtained were slightly turbid at room temperature but became clear at 37-C under agitation. Thus the enzyme was suspended in the organic solvent which contained dissolved substrates at 37-C. Periodically, 3 pl aliquots of the reaction mixture were withdrawn and assayed. The reaction products were separated on a silica gel thin layer plate (Schleicher and Schuell, FRG) with a developing system of chIoroformfmethanol/water (65:25:4, V/V/V). Fractions were visualized by iodine vapor and assayed for radioactivity with an Intertechnique SL 3000 liquid scintillation counter. m activitv determination in aqueous medium Fluorescent substrate (l-palmitoyl 2-(lo-pyrenyldecanoyl)~sn glycero 3-monomethyl phosphatidic acid) was furnished by RSV Chemicals and delipidated BSA by Sigma. The hydrolytic activity of the PLA2 was determinated with a Jobin-Yvon JY3 spectrofluorimeter according to Radvanyi et al. (11).
RESULTS In order to verify the stability with no substrate.
At various
of the enzyme, PLA2 was incubated
times, the solvent was evaporated
at 37°C in solvents
and the residue
dissolved in
10 mM Tris / HCl pH 9, 10 mM CaC12 buffer. The PLA2 activity was then measured. activity could be observed after a 24 h incubation (80:20, VNl or toluene/methanol(80:20,
in benzene,
chloroform,
No loss of
chloroform/methanol
VN) or even after a one week incubation
in toluene.
Up to 6.5% of the LPC was acylated to form PC in the first 24 h (fig. 1) in the presence of PLA2 with toluene or benzene as solvent. No synthesis was observed in the absence of PLA2. The initial
velocity
of the
reaction,
measured
1 nmol h-I Q.tg pro++1. The ratio between
during
the synthetic
activity in water was 1.5X10T4, since the hydrolytic this study was measured as 0.11 pmol min-l Increasing chloroform
resulted
the polarity
5 hours,
activity in toluene
was
approximately
and the hydrolytic
activity of Naju naju venom PLA2 used in
(pg prot)-l.
of the solvent
in a suppression
the first
by adding
methanol
to toluene
or by using
of the PC synthesis as described in other studies (12) for
other synthesis reactions catalyzed in organic solvents. 645
Vol.
168,
No.
2, 1990
BIOCHEMICAL
AND
BIOPHYSICAL
36
40
RESEARCH
0 0
12
24
TIME
COMMUNICATIONS
0 60
72
(hours)
FIGURE 1: Formation of 3H-PC ( 0 venom PIA2. Formation to 10 mM Tris / HCl pH was composed of 70 mM 150 pl of toluene. Values
Use PI&
from
and porcine pancreatic
of bovine Nuju
1 from LPC and 13Hl-oleic acid in the presence of No& najn of [3Hl-PC ( 0 ),in the absence of PLA2. The enzyme was exposed 9, 10 mM CaC12 buffer before lyophilization. The reaction mixture LPC, 700 mM oleic acid, 40 mCi 13Hl-oleic acid, 4.5 U of PIA in are the means of duplicates from a typical experiment.
naju
venom
(table
1). The
PLA2 resulted porcine
enzyme
in a lower yield of PC than with was
less
efficient
in
catalyzing
synthesis than the bovine enzyme, and the activity of bee venom and Streptomyces
PC
violaceoruber
PLA2 in toluene and benzene was minimal. Increasing
the
enhanced the initial possible Higher
concentration
the LPC concentration
(fig. 2B). When the reaction was performed and at an oleic acid&PC synthetic
concentration
and
TABLE
substrates
up
to
140 mM
above 140 mM because of solubility
ratios enhanced both the final yield and the initial
acid&PC
The
of both
in
toluene
velocity of the acylation reaction and the final yield of PC (fig. 2A). It was not
to increase oleie
equimolar
pH
limitations.
velocity of the reaction
in dessicated oleic acid, i.e. in the absence of toluene
ratio of 40, no PC synthesis was observed (fig. 2B). activity of the
of PLA2 preincubation
1: Comparison
of various
displayed and
a strong
lyophilization
dependence buffer.
Initial
sources of PLA2 for the formation PLAz
on the velocity
calcium and
of 13H&PC
source
incubation time
Nqia
nqjb
venom
bovine
porcine
bee
pancreas
pancreas
venom
Streptomyces violaceoruber
3h
1.8 %
0.80 96
0.45 %
0.40 %
0.50 95
24 h
6.5 96
3.3 %
1.3 96
0.60 %
0.70 46
Data are reaction yields; they represent Reaction conditions are as in fig. 1.
the proportion
646
of LPC transformed
into PC.
final
Vol.
BIOCHEMICAL
168, No. 2, 1990
:f
/
20
40
t=24h
60
80 100 concentrations
Substrate
A
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
1
120 140 (mM)
P Oleic
acid/LPC
molor
3
4
5
6
7
8
: 9
: 10
: 11
: 12
PH
ratio
(LPC) - 70 mM
FIGURE 2: A: Effect of substrate concentration on PC synthesis for an oleic acid/LFC molar ratio = 1. B: Effect of oleic acid&PC Other reaction conditions
molar ratio on PC synthesis are as in fig. 1.
for a LPC concentration
of 70 mM.
