Vol. 64, No. 2, 1975
BIOCHEMICAL
Serum Factors
Affecting
Mizue (Zoological
Institute,
April
Received
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
Cathepsin
Wiyamoto Faculty
and
Release Hiroshi
of Science,
from Lysosvmes
Terayama
University
of Tokyo,
Tokyo,
Japan)
1,1975
SUMMARY: Rat or bovine serum exhibited t,he inhibitory effect, on cathepsin release frvm rat liver lysosvmes in vitro. The inhibitory activity of serum was enhanced after dialysis. HeatedO' C, 3-5 min) serum showed the marked stimulatvry effect on the cathepsin release. It was indicated that the adult and infant, rat. or bovine sera contain a large-molecular, heat-labile inhibitory factor and a small-molecular-heat-stable stimulatvry factor. Fetal bovine serum, however, was found to be very poor in these factors. The activity of each of the serum factors was found to change characteristically within a short period after partial hepatectomy. Although biology,
tissue
the real
proliferation
mechanisms
have not yet
we have reported cell
coat
acid
that
of cell
may be responsible inhibitors
mitosis
regeneration
Adams (4)
well
been
temporary
showing
fully
disappearance
proliferation
as that
by Allison
pepstatin)
and migration
nucleic
disappearance
of them from
Copyright o I9 75 by Academic Press, Inc. All rights of reproduction in any form reserved.
acid
of cell
as detected
soon after
the deep inside
617
by the
related
since early
with
cathepsin cathepsin
periods
coat
of liver
accompanying
reports,
the
partial number
the
the report
enzyme activities
in
papers
(RNA, DNA) biosynthesis,
to our previous
a decrease
in
cell
the lysvsomal
during
of some lysosomal
showing
coat,
regeneration
injected
fl-glucuronidase) (5)
of cell
2) and that
In connection
the elevation
of tissue
In the preceding
of liver
the
subjects
seems to be intimately
(1,
the initiation
(3).
control
understood.
stain,
as the temporary
phosphatase,
one of the most attractive
the homeostatic
or retarded
acid
particles
for
and/or
inhibited
as well
DNase,
for
(leupeptin
regeneration
is
mucopolysaccharide
the initiation
liver
regeneration
by
(acid
hepatectomy
as
of lysvsvmal
to the periphery
of liver
Vol. 64, No. 2, 1975
cells within
BIOCHEMICAL
a few hours after
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
partial
hepatectow
seemto be particularly
interesting. During the course of searching for possible humoral or cytoplasmic factor(s)
responsible for the V1lysosomalactivation",
or bovine serum contain factors
affecting
we have found that rat
the permeability
of lysosomal
membranesagainst cathepsin.
MATERIALSAND -___ METHODS Lysosomeswere prepared from the liver 150 g.
A 1:8 (w/v) liver
of male, Wistar rats weighing 100-
homogenatein 20 mMTris (pH 7.2) - 0.25 M sucrese
was subjected to the successive centrifugations,
750 x -g for 10 min, 3,300 x
g for 10 min, and then 15,000 x -g for 20 min, to sediment lysosome-rich pellet, which was Once gently rinsed.
The lysosomal pellet
derived from 10 g liver
was suspendedin 20 ml of 0.25 M sucrose - 1 mMEDTA, pH 7.2 (lysosomal suspension).
Measurements of cathepsin release were carr:c?
,>ut as follWJs.
A 2.0 nit aliyuot
of the lysosomal suspension and a small volume (0.1-0.6
of test material
or 0.9s NaCl as the control
ml)
were mixed and the mixture was
incubated at 37’ C for certain periods of time with gentle shaking, followed by centrifugation
at 15,000 x g for 20 min.
The pellet
was suspendedin 2.0
ml of 0.25 M sucrose - 0.2% Triton X-100 and the suspension was kept at O0 C for 60 min, followed by centrifugation
at 15,000 x -g for 20 min.
supernatant thus obtained was used for the assay of the "residual cathepsin D activityI'.
The "total
cathepsin activity"
2.0 ml of the lysosomal suspension directly D were
with Triton.
carried out by the method of hnson (6).
618
The (unreleased)
was measured by treating Assays for cathepsin
A 0.1 ml aliquot
of supernatant
Vol. 64, No. 2, 1975
from
the
Triton-solubilized
acid-denatured incubated 1.0
solution
at 37O C for
(Total
inhibition)
material
release
control)/$Release
stimulation
and - sign
for
inhibition.
was defined
as the activity
will
The more detailed
be presented
removing
the median Newborn
(8).
in the
and adult
respectively. Science
Bovine
Research
Rockville,
and left
sera
Institute
U.S.A.)
sera were (adult and fetal
was a gift
from Dr.
for
or stimulator
from
and Anderson
and f;-week
the Mitsubishi
serum (Slow
Y. Mizuno
serum factors out by
to Higgins
from 5-day were
of the
was carried
according
prepared
bovine
: + sign
the assay
hepatectomy
and calf)
stimulation
or 10% stimulation,
for
lobes
control
as 100 x
of test
of inhibitor
1% inhibition
lateral
rat
D was defined The percent
in the
Partial
of
acid-soluble
in the presence
conditions
text.
by the addition
at 700 nm by the method
One unit
to cause
prepared
pH 3.2 and
containing
of cathepsin
as 100 x ($Release
the
respectively.
was stopped
activity.
