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Post-heparin phospholipase and post-heparin lipase have different tissue origins
BIOCHEMICAL
Vol. 47, No. 6, 1972
POST-HEPARIN
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
PHOSPHOLIPASE AND POST-HEPARIN
LIPASE
HAVE DIFFERENT TISSUE ORIGINS Franklin
J.
Zieve
and Leslie
Zieve
Department of Medicine, Veterans University of Minnesota, Minneapolis,
ReceivedMay
Hospital, Minnesota
55417
17,1972
SUMMARY: Post-heparin phospholipase activity is reduced by 80% in the hepatectomized rat. In contrast, post-heparin lipase activity is unaffected by hepatectomy. Since the phospholipase originates mainly in the liver and the lipase originates entirely in extrahepatic tissues, these two activities must be different enzymes. A number
of enzymatic
the administration
and Vogel
Among these
A 1 (3).
phospholipase
(4,5)
phospholipase
(l-3).
phospholipase
pancreatic
molecule
are released
of heparin
and post-heparin from
activities
response
and post-heparin et al.
(7) have
was based effects
on their
Lipoprotein is
not
found
strated
that
finding
raised
nate
in
post-heparin
the
plasma
lipase
that
activity
liver
this
could
that were
the
distinguished
(5).
Post-heparin
properties
two activities
(5-71,
are probably This
two activities
detected
and Waite
liver
conclusion
and on the
post-heparin case,
1480
in many tissues (9)
contain
(11,
have recently
a phospholipase phospholipase
post-heparin
not be the same enzyme.
Copyright 0 1972, by Academic Press, Inc.
(1)
states.
Newkirk of rat
lipase
is
substrates.
the
has been
(8).
membranes
If
these
following
on the phospholipid
have many similar
to separate
the possibility
the liver.
inhibitors
and of pathologic lipase
in
to various
on two different
inability
of inhibitors
of attack
concluded
due to the same enzyme acting
activity
site
lipase
the plasma
are lipoprotein
The latter
A2 by its
and by its
into
might
this
demonAl.
phospholipase To test
but
This origiand
possibility,
Vol. 47, No. 6, 1972
BIOCHEMICAL
we have examined
the effects
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
of total
hepatectomy
on the post-heparin
enzyme
activities. MATERIALS AND METHODS Phosphatidyl obtained
from
Searle
International
Corp.,
previously Hormel
14
ethanolamine-l,ZChemical
respectively.
described
and Nuclear
Egg phosphatidyl
(lo),
Institute.
C and glyceryl
and nonradioactive
ChromAR silicic
tri[oleate-l-
14C] were
Corp.
and from Amersham/
ethanolamine
was prepared
triolein
acid-impregnated
was purchased glass
fiber
as from
sheets
were
from Mallinckrodt. Total
hepatectomy
Van Hook (ll), 6 weeks
by removal
inferior the
which
vena
involves of the
5 min after
liver.
procedure
of Bollman
and
vena
cava followed
in
of the inferior
Normal
served
(100 units)
heparin
by the 2-stage
ligation
cava ligation
heparin
liver,
was performed
animals
and rats
as controls.
was injected
administration,
1 hour
subjected after
intravenously.
and the separated
removal Blood
plasma
to of
was drawn
was stored
at
-20". Phospholipase Tris-Cl
(pH 9.01, 14
30 nmoles for
which
for
20 nmoles
(pH 8.0),
scintillation reaction 0.075%
C-triolein,
labeled
fatty
acid-impregnated
phosphatidyl been
with
ethanolamine
applied.
X-100, After
mixture
glass
fiber
ammonium molybdate)
incubation
was applied paper,
mixtures
albumin,
(total
0.075%
released
volume
Triton
50 ~1)
X-100,
Incubation
were
to
developed
and the phospholipid were
cut
out
and counted
was isolated
(12). 1481
contained
0.06
M Tris-Cl
6 ~1 human serum,
was for
to
and lysophosphatidyl
The chromatograms (80:25:3),
0.1 M
counter.
and 10 ul plasma. acid
contained
0.15% Triton
(25 1~1) of the reaction
of silicic
by spraying
30 ~1)
and 5 ~1 plasma.
chloroform-methanol-water
(located
Lipase
volume
taurodeoxycholate,
an aliquot
had previously
a liquid
(total
ethanolamine,
of carrier
10 min with
spots
14
C-phosphatidyl
of a strip
ethanolamine
mixtures
0.2% sodium
15 min at 37",
the origin
in
reaction
15 min at 37",
by TEAE-cellulose
55 nmoles and the
chromatography
BIOCHEMICAL
Vol. 47, No. 6, 1972
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
I
5 10 15 20 ,uL Postheparin Plasma
25
14 Figure 1. Hydrolysis of C-triolein by varying amounts of post-heparin Lipase incubations were carried out as described under MATERIALS plasma. AND METHODS, except that the volume of post-heparin plasma was varied as shown on the abscissa. M-) control rat; o--o, hepatectomized rat.