FIGURE 3: A: Effect of the calcium concentration to which the enzyme was exposed before lyophilization on the PC synthesis. The buffers were 10 mM Tris! HCl pH 9 with 50 mM EDTA or various concentrations of CaCl2 (1 to 100 mM). Other reaction conditions are as in fig. 1. B: Effect of the pH to which the enzyme was exposed before lyophilization on the PC synthesis. The buffers were 10 mM Tris / HCl pH 6 to 9 with 10 mM Ca or 10 mM acetic acid I sodium acetate pH 4 and 5, with 10 mM Ca. Other reaction conditions are as in fig. 1.
yield were maximal in 5mM Caz+ (fig. 3A). PC synthesis was inhibited presence
of EDTA
differently
in the lyophilization
by the pH of the preincubation
pH of the lyophilization dependent
velocity
buffer was between pH 9.0 and 11.0, but the maximal
set of experiments,
[2-I4C]-dipalmitoyl-PC) position
of PC was
PC was
degraded
8 hours
and final yield
PLA2 was added to mixtures
No interesterification
slowly, producing
or transesterification
free palmitic
of incubation.
647
conversion was not
pH 12.0 (fig. 3B). of PC / PE or PC / LPE (1 pCi
in toluene and the fate of the fatty acid initially studied.
were affected
buffer. The rate of PC synthesis was maximal when the
on the pH in the range tested except at the denaturing
In another
after
buffer. Initial
at 100 mM Ca2+ and in the
was
incorporated observed.
acid (data not shown). Equilibrium
in sn2 However
was reached
Vol.
BIOCHEMICAL
168, No. 2, 1990
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
DISCUSSION The present work provides evidence for PC formation
as a result of PL&
catalysis in
organic solvent. First, no PC synthesis was observed in the absence of PLA9 and the initial synthesis
increased
calcium
with
increasing
and pH dependent,
hydrolysis.
Furthermore,
no transacylase 14)(result
substrate
with
the same optimal
after inhibition
Second, the PC synthesis
calcium
concentration
of Naja nuju PLA9 with an anti-PL&
activity in aqueous medium
not shown). Therefore,
concentration.
was observed (as measured
it is unlikely
rate of was
and pH as for antiserum
by Colard’s
that the observed PC synthesis
(131,
method,
is due to a
contaminant.
The present work has implications Water
is absolutely
required
for the role of water in PLA9 activity.
for catalysis
and for maintainance
of the structure
of
enzymes. In the media used in the present study, water was not detectable, but was likely to be present in trace amounts. Since, in toluene, hydrolysis occurred
when
PLA9
was added to phospholipid
necessary to the reaction is enzyme-associated, polarity
medium,
this water
remains
might suppress the enzymatic Zaks and Klibanov
rather than trans- or inter-esterification
mixtures,
we can assume that
which is not eliminated
of enzyme-associated
activity in organic solvent was dependent
of preincubation
phenomenon
active conformation.
is thermodynamically
as “pH-memory”
by increasing
Renaturation
time is sufficient.
aqueous media (171, which renaturation
may explain
by Zaks and
This previously
of the enzyme from an inactive
activity is immediately
of the enzyme
to pH 4.0.
to acid may occur more slowly in organic
that Zaks and Klibanov
for the enzymes they studied. 648
to an
possible but the kinetics may differ in
the pH to 9.0 after a 15 min exposure
of the enzyme after exposure
to which the
show that the catalysis of synthesis is
pH, if the incubation
is possibly due to a transition
This transition
that initial
on the pH of the last aqueous solution
organic solvents and in water. In fact, in an aqueous solution, hydrolytic restored
solvents
as proposed by
water is consistent with the observation
(15,16) for lipase and proteases. Our experiments
undescribed
polar
the linked water molecules,
enzyme was exposed. Such an effect has also been described
possible regardless
In a low
(15).
The importance
Klibanov
by lyophilization.
associated with the enzyme. In contrast,
activity by dissolving
the water
than in
(15, 161, did not observe any
Vol.
BIOCHEMICAL
168, No. 2, 1990 Calcium,
which
microenvironment conformational
is
insoluble
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
in
apolar
solvents,
of the enzyme when in toluene. Moderate
might
concentrations
change in PLA2 in water (18) which may be maintained
hand, high concentrations of the substrates
accumulate
in
the
of calcium induce a
in toluene. On the other
of calcium may create an ionic shield around PLA2 and impede access
to the enzyme.
PLA2 from all the sources we have tested synthesize PC, but we observed differences their catalytic behavior. The differences between porcine and bovine pancreatic due to preparation
procedures,
but they may be due to more fundamental
enzymes. For example, Dijkstra hydrolytic
properties,
et al. (19) demonstrated
in
PLA2 are possibly
differences between the
that these two enzymes exhibit different
due to the presence of two phospholipid
substrate-binding
sites on porcine
PLA2.
In light of the present study, as membranes PLA2 in cell membranes membranes
implies
cannot be ruled out. Tbe very low concentration
a tight
involved in the membrane least three pathways neither
coupling phospholipid
(20,21,22),
between
transacylase
remodeling.
and PLA2
Tbe transacylation
role for
of lysophospholipids activities,
which
in are
can be achieved by at
one of which being ATP and CoA independent
(22). Importantly,
coenzyme A nor ATP are necessary to the synthesis of PC in organic solvents.
Finally, in the present study, esteritication of PLA2
are non aqueous media, a synthetase
to synthesize
Such applications
various phospholipids
of up to 6.5% LPC has been observed. The use
of biological
require a study of the stereospecificity
interest
can therefore
be suggested.
of tbe synthesis.
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