Volume
235, number
FEB 06157
1,2, 229-232
August
1988
Effect of exogenously added acylphosphatases on inositol lipid
metabolism in human platelets
Andrea
Berti, Maurizio
Stefani,
Donatella Degl’Innocenti,
Marco
and Giampiero Ramponi
Ruggiero*,
Vincenzo
Chiarugi*
Istituto di Chimica Biologica and *Istituto di Patologia Generale, University of Firenze, Viale Morgagni SO, 50134 Firenze,
Italy
Received
29 April 1988; revised version received
15 June 1988
In this paper we demonstrate
that human platelets contain an acylphosphatase
isoenzyme. We then investigated the effect
of exogenously
added human muscle and erythrocyte
acylphosphatases
on inositol lipid content in human platelets permeabilized with saponin. Alterations
in the level of the polyphosphoinositides
were observed: in particular,
the levels
of phosphatidylinositol
4,5_bisphosphate,
and of phosphatidylinositol
4-monophosphate
were decreased,
whereas the
level of phosphatidylinositol
was increased. These results suggest that acylphosphatases
promote polyphosphoinositide
dephosphorylation,
possibly through intracellular
Ca2+ mobilization.
Inositol
lipid; Platelet;
1. INTRODUCTION
heart, and brain, although lower amounts were
found in liver, kidney and erythrocytes [4]; on the
other hand, in human the erythrocyte form is more
abundant
in brain, erythrocytes
and blood
platelets. The content of the muscular form in
platelets has not been determined as yet.
Permeabilization
of human
platelets with
saponin has recently been used to investigate the
effect of various compounds on inositol lipid
metabolism and protein phosphorylation
[S-lo].
In the present study we describe the effect of exogenously added acylphosphatases on inositol lipid
metabolism and protein phosphorylation in intact
and permeabilized platelets.
known that muscle acylphosphatase
3.6.1.7)
catalyzes
the
hydrolysis
of
acylphosphates in solution and bound to membrane proteins in several vertebrate species; examples of this activity include the Na+,K+ and
Ca2+,Mg2+-dependent ATPases [ 1,2]. Recently, a
novel acylphosphatase has been purified and sequenced from human erythrocytes.
Although
similar in molecular mass, this enzyme exhibits
greater than 50% variability in amino acid residues
when compared to the muscle isoenzyme; however,
the kinetic parameters are very similar [3]. The two
acylphosphatases
are differently
distributed in
organs and tissues in different species: in horse, the
muscular form is abundant in skeletal muscle,
It is well
(EC
Correspondence
Cancer Institute,
lE24, Bethesda,
Acylphosphatase
2. EXPERIMENTAL
Acylphosphatases
were purified
from human muscle (-SH
form) and erythrocytes
as previously
reported
[3,11- 131.
Acylphosphatase
activity was determined by continuous
optical
test at 238 nm using benzoylphosphate
as substrate,
according
to Ramponi
et al. [14]. Saponin was purchased
from Merck;
carrier-free
“P-orthophosphate
was from Amersham;
precoated
silica
gel plates
were
from
Whatman;
phosphatidylinositol
4,5-bisphosphate
(PIPz) and thrombin
were
address: M.
Ruggiero,
LCMB,
National
National Institutes of Health, Bldg 37, room
MD 20892, USA
Abbreviations:
DTT,
dithiothreitol;
Hepes,
2-(4-hydroxyethyl)-l-piperazinyl)ethanesulfonic
acid;
PAGE,
polyacrylamide gel electrophoresis
Published by Elsevier Science Publishers B. V. (Biomedical Division)
00145793/88/$3.50
0
1988 Federation
of European
Biochemical
Societies
229
Volume 235, number 1,2
FEBSLETTERS
from Sigma; [‘HIPIP
was from New England Nuclear.
All
other reagents were analytical grade, or the best commercially
available.
Preparation
of washed platelets from healthy volunteers, and
pre-labelling
with 32P-orthophosphate
were performed
as
described
[6]. Samples (0.5 ml) of 3*Pi-labelled platelets were
then placed in aggregometer
tubes at 37”C, while stirring, in a
Elvi aggregometer,
in the presence of 1 mM ATP, 0.1 mM
DTT,
and 80 units
of erythrocyte,
or skeIeta1 muscle
acylphosphatase
(-SH form). After 1 min, saponin was added,
and incubation
was carried out for 2 min. Extraction
and
separation
of
thin-layer
inositol
phospholipids
on
chromatography
(TLC) plates were performed
as described
[6,7]. In some experiments,
samples of ‘*Pi-1abelled platelets
were analyzed by polyacrylamide
gel electrophoresis,
performed according to Laemmli [ 151, using a 11% polyacrylamide
continuous
gel. Radioactivity
of the bands was evidenced
by
autoradiography.