RESULTS AND DISCUSSION If,
as reported
then hepatectomy prediction Fig.
should
was confirmed
1, which
plots
amount
of post-heparin
plasma
from
range
(8),
TABLE I.
Effect
Treatment Control Inf.
does not
have no effect in our
plasma
the hepatectomized
in which
liver
incubated. rat
tested.
lipase result
from
Table
I lists were
the
hepatectomized
rats
of hepatectomy
on post-heparin Lipase
is
compared
Activity
shown in
rat
and
throughout
results
the
of a series
with
normal
lipolytic
rats
of and
activities
Phospholipase Activity
7.84
+ 1.25
6.16
k 0.05
.. ..
8.25
f 1.20
5.42
3~ 1.30
.. .. ... ... .. ... .
6.99
k 0.79
1.26
+ 0.05
cava ligation
This
of the
the normal
on the same curve
lipase,
activity.
as a function
Plasma
fell
. ... .. ... .. ... .. .. ..
Hepatectomy
any lipoprotein
A typical
of 14 C-triolein
of Rats
vena
contain
on post-heparin
experiments.
the hydrolysis
of concentrations
experiments
the
Assays were conducted as described under MATERIALS AND METHODS. Lipase activity is expressed as umoles oleic acid formed/ml plasmaihr, and phospholipase activity is expressed as umoles lysophosphatidyl ethanolamine formed/ ml plasmalhr. Each group consisted of 3 rats, and the values are listed as mean + standard error.
1482
Vol. 47, No. 6, 1972
BIOCHEMICAL
with
rats
subjected
cant
differences
in
In contrast
to post-heparin
was sharply
to inferior lipase
reduced
measured further
rats
phospholipase
were
II
indicate
much lower
that than
no signifi-
phospholipase
activity activity
of the controls. activity,
rats
of the reaction,
since
the phosphatidyl
of
Inferior
vena
The decrease
the hepatectomized
in
were
The phospholipase
on phospholipase
was not
in due to
296% of the incu-
ethanolamine
and
spots.
of the possibility than
I).
22% of that
in
three
post-heparin
(Table
activity
of pre-heparin
Table
lipase,
was only
ethanolamine
other
vities
groups.
was recovered
lysophosphatidyl
activities
among the
of the product
radioactivity
Because
There
activity
had no effect
degradation
bated
vena cava ligation,
by hepatectomy
the hepatectomized cava ligation
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
that
those
normally
plasma
were
hepatectomy found
also
the pre-heparin the post-heparin
might
alter
plasma
in post-heparin
measured. lipase
plasma,
The results
and were
the acti-
shown in
and phospholipase
activities,
lipolytic
activities
unaffected
by
hepatectomy. TABLE II.
Effect
Treatment Control Inf.
of hepatectomy
of Rats
on pre-heparin
Phospholipase Activity
Activitv
0.20
+ 0.10
0.57
+ 0.23
.. ..
0.04
f 0.02
0.26
* 0.17
... ... ... .. ... ..
0.34
+ 0.09
0.56
_+ 0.25
cava ligation
Heoatectomv
activities
Lioase
.,......,........,.. vena
lipolytic
Assays were conducted as described under MATERIALS AND METHODS, and results are expressed as described in the legend to Table I. Pre-heparin plasma samples were obtained from the same animals used in the experiments shown in Table I.
It
is
also
possible
in the hepatectomized the Fig.
reaction
rather
that rats
than
2 shows the post-heparin
the reduced
might
post-heparin
phospholipase
be due to the production
to a decreased phospholipase
activity activity
activity
of an inhibitor
of
of the enzyme itself. of normal
plasma,
plasma
BIOCHEMICAL
Vol. 47, No. 6, 1972
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
2
4
(1
,uulPostheparin
8
lo
PLasma
14 Figure 2. Hydrolysis ethanolamine by varying amounts of C-phosphatidyl of post-heparin plasma. Phospholipase incubations were carried out as described under MATERIALS AND METHODS, and the volume of post-heparin plasma was varied as shown on the abscissa. --) plasma from control rat; u, plasma from hepatectomized rat; x----x, mixture of equal amounts of plasma from control and hepatectomized rats.