- $R e 1 ease in
ml of freshly
buffer,
to calorimetry
activity)/Total
was defined
1.9
The supernatant
The percent
- Residual
with
M acetate
The reaction
was subjected
(7).
activity
0.18
acid.
hydrolyzates et al.
was mixed
in
60 min.
ml of 10% trichloroacetic
of Lowry
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
lysosomes
hemoglobin
hemoglobin
(or
BIOCHEMICAL
old
rats,
Life
Laboratories,
in our Institute.
RESULTS AND DISCUSSION ___In Fig. liver Inhibition
l-a
lysosomes
are in
the presence
of cathepsin
more and more marked
illustrated
release
the kinetics
of cathepsin
and the absence by the
as the incubation
of adult
serum appears time
619
increases,
D release rat
to begin
from rat
serum. at 2 h, become
and finally
become
Vol. 64, No. 2, 1975
BIOCHEMICAL
0
Fig.
1
Kinetics presence
1.
2
3
AND BIOPHYSICAL
L
5
0
I
2
RESEARCH COMMUNICATIONS
3
L
5
6
of cathepsin D release from rat liver lysosones of rat serum (a) or of heated rat serum (b).
in the
(a) 0.2 ml of freshly prepared adult rat serum was added to 2.0 ml of lysosomal suspension. As the control, 0.2 ml of 0.25 M sucrose-l mM EDTA was added. (b) Adult rat serum was mixed with an equal volume of 0.9% NaCl, heated at 1OOoC for 5 min, cooled in ice-water and centrifuged. 0.6 ml of the supernatant (equivalent to 0.3 ml of the original serum) was added to 2.0 ml of the lysosomal suspension. As the control, 0.6 ml of 0.9% NaCl was added. 0 : control, l : adult rat serum, and A : heated adult rat serum.
almost
complete
appears
at 4-5
to proceed
experiments, factor.
4.5
almost
bovine
rat
and calf
serum inhibits
serum was found contain
Table sera only
of dialyzed
inhibitory
while
the release
time.
Therefore,
was adopted I,
can inhibit
assaying
cathepsin
dialysis,
which
to increase
be noted
release,
620
in the
the following
serum inhibitory
sera
as well while
the inhibitory suggesting
that
that
the
fetal
the sera
effect
the
inhibitory animals
as the
activity
the
as the age of donor factor
the control
in the
may counteract
should
in
and bovine
Furthermore,
after
It
of a stimulating
rat
the
slightly.
factor.
for
the adult
factor,
serum appears
The presence
with
to be increased
a small-molecular
molecular
linearly
h incubation
As shown in
newborn
h of incubation,
of may
of the largeactivity becomes
serum was more directly
older
BIOCHEMICAL
Vol. 64, No. 2, 1975
Table
Inhibition of cathepsin and dialyzed sera.
I.
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
D release
from lysosomes
by various
sera
0.3 ml of various sera or dialyzed sera (see Fig. 2) from different donors was added to 2.0 ml of lysosomal suspension. As the control, 0.3 ml of 0.9s NaCl was added. The mixtures were incubated at 37' C for 4.5 h.
Addition
Exp. No.
$ Release
None (central)
50.7
----
Adult
36.7 36.2
27.6 28.6
34.5 36.5
32.0 28.0
rat
Newborn
serum
serum a) Bovine serum Calf serum
I
Fetal
rat
bovine
serum
None (control) Adult II
rat
serum
Dialyzed Bovine
adult
rat
serum
serum
Dialyzed
bovine
serum
None (control)
III
a)
kinetics rat
adult
Dial;rzed
newborn
Dialyzed
adult
Dialyzed
calf
Dialyzed
fetal
serum.
by observing
release
Therefore,
serum rat
serum serum a)
bovine bovine
9.1
44.4 32.5
me--
26.8 30.4 15.7
39.6 31.5 64.6
55.7
----
33.0
40.7
41.7 41.8
25.1 32.3 24.9
47.3
15.1
26.8
37.7
serum serum
effects in
of heated
effect
the following
621
In Fig.
and the
effect
of heated
and to become less in
sera.
the presence
to the inhibitory
stimulatory
1 h of incubation
prolonged.
the
In contrast the
rat
46.1
serum was used.
of cathepsin
dialyzate, after
Dialyzed
Immobilized
evidenced
'$ Inhibition
serum marked
experiments
l-b
absence
of whole
we show the
of heated
adult
serum or serum
seems to be most evident as the incubation the
stimulatory
time factor
is
BIOCHEMfCAL
Vol. 64, No. 2, 1975
Table
II.