In another set of experiments,
designed to
study the effect of acylphosphatase
on PIP2 in vitro, 37.5 pg of
PIP2, or 37.5 pg of PIP2 plus 0.375 &i of [3H]PIP2 were
dissolved in chloroform
and the solvent was evaporated
with a
stream of Nz. The residue was dissolved in 0.5 ml of 50 mM
Hepes, pH 7.4, containing
1 mM MgCl2, or in 50 mM acetate
buffer, pH 5.3, containing
1 mM MgClz. The incubation
of the
sonicated
solutions
with
skeletal
human
muscle
acylphosphatase
(100 units)
was carried
out
at room
temperature
for 15 min, and stopped by addition of 2 ml of a
solution of chloroform/methanol/HCl
(100:200: 2, v/v). The
polyphosphoinositides
were then separated
and identified
as
previously described [7].
3. RESULTS AND DISCUSSION
We have determined acylphosphatase content in
human platelets by a non-competitive
enzymelinked immunoadsorbent
assay (ELISA) carried
out with polyclonal anti-(erythrocyte
acylphosphatase) antibodies [ 121. Acylphosphatase content
determination by this method resulted in 23.15 f
0.74 ng/mg protein (mean & SE, n = 3).
In order to investigate the role of the enzyme on
platelet inositol lipid metabolism, we have added
exogenous acylphosphatases to intact and saponinpermeabilized human platelets. Table 1 shows that
addition of acylphosphatases
to permeabilized
human
platelets
caused a modification
of
polyphosphoinositides
level. Both human skeletal
muscle and human erythrocyte isoenzyme produced a loss of PIP2 and phosphatidylinositol
4-monophosphate
(PIP), and an increase of
phosphatidylinositol
(PI). The decreased levels of
PIP2 and PIP with a concomitant increase of PI indicate phosphomonoesteratic
cleavage of the
phosphates in position 4 and 5 of the inositol moiety of the polyphosphoinositides.
It should be noted
230
August 1988
Table
Effect
1
of acylphosphatases
and calcium on inositol
in permeabilized
human platelets
Treatment
None
HSM-AP
E-AP
Ca*+
PIP2
5226
4668
4969
1201
+ 38
k 90;
+ 28’
+ 107’
lipid levels
PIP
10450
6555
I3495
2612
+
+
+
+
PI
741
348*
176*
128*
6257
7239
6707
7408
+ 225
+ 164*
+ 97
+ 112*
Washed human platelets, pre-labelled
with “Pi, were treated
with 20pg/ml
of saponin,
and with human skeletal muscle
acylphosphatase
(HSM-AP),
erythrocyte
acylphosphatase
(EAP), or Cazf (500/M)
for 2 min. Phosphoinositides
were
extracted
and separated
on thin-layer
chromatography
plates.
phosphatidylinositol
4,5-bisphosphate;
PIP,
PIP2,
phosphatidylinositol
4-monophosphate;
PI,
phosphatidylinositol.
Results, expressed as cpm, are means +
SE of six replicate samples in a single experiment,
one out of six
that gave almost identical results. Statistical
significance
was
assessed by Student’s
t-test. * P < 0.02 versus control
(no
addition).
Control
experiments
have shown that addition
of
saponin alone does not affect the metabolism
of inositol lipids,
in agreement
with previous reports [6,7]
that the effect of the muscular isoenzyme seemed
more pronounced. Addition of acylphosphatases
to intact platelets did not induce any change in
platelet aggregation or inositol lipid metabolism
(not shown). Addition of 500 PM Ca2+ to platelets
pre-treated with saponin, produced a marked
dephosphorylation
of PIP2 and PIP with a resultant accumulation of PI. The smaller increase of
32P radioactivity in PI compared with the loss in
PIP2 and PIP could be explained by the observation that [32P]PI has a specific activity 15 and 30
times lower than that of PIP2 and PIP, respectively
[161.
Alterations in inositol lipid metabolite levels
were not accompanied by any modification of the
pattern of platelet protein phosphorylation,
as
determined by SDS-PAGE analysis of samples incubated in the presence of 32Pi and treated with
saponin, or with saponin and the muscular or the
erythrocyte acylphosphatase
isoenzymes (fig. 1).
The effects reported in table 1 were not due to a
direct hydrolytic action of acylphosphatases
on
PIP2. Indeed, the [3H]PIP2 content in samples
containing this inositol lipid, incubated with the
two acylphosphatase isoenzymes, at pH 7.4 and
5.3, was the same as that of controls, as monitored
by autoradiography
of TLC separation of the
polyphosphoinositides
(table 2).
Volume 235, number 1,2
FEBS LETTERS
+40
K
-20
K
1234567
Fig.1. Effect of acylphosphatase
and thrombin
on protein
phosphorylation
in human
platelets
prelabelled
with “Pi.