from hepatectomized plasma. that
rats,
The mixture
the decreased
and a mixture
gave an intermediate phospholipase
activity
of equal
amounts
value
throughout,
was not
of the two types which
of
indicated
due to an inhibitor
of the
reaction. We conclude liver,
while
Since
these
lipase
two activities
have
enzymes
We are
currently their
post-heparin
post-heparin
ferent
study
that
rather
than
originates different
entirely tissue
the same enzyme acting
attempting
respective
phospholipase
to separate
roles
in fat
and purify
originates
mainly
in
in extrahepatic origins,
they
on different these
the tissues.
must be difsubstrates.
enzymes
in order
to
transport.
ACKNOWLEDGMENTS We thank ing
Ann M&ale
for
technical
assistance
and Orin
Shuck for
the hepatectomies. REFERENCES
1. 2. 3.
Robinson, D.S., Advan. Lipid Res., 1, 133 (1963). Shore, B., and Shore, V., Amer. J. Physlol., 201, 915 (1961). J. Lipid Res., 5, 177 (1964). Vogel, W.C., and Zieve, L.,
1484
perfonn-
Vol. 47, No. 6, 1972
4. 5. 6.
7. 8. 9. 10. 11. 12.
BIOCHEMICAL
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
Vogel, W.C., and Biennan, E.L., J. Lipid Res., 8, 46 (1967). Doizaki, W.M., and Zieve, L., Proc. Sot. Exp. Biol. Med., 129, 182 (1968). Vogel, W.C., and Bierman, E.L., Lipids, 2, 385 (1970). Vogel, W.C., Brunzell, J.D., and Bierman, E.L., Lipids, 6, 805 (1971). Grafnetter, D., and Zemplenyi, T., Z. Physiol. Chem., 316, 218 (1959). Newkirk, J.D., and Waite, M., Biochim. Biophys. Acta, 225, 224 (1971). Doizaki, W.M., and Zieve, L., J. Lab. Clin. Med., 63, 524 (1964). Bollman, G.L., and Van Hook, E., J. Appl. Physiol., 24, 722 (1968). submitted for publication. Zieve, F.J.,
1485
Vol. 47, No. 6, 1972
POST-HEPARIN
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
PHOSPHOLIPASE AND POST-HEPARIN
LIPASE
HAVE DIFFERENT TISSUE ORIGINS Franklin
J.
Zieve
and Leslie
Zieve
Department of Medicine, Veterans University of Minnesota, Minneapolis,
ReceivedMay
Hospital, Minnesota
55417
17,1972
SUMMARY: Post-heparin phospholipase activity is reduced by 80% in the hepatectomized rat. In contrast, post-heparin lipase activity is unaffected by hepatectomy. Since the phospholipase originates mainly in the liver and the lipase originates entirely in extrahepatic tissues, these two activities must be different enzymes. A number
of enzymatic
the administration
and Vogel
Among these
A 1 (3).
phospholipase
(4,5)
phospholipase
(l-3).
phospholipase
pancreatic
molecule
are released
of heparin
and post-heparin from
activities
response
and post-heparin et al.
(7) have
was based effects
on their
Lipoprotein is
not
found
strated
that
finding
raised
nate
in
post-heparin
the
plasma
lipase
that
activity
liver
this
could
that were
the
distinguished
(5).
Post-heparin
properties
two activities
(5-71,
are probably This
two activities
detected
and Waite
liver
conclusion
and on the
post-heparin case,
1480
in many tissues (9)
contain
(11,
have recently
a phospholipase phospholipase
post-heparin
not be the same enzyme.
Copyright 0 1972, by Academic Press, Inc.
(1)
states.
Newkirk of rat
lipase
is
substrates.
the
has been
(8).
membranes
If
these
following
on the phospholipid
have many similar
to separate
the possibility
the liver.
inhibitors
and of pathologic lipase
in
to various
on two different
inability
of inhibitors
of attack
concluded
due to the same enzyme acting
activity
site
lipase
the plasma
are lipoprotein
The latter
A2 by its
and by its
into
might
this
demonAl.
phospholipase To test
but
This origiand
possibility,
Vol. 47, No. 6, 1972
BIOCHEMICAL
we have examined
the effects
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
of total
hepatectomy
on the post-heparin
enzyme
activities. MATERIALS AND METHODS Phosphatidyl obtained
from
Searle
International
Corp.,
previously Hormel
14
ethanolamine-l,ZChemical
respectively.
described
and Nuclear
Egg phosphatidyl
(lo),
Institute.