Stimulation sera.
of cathepsin
AND BIOPHYSICAL
D release
from
RESEARCH COMMUNICATIONS
lysosomes
by various
heated
Heated sera were prepared from various sera from different donors as 0.6 ml of heated serum (equivalent to 0.3 ml of original described in Fig. l-b. serum) was added to 2.0 ml of lysosomal suspension and the mixture was incubated at 37O C for 1 h. As the control, 0.6 ml of 0.9s NaCl was added.
Addition
$ Release
8.2
None Heated
adult
Heated
newborn
Heated Heated
bovine serum a) calf serum
Heated
fetal
a)
rat
serum rat
was assayed activities
serum
bovine
Immobilized
25.9
210
21.4
161
22.9 21.4
161
179
8.7
serum
at 1 h of incubation.
except
---
6
serum was used.
of various
the sera
$ Stimulation
the
sera fetal
In Table
heated
at 100"
bovine
II
C for
serum contain
are
compared
5 min.
It
the
s'cimulatory
appears
the heat-stable
that
all
of
stimulatory
factor.
for
AS
shown in Fig.
2, almost
both
the inhibitory
factor
heated
serum within
a rational
basis
Based factors
for
in dialyzed
below
in
Table
at various
assay
III,
after
partial
higher
the initial
the
factor
hepatectomy, level
of the
after
stimulatory
reach
at around
stimulatory
factor
partial
of the rat
hepatectomy.
was found a maximal
level
partial
serum As
to increase almost
in
affording
serum factors.
activities
Z-3 h after
622
can be observed
or 140% stimulation,
relation,
times
relation
serum and the
354 inhibition
the quantitative
measured
immediately than
dose-response
on the above dose-response
were
sum?larized
a range
linear
Lfold
hepatecttomy
and
BIOCHEMICAL
Vol. 64, No. 2, 1975
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
50
1 - 30 =II: 20 f: IO LO
0
01
02
Dialyzed
Fig.
2.
or
Dose-dependent inhibition lysosomes by dialyzed rat
03
OS
heated
5erum
05
06
I ml I
or stimulation of cathepsin serum or heated rat serum.
D release
from
Various volumes of adult rat serum dialyzed against 0.9s NaCl at 4oC overnight or heated adult rat serum prepared as described in Fig. l-b were added to 2.0 ml of lysosomal suspension. The final volume was adjusted to 2.6 ml by adding 0.9% NaCl. The mixture was incubated at 37oC for 4.5 h in case of dialyzed serum or for 1 h in case of heated serum. Percent inhibition or stimulation was measured as described in Materials and Methods.
Table
III.
Change
of activities
of the
serum factors
after
partial
hepatectomy.
Serum was prepared from 2 rats at various times after partial hepatectomy. The 0 h serum was prepared from 2 sham-operated rats. Dialyzed or heated serum was prepared as described earlier (Fig. 1, Fig. 2) and used for the assay of the inhibitory factor or the stimulatory factor, respectively. In each assay, two different volumes of serum (usually 0.1 and 0.2 ml) were added to 2.0 ml of lysosomal suspension in order to check the linear dose-dependency. The mixtures were incubated for 1 h or 4.5 h to measure the stimulatory factor or the inhibit,ory fact,or, respectively.
Time after hepatectomy
decline remained
partial (h)
Activity Stimulatory
0
62
3
136
6
93
gradually unaltered
thereafter. for
the first
On the
(units/ml factor
factor 126
12-7 180
other
few hour
623
serum) Inhibitory
hand,
period,
the inhibitory followed
by the
factor gradual
BIOCHEMICAL
Vol. 64, No. 2, 1975
increase. (5,
Taking
6) as well
accomplished in this
paper
as the within
partial
early
to conclude
factors
considerations initiating
a very
showing
after
regeneration.
into
the
hepatectov that
that
stimulus
early
period
temporary
this
the apparent for
as well
partial
of the
interesting,
may be an actual
lysosomal
hepatectomy,
serum
chemical
the results
stimulatory
although
it
trigger
as the mode of actions
activation
(9) seem to be
the regeneration
after
surge
seems quite
The nature
are now under
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
factor may be still
for
of these
soon too
the liver serum
investigations. REFERENCES
1. 2. 3. 4. 2:
7. 8. 9.
Yamamoto, K., Omata, S., Ohnishi, T., and Terayama, H. (1973) Cancer Res. 33, 657-572. Ohnishi, T., Yamamoto, K., and Terayama, H. (1973) Histochemie 2, 15-20. Miyamoto, M,, and Terayama, H. (1973) Biochem. Biophys. Res. Commun. 2, 84-90. Adams, R. L. J'. (1963) Biochem. J. 87, 532-536. Allison, A. C., and Mallucci, L. (lg4) Lancet 26, 1371-1373. Anson, M. L. (1938) J. Gen. J)hysiol. 22, 79-89. N. J., Farr, A. L., and Randall, R. J. (1951) Lowry, 0. H., Rosebrough, J. Biol. Chem. 193, 265-275. Higgins, G. M., and Anderson, R. M. (1931) Arch. J'athol. 12, 186-202. Fuji&a, M., Koga, M., and Lieberman, I. (1963) J. Biol. mem. 238, 34°1-3406*
624