Washed human platelets prelabelled with ‘*Pi were incubated in
the aggregometer
tubes as in table 1. Autoradiography
shows
the
“P-1abelled
protein
separated
on
an
11%
SDSpolyacrylamide
gel. Each sample is equivalent
to 0.015 ml of
the original 0.5 ml platelet suspension.
Lanes: 1,2, control (no
addition);
3,4,
saponin;
5,6,
human
skeletal
muscle
acylphosphatase
plus saponin; 7, thrombin (0.5 units/ml).
It is
worth
noting
that acylphosphatase
does not induce
any
modification
in
apparent
pattern
of
protein
the
phosphorylation
in permeabilized
platelets. Thrombin
induces
the phoshorylation
of the 20 and 40 kDa proteins which are,
respectively,
the substrates
for myosin light chain kinase and
protein kinase C.
The results described above indicate that: (i)
human platelets contain an acylphosphatase isoenzyme as determined
by ELISA; (ii) added
acylphosphatases are able to modify the pattern of
inositol lipid content in human platelets; (iii) this
effect is not accompanied by modifications of the
protein phosphorylation pattern; (iv) this effect is
not caused by direct hydrolytic action of the two
isoenzymes on PIP2.
Previous studies [1,2] have demonstrated that
acylphosphatase
is able to hydrolyze
the
phosphorylated
intermediate formed during the
August 1988
activity of the Na+,K+- and Ca*+,Mg*+-dependent
ATPases, the latter in sarcoplasmic reticulum
the
well
known
that
vesicles.
It
is
Ca*+,Mg*+-dependent ATPase is also present in
the dense tubular system [17], that represents one
of the major Ca*+ stores in platelets; consequently,
it is conceivable that acylphosphatase
might
catalyze the hydrolysis of the phosphorylated intermediate which is formed during Ca*+ transport
in the dense tubular system. Indeed, functional
similarity between the Ca*+,Mg*+ ATPase from
sarcoplasmic reticulum vesicles and that from
membrane of the dense tubular system, has been
demonstrated.
Thus, acylphosphatase action on
the phosphorylated intermediate might raise the
level of free Ca*+, promoting the phosphomonoesteratic cleavage of polyphosphoinositides
[6,18]. In this regard, it is important to note that
the protocol for purification of the enzymes,
described in detail in [3,12,13], excludes the
possibility of contamination by Ca*+.
Results shown in table 1 indicate that Ca*+ promotes a more drastic dephosphorylation
of PIP2
and PIP in comparison to acylphosphatases.
However, it should be noted that the amount of
Ca*+ used in those experiments (500 PM) is
presumably much higher than that possibly raised
by the action of acylphosphatases
on the
Ca*+,Mg*+-dependent ATPase.
The amount of acylphosphatases used in the experiments reported above is higher than that
physiologically
present
in human
platelets;
however, it should be considered that, in a
permeabilized system, one is forced to administer
large quantities of a compound in order to observe
an effect. This holds true, as an example, for the
intracellular
Ca*+-mobilizer,
inositol
(1,4,5)trisphosphate, that has to be administered in the
15-45 PM range in order to induce platelet activation [8].
In conclusion, this study demonstrates that
human platelets do contain an acylphosphatase
isoenzyme and that acylphosphatase might play a
role in the metabolism of inositol lipids; the exact
nature and extent of this involvement require further study.
This work was supported
by grants from
the Consiglio Nazionale delle Ricerche, Minister0 Italian0 della
Pubblica
Istruzione,
and Associazione
Italiana per la Ricerca
sul Cancro (to M.R.).
Acknowledgements:
231
Volume
235, number
FEBS LETTERS
1,2
August
1988
Table 2
Effect of human
skeletal muscle acylphosphatase
on phosphatidylinositol4,5-bisphosphate
vitro
pH 5.3
Control
[‘HIPIP
78265
+ 692
in
pH 7.4
HSM-AP
78423
? 897
Control
70817
+ 971
HSM-AP
71690
k 1073
37.5 pg of PIPz, or 37.5 pg of PIP2 plus 0.375 pCi of [3H]PIPz were dissolved in chloroform
and the solvent was evaporated
with a stream of Nz. The residue was dissolved in 0.5 ml of
50 mM acetate buffer,
pH 5.3, containing
1 mM MgClz, or in 50 mM Hepes, pH 7.4,
containing
1 mM MgC12. Incubation
of the sonicated solutions with human skeletal muscle
acylphosphatase
(HSM-AP,
100 units) was carried out at room temperature
for 2 h, and
stopped by addition of chloroform/methanol/HCl
(100:200:2,
v/v). Inositol phospholipids
were separated by thin-layer chromatography
as described [7]. Results expressed as cpm, are
means + SE (n = 3). It should be mentioned
that 99.7 + 0.02% of the total recovered
radioactivity
was found associated
with PIP2 zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQ
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