C and glyceryl
and nonradioactive
ChromAR silicic
tri[oleate-l-
14C] were
Corp.
and from Amersham/
ethanolamine
was prepared
triolein
acid-impregnated
was purchased glass
fiber
as from
sheets
were
from Mallinckrodt. Total
hepatectomy
Van Hook (ll), 6 weeks
by removal
inferior the
which
vena
involves of the
5 min after
liver.
procedure
of Bollman
and
vena
cava followed
in
of the inferior
Normal
served
(100 units)
heparin
by the 2-stage
ligation
cava ligation
heparin
liver,
was performed
animals
and rats
as controls.
was injected
administration,
1 hour
subjected after
intravenously.
and the separated
removal Blood
plasma
to of
was drawn
was stored
at
-20". Phospholipase Tris-Cl
(pH 9.01, 14
30 nmoles for
which
for
20 nmoles
(pH 8.0),
scintillation reaction 0.075%
C-triolein,
labeled
fatty
acid-impregnated
phosphatidyl been
with
ethanolamine
applied.
X-100, After
mixture
glass
fiber
ammonium molybdate)
incubation
was applied paper,
mixtures
albumin,
(total
0.075%
released
volume
Triton
50 ~1)
X-100,
Incubation
were
to
developed
and the phospholipid were
cut
out
and counted
was isolated
(12). 1481
contained
0.06
M Tris-Cl
6 ~1 human serum,
was for
to
and lysophosphatidyl
The chromatograms (80:25:3),
0.1 M
counter.
and 10 ul plasma. acid
contained
0.15% Triton
(25 1~1) of the reaction
of silicic
by spraying
30 ~1)
and 5 ~1 plasma.
chloroform-methanol-water
(located
Lipase
volume
taurodeoxycholate,
an aliquot
had previously
a liquid
(total
ethanolamine,
of carrier
10 min with
spots
14
C-phosphatidyl
of a strip
ethanolamine
mixtures
0.2% sodium
15 min at 37",
the origin
in
reaction
15 min at 37",
by TEAE-cellulose
55 nmoles and the
chromatography
BIOCHEMICAL
Vol. 47, No. 6, 1972
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
I
5 10 15 20 ,uL Postheparin Plasma
25
14 Figure 1. Hydrolysis of C-triolein by varying amounts of post-heparin Lipase incubations were carried out as described under MATERIALS plasma. AND METHODS, except that the volume of post-heparin plasma was varied as shown on the abscissa. M-) control rat; o--o, hepatectomized rat.
RESULTS AND DISCUSSION If,
as reported
then hepatectomy prediction Fig.
should
was confirmed
1, which
plots
amount
of post-heparin
plasma
from
range
(8),
TABLE I.
Effect
Treatment Control Inf.
does not
have no effect in our
plasma
the hepatectomized
in which
liver
incubated. rat
tested.
lipase result
from
Table
I lists were
the
hepatectomized
rats
of hepatectomy
on post-heparin Lipase
is
compared
Activity
shown in
rat
and
throughout
results
the
of a series
with
normal
lipolytic
rats
of and
activities
Phospholipase Activity
7.84
+ 1.25
6.16
k 0.05
.. ..
8.25
f 1.20
5.42
3~ 1.30
.. .. ... ... .. ... .
6.99
k 0.79
1.26
+ 0.05
cava ligation
This
of the
the normal
on the same curve
lipase,
activity.
as a function
Plasma
fell
. ... .. ... .. ... .. .. ..
Hepatectomy
any lipoprotein
A typical
of 14 C-triolein
of Rats
vena
contain
on post-heparin
experiments.
the hydrolysis
of concentrations
experiments
the
Assays were conducted as described under MATERIALS AND METHODS. Lipase activity is expressed as umoles oleic acid formed/ml plasmaihr, and phospholipase activity is expressed as umoles lysophosphatidyl ethanolamine formed/ ml plasmalhr. Each group consisted of 3 rats, and the values are listed as mean + standard error.
1482
Vol. 47, No. 6, 1972
BIOCHEMICAL
with
rats
subjected
cant
differences
in
In contrast
to post-heparin
was sharply
to inferior lipase
reduced
measured further
rats
phospholipase
were
II
indicate
much lower
that than
no signifi-
phospholipase
activity activity
of the controls. activity,
rats
of the reaction,
since
the phosphatidyl
of
Inferior
vena
The decrease
the hepatectomized
in
were
The phospholipase
on phospholipase
was not
in due to
296% of the incu-
ethanolamine
and
spots.
of the possibility than
I).
22% of that
in
three
post-heparin
(Table
activity
of pre-heparin
Table
lipase,
was only
ethanolamine
other
vities
groups.
was recovered
lysophosphatidyl
activities
among the
of the product
radioactivity
Because
There
activity
had no effect
degradation
bated
vena cava ligation,
by hepatectomy
the hepatectomized cava ligation
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
that
those
normally
plasma
were
hepatectomy found
also
the pre-heparin the post-heparin
might
alter
plasma
in post-heparin
measured. lipase
plasma,
The results
and were
the acti-
shown in
and phospholipase
activities,
lipolytic
activities
unaffected
by
hepatectomy. TABLE II.
Effect
Treatment Control Inf.
of hepatectomy
of Rats
on pre-heparin
Phospholipase Activity
Activitv
0.20
+ 0.10
0.57
+ 0.23
.. ..
0.04
f 0.02
0.26
* 0.17
... ... ... .. ... ..
0.34
+ 0.09
0.56
_+ 0.25
cava ligation
Heoatectomv
activities
Lioase
.,......,........,.. vena
lipolytic
Assays were conducted as described under MATERIALS AND METHODS, and results are expressed as described in the legend to Table I. Pre-heparin plasma samples were obtained from the same animals used in the experiments shown in Table I.
It
is
also
possible
in the hepatectomized the Fig.
reaction
rather
that rats
than
2 shows the post-heparin
the reduced
might
post-heparin
phospholipase
be due to the production
to a decreased phospholipase
activity activity
activity
of an inhibitor
of
of the enzyme itself. of normal
plasma,
plasma
BIOCHEMICAL
Vol. 47, No. 6, 1972
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
2
4
(1
,uulPostheparin
8
lo
PLasma
14 Figure 2. Hydrolysis ethanolamine by varying amounts of C-phosphatidyl of post-heparin plasma. Phospholipase incubations were carried out as described under MATERIALS AND METHODS, and the volume of post-heparin plasma was varied as shown on the abscissa. --) plasma from control rat; u, plasma from hepatectomized rat; x----x, mixture of equal amounts of plasma from control and hepatectomized rats.
from hepatectomized plasma. that
rats,
The mixture
the decreased
and a mixture
gave an intermediate phospholipase
activity
of equal
amounts
value
throughout,
was not
of the two types which
of
indicated
due to an inhibitor
of the
reaction. We conclude liver,
while
Since
these
lipase
two activities
have
enzymes
We are
currently their
post-heparin
post-heparin
ferent
study
that
rather
than
originates different
entirely tissue
the same enzyme acting
attempting
respective
phospholipase
to separate
roles
in fat
and purify
originates
mainly
in
in extrahepatic origins,
they
on different these
the tissues.
must be difsubstrates.
enzymes
in order
to
transport.
ACKNOWLEDGMENTS We thank ing
Ann M&ale
for
technical
assistance
and Orin
Shuck for
the hepatectomies. REFERENCES
1. 2. 3.
Robinson, D.S., Advan. Lipid Res., 1, 133 (1963). Shore, B., and Shore, V., Amer. J. Physlol., 201, 915 (1961). J. Lipid Res., 5, 177 (1964). Vogel, W.C., and Zieve, L.,
1484
perfonn-
Vol. 47, No. 6, 1972
4. 5. 6.
7. 8. 9. 10. 11. 12.
BIOCHEMICAL
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
Vogel, W.C., and Biennan, E.L., J. Lipid Res., 8, 46 (1967). Doizaki, W.M., and Zieve, L., Proc. Sot. Exp. Biol. Med., 129, 182 (1968). Vogel, W.C., and Bierman, E.L., Lipids, 2, 385 (1970). Vogel, W.C., Brunzell, J.D., and Bierman, E.L., Lipids, 6, 805 (1971). Grafnetter, D., and Zemplenyi, T., Z. Physiol. Chem., 316, 218 (1959). Newkirk, J.D., and Waite, M., Biochim. Biophys. Acta, 225, 224 (1971). Doizaki, W.M., and Zieve, L., J. Lab. Clin. Med., 63, 524 (1964). Bollman, G.L., and Van Hook, E., J. Appl. Physiol., 24, 722 (1968). submitted for publication. Zieve, F.J.,
1485